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Key words: expression analysis, expressed sequence tags, large-scale sequencing, molecular breeding, Phaseomics
consortium, P. vulgaris, Rhizobium
Abstract
Globally, 800 million people are malnourished. Heavily subsidised farmers in rich countries produce sufficient
surplus food to feed the hungry, but not at a price the poor can afford. Even donating the rich world’s surplus to
the poor would not solve the problem. Most poor people earn their living from agriculture, so a deluge of free food
would destroy their livelihoods. Thus, the only answer to world hunger is to safeguard and improve the productivity
of farmers in poor countries. Diets of subsistence level farmers in Africa and Latin America often contain sufficient
carbohydrates (through cassava, corn/maize, rice, wheat, etc.), but are poor in proteins. Dietary proteins can take
the form of scarce animal products (eggs, milk, meat, etc.), but are usually derived from legumes (plants of the bean
and pea family). Legumes are vital in agriculture as they form associations with bacteria that ‘fix-nitrogen’ from the
air. Effectively this amounts to internal fertilisation and is the main reason that legumes are richer in proteins than
all other plants. Thousands of legume species exist but more common beans (Phaseolus vulgaris L.) are eaten than
any other. In some countries such as Mexico and Brazil, beans are the primary source of protein in human diets. As
half the grain legumes consumed worldwide are common beans, they represent the species of choice for the study
of grain legume nutrition. Unfortunately, the yields of common beans are low even by the standards of legumes,
and the quality of their seed proteins is sub-optimal. Most probably this results from millennia of selection for
stable rather than high yield, and as such, is a problem that can be redressed by modern genetic techniques. We
have formed an international consortium called ‘Phaseomics’ to establish the necessary framework of knowledge
and materials that will result in disease-resistant, stress-tolerant, high-quality protein and high-yielding beans.
Phaseomics will be instrumental in improving living conditions in deprived regions of Africa and the Americas. It
will contribute to social equity and sustainable development and enhance inter- and intra-cultural understanding,
knowledge and relationships. A major goal of Phaseomics is to generate new common bean varieties that are not
only suitable for but also desired by the local farmer and consumer communities. Therefore, the socio-economic
dimension of improved bean production and the analysis of factors influencing the acceptance of novel varieties
will be an integral part of the proposed research (see Figure 1). Here, we give an overview of the economic and
nutritional importance of common beans as a food crop. Priorities and targets of current breeding programmes
are outlined, along with ongoing efforts in genomics. Recommendations for an international coordinated effort to
join knowledge, facilities and expertise in a variety of scientific undertakings that will contribute to the overall
goal of better beans are given. To be rapid and effective, plant breeding programmes (i.e., those that involve
crossing two different ‘parents’) rely heavily on molecular ‘markers’. These genetic landmarks are used to position
Economic and nutritional importance they have been adapted, and morphological variability.
They are found from sea level up to 3000 m above sea
Introduction level, are cultivated in monoculture, in associations, or
in rotations. Beans are consumed as mature grain, as
Beans (Phaseolus spp. L.) are one of the most ancient immature seed, as well as a vegetable (both leaves and
crops of the New World. Together with maize and pods). Their genetic resources exist as a complex array
cassava, they have been a dominant staple in the low of major and minor gene pools, races and intermedi-
to mid-altitudes of the Americas for millennia. Beans ate types, with occasional introgression between wild-
are extremely diverse crops in terms of cultivation and domesticated-types.
methods, uses, the range of environments to which Beans are thus a crop that is adapted to many
niches, both in agronomic and consumer preference
57
Figure 1. Flow-chart showing the relationships between the various components of Phaseomics.
terms. Since fruits (pods) can be obtained in as little as tivated on a commercial scale, and are now offered in
2 months, rotations are possible with other crops dur- national, regional and international markets.
ing short growing seasons. Short bush growth habits
offer minimal competition and permit inter-planting
Importance of beans
with other species, for example, in reforestation pro-
jects or among fruit trees or coffee plantations during Production
the early years until the main crop can be exploited.
Beans are the most important grain legumes for di-
At the other extreme, aggressive climbers are found
rect human consumption in the world. Total produc-
at higher altitudes on subsistence farms where a few
tion exceeds 23 million metric tonnes (MT) (Tables
plants are maintained as a sort of insurance and are
1,2), of which 7 million MT are produced in Latin
continually harvested for about 6 months. Over the
America and Africa. Bean production is almost twice
past 20 years, beans have also been increasingly cul-
that of chickpea, which is the second most important
grain legume. Social factors and ecological constraints
58
Table 1. Bean production in Latin America
determine whether beans are grown in a particular re- Table 2. Bean production in Africaa
gion. As agriculture and social systems have evolved Region Area Area
together, the current state of farming systems is the (%) (ha×10−3 )
result of the interaction of climatic, edaphic, biotic and
social factors. Eastern Africa – highland and mid-altitude
A large part of bean production in Latin America (Burundi, DR Congo, Ethiopia, Kenya,
(Table 1) takes place on small farms ranging from 1 Rwanda, Tanzania, Uganda) 62 2490
Southern Africa (Lesotho, Madagascar,
to 10 ha in size, often on sloping land of low fertility.
Malawi, Mozambique, South Africa,
Some estimates suggest that as much as 80% of the
Swaziland, Tanzania, Zambia, Zimbabwe) 31 1290
area planted with common beans in Latin America
Western Africa (Angola, Cameroon, Cape
is found on hillsides. Moreover, these smallhold- Verde, Togo) 3 135
ings are dispersed, making it difficult to define main Lowlands-winter season (Algeria, DR Congo,
production areas. Egypt, Mali, Malawi, Mauritius, Morocco,
Except for Argentina, where most beans are pro- Nigeria, Sudan, Tunisia) 4 200
duced on large holdings in modern production sys-
tems, small landholders usually cultivate beans in TOTAL 100 4025
Latin America. In Brazil, about one-third of total bean a Modified from ‘Atlas of Common Bean Production in Africa’
output is produced on farms of less than 10 ha. In (Wortmann et al., 1998).
Mexico, an estimated 67% of production comes from
even smaller farms (<5 ha). Even in Chile, which ex-
ports much of its production, beans are grown by some
million hectares produced in more than 20 countries
50 000 farmers whose plots vary from 2 to 6 ha and
(Table 2). As in Latin America, resource-poor farmers
a smaller number of medium-size growers who plant
with very few inputs, grow beans primarily on small-
20–30 ha. Regionally, more than half the production
scale, marginal farms. In Africa, women farmers, who
occurs on farms smaller than 20 ha and more than
have little access to fertiliser compared to men far-
20% on farms of less than 5 ha. The extreme cases
mers, more often grow beans. Intercropping of beans
are represented by countries like Haiti, the Lesser
with cereals (maize, millet or sorghum), bananas and
Antilles, and Paraguay where production is almost
plantains or root and tuber crops is common practice.
exclusively in the hands of small-farm families. In
Given these problems, it is not surprising that average
Mexico, Brazil, Chile and Cuba, it is possible to find
yields are low. Much of the bean crop is lost to diseases
small, medium and large-scale bean producers. Even
as well as insect pests or drought, low soil-fertility and
in Brazil where large-scale agriculture has been widely
other abiotic stresses. Higher-yielding climbing beans
promoted, only about 4% of the area and 15% of the
have been adopted in some areas of greater population
bean production is derived from high input irrigated
density. Many varieties of beans are grown in Africa,
systems.
with notable diversity in seed types and adaptation.
Beans are also a very important food crop in many
Local and market preferences as well as the variability
parts of Eastern and Southern Africa with over four
in climatic and agronomic conditions generally dictate
59
Table 3. Per capita bean consumption in several Latin American and
African countries – by region and/or economic strata where data are
however, which is well above the 1.7% population
available growth rate. As virtually all beans produced are con-
sumed within the region, this suggests per capita
Country/region Average annual Range over consumption has increased modestly. Production has
consumption economic increased by 16% in the Andean zone demonstra-
(kg) strata ting the potential of augmenting consumption through
Latin America greater production. Bolivia is another case in point.
Mexico 16+ Consumption in rural areas around Santa Cruz was
Durango 18∗∗ 10–26∗∗ low but since beans have become an important cash
Honduras 13.0 11.2–15.9 crop in the region, consumption has reached one of
Nicaragua 14 the highest levels in the Americas (24 kg yr−1 ). Docu-
Guatemala 10 mentation of consumption through household surveys
Costa Rica 11 shows that it continues to be high in traditional bean
El Salvador 13.5 consuming countries. For example, in Brazil, the two
Colombia 4.3∗ regions with the highest consumption are the northeast
Cali (lowest strata) 9.8 (20.8 kg yr−1 ) and the southeast (18.2 kg yr−1 ), which
Medellin 12.8 11.5–14.3 are, respectively, the least and the most developed re-
Ecuador 6 gions. Those few cases in which consumption has been
Bolivia
broken down into family incomes show that consump-
Sta. Cruz (urban) 6
tion in lower income strata is as much as 20% higher
Sta. Cruz (rural) 24
than average figures would indicate. In this sense,
Brazil 17.2
beans are the ‘poor man’s meat’ and play a particu-
Southeast 18.2
Northeast 20.8
larly important role in the diet of the underprivileged.
Africa
Available consumption data are presented in Table 3.
Kenya 12 Beans are a major staple of eastern and southern
Kisii 66 Africa. In these areas, yearly bean consumption is as
Rwanda 48 high or higher than in Latin America reaching up to
Uganda 11 66 kg per person in some rural areas of Kenya. In both
Mbale 58 Rwanda and Burundi statistics show that the average
national consumption exceeds 40 kg per person per
+ High end value, although consumption fluctuates from 10 to 16
kg/yr as availability varies from year to year.
year. Beans are estimated to be the second most im-
∗ Based on national production figures but ignoring importations. portant source of dietary protein and the third most
∗∗ Estimated from reported family consumption.
important source of calories in the region. Beans are
often combined with such energy sources as maize,
plantains or root crops. The high nutritional quality of
which varieties are most popular. There is some bias beans in terms of percentage protein is an important
towards the large-seeded types however, especially in complement to these starchy foods. In addition the
the Great Lakes and highland regions of Eastern and high mineral content of beans, especially of iron and
Central Africa, where many farmers grow and main- zinc, are advantageous in regions where there is a high
tain seed mixtures of all sizes and colours. The grain prevalance of micronutrient deficiencies such as iron
is an important cash-crop. Marketing of beans occurs deficiency anemia.
locally and across established trade routes usually to
urban areas within the same country of production. Dietary proteins
Bean leaves are also an important vegetable in parts Beans provide dietary proteins that play an essential
of the African continent. role in human nutrition by complementing other foods
(e.g., maize in the Latin American highlands and East-
Consumption ern Africa and rice in Brazil) that are primarily sources
It is commonly believed that demand for beans is of carbohydrates. Bean seeds contain between 20 and
income-inelastic, and that consumption drops as eco- 25% proteins, much of which is made up of the storage
nomic levels rise. Bean production in Latin America protein phaseolin (Ma and Bliss, 1978). Phaseolin is
has increased by 3% per year over the past decade a major determinant of both quantity and nutritional
60
Table 4. Mineral contribution of beans assuming 15 kg per capita
quality of proteins in bean seeds (Bliss and Brown, annual consumption
1983; Gepts and Bliss, 1984). Like other seed pro-
teins of the legume family, phaseolin is deficient in Nutrient Content of Adult male % Adult
sulphur-containing amino acids such as methionine. average daily requirement requirement
Seed proteins of cereals generally contain sufficient serving (125 g (mg) in one serving
sulphuryl amino acids but are themselves deficient in cooked)
other essential amino acids such as lysine. Combined Sodium 0 mg 2200 0
consumption of cereals and legumes generally allevi- Potassium 475 mg 3900 12
ates these mutual deficiencies ensuring a balanced diet Calcium 65 mg 800 8
when cereals and legumes are consumed in the ratio Phosphorus 161 mg 800 20
of 2:1 (Bressani, 1983). Unfortunately, this is seldom Magnesium 56 mg 350 16
the case as legume yields are generally low. Thus, Iron 2.78 mg 10 27
increasing legume yields has important repercussions Zinc 1.24 mg 15 8
on improving nutrition and health of hundreds of mil- Copper 0.307 mg 2.5 12
lions of bean consumers in the world, especially in Manganese 0.668 mg 3.75 18
developing countries. Selenium 0.002 mg 0.05–0.2 1–4
Iodine 0.032 mg 150 0
Vitamins and minerals Starch 22.1 g 570 g (2750 kcal.) 4
Protein 8.5 g 69 g 12
Vitamins Biotin is an essential cofactor for a variety
of carboxylases and decarboxylases found in diverse Adapted from Pennington and Young (1990a,b) and Robinson
metabolic pathways of all organisms (Knowles, 1989). (1987).
It plays a central role in membrane biogenesis, cata-
bolism of some amino acids and the production of
oxaloacetate. Despite this ubiquitous requirement for • All the known bacterial intermediates of bi-
biotin, its de novo synthesis is restricted to plants and otin synthesis including the novel metabolite 9-
some microbes. In order to meet the daily requirement mercaptodethiobiotin, have been found in plants
in the human diet, the vitamin is routinely added to (Baldet et al., 1993a, 1997).
fruit juices and other food products. It is also added • Two embryo-arrested mutants of Arabidopsis
to many health care and cosmetic products. A major thaliana were found to be biotin auxotrophs: the
part of the biotin produced is used directly to increase bio1 mutant is defective in DAPA aminotransferase
the biotin contents of livestock and other animal feeds. (Schneider et al., 1989) and could be complemen-
Most of the biotin commercially available is currently ted by the E. coli bioA gene (Patton et al., 1996a);
synthesised in a chemical process that is complex the second Arabidopsis mutant, bio2 was found to
(comprising 13 steps), requires large energy inputs and be defective in the final step of biotin synthesis,
generates considerable waste. An alternative way of i.e., the conversion of dethiobiotin to biotin (Patton
supplementing dietary biotin would be through the de- et al., 1998). Furthermore, a cDNA clone encoding
velopment of plants, fruits and seeds with high biotin an A. thaliana homologue of the bacterial bioB has
contents. been isolated (Patton et al., 1996b; Weaver et al.,
Preliminary data suggest that the biotin status va- 1996). In general, bacteria use all of their biotin
ries among plants, during the stage of development to biotinylate biotin-containing proteins (Cronan,
and with conditions of growth. Accordingly, the isol- 1989), whereas, plants accumulate most of their
ation, identification and characterisation of the bio- biotin as the protein-free molecule (Baldet et al.,
genes of P. vulgaris will widen our understanding of 1993b; Shellhammer and Meinke, 1990; Wang et
the optimal conditions and of the most suitable plant al., 1995). In pea leaves, the free biotin pool in
tissue for obtaining large amounts of free biotin. Inclu- the cytosolic compartment accounts for 90% of
sion of these foods in human and livestock diets would the total (free plus protein-bound) biotin (Baldet et
provide important benefits for general health. al., 1993a). Biotin synthesis may thus occur in the
Recent evidence suggests that the biotin biosyn- cytosol (Baldet et al., 1993b; Shellhammer, 1990).
thetic pathway may be very similar in plants and
bacteria (reviewed by Streit and Entcheva, 2003) and Minerals Most measures of health in the develo-
includes: ping world have shown gradual improvement over
61
the last 50 years. Micronutrient deficiencies (espe- stances and tannins (Kigel, 1999). Tannin contents
cially iron) have become more common however, even change with seed colour. The anti-nutritive proper-
in developed countries. Cereals normally make up ties of phytates stem from their ability to chelate
the bulk of diets composed of basic grains and sup- calcium, iron, magnesium and zinc. A multi-gene fam-
ply the greater energy component. Legumes on the ily encodes lectins. Interestingly, although arcelins
other hand contribute more of the other components of and α-amylase inhibitors render bean seeds less pa-
diet. Legumes are much superior to cereals as sources latable, they serve protective functions in the fruit.
of micronutrients (Welch et al., 2000) first because Arcelins and α-amylase inhibitors have insecticidal
legumes have a higher initial content of minerals, and properties, while α-amylase inhibitors confer resist-
second since many cereals are polished before eating ance to bruchid beetles since these glycoproteins are
(for production of white rice or wheat flour for white toxic to their larvae (Shade et al., 1994). Thus, given
bread, etc.). As a significant proportion of the minerals the wide-variations present in the gene pools, breed-
are found in the seed coat (or bran) they are discarded ing for lower contents of anti-nutritional compounds
during processing. Most legumes, including common should be possible in beans. It is necessary however,
beans, are consumed whole. As a result their min- to avoid weakening the plant by decreasing some of
eral content is conserved. Consumption of beans in its protective functions.
Latin America thus represents a significant contribu-
tion to human nutrition. Beans are an important source Phytic acid Phytic acid is the main seed storage
of iron, phosphorus, magnesium, manganese, and in molecule for phosphorous. Phytic acid is necessary
lesser degree, zinc, copper and calcium (Table 4). At for normal seed development and germination al-
levels of consumption commonly found in people of though its concentration in different bean varieties
restricted economic means (15–20 kg yr−1 ), beans is variable (Lolas and Markakis, 1975). Phytic acid
provide 10–20% of the adult requirement for a number (myo-inositol hexaphosphate) and its salts (phytates)
of nutrients. represent between 54 and 82% of the phosphorous
content of the bean i.e. between 0.5 and 1.6% of the
Culinary and nutritional quality seed weight (Lolas and Markakis, 1975). Embryogen-
Unfortunately, the culinary and nutritional quality of esis continues up to 36 days but the peak accumulation
many bean varieties leave much to be desired. Gen- is between 24 and 30 days, which normally coin-
erally, bean seeds need to be soaked and must be cides with the high level of inorganic phosphorous
cooked to render them palatable. Cooking inactivates in the cotyledon (Walker, 1973). Walker (1973) also
heat-labile anti-nutritional compounds as well as per- showed that phytase was undetectable during the same
mits the digestion and assimilation of proteins and period and that the activity of this enzyme was first
starch (Kigel, 1999). Cooking beans also solubilises measurable two days after germination.
the proto-pectin within the middle lamella forming Phytic acid chelates various divalent metal ions and
soluble pectin that depolymerises rapidly during heat- is implicated in their reduced absorption leading to
ing, allowing water to enter cells of the cotyledon deficiency symptoms in animals and humans in di-
(Stanley and Aguilera, 1985). Modifying the compos- ets predominated by legume seed proteins (Sandberg
ition of the middle lamella may thus render bean seeds et al., 1993). The catabolism of phytate is controlled
easier to cook (see Table 8, p. 75). by phytase and some other acid phosphatases that al-
Flatulence in humans is often the result of ingest- low the phosphorous to be assimilated. Without these
ing foods high in raffinose, stachyose and verbascose. enzymes, phytate passes through the intestinal sys-
Although these sugars are indigestible, micro-flora of tem without being degraded, so contributing to the
the lower intestine ferment them producing gas (Ki- P load of the resulting manure (Lott et al., 2000).
gel, 1999). As considerable variability among various This is already becoming a problem in Europe and
bean genotypes for their propensity to induce flatu- North America, where it accelerates the eutrophica-
lence exists, it seems likely that molecular breeding tion of waterways and reservoirs. On the other hand,
programmes should help alleviate this problem (Table recent research has revealed the possible therapeutic
8). properties of phytate in the prevention of cancers of
Anti-nutritional factors are present in the seeds the breast and colon, probably due to its anti-oxidant
of many legumes. Amongst them are α-amylase in- properties. Phytate has also been implicated in the
hibitors, arcelins, lectins, phytates, phenolic sub- reduction of cholesterol and other lipids due to its
62
presence in high fibre diets (Midorikawa et al., 2001; work. Introduction of Brazilian types of beans for ex-
Reddy, 1999; Shamsuddin et al., 1997; Thompson and port offered the possibility of winter cultivation that
Zhang, 1991). has been widely accepted. Although these grain types
Various phytic acid mutants have been described earn lower prices than the traditional varieties, net in-
in the literature for rice, Larson et al. (2000), maize, comes were estimated at $113 ha−1 , (or $248 when
Raboy et al. (2000) and wheat, Raboy et al. (1991). family labour is not counted). Given that a farmer
Basically, a reduction in the phytate content was ob- would otherwise have to abandon his home for sev-
served in all the mutants but the overall concentration eral months, the higher figure reflects more accurately
of phosphorous was not reduced and was compensated the value of the bean crop. Farmers attribute their im-
by a corresponding increase in the inorganic phosphor- proved wellbeing to income from beans, and cite such
ous content. Analysis of the levels of phytic acid and additional benefits as improved nutrition for the family
total protein content of the seeds revealed that there ex- as well as increased educational opportunities for their
ists a positive correlation between these two variables children.
(Raboy et al. 1991). The authors suggest that a se-
lection for low phytic acid could result in undesirable
reductions in the protein level. Globalisation
The past decade has seen the development of an inter-
Economics of bean production national market for beans that now exceeds 2.4 million
MT. According to the Food and Agricultural Organisa-
Cost analysis tion of the United Nations (FAO), China and Myanmar
Much poverty in Africa and Latin America is found are the largest exporters (19% each of total exports!)
in rural areas, and thus the success of agriculture but part of this volume undoubtedly represents other
is a central issue in ameliorating living conditions. legumes. Nonetheless, these two countries stand out
Legumes in general are considered to be relatively for their low costs of production. Other important
profitable crops compared to other options such as exporters include the United States (18%), Argen-
cereals, and beans are no exception. For example, tina (12%) and Canada (6%). Within Latin America,
in Brazil, large-scale farmers who recover their in- Mexico, Brazil, Venezuela and Cuba are major im-
vestment on irrigation systems count on beans for a porters. Costa Rica, a traditional bean producer, now
quick profit. In Central America small farmers re- imports 50% of the beans consumed. At present the
port that among the traditional field crops, beans are most widely traded cultivars are pinto and black beans,
the best income generator. Recent cost analyses of but other classes may be produced shortly for markets
bean production confirm that beans remain profit- such as Central America. In a very real sense, this re-
able. In Nicaragua, farmers were separated into two presents a challenge to both large and small producers
groups; those using a landrace variety and those us- in the developing world, and draws them directly into
ing an improved variety. Unsurprisingly, farmers using the arena of world competition. Competition heigh-
the improved variety enjoyed much greater profits tens the problems of small bean producers that have
($390 vs. $136 ha−1 ) that are due to higher yields. few other sources of income. Thus, competitiveness is
Thanks to these higher yields, production would still a major concern for bean production in Latin Amer-
be profitable even if prices were 40% lower. ica, and in most cases must be met by higher yields.
In Colombia, large-seeded Andean types are pre- In a study of competitiveness by Hertford and Garcia
ferred because they command better prices and higher (1999) of several crops in Latin America, beans were
profits than the small-seeded types used in Central found to be reasonably competitive across most of the
America. Small farmers in the Santander department region (with notable exceptions of Mexico and Brazil,
earned from US$960 to $1153 ha−1 yr−1 (over two which probably have large internal differences in com-
production seasons) with improved varieties compared petitiveness). Argentina was by far the most compe-
to $260 ha−1 yr−1 earned with the local cultivar Radi- titive of all bean-producing countries. Guatemala was
cal. Again, increased income was due largely to higher generally a very poor competitor in agriculture, but
yields. Immigrant families from the Sierra have co- beans are one of the few crops in which it did well. In
lonised the eastern plains of Bolivia that previously Africa, most trade in beans is within a single country
enjoyed only one planting season per year. Winter was or informally between countries. As a result, imports
a time of want and migration to other regions to seek are generally small.
63
Table 5. Grain legumes: tribal affiliations, genome organisation and world productiona
ten result in excessive pesticide use (e.g., with snap species will have valuable repercussions on other
beans). Access to the necessary infrastructure that in- species of the same tribe.
cludes markets is another limitation, especially for In recent years, several studies have clarified the
small farmers who are familiar with their traditional phylogenetic relationships among Phaseolus species
bean varieties. in general, and P. vulgaris, in particular. Studies
on cpDNA (Delgado-Salinas et al., 1993) and ITS
sequences (Delgado-Salinas et al., 1999) have es-
Genetic improvement tablished a phylogeny for the entire genus. A basal
species has been identified, P. microcarpus. P. vul-
Phylogeny garis belongs to a complex of species, that include
P. acutifolius, P. coccineus, and P. polyanthus. Mo-
The legume family (Leguminosae) is very large with lecular data have thus confirmed that all these species
643 genera (18 000 species) grouped into 40 tribes that can be inter-crossed, although the degree of difficulty
are found in both tropical and temperate environments and the viability of reciprocal crosses vary (Hucl and
(Lavin et al., 1990; Mabberley, 1998; Pollhill, 1981, Scoles, 1985; Waines et al., 1989). Remarkable di-
1994). The tribe Phaseoleae [common beans (P. vul- versity of morphology occurs within this group of
garis), long-beans/cowpeas (Vigna unguiculata), and species (bushes to climbers, seed colour and colour
soybeans (Glycine max)] is by far the most important patterns), adaptation (from hot deserts to cool moun-
economic group, and contains 75% of the legumes tain environments), and reproductive systems (from
traded in the world (Table 5). Other tribes inclu- cleistogamy to out-crossing). In this sense too, the P.
ding Aeschynomeneae [peanuts (Arachis hypogaea)], vulgaris complex provides a model to study the mo-
the galegoid group that contains the Cicereae [chick- lecular basis of agronomically important phenotypes
peas or garbanzo (Cicer arietinum)], Trifolieae [al- among closely related lines.
falfa (Medicago sativa), lentils (Lens culinaris), peas Intra-specific organisation of genetic variation in P.
(Pisum sativum), and Vicieae [field beans (Vicia faba)] vulgaris has been well studied. A nucleus of diversity
are also widely cultivated. Of these tribes, only those is located in Ecuador and northern Peru (Coulibaly,
of the galegoid group are temperate. As the tropical 1999; Kami et al., 1995), from which wild beans dis-
and temperate tribes diverge markedly it remains to persed both northwards and southwards to form two
be demonstrated whether a legume such as Medicago geographically distinct gene pools in Mesoamerica
truncatula, which is touted as a model species for the and the southern Andes (reviewed in Gepts, 1998). In
entire legume family, can reach across this divide and turn, post-domestication divergence gave rise to three
provide useful information relevant to tropical grain domesticated races in each of these two gene-pools
legumes. Co-linearity or synteny among all legume (Singh et al., 1991; see Figure 2).
genomes is essential if one legume is to serve as the These geographically distinct gene-pools qualify
model for all others. Although high-levels of syn- as sub-species based on the existence of partial re-
teny have been found in the Trifolieae (Weeden et productive isolation between them. Pairs of comple-
al., 1992) as well as amongst diploid species of the mentary genes that influence either the F1 (dominant
Phaseoleae, co-linearity is much less evident with alleles) or later generations (recessive alleles) are ge-
more distantly related genera such as soybeans (Boutin netically responsible for the isolation (Gepts and Bliss,
et al., 1995). It is thus too early to say whether 1985; Koinange and Gepts, 1992; Shii et al., 1981;
the extensive synteny found in the Gramineae (Ben- Singh and Molina, 1996). Furthermore, it has of-
netzen and Freeling, 1997) also applies to legumes, ten been difficult to obtain high-yielding genotypes
highlighting the need to study nodal species in the in Andean×Mesoamerican crosses because of out-
Leguminosae, including P. vulgaris. In addition to breeding depression (Beaver and Kelly, 1994; Johnson
food plants such as beans, long-beans, pigeon peas, and Gepts, 1999; Kelly et al., 1998; Welsh et al.,
soybeans, jícama (Pachyrhizus erosus), and several 1995). Preliminary estimates show a divergence time
locally important species such as Bambara groundnut of some 500 000 years between these two gene-pools
(Vigna subterranea), this tribe also contains forage (Coulibaly, 1999). Thus, P. vulgaris is, at this stage,
and ornamental species. Thus, a co-ordinated and unique among crops in that two evolutionary lineages
integrated genomics/transcriptomics/proteomics pro- tracing back to the same ancestral populations have
gramme in the economically most important legume been identified. Similar information for other species
65
Cali, Colombia, and elsewhere (pp. 88–89). At CIAT, nuclear genome of P. costaricensis (Debouck, 1999).
the numbers of accessions belonging to the secondary P. coccineus and its allies may thus be the reservoir
and tertiary gene pools, respectively, are 1049 and 335. of diversity with greatest potential once the primary
In spite of this diversity, the genetic base of commer- gene pool and the P. vulgaris phylum have been fully
cial cultivars of specific market classes is narrow. In exploited.
fact, only a small portion (<5% of the available ge- ‘Congruity backcrosses’ coupled with the careful
netic diversity) has been used globally despite nearly choice of donor parents amongst P. vulgaris and P.
a century of organised bean improvement. acutifolius (a species belonging to the tertiary gene
Systematic evaluation of wild common bean as pool of P. vulgaris) accessions are a promising new
well as wild and domesticated germplasm of alien spe- method of improvement (Mejia-Jiménez et al., 1994).
cies for resistance to pests, diseases and other useful Nonetheless, embryo rescue techniques are needed
traits has been limited. Nevertheless, alien germplasm and F1 hybrids are completely male-sterile. P. fili-
seems to be a promising source of common bean im- formis and P. angustissimus have also been crossed
provement as resistance to bruchids was found in wild with common bean but rescued hybrid plants were
P. vulgaris. P. polyanthus is well known for its resist- completely sterile (Baudoin, 2001). Chromosome
ance to ascochyta blight as well as to BGYMV (Bean doubling has been attempted to overcome incompa-
Golden Yellow Mosaic Virus). P. costaricensis might tibility barriers but may not be very useful given the
also be a source of BGYMV resistance genes. P. coc- difficulty of exploiting amphidiploids. P. parvifolius
cineus is a source of resistance to anthracnose as well crosses easily with P. acutifolius, and has been crossed
as root rots, white mold, BYMV (Bean Yellow Mosaic with P. vulgaris (using embryo rescue). In spite of
Virus) and BGYMV. Tolerance to leaf-hoppers exists this work, its potential usefulness for common bean
in P. acutifolius, and high levels of resistance to CBB improvement has yet to be determined. An unrealised
(Common Bacterial Blight) and bruchids are found dream of combining the potential of Lima bean (part
in some accessions of tepary bean (Baudoin, 2001; of the quaternary gene pool) that is well adapted to tro-
Debouck, 1999; Schmit, Baudoin, 1992; Singh, 1999). pical conditions, with the genome of the common bean
Thus, major production constraints, lack of re- has failed to produce fertile hybrids. The reciprocal
sistance to diseases/pests, as well as slow progress cross, P. lunatus×P. vulgaris was even less successful,
in identifying useful genes in related species have confirming the taxonomic positions of common and
led to the widespread adoption of interspecific hy- Lima beans in the genus (Baudoin et al., 1995).
bridisations among Phaseolus species. From 1940 to The major reproductive barrier to interspecific hy-
1985, P. vulgaris and P. coccineus were frequently bridisation amongst the genus Phaseolus occurs post-
intercrossed. It was observed however, that in recip- fertilisation, especially during early embryo develop-
rocal crosses using P. coccineus as the female parent, ment (Baudoin et al., 1995). When maintained in vivo,
segregants naturally reverted to the cytoplasm donor embryos resulting from P. polyanthus (female)×P. vul-
parent after a few generations (Baudoin et al., 1995). garis crosses develop poorly despite the close phylo-
Major genes have established a barrier between these genetic relationship of these species. Infertility in
two species, and chromosome pairing is not perfect. P. polyanthus×P. vulgaris crosses results from early
Since reproductive isolation may be due to domestica- nutritional barriers that are related to a deficient endo-
tion, attempts were made to cross P. vulgaris with wild sperm tissue development while in reciprocal crosses,
variants of P. coccineus. Nevertheless few commercial endothelium proliferation, and to some extent, hyper-
cultivars have been created this way. P. polyanthus trophy of the vascular elements are causes of early
crosses more easily with P. coccineus and related embryo abortion (Geerts, 2001; Geerts et al., 1999,
forms than with P. vulgaris, particularly if the latter is 2002; Lecomte et al., 1998). To a large extent, the
the pollen donor (Baudoin et al., 2001). P. polyanthus importance of these abnormalities depends on the
belongs to the P. vulgaris clade, but its nuclear genome compatibility between the genotypes used as parents.
has been introgressed with P. coccineus genes and this Although several hybrids between P. vulgaris and spe-
limits its use in interspecific hybridisations. Especially cies belonging to its tertiary gene pool can only be ob-
when P. vulgaris is used as the female parent, crosses tained by embryo rescue, most infertility results from
between P. vulgaris and P. costaricensis are simple male sterility which is caused by incomplete chromo-
to perform without embryo rescue, but it is not clear somal pairing in Metaphase I (Baudoin et al., 1995).
whether P. coccineus genes have contaminated the Where sterility of hybrids precludes any form of in-
67
Figure 3. Chromosomal location of genes involved in the domestication syndrome in beans. Genes in bold are those that are presumably
important in a conversion programme: Ppd – photoperiod sensitivity; fin – determinancy; St – presence of pod suture fibres; DO: QTL for seed
dormancy; SW: QTL for seed weight (after Gepts, 1999b).
by a combination of traits comprising determinacy, areas at higher latitudes led to a selection of geno-
non-twining branches, few vegetative nodes, and long types that are insensitive to daylength compared to the
internodes. Less evolved growth habits may show wild progenitor, which will only flower under short
some or only one of these traits. Selection by hu- days. In concert with the changes in growth habit and
mans has also led to pods and seeds that are larger photoperiod sensitivity, common bean cultivars gen-
(‘gigantism’) and show different or no anthocyanin erally flower earlier than their wild ancestors. The
pigmentation. The dissemination of cultivated beans genetic control of this complex arrray of traits has
from their domestication centres in the tropics to new been super-imposed on a linkage map (Figure 3, Fig-
69
ure 4). Genetic control of the domestication syndrome yields in developed countries between 1959 and 1990
involves genes that have a major effect and account for are directly attributable to a 10-fold increase in N
most of the variation observed (>60%). As domestica- fertiliser application. Concomitant with high rates of
tion of the common-bean probably proceeded rapidly, application of N fertilisers in developed countries are
adaptation to rapidly changing environmental condi- volatilisation of N oxides (greenhouse gases) into the
tions must have involved genes with major phenotypic atmosphere, depletion of non-renewable resources,
effects (Koinange et al., 1996). an imbalance in the global N cycle, and leaching
of nitrate to groundwater. By contrast, in develop-
Genome ing countries the high cost of N fertiliser, the energy
Among the species recognised as major crops by requirements for production, and the suboptimal trans-
the USDA, the genome size of beans (450–650 portation capabilities limit its use, especially on small
MBp/haploid genome – Bennett and Leitch, 1995) farms (Vance, 1997).
is small and comparable to that of rice (340–560 One of the driving forces behind agricultural sus-
MBp/haploid genome – Bennett et al., 2000), which tainability is effective management of N in the envi-
is generally considered to be the economically import- ronment. Successful manipulation of N inputs through
ant plant with the smallest genome. Cytogenetically, the use of biologically fixed N results in farming
common bean is a true diploid with 11 chromosomes. practices that are economically viable and environ-
There is no evidence for poly-ploidisation. During cer- mentally prudent. Although many diverse associations
tain stages of development, polytene chromosomes ap- contribute to symbiotic N fixation, in most agricultural
pear in such readily accessible tissues as the pulvinus. settings the primary source (80%) of biological fixed
From the limited molecular research that has been N is through the soil bacteria Rhizobium-legume sym-
published, the gene families tend to be small. The actin biosis (Vance, 1997). Legumes provide 25–35% of the
gene family has six members, and traditionally large worldwide protein intake. Important agricultural goals
families such as resistance gene analogues (Rivkin et include enhancing the use of and improving the man-
al., 1999) and protein kinases (Vallad et al., 2001) are agement of biologically fixed N by legumes for both
of moderate size. A large resistance gene-cluster has humanitarian and economic reasons (Vance, 1997).
also been identified (Geffroy et al., 1999). Many genes Nitrogen fixing species have played an integral role
are well characterised. In particular, a detailed molecu- in cropping systems since the domestication of plants
lar genetic analysis of the important seed coat colour and have prominently featured in rotations and inter-
and pattern genes that lead to the nutritionally import- cropping systems, as alley crops, in pasture systems,
ant isoflavones was recently completed (Bassett et al., as green manures, in agroforestry, and cover crops.
1999a,b, 2000; Bassett and McClean, 2000; Brady More than 50% of the crops grown in Africa, India and
et al., 1998). Detailed phylogenetic analyses point Latin America are either intercropped or rotated with
to the origin of many of the domestication traits that N fixing species. Nevertheless, improved strategies
are important in current agronomic production. These must be developed and transmitted to growers to more
studies provide much of the data necessary for the in- efficiently exploit biological N fixation (Vance, 1997).
trogression of traits that may broaden the genetic base Roots of leguminous plants often associate with
of the current breeding pool. It should be emphasized bacteria of the family Rhizobiaceae to generate highly
that most of the work on breeding has been performed specialised structures – nitrogen-fixing nodules. Bac-
on a limited set of mapping populations. These are terial cells within the nodule fix the atmospheric ni-
shown in Table 6. Finally, it should be emphasized trogen and produce ammonium that is assimilated by
that Phaseolus, in comparison to Lotus japonicus and the plant. In return, the plant supplies carbon com-
Medicago truncatula, is a tropical legume species. pounds derived from photosynthesis, for maintenance
of the bacteria. Many genes from both organisms are
Rhizobium-legume symbioses required for the establishment and optimal functioning
Nitrogen is the major limiting nutrient for most crop of this symbiosis.
species. Acquisition and assimilation of N is second The original microsymbiont of Phaseolus vulgaris
in importance only to photosynthesis for plant growth is Rhizobium etli (Segovia et al., 1993). The gen-
and development. Production of high-quality, protein ome of this Gram-negative bacterium is distributed
rich food is thus completely dependant upon the avail- among several replicons: one chromosome with the
ability of nitrogen. The large rises in cereal grain genes for maintenance and growth and from one to
70
Figure 4. Distribution of genes with a biochemical function, major genes coding for external phenotypic traits, and QTLs in the genome of Phaseolus vulgaris. To the left of each linkage
group, are the framework molecular markers (smaller font) and the genes with known biochemical function (larger font). Major phenotypic trait genes are shown in shaded boxes (see Gepts,
1999a). To the right (boxed two-letter symbols), are QTLs. CBB: common bacterial blight resistance, DF and DM: number of days to flowering and to maturity, DO: seed dormancy, HI: harvest
index, L5: length of the 5th internode, NM: number of nodes on the main stem, NN: Rhizobium nodulation, NP: number of pods, PD: photoperiod-induced delay in flowering, PL: pod length,
SW: seed weight. Location of most genes is only approximate as most were not directly mapped in the BJ population (see Table 6).
71
eight large plasmids, ranging from 100 to 700 kbp in formed and non-transformed sectors and only 0.5%
size. The genetic information present in these plas- of the explants produced transgenic seeds. Recently,
mids may constitute up to 50% of the total bacterial Aragão et al. (2002) again used bombardment tech-
genome. The plasmid carrying most of the informa- niques to transform the P. vulgaris cultivars Carioca
tion that is indispensable to an effective symbiosis is and Olathe with the phosphinothricin acetyl trans-
known as the symbiotic plasmid (pSym). R. etli strain ferase gene (bar) that encodes tolerance to the herbi-
CFN42 carries six plasmids, the pSym of which is cide ‘glufosinate’. Only 0.5% of the regenerated plants
390 kbp in size (Davila et al., 2000). Complete se- (To ) were resistant to the herbicide, and of these plants
quencing of the CFN42 genome as well as analysis only two were tolerant in the first, sexual generation
of its expression under symbiotic conditions (by tran- (T1 ). Nevertheless, these plants were resistant to herbi-
scriptomics and proteomics) has been initiated at the cides in both glass-house and field trials and have been
CIFN in Cuernavaca. So far, the complete sequence included in a Brazilian breeding programme.
of the pSymCFN42 has been obtained and annotated Dillen et al. (1997a) were the first to use
(González et al., 2003). Agrobacterium-mediated transformation of Phase-
olus. They transformed P. acutifolius A. Gray (tepary
Transformation systems beans) and provided evidence for the transmission of
Transformation of leguminous species, and of large- the transgenes to the progeny. The procedure included
seeded legumes (grain legumes in particular), is often the co-cultivation with A. tumefaciens of green nodu-
difficult. Proof of transformation requires that, at least lar callus from bud explants, and the regeneration of
for sexually propagated species, transmission of the shoots in the presence of antibiotics (kanamycin or
introduced DNA be confirmed by molecular analysis geneticin). In addition, only one transformed callus-
of the offspring of primary transformants. In addition, line yielded transgenic plants of clonal origin that were
to be of practical value, transformants should cor- fertile. Nevertheless, it should be possible to introduce
rectly express all the introduced genes. A drawback of P. vulgaris genes into P. acutifolius using this proced-
biolistic gene delivery is the often complex and unpre- ure then cross them back into P. vulgaris since the two
dictable pattern of DNA integration. As a result, there species are compatible (Dillen et al., 1997a). In the
is a growing consensus among breeders that the pre- meantime the authors, as well as CIAT (see pp. 88–
cision of the Agrobacterium tumefaciens-mediated in- 89) have improved the protocol, so that three different
tegration mechanism and its tendency to produce low- varieties of P. acutifolius can now be transformed, and
or single-copy insertions constitutes a considerable the efficiency is such that between 5–10 transformants
advantage over direct gene-transfer techniques. can be obtained in one experiment (De Clercq et al.,
Stable genetic transformation of beans via particle 2002; Zambre et al., 2003). Within the grain legumes,
bombardment has been reported, albeit at very low P. acutifolius is thus now one of the few species for
frequencies. Transgenic navy bean plants (P. vulgaris which the number of transformed plants that can be
cv. Seafarer) have been obtained following electric- generated is large enough to enable the application of
discharge mediated particle acceleration. After bom- transgenic techniques. It is possible to introduce P.
bardment, bean-seedling meristems were subjected to vulgaris genes into P. acutifolius using this proced-
a time-consuming tissue culture protocol involving ure (Mejía-Jiménez et al., 1994). Of course we realise
shoot-induction. Nevertheless, transformed plants that the deliberate release of transgenic plants is con-
were recovered at a very low frequency (0.03%)(Rus- troversial and for this reason, transformed beans will
sell et al., 1993). Aragão et al. (1996) reported the only be used to answer biological questions in the first
regeneration of transgenic beans via particle bombard- instance.
ment using a high-pressure helium device to introduce
DNA into embryogenic axes. Shoot formation was in- BAC and cDNA libraries
duced from bombarded explants, shoots were rooted Several BAC libraries exist or are being developed
and stable transgenic plants were generated at an aver- for various genotypes – Sprite (see Vanhouten and
age frequency of 0.9%. Kim and Minamikawa (1996) MacKenzie, 1999; and p. 111 – UN/L – Sally MacK-
reported the regeneration of transgenic P. vulgaris enzie), Phaseolus lunatus cv. Henderson (lima bean),
cv. Goldstar plants via gold particle bombardment of P. vulgaris DGD 1962, P. vulgaris cv. BAT93, and
shoot apexes of embryogenic tissues. Unfortunately, P. vulgaris G02771 (see pp. 104–108. ARS/UC –
most shoot apexes were chimeric showing both trans- Paul Gepts). Many cDNA libraries have been or
72
are being constructed (see Table 9, p. 78). Seeds Marker assisted selection (MAS)
of the recommended cultivar BAT93 (BAT93 is Marker assisted selection has been implemented in
one parent of the main linkage-mapping population various bean-breeding programmes especially for se-
BAT93×Jalo EEP558) are available from P. Miklas lection of the bgm-1 gene that codes for resistance to
[pmiklas@betatricity.wsu.edu or s.beebe@cgiar.org]. Bean Golden Yellow Mosaic Virus (BGYMV). This
programme is based on: (1) the critical importance of
Breeding objectives BGYMV in tropical America; (2) the fact that this par-
ticular gene is the most important and effective gene
Plant improvement implies selection among gene- available; and (3) that while greenhouse inoculation is
tically variable individuals or populations to obtain possible on a limited scale (Morales and Singh, 1991),
superior expression of a desired trait. Many disciplines massive resistance screening is not practical. Once
have evolved over the past century that contribute to such genes are identified, and reliable PCR-based
this end: e.g. Mendelian genetics, quantitative ge- markers are available for massive screening, they can
netics, statistics, molecular genetics, pathology, and be manipulated with greater confidence through MAS.
physiology. In particular, tools derived from biotech- Other genes that are foreseen as priorities for selection
nology have led some to refer to ‘molecular breeding’. by MAS include an important QTL for BGYMV resis-
Attempts to classify breeding as traditional, conven- tance, the bc-3 recessive gene for BCMV resistance,
tional, modern, molecular or participatory lose sight of and possibly genes for P use efficiency.
the fact that these approaches are complementary. The
plant breeder is faced with the challenge of drawing Quantitative trait and other functional analyses
upon and coordinating the use of the many tools de- Molecular analysis has proven to be a useful tool
veloped for the common purpose of improving a crop even when the genes or QTL identified are not candi-
species for the benefit of the farmer. Some of these dates for MAS. QTL analysis has been used as a tool
tools are described below. for revealing the inheritance of complex traits such
as biological nitrogen fixation (BNF), root structure
Field breeding for nutrient uptake, and drought tolerance. Combined
Most traits are still selected by conventional means with physiology, QTL analysis can reveal physiolo-
at field sites where the most important diseases, eda- gical relationships and interactions with greater pre-
phic constraints and drought are found (cf Singh, cision than was possible previously. These methods
1999). Gene-banks of ∼ =25 000 and 13 000 accessions could be combined with a candidate gene approach to
of common bean that have been the source of disease seek underlying mechanisms of P use efficiency. At
resistance, abiotic stress tolerance and increased yields present primers for a ferritin gene are being employed
are available at CIAT (in Cali, Colombia) and the to seek QTL for higher seed iron content and improved
USDA Western Regional Plant Introduction Station nutritional value. Beans have been transformed with
(in Pullman, WA, USA), respectively. genes aimed at control of BGYMV through biolistics
at the University of Wisconsin and subsequently in
Participatory plant breeding (PPB) CENARGEN–Brazil, but unfortunately the genes did
In Africa, but not Latin America, participatory plant not have the expected effect on resistance.
breeding of beans has a long history. PPB has im-
portant applications in a small farmer crop like beans Objectives for specific environments
with well-defined production and market niches, and
Mono-cropped beans in favourable environments
will serve to deliver the outputs of breeding to end-
users more rapidly. Target regions in both the Andean Mono-cropping is the favoured system of large, input-
zone and in Central America would be logical areas rich farmers in Latin America such as those in Argen-
in which this activity could be developed. Sites and tina and Brazil, but is also practiced by small farmers
farmers should be identified that are representative of in the north of Ecuador and medium-sized farmers
environments and market criteria of a broader sector in the Dominican Republic. Beans in this system are
of the target region. largely commercial crops. Modest to high inputs are
used and thus soil fertility is not usually an issue, but
farmers seek to protect their investment with disease
resistant cultivars. IPM is an important component
73
Table 7. Percentage of total bean production areas poten-
since mono-cropping can favour the build-up of pests. tially affected by P deficiency and Al toxicity in countries
As a result, pesticide abuse is common. Soil compac- and regions of the developing world
tion is a serious problem in Brazil due to excessive
tillage. Regions or countries % Total bean area affected by:
P-deficiency Al-toxicity
Associated beans as a crop of primary importance Brazil 51 61
This system, particularly the maize-bean association, Mexico 55 2
is the most common traditional system in both Latin Central America 62 19
America and Africa. It is practiced in one or another Southern Zone 22 13
form in Central America, Southern Brazil, the Andean Andean Zone 66 26
Zone, and Eastern and Southern Africa, where most Eastern Africa 65 52a
bean producers are small, resource poor farmers. In Southern Africa 80 42a
this production context, beans are both a product for a Acid soils with pH 5.2 and below as well as a higher
home consumption and an important income source. prevalence of Al toxicity (Wortmann et al., 1998).
Although the biophysical features of the environment
are far from optimal, they are not critically limited by
abiotic stresses. Thus the possibility exists of impro- Pest resistance
ving productivity through a combination of genetic Diseases and insects represent some of the most im-
and resource management solutions that are accessible portant risks that farmers confront. All farmers, both
to farmers who do not have the capital to resolve these large and small, are risk-averse – some more than
problems through inputs. Lack of capital for pesticides others. Breeding for disease resistance avoids risk of
and the dangers of pesticide toxicity also make bree- yield losses, and farmers are very appreciative of re-
ding for disease resistance a desirable goal. Similar sistant varieties to protect their profit margin. Some of
rationale applies to bean–banana and bean–root crop the most significant successes in bean breeding have
associations in Africa. been in the area of disease resistance. At least five ma-
jor diseases [anthracnose, angular leaf spot, common
Associated beans as a secondary crop bacterial blight, BGYMV, and bean common mosaic
One of the strengths of beans is their ability to adapt virus (BCMV)] are widespread, and several others
to a variety of niches. Short or medium season bush are important locally or regionally. Central America
types that offer minimum competition to the primary is a case in point. BGYMV is the single most im-
crop are used as secondary crops. Inter-cropping with portant disease in the region, and varieties resistant
coffee after pruning is an excellent example of this to BGYMV are widely grown in several countries.
system. Given the favourable environment chosen for Adoption studies suggest that about 40% of the area
the primary crop, abiotic stresses are usually minimal. in the region is planted to improved varieties. Yet in
Disease resistant varieties are desirable but these are a 20-year period, region-wide yields have risen by
usually obtained as spin-offs from the work with other only 100 kg ha−1 , from 550 to 650 kg ha−1 . If the
systems. yield increase could be attributed entirely to the area
planted with improved varieties, one would predict
Mono-cropped or associated beans in fragile niches yields of 800 kg ha−1 for improved cultivars – still
In some important agricultural settings, the environ- far below the potential of the crop. Thus, breeding for
ment is so harsh that few crops are productive. This disease resistance has minimised crop losses by main-
is the case of the dry highlands of Mexico and the taining production and yield stability in areas where
northeast of Brazil for example. As a result of its the crop would otherwise have been abandoned. It has
adaptable physiology and its indeterminate flowering not however increased yield potential dramatically.
pattern, beans still produce (albeit 400 kg ha−1 or
less) in environments where other crops like maize fail Abiotic stresses
completely. Although stress resistance has modestly Drought stress is another problem that farmers fre-
increased through breeding, these are problems that quently face. Beans require between 200 and 400 mm
are best addressed through crop and resource manage- of rainfall or comparable residual soil moisture dur-
ment. In these environments, breeding will be mostly ing growth and development. It is estimated that up to
targeted towards disease resistance. 73% of the total Latin American and 40% of the total
74
African bean production occurs under micro-climates 800 kg ha−1 . Improving yields is therefore an imper-
that have moderate to severe mean water-deficits at ative. Data on the profitability of beans in Nicaragua
some time during the cropping season. Recent stu- and in Colombia (see above) clearly show that im-
dies suggest that only 7% of the bean-growing area proved varieties produce higher yields and result in
is well watered. Except for a few highland areas with increased incomes. Several strategies are being pur-
abundant and well-distributed precipitation, and re- sued to improve yield potential, including the use
gions where irrigation is available, bean production of wild germ-plasm through the advanced backcross
is exposed to the risk of drought. Soil problems due method as well as crosses among gene pools and
to toxicities and/or nutritional deficiencies limit pro- races. Another promising development is the renewed
ductivity. Beans are frequently produced on acid soils effort to improve climbers for the very small and land-
that are low in available P and/or high P-fixing capacit- limited farmer. Interestingly, increased productivity
ies. Over 50% of bean-growing areas in Latin America may be emerging in an unexpected way – from work
and 65–80% of these areas in Africa are thought to on edaphic resistance. Lines that were selected un-
be critically deficient in P. Such soils are often high der moderate aluminum and phosphorus stress also
in Al and beans are affected by Al toxicity. Details perform well under optimal soil conditions, yielding
of bean growing areas in Latin America affected by as much as 40% more than the standard high yield-
P deficiency and Al toxicity are shown in Table 7. A ing controls. It is possible that selection has led to
major portion of both Africa and Latin America are improved root systems that perform well under any
also affected by Mn toxicity and low availability of N conditions.
in soil. Although very little is known of the extent and
significance of these micro-nutrient balances in bean Nutritional quality
production systems, preliminary observations indicate More nutritious beans serve both rural and urban con-
that it is also about the same as for potassium. sumers independently of how and where they are
Small farmers also do not have the capital to produced. As noted above, beans are especially rich
solve edaphic limitations through inputs. Moreover, in iron and protein. When bean consumption patterns
soil problems differ from disease and drought as con- are compared to iron deficiencies and the frequencies
straints in the sense that they are largely invariable. A of anaemia in women (27% of whom exhibit iron
producer knows what yield to expect under the par- deficiencies) within Latin America, it is clear that
ticular fertility conditions, and can adjust investment iron-rich beans could make a particularly important
of other inputs accordingly. Although the extent that contribution to health in this region. In Sub-Saharan
edaphic problems can be resolved through breeding Africa, the situation is even worse, with 40% of wo-
programmes is uncertain, their effect will be mostly men suffering from iron deficiency. Often the bean
on yield. farmers are women, and even in areas in which male
family members cultivate beans commercially, such
Yield potential as Uganda, women tend their own plots of beans for
Globalisation of trade in agricultural products will in- home consumption. Thus, women are in a position to
crease the pressure to improve bean yields. Yet, in a receive and apply technology in the form of new bean
crop as diverse as beans, yield potential must be taken varieties. Raising the zinc content is another possi-
in a very relative sense. We have seen that bean envir- bility – nutritional studies have shown that high zinc
onments vary widely in their productivity. Often the beans contribute zinc to the human body. In all cases,
cropping system itself limits the yield potential if for maintaining a reliable supply is a crucial element in
example, only early varieties (hence, lower yielding) exploiting their nutritional potential and should not be
are acceptable. A given yield level (e.g., 1000 kg ha−1 ) overlooked.
may be totally acceptable in high value grain type but
not in a lower value grain, or in a production sys-
tem with high production costs. Thus goals for yield Genomics, transcriptomics and proteomics
potential must be seen in the context of a given re-
gion, production system and grain type. Nevertheless, Molecular techniques are radically altering the way
yields throughout Africa and Latin America are well that plant breeding is being performed. In a sense this
below the potential of the crop by any standard. Most is surprising for the individual methods that derive
countries register national averages between 500 and from biochemistry, physiology, genetics, structural
75
Table 8. Some desired characteristics of ‘new beans’
Low protein Phaseolin and APA gene families Single, complex loci ARS/UC (pp. 104–108)
seed levels
Plant type Determinancy genes fin locus ARS/UC (pp. 104–108); Kelly (2000)
Sensitivity to Several major loci and QTLs for resistance COK-4 (protein kinase), Melotto and Kelly (2001)
Colletotrichum B4 cluster and others
biology, and informatics are hardly new. What has of the white mould pathogen that causes a disease to
changed however is the scale at which genes can be which all known bean cultivars are susceptible. It is
sequenced, their expression analysed, and proteins thus possible that in studying the biology of flower
identified. Genomics, transcriptomics, and proteom- development, ‘Phaseomics’ would help unravel the
ics (when applied to beans we call them Phaseomics) mysteries of the white mould and provide avenues to
permit the study of many (and sometimes all) genes increase resistance to this disease.
of a particular organism. Significant discoveries con- Perhaps the most important information necessary
cerning the inter-relationships between some of the to address both fundamental and applied questions in
basic metabolic functions of an organism have been the agricultural and biological sciences is the basic
made this way. As a consequence, an integrated, al- DNA sequence. Although this information is complete
most holistic view of the organism is evolving. What for some species (e.g., Arabidopsis thaliana and rice),
were once thought to be separate, unrelated functions public databases hold relatively few entries for Phase-
are now seen as part of a complex network of in- olus (<500 nuclear-encoded genes). There are several
teracting genes and their products. From an applied ways of obtaining molecular markers and one of the
perspective, it is possible that studying what seem to cheaper is to sequence messenger RNA’s extracted
be unrelated problems, such as floral biology and dis- from tissues of interest (e.g., developing pods). These
ease resistance, may unveil previously unrecognised so-called expressed sequence-tags (ESTs) are short
relationships. For example, in P. vulgaris a series of (450–600 bp) sequences that are like milestones on a
clearly defined genes are necessary to paint the flower chromosome (see Figure 5). Breeders can use them to
a specific colour. Yet flowers are also the point of entry position other genes. Judicious selection of the type
76
of tissue from which to isolate the mRNA (and hence quenced from the 5 -end and annotated. Selected ESTs
prepare a cDNA library) provides valuable informa- will be spotted on to micro-arrays. Expression ana-
tion not only on the type of genes found in a particular lyses will be used to identify stress-related genes. The
plant, but also on the conditions in which they are roles of individual genes identified in this manner will
expressed. EST projects thus permit ‘skimming’ of then be analysed in plants using standard molecular
the genome. How much information they gather is and genetic techniques.
dependent on pre-existing information as well as on
the abundance of mRNAs, their stability and so on. Australia (AgWA – Sonya Broughton, Francis De
Nevertheless, they are an efficient way of generating Lima)
data that can be directly applied in traditional breeding
programmes. Another, more thorough technique, is to Development of a standard insect screening system
completely sequence the genome. A large proportion for beans
of the funds donated to Phaseomics will be used to pay Beans are host to a wide range of insect pests including
for the commercial sequencing of BACs and ESTs. Aphidae (aphids, e.g. Aphis fabae, Myzus persicae),
BACs will be screened for novel genes, SSRs, etc. Hemiptera (bugs, e.g. Nezara viridula), Coleoptera
and placed on the existing linkage maps. ESTs will (beetles, e.g. Acanthoscelides obtectus, Apion god-
be obtained from cDNA libraries made from various mani, Zabrotes subfasciatus), Homoptera (whiteflies,
tissues (Table 9). e.g. Bemisia argentifolii, B. tabaci), Diptera (flies,
e.g. Ophiomyia phaseoli, Liriomyza trifoli), Lepid-
optera (moths, e.g. Helicoverpa zea, Helicoverpa
The Phaseomics Consortium armigera) and Thysanoptera (thrips, e.g. Franklinella
schultzei, Thrips tabaci). Mites also damage beans
Argentina (UNLP – Mario Aguilar) (e.g. Aphis fabae, Tetranychus urticae). Insect damage
is caused by direct feeding on leaves (aphids, flies,
Effects of soil stresses on nodulation thrips, moths), damage to developing pods (beetles,
Argentina produces about 280 000–300 000 tonnes of bugs, moths), damage to the stem (flies) and through
common beans per year (about 98% of which are ex- the transmission of viruses such as bean golden yel-
ported) primarily in the Northwest region (NWA) of low mosaic virus and bean dwarf mosaic virus (aphids,
the country. The recent expansion of bean cultivation thrips). Once harvested, beans are also susceptible to
occurred in deforested areas where successive crop- damage by seed-feeding beetles, which can begin their
ping leads to decreased nutrient contents of the soil. As infestation before harvest.
a result, bean production is not sustainable and yields Methods used to control insects in beans include
are below potential. The availability of nitrogen, either the use of pesticides, cultural control and biological
from fertilisers or biological nitrogen fixation, limits control. Pesticides only poorly control aphids, thrips
productivity. Strains of R. etli predominate in both and whiteflies, due to the rapid development of insect-
the soil and in nodules of beans growing in the NWA icide resistance (De Barro, 1995; Lewis, 1997). In-
(Aguilar et al., 1998), and these indigenous strains creasingly, varieties of plants resistant to insect attack
limit the effectiveness of introduced strains. Neverthe- are being used as a method to reduce losses caused by
less, inoculation with high levels of selected rhizobial insect feeding and to reduce the population density of
strains can increase in yields. Unfortunately, the res- pests developing on crops (Carozzi and Koziel, 1997).
ults are not consistent in successive cropping seasons Resistant plant varieties can be used as the primary
(Aguilar et al., 2001), and optimisation of inocula- method of insect control, or as a component of an in-
tion requires detailed knowledge of the interaction tegrated pest management program (Wiseman, 1994).
between bean varieties and selected rhizobial strains. Insect resistant varieties have been developed for corn
In Phaseomics, we will identify and characterise sym- (Wiseman, 1994; Wiseman et al., 1996), rice and soy-
biotic genes that code for resistance to environmental bean (Carozzi and Koziel, 1997). For common beans,
stresses (Batista et al., 2001), and use this informa- varieties resistant to pre- and postharvest damage by
tion to breed new bean varieties that yield well under beetles (Beebe et al., 1993; Ishimoto et al., 1999;
marginal conditions. To do this, cDNA libraries will Kornegay and Cardona, 1991) and varities showing
be constructed (in La Plata) from the tissues shown in multiple resistance to insect attack (Bueno et al., 1999)
Table 9. Several thousand of these clones will be se- are being selected.
78
Table 9. Existing/planned cDNA libraries for the production of expressed sequence tags (ESTs) in P. vulgaris and related species
Our aim is to develop a standardised system for have been analysed this way, along with bacteria that
screening insect resistant and tolerant varieties of interact with them (see Nouwends et al., 2000). As a
beans. This will involve three stages: result, greater understanding of the biochemical reas-
1. Review of the literature to determine which insects ons for higher productivity, temperature and salinity
are present on the common bean and are con- tolerance, disease resistance as well as the reasons
sidered to be the main economic pests. From this for the plants having particular properties (e.g., for
review, a list of groups of insects for testing will be dough formation) has been achieved. These results
developed. A field review will be required to de- have provided plant breeders with readily applicable
termine the economic injury levels under different markers for use in breeding programmes to select for
climates (e.g. 10 aphids/plant in a dry climate has a variants with even more desirable qualities.
greater impact than in a wet climate because of rate Our approach at APAF uses two-dimensional gel
of plant growth and compensation for damage). electrophoresis to separate proteins in extracts. Des-
2. Laboratory screening. A protocol will be de- pite a great deal of investigation into alternate meth-
veloped to determine at what insect pressure beans odologies, 2-D PAGE still has the highest resolution
are able to recover from damage. This involves of any analytical protein separation technology. APAF
determining exposure time and will be tested at has the capacity to run more than 200 such gels per
different stages of the plant lifecycle to obtain week. APAF has also paid particular attention to the
a tolerance/resistance rating for specific insects. study of hydrophobic membrane proteins. Here, the
Based on this information, lines that are resist- challenge has been to develop means to solubilise
ant to particular insect groups will be determined these proteins in a form that is compatible with the
and fed back to the Phaseomics group for further first dimension separation step. It is not possible to use
development. powerful ionic detergents for this solubilisation as they
3. Field trials. Resistant and tolerant varieties will alter the isoelectric point of the proteins. APAF has
be tested in the field under different climates to developed a range of solutions that can be utilised par-
determine yield and performance. To cover vari- ticularly to dissolve these intractable proteins without
ations in climate and growing conditions, several interfering with their isoelectric point (pI).
countries will be selected. Extensive screening of Once solubilised, the proteins are separated by
several lines grown in comparison with standard isoelectric focussing on any of a large range of im-
varieties of beans will be used in each country. mobilised pH gradient (IPG) strips. APAF has assisted
Scoring for damage and yield will be done in in the development of a range of such strips that cover
collaboration with local field staff. any of a large selection of pH ranges. Strips are now
available that cover from broad ranges (pH 3–10) to
Australia (APAF – Gary Cobon) ranges as narrow as one pH unit. The advantage of the
narrow pH range strips is that each strip can be loaded
Proteomic analyses of beans with a large amount of protein. The lower abundance
The Australian proteome analysis facility (APAF) has proteins within that range can be observed.
extensive experience in analysing proteins that are ex- Separation in the second dimension is based on
pressed by plant varieties of varying characteristics. the size of the proteins. The resulting 2- D polyac-
Leaf, fruit and roots of wheat, rice, cotton and corn rylamide slab can contain up to 3000 different spots,
80
each of which is a different protein or a variant of the protein by gene sequencing. Once the individual pro-
same protein that has been post-translationally modi- teins have been identified, it is possible to identify
fied. Usually triplicate gels are run of the variants of the biochemical pathways that have been modified in
the plant that are to be compared. In instances where the variants. In many cases, the identification of the
the availability of material is not a limitation (which variant pathway has come as a complete surprise that
is the case with most plants) the more gels that are could not have been predicted in the absence of the
run the less likely that investigations will result in the proteomics information.
identification of variations that are due to gel-to-gel Here we will analyse bean varieties at the protein
variations. level. Our experience suggests that analysis of tran-
Gels are stained with very sensitive fluorescent scription patterns is in itself not sufficiently accurate
dyes as the intensity of the fluorescence is proportional to reflect the level of the proteins within a cell at any
to the amount of protein in the gel over at least a particular time. To do this, it will be important to have
two-log range. This enables not only the presence or access to genome sequence information for the bioin-
absence of a particular protein in a gel to be identified formatics segment of the programme. Combination of
but variations of as little as two-fold in the amount of proteomics and transcriptome analysis will give new
a protein between gels can be determined. Subtle dif- insights into symbiotic development of legumes.
ferences in the amounts of particular proteins in plant
varieties make the difference: it is less common that a Australia (UWA/P – Craig Atkins and Penny Smith)
particular protein is absent.
Computer images of the gels are obtained by scan- Assimilation of fixed N
ning the fluorescent gels. The replicate computer im- Crop legumes fall into two groups on the basis of the
ages are combined using imaging software programs. pathways used to assimilate fixed N in nodules and
Comparison of the images allows identification of the the N-solutes that translocate this N to the host plant.
proteins that vary in abundance in the extracts. Once Apparently all assimilate ammonia initially as the
identified, the proteins that differ between the extracts amide group of glutamine through cytosolic glutam-
are excised and placed into microtitre plates. At APAF, ine synthetase (GS) in the infected cells. While most
we utilise a robotic spot cutter for this purpose, as it is temperate legumes (e.g. peas, lupins, clovers, medics,
often necessary to pick several hundred from one gel. etc.) translocate this glutamine (or asparagine), in xy-
The proteins from the extract are then digested with lem to the host shoot, species of tropical origin form
an endoproteinase. Trypsin is commonly utilised as and translocate the ureides, allantoin and allantoic
it gives a reasonable number of fragments from most acid. The formation and translocation of fixed N as
proteins that are within the size range suitable for sub- ureides is restricted, almost exclusively, to species of
sequent analysis by mass spectrometry. The resulting the tribes Desmodieae, Indigofereae and Phaseoleae
digests are then analysed by mass spectrometry. If the (Atkins, 1991) within the Phaseoloid group. This
complete gene sequence is of the organism or a close group includes important crops like soybean, cowpea,
relative is known, MALDI-TOF mass spectrometry and mung bean as well as members of the genus
works well as the output of this analysis, the mass Phaseolus.
of the trypsin peptides, is sufficient to give an iden- Roots and other tissues of the ‘ureide-forming
tification of the protein (particularly when combined legumes’ assimilate soil mineral N (NO− 3 or NH4 )
+
with the approximate molecular weight and/or isoelec- into glutamine and asparagine (Atkins and Smith,
tric point information that can be obtained from the 2000) and these are the translocated forms of N in
gel). If detailed sequence information is not available, both xylem and phloem. Thus, elevated expression of
tandem mass spectrometry can be used even though the ureide synthetic pathway is a specific metabolic
it is slower, more labour intensive and consequently feature of the symbiosis. In fact, the unique associ-
more expensive. The resultant amino acid sequence ation of ureide synthesis with nodules is sufficiently
information that is obtained more than compensates specific that an assay for xylem-borne N-solutes has
for this added expense as it enables identification of been developed as the basis of a practical field method
the protein in situations where genome sequence in- to estimate relative proportions of fixed and soil-N in
formation is less reliable, or it enables the design of soybean (see Hardarson et al., this volume).
oligonucleotide primers for the PCR amplification of Ureides are oxidation products of purines (xanth-
cDNA fragments and subsequent identification of the ine and hypoxanthine) formed through the de novo
81
purine pathway, initially as the nucleotide inosine synthesis in nodules and roots of bean. The close as-
monophosphate (IMP). To accommodate this flux of sociation between the rate of nitrogenase activity and
fixed N in nodules, activity of the ten enzymes in the purine synthesis that we have found indicates that one
pathway is enhanced considerably (at least 100-fold) factor in enhancing the effectiveness of fixation in
compared to other tissues, including active meristems Phaseolus bean may be the levels of pur gene expres-
where de novo synthesis of purines is essential for sion and regulation of protein targeting to organelles
DNA replication (Atkins and Smith, 2000). For this in infected cells.
reason, nodules have been exploited as the tissue of
choice in which to study the enzymology of pur- Australia (VCP/M – Helen Irving and Marilyn Kelly)
ine biosynthesis in plants. We have cloned the nine,
purine-(pur) encoding genes from Vigna unguiculata Signal transduction in host plants in response to Nod
and have initiated studies to characterise their pro- factors
moter regions with a view to identifying the effectors Our group is interested in signal transduction path-
that lead to enhanced expression. ways. Recent work has focused on signalling path-
The localisation of the purine biosynthesis path- ways initiated in beans in response to Nod-factors
way in plants is different to that of all other organisms isolated from Rhizobia sp. NGR234. The root hairs
in that it is organelle-based. All nine pur genes carry of the host plant are particularly responsive to Nod-
pre-sequences that in general have features consistent factors. Critical to optimising this interaction is un-
with targeting to plastids. Both plastids and mitochon- derstanding of the cellular signalling events that occur
dria of Vigna nodules are capable of IMP synthesis in this dynamic interaction at both a molecular and
from R5P or PRPP and the activities of a number of biological (in planta) level. Chronicling Ca2+ changes
pathway enzymes have been confirmed in both or- and their role in the signalling cascade (Gehring et
ganelles (Atkins et al., 1997). Furthermore, a single al., 1997) has been energetically followed by several
gene in each case encodes eight of the pathway en- other groups and is beyond the capacity of our current
zymes and we have confirmed that one of these, (AIR imaging facilities. As a consequence, we have turned
synthetase, pur5), encodes a protein that is dual tar- to pharmacological and biochemical approaches to
geted (Smith et al., 1998). We expect that each of the investigate the possible signaling events that are ac-
products of the pur genes will be confirmed as dual tivated upstream and downstream of the Nod-factor
targeted and that this feature may be exclusive to nod- induced Ca2+ changes. We believe that our approach
ules. The mechanisms that achieve these outcomes are not only complements that of other workers but also
not yet known. falls into a niche where we can make a significant
There is a noteworthy link between N2 fixation and contribution to the understanding of Nod-factor sig-
the assimilation of fixed-N. When purine biosynthesis nalling at a functional level. We have developed the
is blocked by allopurinol (an inhibitor of xanthine biochemical, cell and molecular biological techniques
dehydrogenase), fixed-N is not assimilated via altern- necessary to dissect and functionally characterise the
ative pathways, such as those that form asparagine signalling events upstream and downstream of these
(even though asparagine synthetase is expressed in changes in Ca2+ in legume root hairs that occur in
roots). N2 fixation is inhibited and the nodules begin response to Nod-factors (Irving et al., 2000; Kelly
to senesce after 24 h (Atkins et al., 1988). Simil- and Irving, 2001, 2002). We have begun the ini-
arly, where ureide synthesis is blocked by anti-sense tial pharmacological and biochemical characterisation
expression of uricase (activity reduced by 80%), the of phospholipase C (Irving et al., 2000; Kelly and
transgenic plants show symptoms of N deficiency. Irving, 2001) and G-proteins (Kelly and Irving, 2002)
These results indicate that N2 fixation is only ef- that are activated in the legume host in response to
fective and is only maintained at high rates when the Nod factors. We have shown that both heterotrimeric
assimilatory pathway for purines is active and access- and monomeric G-protein components are activated
ible to fixed N. Although the nature of the connection in root hairs in response to Nod factors. One of the
is not clear it suggests that understanding regulation earliest physiological changes in the host plant in re-
of pur gene expression could be a route to enhance sponse to Nod-factors (or rhizobia) is initiation of root
symbiotic effectiveness. hair deformation, which in turn means that the un-
We will use the molecular tools developed using derlying structure of the cytoskeleton of these hairs
V. unguiculata to study the regulation of purine/ureide is rearranged. In eukaryote systems, including plants,
82
We have developed an improved P. acutifolius 2003) and we have developed a protocol to regenerate
agrobacterium based transformation protocol (De shoots from P. polyanthus (Zambre et al., 2001).
Clercq et al., 2002; Dillen et al., 1997a; Zambre et
al., 2003). With this protocol, P. acutifolius can be Belgium (LTCHH/G – Jean-Pierre Baudoin and Alain
routinely transformed. As P. acutifolius can be hy- Maquet)
bridised (through embryo-rescue to P. vulgaris) this is
an indirect way of genetically improving the common Inter-specific hybridisation among Phaseolus species
bean. Our studies have focused on the seed storage (see p. 66)
proteins known as arcelins. These are very abundant The Laboratory of Tropical Crop Husbandry and Hor-
seed storage proteins found in some wild P. vulgaris ticulture at Gembloux Agricultural University invest-
genotypes. Seeds of A. thaliana and P. acutifolius igates the following:
plants transformed with arcelin-5 gene constructs, • The genetics of domestication and evolution of
synthesised arcelin-5 to levels of 15 and 25% of the beans (molecular systematics).
total protein content, respectively (Goossens et al., • The effects of in situ wild Phaseolus populations
1999a,b). This high expression level of arcelin5 is be- on the genetic structure at both inter- and intra-
ing exploited in a project aimed at expressing arcelin5 population levels. Special attention will be given to
genes modified to contain extra methionine codons. the influence of gene-flow and breeding systems.
Legume seeds are known to be low in sulphur con- • The mechanisms of genetic incompatibility. Com-
taining amino acids, including methionine (p. 60). parison of the mapping order of molecular markers
High-level accumulation of these modified arcelin5 will indicate if rearrangements of chromosomes
proteins should result in increased seed methionine have occurred during development of the different
levels and thus improved nutritional balance. As the Phaseolus species.
crystal structure of Arcelin5 has been determined, the • The biochemistry of embryogenesis and the mech-
influence of substitutions and insertions of methionine anisms of abortion. In particular, histology of in-
codons on protein stability can be evaluated through terspecific embryos and search of candidate genes
computer simulations. Six modified arcelin5 genes, in embryo development (probing with genes of
each containing three to five extra methionine codons, model species) will help overcome incompatibility
were constructed and four of these were found to yield barriers and refine methods of introgression.
stable proteins in Arabidopsis accumulating to levels
similar to those of unmodified Arcelin5. One of the Belgium (LoGT/UL – Guido Volckaert)
constructs (with four methionine residues) was intro-
duced into P. acutifolius. Ten independent lines were The contribution of the Laboratory of Gene Techno-
generated, all of which show stable protein accumula- logy (LoGT) of the Katholieke Universiteit Leuven
tion to levels similar to the unmodified Arcelin5 (De to Phaseomics is in genome sequencing. LoGT will
Clercq et al., 2002). To enhance the methionine con- provide a niche for efficient and cost-effective sequen-
tent of Phaseolus beans to that of the FAO reference cing of selected BACs (or clones of similar large-sized
protein, an Arcelin5 gene with at least ten additional genomic segments) and will operate in partnership
methionine codons, expressed at the same level as with other laboratories of the consortium. So far, gen-
the unmodified protein, is required. Therefore, vari- ome sequencing has been based either on full-genome
ous combinations have been made of the modifications shotgun cloning libraries, or on physical maps of cos-
that yield stable proteins in Arabidopsis. These new mid/phage/BAC clones arranged in a minimal tiling
constructs are currently being tested in Arabidopsis path. The former approach requires large-scale fund-
and P. acutifolius. ing ab initio (at least $US 50 million) and substantial
We are also continuing to improve the regenera- computing power for assembly; the latter approach
tion and transformation protocols for Phaseolus, and involves a time-consuming and costly mapping phase.
particularly P. vulgaris. P. vulgaris can be regenerated With the current approaches in transcriptomics and
using a callus-based protocol (Zambre et al., 1998) proteomics, it is clear that much information from
which we hope will yield stable transformants. To this functionally interesting regions of Phaseolus can be
end we are looking at factors that influence transform- rapidly gathered. Using the available BAC libraries,
ation efficiency (Dillen et al., 1997b; Zambre et al., the genomic equivalent regions are readily obtained.
Sequencing such BACs will yield a genomic frame-
84
work of target sites that can be expanded to fill-in the farmers whose properties are small (less than 10
gaps between targets systematically, and eventually ha) and located in areas of sub-optimal soil condi-
lead to the complete genome sequence. tions (mainly low pH and phosphorus availability and
LoGT has participated in many of the major gen- phytotoxic levels of aluminium). It is estimated that
ome sequencing projects of the past decade: Sacchar- only 4% of the planted areas are occupied by large-
omyces cerevisiae, Arabidopsis thaliana, Schizosac- scale irrigated farms and produce only 15% of the
charomyces pombe, etc. (see, e.g., Arabidopsis Gen- annual production.
ome Initiative, 2000; Winzeler et al., 1999). In addi- In many parts of the world, including Brazil, beans
tion to contributing sequences to these projects, we provide the primary source of dietary proteins and car-
have specialised in problem-solving approaches and bohydrates as well as other minerals such as Fe (Lott
quality-control procedures (Voet et al., 1997). This et al., 2000; Sandberg et al., 1993; Sathe et al., 1984).
includes error checking and solving conflict-positions The main storage protein is phaseolin and like all other
directly from genomic templates; solving complex and seed proteins of legume family is deficient in sulphur-
imperfect repeat structures and regions of low A+T containg amino acids, principally methionine. This
content or containing homopolymeric tracts; sequen- deficit is made up by including cereal seed storage
cing unclonable regions; reading through polymerase- proteins in the diet, which are themselves deficient in
pausing and other (e.g., secondary structure) stops. lysine.
This expertise results in a gapless sequence that is In Phaseomics, we will use strictly Brazilian vari-
essential for diversity analyses, and a prerequisite in eties or varieties that are currently accepted for plant-
Phaseolus genomic sequencing. ing in Brazil because of their better performance in
The BAC sequencing process (around 100 kb/BAC) this country. We will construct cDNA libraries from
can be divided into two phases: (1) the ‘routine the roots and endosperm of various bean varieties,
phase’: an initial collection of sequence reads made stressed and un-stressed (see Table 8), to allow the se-
by systematically sequencing shotgun clones; (2) the quencing of at least 50 000 Expressed Sequence Tags
‘finishing phase’. Here the ‘reads’ are assembled (ESTs). Libraries will be constructed at CENA/USP
into contigs and finalised by closing any remaining and the sequences made publicly available, after
gaps (using primer-walking) and making the entire proper evaluation, through public databases such as
sequence double-stranded. The routine phase can be the NCBI Genebank and BeanGenes.
subcontracted to so-called ‘sequencing companies’
with proven record of high-quality, large-scale sequen- Phytic acid and aluminium tolerance
cing, as this routine phase is more cost-effective in a After the isolation of the poly-A mRNA fraction using
specialised facility with automated processing rather commercially available kits, we will use the OR-
than in a purely academic environment. The finishing ESTES system (Neto et al., 1997) for the construction
phase, however, requires more personal involvement, of the cDNA libraries for two main reasons: the first
including specialised manual operations that can be is the reduced quantity of poly-A mRNA necessary
more efficiently performed in research laboratories. to synthesise the cDNA and secondly this method-
Thus, competitive offers for 800 reads per BAC to ology greatly compensates for the unequal message
be provided on CD-ROM will be requested from se- abundance that avoids the need to construct com-
quencing companies and finishing will be done at the plex normalised libraries. To study the production of
LoGT, including basic bioinformatic analysis. Mem- phytate in beans the material used will be endosperm
bers of the Phaseomics network at their discretion may for varieties with high and low phytate content at
provide BACs, or BACs will be selected from libraries various developmental stages and under various nu-
based on EST data. tritional conditions and following the developmental
stages indicated by Walker (1973) The candidate vari-
Brazil (CENA/USP – S.-M. Tsai and D.H. Moon; eties are Rio Tibagi, Carioca 80SH, G19833, G21212,
LICR – A. Vettore and A.G. Simpson) G4000, BAT 477 and A774. To study aluminium tol-
erance, root-tip material from sensitive and tolerant
Brazil has approximately five million ha of land varieties, under stressed and un-stressed conditions,
planted with P. vulgaris varieties and produces three will be used to isolate mRNA (Carioca 80SH and Car-
million Mt of beans (see p. 58). Economically, beans gamanto varieties). To generate 50 000 clones we need
are an important cash crop for the many Brazilian a total of approximately 100 ng of poly-A mRNA from
85
each tissue type to generate 10 000 ESTs (4ng/cDNA databases and subjected to motif analysis using a vari-
synthesis and each synthesis generates 20 AP-PCR re- ety of computational tools. Sequence annotation will
actions and each mini-library generates on average 25 also include clustering analysis. Finally, clones, se-
clones or approximately 500 clones for each 4ng of quences and derived information will be deposited in
mRNA, data from Neto et al. (1997). publicly accessible databases and individual clones
will be available to researchers upon request.
Brazil (ESALQ/USP – Maeli Melotto, Luis E.A. Later, we will focus on studying disease resistance
Camargo) genes (see Table 9). cDNA clones showing homo-
logy to resistance genes will be mapped and those that
Bean EST project – BEST co-segregate with known genes will be selected for ge-
Expression libraries are needed to accelerate bean ge- netic complementation experiments. This work will be
nomics. The information provided by the BEST (Bean developed in collaboration with CIAT (see COLOM-
EST) Project can be exploited in many different ways. BIA (CIAT – Steve Beebe, Matthew Blair, Joe Tohme,
We will construct more cDNA libraries, sequence pp. 88–89), with the additional aim of developing new
10 000 expressed sequence tags (ESTs) and build an micro-satellites from the EST sequences. Ultimately,
annotated database for the sequences. many of these ESTs will be genetically mapped us-
A cDNA library has been constructed from total ing RFLP or SNP (single nucleotide polymorphism)
mRNA extracted from above ground vegetative parts based assays, especially as bean micro-arrays become
of adult plants of the Andean common bean variety available.
G19833. The source genotype, G19833, is tolerant
to low phosphorus levels in soils, is resistant to an- Brazil (LCV/UFPE – Andrea Pedrosa, Marcelo
thracnose, angular leafspot as well as Ascochyta but Guerra)
is susceptible to bean golden yellow mosaic virus
and bean common mosaic virus. G19833 is also one Cytogenetic-based physical map of P. vulgaris
parent of the principal mapping population used at Cytogenetic analysis in beans has long been hampered
CIAT which consists of 87 recombinant inbred lines by the small size and similar morphology of its
(F-11 generation) from the cross DOR364×G19833. 22 chromosomes. Although some progress has been
QTLs for low phosphorus tolerance and disease resist- achieved by using giant, polytene chromosomes of
ance have been mapped in this population. At a later the embryo suspensor (Schweizer and Ambros, 1979),
date, cDNA clones from leaf tissue of the bean line identification of these chromosomes remained con-
SEL1308 stressed with Colletotrichum lindemuthi- troversial. Recently, most of the common bean mi-
anum (the causal agent of anthracnose) will also be in- totic metaphase chromosomes could be identified by a
cluded in the analysis. This is a black bean line derived combination of chromosome morphology, heterochro-
from the landrace G2333 (Colorado de Teopisca) and matin distribution and fluorescent in situ hybridisation
possesses the Co-4 gene for anthracnose resistance. (FISH) with rDNA probes (Moscone et al., 1999).
Two cDNA libraries will be constructed from seed- Our group is interested in establishing a cytogenetic-
lings non-inoculated and inoculated with the fungal based physical map of common bean. As a first step,
pathogen Colletotrichum lindemuthianum that causes we have integrated the genetic map and the chromo-
anthracnose in common bean. The black bean geno- somal map of the species. For this purpose, a new
type, SEL1308 was chosen in this study as it carries strategy was used in which clustered or linked RFLP
the Co-42 gene for anthracnose resistance (Melotto clones were combined and directly used as probes for
and Kelly, 2001; Young et al., 1998). Black beans FISH experiments (Pedrosa et al., 2001). This allowed
have been described as the best to study nodulation the assignment of all linkage groups of the University
and bean/Rhizobium interactions. These libraries will of Florida map (Vallejos et al., 1992), and indirectly of
be normalised and directionally cloned into plasmid the core map (Freyre et al., 1998), to the chromosomes
vectors for 5 -end sequencing. Approximately 10 000 of the species (Pedrosa et al., 2002b). Furthermore,
randomly selected cDNAs will be partially sequenced. cytogenetic markers for identifying each bean chro-
Libraries will be stored at the Department of Plant mosome are now available. No correlation between
Pathology, ESALQ, University of São Paulo, Brazil. linkage group sizes and chromosome sizes was ob-
All sequences will be analysed for possible function served, suggesting a high variability in Mbp/cM ratios
by similarity to known genes represented in public along different linkage groups and emphasising the
86
importance of a detailed correlation of genetic and SAR. SAR is expressed to a maximum level when
physical distances throughout the bean genome. the inducing organism causes necrosis whereas PGPR
As a result, our present aim is to: typically do not cause necrotic symptoms. Both SAR
• Improve the correlation of genetic and chromo- and ISR involve the activation of latent resistant mech-
somal maps by hybridising BAC clones selec- anisms that are expressed afer challenge inoculation
ted with genetically mapped-markers distributed by a pathogen.
throughout the genome to pachytene chromo- Some PGPR strains affect the growth of beans
somes of common bean. As demonstrated for other (Peix et al., 2001). Petersen et al. (1996) showed
model legumes, this approach allows comparison that co-inoculation of beans with Bacillus polymyxa
of both maps in multiple regions (Pedrosa et al., and Rhizobium etli increased lateral root formation
2002a) and generates a high-resolution physical and nodule number. These effects were not linked to
map (Kulikova et al., 2001). The use of pachytene the ability of the Bacillus isolates to produce indole
chromosomes will also allow the assignment of acetic acid in vitro (Srinivasan et al., 1996). Inocu-
BACs to the eu- or hetero-chromatin domains. lation of bean seeds with the PGPR strain Pseudo-
• Expand the maps by integrating groups of unlinked monas fluorescens S97 suppressed attack by the leaf
markers through BAC FISH. pathogen Pseudomonas syringae pv. phaseolicola (Al-
• Assist the development of a contig physical map, strom, 1995). Growth and yield of water stressed
by supplying anchoring clones, joining non- bean plants were improved by inoculation of with the
overlapping contigs and characterising the gaps. vesicular arbuscular mycorrhizal fungus Glomus in-
Development of this physical map will not only traradices (El-Tohamy et al., 1999). Inoculation of
contribute to the understanding of the common bean beans with Glomus mossae also significantly reduced
genome, but also provide additional markers for fu- root infection with Fusarium solani (Dar et al., 1997).
ture comparative cytogenetic analysis within the genus Here we will characterise and clone novel bean
Phaseolus. genes (using cDNA techniques) that are expressed
during the interaction with:
Canada (UL/RSVS and AgCAN – Hani Antoun, Serge • PGPR, including non-homologous rhizobia: Phase-
Laberge) olus vulgaris appears to be a non-selective host
for nodulation because it is able to perceive sig-
Novel genes induced by PGPR, mycorrhizae and cold nals for nodulation from many rhizobia (Michiels
stress et al., 1998). Although most of these interac-
Plant growth promoting rhizobacteria (PGPR) are a tions produced ineffective nodules, we have pre-
very small portion (2–5%) of rhizosphere inhabit- viously observed that inoculation of Medicago
ing bacteria that are able to promote plant growth or sativa with some combination of homologous and
health when reintroduced in large numbers by inocu- non-homologous rhizobia produced a significant
lation (Antoun and Kloepper, 2001). PGPR use one or synergistic effect on yield (Antoun et al., 1979).
more of several mechanisms to promote plant growth. Many rhizobia also act as PGPR with non-legumes
Some examples are the production of phytohormones (Antoun et al., 1998).
or the improvement of plant nutrition through biolo- • Vesicular arbuscular mycorrhizae.
gical nitrogen fixation. Indirect mechanisms of action
• Low temperatures. This part of the work will
are by far the most important and they include biolo-
also indicate if there is a connection between re-
gical control of plant pathogens and induced systemic
sponses to biotic and abiotic factors as observed
resistance.
in Arabidopsis thaliana (Timmusk and Wagner,
Induced resistance is defined as an enhancement
1999).
of the plant’s defence capacity, against a broad spec-
trum of pathogens and pests (see Ramamoorthy et • Gene expression will be studied in relation to bean
al., 2001). The resulting elevated resistance due to cultivars, plant age, co-inoculation with more than
an inducing agent upon infection by a pathogen is one organism as well as the involvement of biotic
called induced systemic resistance (ISR) or systemic and abiotic stresses (see Table 9).
acquired resistance (SAR). Induction of systemic res-
istance by rhizobacteria is referred as ISR, whereas
that by other agents (pathogens or chemicals) is called
87
Canada (CDC/USS – K. Bett, B. Tar’an, A. of the site (see USA (NDSU/F – Phil McClean, pp.
Vandenberg and P. Balasubramanian) 109–110).
wild common bean lines as well as related species existing integrated maps for the species. A set
from all major growing regions. Seeds from these lines of micro-satellites is being put together to effi-
are held in trust under the auspices of the Food and ciently map other populations (see below). Several
Agriculture Organisation of the United Nations (FAO) other mapping populations have been developed
designated world collection. This gene bank is used at CIAT and are used to tag quantitative trait loci
as the source of novel traits for breeding improved (QTL) for characteristics of interest to CIAT plant
genotypes. breeders. These include abiotic stress tolerance
(low phosphorous, aluminum toxicity and drought
Research focus tolerance), micronutrient content (iron and zinc),
CIAT works on many aspects of breeding, genetics, as well as insect and disease resistance. Several
pathology, nutrition and physiology, the focus has of these CIAT populations are being analysed by
been on breeding beans for biotic stress resistance, other groups involved in studying the molecular
especially for disease and insect pests of the lowland genetics of common beans in Brazil, Belgium,
and highland tropics. New emphasis is being placed France, Germany, Mexico and the United States.
on breeding varieties for higher nutrition that are adap- 3. Genomic libraries: As part of the process to de-
ted to abiotic stresses. Beans are frequently produced velop additional micro-satellite and SCAR mark-
on acid soils that are low in available phosphorous ers, the biotechnology unit at CIAT has made
and high in aluminum. Symbiotic nitrogen fixation is several types of genomic libraries including one
affected by phosphorous availability. In some areas enriched in micro-satellites.
beans are grown on alkaline soils where iron avail- 4. cDNA libraries and EST sequencing: Three cDNA
ability is low. Meanwhile, many soils are deficient libraries have been made from bean tissues at
in nitrogen, potassium and zinc or have high levels CIAT. The first was a leaf cDNA library construc-
of manganese. All these soil conditions affect the ted from total mRNA extracted from leaves of
nutritional status of the plant, which in turn affects adult plants of the Andean variety G19833 (see
accumulation of nutrients in the grain and total yield. Table 8). This library was made in the pCMV
There is thus a direct link between crop nutrition and Sport 6.0 vector. A total of 64 000 clones have
human nutrition. been plated and picked into 384-well plates that
were arrayed onto high-density filters and stored as
Bean biotechnology at CIAT glycerol stocks. The clones have an average insert
1. Marker development: One priority of CIATs bio- size of 1.3 kb. The source genotype, G19833 is
technology efforts for common bean has been the tolerant to low phosphorous levels in soils and has
development of PCR-based markers. Two main multiple disease resistance including anthracnose,
marker types have been emphasized: sequence angular leaf spot as well as Ascochyta leaf blight.
characterised amplified region (SCAR) markers G19833 is also one parent of the principal map-
and micro-satellites or simple sequence repeats ping population used at CIAT which consists in 87
(SSRs). These markers have been essential for recombinant inbred lines (F-11 generation) from
mapping and tagging genes of agronomic import- the cross DOR364×G19833. DOR364 is a popu-
ance and for their eventual selection in marker- lar Central American variety that is high yielding
based breeding schemes. Other marker systems, and adapted to conditions in the region. QTLs for
such as AFLPs and RAPDs have been used to low phosphorous tolerance, agronomic perform-
study the diversity within different species of the ance and disease resistance have been mapped in
genus Phaseolus and the many accessions that are this population. Two root cDNA libraries have also
stored in the germ-plasm bank. been made from mRNA extracted from adventi-
2. Genetic mapping: All new markers are mapped tious and basal roots grown under phosphorous
onto CIAT’s principal mapping population as men- deficiency stress for the genotypes G19833 and
tioned earlier, which now contains over 500 mark- DOR364. Both libraries were made in a high
ers including AFLPs, micro-satellites, RAPDs and efficiency phagemid vector from Stratagene Clon-
RFLPs. Probes from both the University of Cali- ing Systems (Uni-Zap XR). An additional 32 000
fornia at Davis (see ARS/UC) and the University clones will be picked from each of the root librar-
of Florida have been used in this mapping pop- ies. About 4000 clones have been sequenced so far
ulation to correlate the CIAT genetic map with and the ESTs are being used to develop molecular
90
markers. Many of the bean ESTs have homologues chromosomes and in situ hybridisation. This work led
in the soybean database. to the localisation of seed storage protein genes on
5. Resistance gene analogues: Analogues to resist- Vicia faba chromosomes (Macas et al., 1993a,b), to
ance genes have been analysed using degenerate the development of methods for fluorescent labelling
primers to amplify their NBS-LRR, TIR and P- of specific sequences on chromosomes in suspension
loop regions. Amplification products have been (Macas et al., 1995; Pich et al., 1995), and to the
cloned, sequenced and used as probes to map construction of the first complete set of chromosome-
the homologous loci in the bean genome and to specific DNA libraries in plants (Macas et al., 1996).
identify BACs containing the sequences. The in- Recently, most of the research has been focused on
formation gained will be used to develop mark- repeated DNA sequences, especially in species pos-
ers for the selection of the resistance genes that sessing large genomes (Vicia spp. and Pisum sat-
co-segregate with the cloned fragments. ivum) (Macas et al., 2000; Neumann et al., 2001;
6. Transformation and tissue culture: Biolistic and Nouzová et al., 1999, 2001). In order to isolate DNA
Agrobacterium tumefaciens-mediated transforma- repeats from complex genomes efficiently, several
tion strategies are being tested for transformation. novel methods were introduced or adapted, includ-
Inter-specific hybrids with tepary bean have been ing DNA microarrays (Nouzová et al., 2001), and
developed at CIAT through congruity backcross genomic self-priming PCR (Macas et al., 2000). A
and embryo rescue methods. These have proven database of plant satellite repeats has been established
useful since they are more amenable than com- (Macas et al., 2002) which is accessible via internet
mon bean to transformation. Greenhouse testing of (http://w3lamc.umbr.cas.cz/PlantSat).
beans transformed with GUS has been undertaken Although the genome of Phaseolus is one of the
and field-testing will be performed once permis- smallest among legumes (Bennett and Leitch, 1995),
sion of the Colombian bio-safety authorities has it is still expected to contain considerable proportion
been granted. of repetitive sequences. Only a very limited number
7. Bio-informatics and databases: CIAT is part of a of Phaseolus repeats have been isolated and character-
consortium of CGIAR centres that are developing ised so far, including rDNA genes, a family of retro-
bio-informatics tools for linking mapping, QTL transposons Tpv2 (Garber et al., 1999), and a mini-
analysis and germplasm evaluation. Emphasis will satellite sequence OPG9-130 (Metais et al., 1998).
be placed on creating databases for managing gen- Thus, we propose to screen for repetitive sequences to
otype and genetic mapping information as well isolate representative collections of both dispersed and
as establishing sequence storage and processing tandemly organised repeats. The following techniques
capacities. Molecular marker data is continually will be used:
updated in the BeanGenes AceDB database. • Screening short-insert shotgun genomic libraries
8. Future plans: CIAT has established a DNA micro- of total genomic DNA using the rapidly renaturing
array facility that will be used to develop new fraction (Cot-1) of genomic DNA as probe. Since
genetic marker systems based on the diversity ar- size-fractionated DNA will be used for the library
ray system that was developed at CAMBIA. DNA construction, the hybridisation signals will reflect
chips to follow gene expression will be developed copy numbers of cloned fragments in the genome
with clones from the cDNA libraries described and will be used for identification of repetitive
above. In addition, single nucleotide polymorph- sequences.
ism markers will be constructed using sequence
• Cloning and shotgun sequencing of Cot-1 and Cot-
data generated from projects described above.
0.1 fractions of genomic DNA in order to identify
the most abundant classes of genomic repeats
Czech Republic (IPMB/CB – Jiri Macas, Vit Našinec) (usually satellite DNA sequences).
Analysis of repetitive sequences • Performing genomic self-priming (GSP-) PCR as
The laboratory’s long-term interest is focused on the described by Macas et al. (2000). This tech-
molecular structure and evolution of legume genomes. nique is designed to specifically amplify and clone
In collaboration with several other groups, the laborat- tandemly organised repeats.
ory has been developing new techniques for physical • Computer analysis of novel sequences obtained
genome mapping using micro-isolated or flow-sorted above.
91
The newly isolated repeats will be sequenced and char- using a set of molecular markers representing each ge-
acterised with respect to their copy numbers, genomic netic linkage group, including existing RFLP, as well
organisation and distribution in Phaseolus and other as EST and SSR markers that will be generated within
legume species. Full-length clones of very long re- this project. Markers evenly distributed throughout the
peats (mostly retro-elements) will be isolated from genome and/or linked to important genes will be used.
available BAC or phage libraries based on their par- Depending on the type of marker, the screening will
tial sequences obtained in the primary screening. The be performed either by hybridisation to DNA arrays
data obtained from these experiments will be used in or by PCR using a pooling strategy. Positive BAC
several ways: clones will be fingerprinted to confirm the copy num-
• A computer database containing Phaseolus repeats ber of the probe loci and to eliminate false positives.
will be established and made available through the Sequence-tagged BAC clones will be localised on mi-
Internet, so that the repeat sequences can be used totic chromosomes using FISH. In cases where two
by other participating groups for identification of BAC clones localise to the same site on a chromo-
ESTs or other clones bearing repetitive sequences, some, physical distance will be estimated by FISH
and for masking DNA repeats during the assembly on stretched mitotic chromosomes or by fibre-FISH
of contigs from sequenced clones. and compared to the genetic distance. Mapped BAC
• Clones of selected repetitive elements will be clones will be hybridised to cDNA arrays to identify
made available as probes for in situ hybridisa- gene-rich clones. The arrays will be prepared from
tion on mitotic or polytene chromosomes and for existing cDNA libraries and from cDNA libraries ob-
DNA fingerprinting of various Phaseolus species. tained within this project (see Table 8). Selected BAC
This should provide tools for cytogenetic charac- clones will be candidates for preferential sequencing
terisation of karyotypes and for assessing phylo- and gene discovery.
genetic relationships among individual species and This work will result in:
cultivars, respectively. • Generation of chromosome- and arm-specific
• The repetitive sequences will be studied with re- cytogenetic markers.
spect to their evolutionary dynamics and possible • A framework of sequence-anchored ‘seed’ BAC
role(s) in the genome. Comparative analyses of clones covering the whole genome.
Phaseolus repeats with those from other well- • Integration of genetic linkage and physical cyto-
studied legumes (Vicia, Pisum) will be performed. genetic maps.
• Orientation of genetic linkage groups with respect
Czech Republic (IEB/O – Jaroslav Dolezel) to chromosome arms.
• Comparison of genetic and physical distance for
Physical and cytogenetic mapping selected markers.
Identification of individual chromosomes in Phaseolus • Identification of gene-rich BAC clones for rapid
is difficult due to similar morphology and lack of dis- gene discovery.
tinct chromosomal landmarks. In some plant species, • Determination of the chromosomal distribution of
fluorescence in situ hybridisation (FISH) has been em- interesting genes.
ployed using repetitive DNA sequences as probes to Knowledge gained this way will allow comparative
identify individual chromosomes. This application re- analysis of chromosome structure, gene synteny, do-
lies on the availability of repetitive sequences with mestication and evolution within the genus Phaseolus
specific distributions. BAC and FISH clones can be and to analyse the extent of colinearity with other
used to generate chromosome- or arm-specific probes. species.
The main advantages of using large-inserts are easy
detection, strong signals, and the possibility of com- France (INRA/M – Jean-Jacques Drevon)
parative studies. Thus physical mapping of molecular
markers via BAC and FISH can play critical roles Tolerance of symbiotic nitrogen fixation to
in mapping wild relatives and progenitor species for phosphorus deficiencies
which linkage maps do not exist. In both tropical and mediterranean regions of Africa
The availability of BAC libraries of P. vulgaris and Latin America, symbiotic nitrogen fixation (SNF)
makes possible the construction of a physical cytogen- is often limited by such soil constraints as low phos-
etic map. We will screen an existing bean BAC library phorus availability, drought or salinity. Although
92
beans are often considered poor N2 -fixing legumes, France (LPPM/O – Thierry Langin, Valérie Geffroy)
high N2 -fixing lines have been found in Latin Amer-
ica. Some can express their full SNF potential despite Anthracnose and beans
low soil P particularly by increasing the permeability Our laboratory is involved in the genetic and molecu-
of nodules to O2 diffusion and proton efflux. Low per- lar analysis of the interaction between Phaseolus vul-
meability of nodules to O2 diffusion and ion-exchange garis and the pathogenic fungus Colletotrichum linde-
is associated with P deficiencies. Cytological obser- muthianum, causal agent of anthracnose (see Geffroy
vations suggest that variations in nodule permeability et al., 1998, 1999, 2000). Independent and comple-
are due to reversible, osmoregulated contractions of mentary projects are underway on both the host plant
inner-cortical cells of nodules that fine-tune the N2 and the pathogen. In P. vulgaris, we are mostly inter-
fixation process. We have initiated a search for genes ested in the evolution of disease resistance (R) genes
that control SNF under conditions of low soil P. Lines in response to pathogen selection pressure. The in-
possessing high phosphorus use efficiency (PUE) and teraction between P. vulgaris and C. lindemuthianum
good SNF ability have been selected and crossed with constitutes a good model system for the study of the
widely grown cultivars. A group of 20 RILs (Recom- molecular mechanisms underlying the evolution of R
binant Inbred Lines F8) from one of these crosses, genes because of:
BAT477 (high SNF and PUE) x DOR364 (well adapt- • The existence of divergent and well characterised
ated in Central America and the Caribbean as well as bean gene pools.
tolerance to BGMV virus), were selected in multi-year • The occurrence of many specific resistance genes.
field trials. These lines have been genotyped using • The existence of co-evolution phenomenon between
RAPD, SCAR and microsatellite markers and pheno- the fungus and its host at the level of the centres of
typed in field trials under drought conditions at various diversity of the plant.
sites across Cuba and Mexico. A genetic map has been
constructed for this population and a set of quantitative
trait loci (QTLs) have been identified that affect PUE A – Identification of the B4 resistance-gene cluster
and SNF. Future work will determine which candidate The genomic distribution of both specific R genes and
genes are responsible the variation in PUE and SNF. R QTLs was studied using a recombinant inbred line
The candidate genes will be sought using differential (RIL) population derived from a cross between parents
display techniques as well as from the analysis of nod- chosen to represent the two major P. vulgaris gene
ule cortex cDNA libraries and DNA microarrays that pools: BAT93 (Mesoamerican) and Jalo EEP558 (An-
prepared during the course of the project (see Table 9). dean). This RIL population, developed by the group
Phenotypic characterisation will use the following of P. Gepts (pp. 104–108), is being used to construct
methods: an integrated linkage map of common bean. Seven
specific R genes (four Andean and three Mesoamer-
• Measurement of gas and ion exchange on in-
ican) were identified and mapped to four loci (Geffroy
tact nodulated roots of hydro-aeroponically grown
et al., 1999). Ten genomic regions involved in partial
plants to quantify the nodule conductance to O2
resistance against two different strains were identified
(gno ), (which is linked to nitrogenase activity and
(Geffroy et al., 2000). Four QTLs co-localise with
proton efflux) will be made. This way we will be
specific R genes. These QTLs may therefore share
able to relate the kinetics of changes in gno and
structural and functional relationships with specific R
H+ eflux to variations in rhizospheric O2 concen-
genes. Co-localisation of QTLs with defence genes
trations (Drevon and Hartwig, 1997; Ribet and
was also observed. Clustering of resistance specificit-
Drevon, 1996; Tang et al., 2001).
ies against C. lindemuthianum, against other patho-
• Image analysis of nodule cortex parenchyma in gens and QTLs, as well as clustering of resistance
order to correlate cell structural and morphomet- gene analogs (RGA) provided evidence that R loci are
ric features with immunolocalisation and in situ complex at both the genetic and molecular level.
hybridisation of molecular probes. This way we A particularly complex locus was identified on
will be able to correlate expression of genes in linkage group B4. This cluster, named the B4 R gene
the nodule cortex with the physiological measure- cluster, contains:
ments described above (Serraj et al., 1998; Vadez • Two Andean and one of Mesoamerican R spe-
et al., 1999). cificity.
93
• A family of RGAs of the nucleotide binding site diversity. Study the B4 R cluster in Medicago trun-
type (PRLJ1 family). catula in order to assess the evolution of the B4 R
• Two QTLs of Andean and Mesoamerican origin. cluster on a longer timescale.
Co-localisation of Andean and Mesoamerican R
specificities suggests that this locus existed prior to the France (CERMAV-CNRS – Eric Samain, Hugues
separation of the two major P. vulgaris gene pools. The Driguez)
molecular dissection of this locus in both BAT93 and
JaloEEP558 should bring improved understanding of The micro-symbionts of P. vulgaris constitute a het-
co-evolution phenomenon at the molecular level. erogeneous group of bacteria. At least five differ-
ent species belonging to the genera Rhizobium and
B – Molecular tools to study the B4 resistance-gene Sinorhizobium have been identified from bean nod-
cluster ules. These different species produce different nodu-
In order to isolate expressed resistance gene ana- lation factors that show important structure dissimil-
logues (RGAs) corresponding to the R specificities of arities. For example R. etli produce acetyl-fucosylated
the B4 locus, cDNA libraries have been constructed Nod-factors whereas R. tropici factors are sulphated
from infected leaves for both BAT93 and JaloEEP558 at the same position. It is thus of great interest to
(Ferrier-Cana et al., 2003). Genomic libraries were have library of pure Nod-factors that are recognised
also constructed from the BAT93 and JaloEEP558 by Phaseolus spp.
DNA partially digested with Sau3A and inserted into In the last few years we have developed a method
the lambda FIX vector phage (inserts from 15 to 20 for the synthesis of structurally defined Nod-factors.
kb). These four libraries have been screened with the To do this, the chito-oligosaccharide backbone carry-
PRLJ1 probe specific to the B4 R gene cluster. ing suitable decorations is first produced by genetic-
ally engineered Escherichia coli strains that express
C – Molecular basis of host-pathogen co-evolution heterologous rhizobial nodulation genes (Samain et
Sequencing has revealed that the R genes present at the al., 1997, 1999). Then the lipid chain is added
B4 R cluster encode putative R factors belonging to the by chemical acylation yielding synthetic Nod-factors
Nucleotide Binding Site–Leucine Rich Repeat (NBS– (Gressent et al., 1999). This process can be scaled-
LRR) class of disease R proteins. This is the prevalent up for the synthesis of large quantities for agricultural
class of disease R genes identified in plants. Currently, applications.
30 NBS-LRR-encoding R genes located at the B4 R This method has been used to synthesise sulphated
cluster have been completely sequenced from BAT93 tetramers that are analogues of R. meliloti Nod-factors.
and JaloEEP558. The comparative analysis of these Here we propose to synthesise the main structures that
sequences is underway. No molecular signature of An- are produced by the different microsymbionts of P.
dean and Mesoamerican R-like genes was identified. vulgaris.
Consequently, the co-evolution process seems to be
governed by minor molecular changes. Furthermore, Synthesis of sulphated pentamers
family members within one haplotype (paralogues) are We have shown that co-expression of R. meliloti
not more similar to each other than they are to those nodBC and nodH genes in E. coli results in the bio-
from the other haplotype (orthologues). Therefore, synthesis of sulphated chitotetraose that is specifically
concerted evolution did not lead to homogenisation of N-deacetylated on the non-reducing residue. By us-
sequences within a particular haplotype. ing a nodC gene from a pentamer producing rhizobia
(such as Azorhizobium caulinaudans, or Rhizobium
D – Future directions sp. NGR234) we should be able to obtain the sulphated
• Test the functionality of the candidate disease R pentameric precursors for the synthesis of R. tropici
genes using an Agrobacterium tumefaciens transi- Nod-factors. Chemical acylation with an appropriate
ent expression assay. fatty-acid chain will yield the target molecules.
• Screen a P. vulgaris BAC library for the B4 R gene
cluster. Synthesis of acetyl-fucosylated pentamers
• Study the molecular diversity of the second half We have recently shown that E. coli can be meta-
of the LRR encoding region of the gene in wild bolically engineered to allow the in vivo synthesis of
genotypes of P. vulgaris from the three centres of fucosylated oligo-saccharides (Dumon et al., 2002).
94
Co-expression of the nod-gene that is responsible for • UNIAN (Papa) focuses on gene flow between wild
fucosylation (nodZ) with nodBC should thus result in and domesticated populations. Loci of interest in-
the production of chito-oligo-saccharides fucosylated clude those involved in domestication and disease
at the reducing terminus. Introduction and expres- resistance, natural selection mapping and influ-
sion of acetyltransferase gene nolL should lead to the ence of mating system on shaping the genetic
synthesis of R. etli Nod-factor precursors. diversity in beans. Here they will develop STSs
and SSRs from appropriate populations.
Availability of Nod-factors • UNIBAS (Zeuli) works on plant genetic resources
All synthesised Nod-factors will be made available to and population genetics. In Phaseomics their focus
the Phaseomics community. will be on biochemical and molecular characterisa-
tion of genetic diversity in local bean populations.
Germany (UG/G – Wolfgang Streit) • UNITUS (Soressi) studies the genetics of resist-
ance to biotic and abiotic stresses. In Phaseomics,
A description of our work and the importance of the team will work on the in vitro induction of ad-
vitamins in bean nutrition is given on page 60. In ventitious shoots from meristematic explants of P.
Phaseomics, we will: vulgaris and P. coccineus as part of an on-going
• Isolate and biochemically characterise bio1 and effort to set up reliable genetic transformation
bio4 genes coding for DAPA-aminotransferase and protocols.
dethiobiotin synthase, respectively, from Phase- • UNITUS (D’Ovidio) works on the characterisa-
olus plants. This will be done by hybridisation tion of the molecular events underlying plant re-
with known bio-genes and sequencing of complete sponses to biotic stresses. In particular, the re-
BAC clones. search is focused on clarifying the involvement
• To characterise the expression patterns of the isol- of the Polygalacturonase Inhibiting Protein (PGIP)
ated genes and evaluate the basic environmental that limits fungal colonisation of plant tissue.
factors affecting their expression. In Phaseomics the research group, in collabora-
• Explore the role of biotin bean metabolism, by tion with the University of Rome ‘La Sapienza’
identifying other biotin regulated genes such as (Cervone and De Lorenzo), will concentrate their
biotin transport genes, a biotin biosynthesis reg- efforts on defining the structural and functional
ulator (birA), biotin dependent carboxylases, etc. characteristics of the bean PGIP locus.
• ISCI (Carboni) are concerned with the quality of
agricultural products and the environmental com-
Italy (PIN – Roberto Papa and Phaseolus Italian
patibility of agricultural techniques. Beans suit-
network)
able for mechanical harvest, for freezing and for
The Phaseolus Italian Network (PIN) has been formed fresh market have been produced (varieties ‘bor-
to integrate and promote the different perspectives and lotto’ and ‘cannellino’). In Phaseomics, the group
projects among six Italian laboratories working on mo- will study the genetic basis of inheritance of dis-
lecular biology, genetics of conservation, evolution, ease resistance. The occurrence of physiological
population genetics, plant breeding and agronomy races will be investigated, the source of resistance
of Phaseolus spp. Groups involved include those of identified, and screening of segregant populations
Roberto Papa in Ancona (UNIAN) who co-ordinates for resistance to nematodes (Meloidogyne incog-
the project, Pierluigi Spagnoletti Zeuli, in Potenza nita and other spp), halo blight (Pseudomonas
(UNIBAS), Gian Piero Soressi and Renato D’Ovidio savastanoi pv. phaseolicola), Rhizoctonia, etc.
in Viterbo (UNITUS), Andrea Carboni in Bologna will be performed.
(ISCI), Valeria Negri in Università degli Studi di Per- • UNIPG (Negri) has a relational database and a
ugia (UNIPG), and Giovanna Attene in Sassari (UN- collection of over 100 Phaseolus accessions that
ISS). Phaseomics activities will be conducted by all have been almost completely characterised. Their
partners and organised in five work-packages (STSs, experience is in the genetics of reproduction and
ESTs, transformation, resistance to biotic stresses, and in the molecular characterisation of the pulses (in-
mapping populations), each one under the responsib- cluding Phaseolus). In Phaseomics they will focus
ility of a different partner. on the assessment of genetic variation of landraces
95
during on-farm conservation and reproduction un- identification and screening. The main target traits
der different environmental conditions. are shattering, dormancy and drought tolerance. In
• UNISS (Attene) focuses on plant genetic re- collaboration with others of the PIN and Phaseomics
sources, population genetics as well as the co- consortia, we will study the expression and function of
evolution of plants and pathogens. In Phaseomics gene sequences (ESTs) of interest, cloned from Phase-
they will work on the biochemical and molecular olus, in homologous (depending on the availability of
characterisation of genetic diversity in Italian bean an efficient transformation protocol), and heterologous
populations. (e.g., Lotus japonicus) model systems.
and ARP have studied the biodiversity in the libraries from various stages of flower and pod devel-
Phaseolus genus for several years (Limongelli et opment (see Table 9). After random sequencing a large
al., 1996; Lioi and Hammer, 1993). These stud- number cDNAs, selected clones will be used to fabric-
ies have been carried out in collaboration with ate micro-arrays. These micro-arrays will be used to
regional organisations and are aimed at the charac- examine gene expression during each stage of flower
terisation, evaluation and promotion of ‘on-farm’ and pod development by hybridisation against labelled
maintenance of local populations (Piergiovanni et RNA/cDNAs. Genes that are stage-specific will be
al., 2000). Our involvement is aimed at the ge- identified. Once a base line of modulation of gene-
netic recovery of local and old varieties threatened expression during each stage of development has been
with extinction. Analysing seed storage protein obtained, changes in gene expression that result from
profiles and RAPD or SSR markers will be used various environmental stimuli will also be analysed.
to investigate the genetic variability present in the
populations of each cultivar.
Pest and fungal resistance
Malaysia (MBC/M – Farida Shah) All plants possess a certain degree of resistance to
insects. This inherent resistance results from vari-
The Malaysian beans genomic group (MBGG) con- ous defence mechanisms, including a wide range of
sists of five laboratories – from the Melaka Bio- noxious secondary metabolites produced by the plant
technology Centre (MBC), the Universiti Kebangsaan (Schuler et al., 1998). Individual plants within one
Malaysia (UKM), from the Universiti Putra Malaysia genus, or even within one species, vary in their level
(UPM), from the Universiti Sabah Malaysia (USM) of insect resistance, a fact long used by plant breed-
and from the Malaysian Agricultural Research Insti- ers to increase the insect resistance of crop cultivars.
tute (MARDI). Within MBGG we have expertise in These different levels of resistance are related to ex-
molecular studies of the expression of genes involved pression of the specific genes in particular plants.
in fatty acid biosynthesis in oil palms (Shah et al., Many different genes confer insect resistance in vari-
2001), genetic manipulation of the fatty acid com- ous plant species, and many more are expressed after
position (Shah et al., 2002), genetic enhancement of insect attack (Pickett et al., 2001). Identification and
disease and pest resistance in oil palms, genetic ana- manipulation of insect resistance genes in plants, of-
lysis of floral development (especially to look for floral fers certain advantages over conventional insecticides,
abnormalities in oil palms), as well as EST analyses such as more-effective targeting of insects protected
of genetic traits in oil palms, bananas and melons. within plants, greater tolerance of adverse weather
Differential display has been used to isolate tissue conditions, fast biodegradability, reduced operator
specific clones and genes from mesocarp (Shah and exposure to toxins and financial savings (Mitchell-
Cha, 2000), kernel (Shah and Cha, 2001), leaves, olds et al., 1998). Widespread use of bioinsecticides
flowers, etc. of oil palms. Micro-arrays have been used should also lead to a reduction in the use of broad-
to follow expression of ESTs in flowers and roots. spectrum insecticides, thereby extending the useful
Membrane-based micro-arrays have already been used life of these compounds and reducing the ecological
to study the expression of oil palm genes. Currently, damage they cause (Baldwin et al., 2001). Here we
we have the capacity to sequence 800 clones (ESTs) a will identify genes expressed during pest and fungal
day (64 000 bp). MARDI is involved in the breeding attack of flowers and pods using micro-arrays.
of the beans. Beans will be treated with jasmonic acid, salicylic
In Malaysia, common beans (80% of which are acid or wounding prior to mRNA extraction since
destined for the export market) are only grown on a these agents have been reported to mimic insect and
small scale. Dwarf varieties have been cultivated but pathogen attacks. A DDRT-PCT method will be used
without much success. The main commercial cultivars to identify transcripts specifically expressed in treated
are MK12 and MK13. plants. mRNA obtained from untreated plants will be
used as a control. cDNA clones whose expression is
Floral and pod development up-regulated in an insect resistant manner will be se-
Little is known about the molecular basis of floral and quenced, and analysed. Northern/micro-array analyses
seed development in Phaseolus spp. Accordingly, our will then be used to study the expression pattern of the
contribution to Phaseomics will be to construct cDNA ESTs.
98
isoforms of root-actin resemble those of bean root– Most LEA proteins are part of a more wide-
nodules approaching senesce. Mono-ubiquitylation of spread group, which we call “hydrophilins”. The
actin is common in Rhizobium–legume interactions as criteria that are used to define hydrophilins are an ex-
well as in a wide range of plant–pathogen infections cellent predictor of responsiveness to hyper-osmosis
(Dantán-González et al., 2001). Since this modific- (Colmenero-Flores et al., 1997). Hydrophilins rep-
ation augments the stability of actin micro-filaments, resent analogous adaptations to common stresses in
we suggest that actin mono-ubiquitylation plays a key such diverse organisms as prokaryotes and eukaryotes.
role in "immunity" against microbial infections. This Questions that are being addressed include: (a) how
is an ancient strategy shared by plants, insects and do the structural and physicochemical characteristics
vertebrates (Nümberger and Scheel, 2001). Recently, of hydrophilins relate to the largely unknown func-
we reported (in bean nodules), that a single profilin tions of LEA proteins?; (b) are hydrophilins a solution
transcript gives rise to multiple isoforms (at least four) to a plant-specific problem, or to a more general
which are generated by phosphorylations on tyrosine one?; (c) do LEA proteins have protective properties?,
residues (Guillén et al., 1999), providing strong evi- and; (d) if they do, what protective function(s) are
dence for the existence of tyrosine protein kinases in exerted during dehydration? Some answers to these
plants. Here we will examine some of these pathways questions have already been provided. One observa-
using beans as the model system. tion is that expression of a lea gene that we identified
in P. vulgaris (Pvlea-18) (Colmenero-Flores et al.,
1999; Garay-Arroyo et al., 2000) during dehydration
Nod-factors and signal transduction (Carmen Quinto) is mostly independent of absicic acid (ABA). It is
We study the early signal transduction events induced also the first published example of where the 3 -region
by Rhizobium etli on the roots of Phaseolus vulgaris of a gene specifically participates in modulation of
(Cárdenas et al., 1995). Of special interest are the gene-expression in response to dehydration (Moreno-
dynamics of actin cytsokeleton, as well as oscilla- Fonseca et al., 2001). Additionally, in collaboration
tions in Ca2+ and other ions (Cárdenas et al., 1998, with J.-P. Vielle-Calzada (see México CIFN/UNAM),
1999, 2000). Among the most rapid responses of bean we are employing the “gene trap” system in Arabidop-
roots to the Nod-factors produced by rhizobia are the sis to identify and isolate genes whose expression is
changes in membrane potential and oscillation of cer- modulated by water deficits. Two proline-rich proteins
tain ions, notably Ca2+ , Cl− and H+ . To gain a better (p33 and p36), which accumulate in response to water
understanding of these ion fluxes in the P. vulgaris–R. deficits, have been identified that bind leaf protoplasts
etli symbiosis, we will examine different ion channel and PM vesicles. This binding can be inhibited by
populations using planar lipid bilayers, and determine a peptide that contains the RGD motif, as well as
their role in Nod-factor signalling. fibronectin, suggesting that the PM binding protein has
an integrin-like function whose natural ligands are p33
and p36. Characterisation of this interaction will be
México (IBt-UNAM/Alejandra Covarrubias;
continued by isolating and identifying those proteins
INIFAP/Jorge Acosta)
that bind p33/p36 to elucidate their roles in adapt-
Molecular and cellular bases of the plant adaptive ive stress. Finally, in collaboration with Jorge Acosta
responses to water deficit. at the Instituto Nacional de Investigaciones Agrícolas
y Forestales (INIFAP), and with June Simpson at
We study the molecular mechanisms of adaptive re- CINVESTAV [see México (CINVESTAV)], we are
sponses to osmotic stress, adverse conditions that are involved in the identification of molecular markers
frequently encountered and restrict growth of land associated with drought resistance.
plants. Our interest focuses on two main areas: (a)
the functional characterisation of genes (and their pro-
teins) involved in drought tolerance, especially the so-
called LEA genes (Ingram and Bartels, 1996); and (b)
the interaction between cell-wall proteins and plasma- Participation in Phaseomics
membranes (PM) in response to osmotic stress. Two
experimental models, are used – Phaseolus vulgaris Our main focus will be the analysis of pathogenic
and Arabidopsis thaliana. interactions and adaptation to drought.
100
Mexico (CINVESTAV – June Simpson, Luis 5000 ESTs of cDNA libraries constructed from mRNA
Herrera-Estrella) obtained from plants grown under phosphate limit-
ing conditions and plants infected with Colletotrichum
An increasing number of viral, bacterial and fungal lindemuthianum, the causal agent of anthracnose.
diseases can be effectively controlled using trans- Phosphate-limiting conditions and anthracnose have
genic strategies. For this reason, our research at the been chosen since they have been identified as two
Irapuato-Unit of the Centro de Investigacion y Estu- of the most important constraints limiting bean pro-
dios Avanzados is centred on the development of an ductivity worldwide.
efficient transformation system for beans as well as
the sequencing of ESTs from plants grown under The Netherlands (PRI/W – Jan-Peter Nap)
phosphate limiting conditions or from plants infec-
ted by Colletotrichum lindemuthianum. Progress in Plant Research International (PRI) is part of the Plant
bean improvement has been slow since an efficient Sciences Expertise Group of Wageningen Univer-
and reproducible transformation system has yet to sity and Research Center. Its research topics cover
be developed. The only well documented reports are the whole chain from gene and genome analysis to
those based on the bombardment of apical meristems. design and implementation of agricultural produc-
Unfortunately, these methods are intensive and have tion schemes. The co-ordinator’s interests include
dramatically low efficiencies – only 0.02% of the the stability of plant (transgene) expression (Mlyn-
regenerated plants transmit the introduced DNA to árová et al., 1996), gene silencing (Hutvágner et al.,
their progeny. Genetic engineering strategies, which 2000), development of novel approaches in statist-
rely upon the production of a relatively large num- ical plant breeding (Nap et al., 1997), as well as
ber of transgenic plants are thus severely restricted. genomics (Jansen and Nap, 2001). PRI operates a
Efficient transformation protocols are therefore essen- high-throughput DNA sequencing facility (Greenom-
tial; especially for studying e.g. the cis-acting DNA ics) that has been involved in the completion of the
sequences involved in tissue specific and environment- Arabidopsis genome sequence (Arabidopsis Genome
ally induced gene-expression. In turn, these promoter Initiative, 2000) and a successful microarray facility
sequences will be necessary to successfully produce (Aharoni et al., 2000).
transgenic plants with new agronomically important Although PRI has no prior experience with P. vul-
traits. garis as object of research, its facilities, expertise
We have developed a tissue culture system that al- and international focus well complement existing ex-
lows the production of embryogenic bean cell lines pertise and initiatives, notably those in the Genomics
from which mature plants can be obtained at high part of the Phaseomics framework. Contributions will
frequency. Using these embryogenic cell lines and include:
particle bombardment or Agrobacterium-based trans- • EST analysis: production and EST analyses of
formation systems, we have been able to produce a normalised library of aluminium-grown BAT93
Basta and hygromicin resistant plants. Although our Phaseolus roots/flowers; EST analyses of nor-
results suggest that the transformation of embryogenic malised and subtracted libraries (±high Al3+ ;
cell lines could become an attractive alternative for ±pathogen). From 1000 to 3000 EST’s per library
bean transformation, we still need to carry out South- will be sequenced and annotated.
ern blot analysis of the T1 progeny of the resistant • Bioinformatics: PhaseoBase, a central database of
lines to confirm that the resistant plants are indeed all data generated by the consortium will be gener-
transgenic. We will continue these efforts to develop ated and made ready for mining by the consortium
a high efficiency bean transformation system based on members. This database will be modelled on the
embryogenic cell lines. XGI/ISYS system of the US-based National Cen-
Since ESTs represent genes that are transcribed ter for Genomic Research (NCGR) with whom
under specific physiological or developmental condi- PRI collaborates.
tions, one way to identify genes involved in particular • Expression profiling: PRI will produce micro-
processes is the construction of gene libraries from arrays for the consortium-selected ESTs for ex-
RNA extracted from plants subjected to specific en- pression profiling of Phaseolus materials, prefer-
vironmental conditions and/or exposed to pathogens. ably also using the available RIL populations.
As part of the Phaseomics initiative we will sequence QTL analysis of microarray data combined with
102
mapping data is a novel approach to the isolation in the laboratory to isolate new Rhizobium phages.
of QTL genes and possible identification of other Their host range and biological characteristics will be
QTL determining genes. scrutinised.
• Genome sequencing: sequencing and annotation Biological factors (including root diseases, micro-
of two BACs selected by other partners for the bial antagonism, predators, etc.) affect nodulation and
presence of interesting markers/genome regions. nitrogen fixation. Co-inoculation studies of beans with
• Proteo-Metabolo-Phaseomics: detailed analysis of rhizobia and other beneficial bacteria are promising
the proteome and metabolome spectrum of selec- (Burdman et al., 2000). Some bacteria show excel-
ted Phaseolus materials. lent biocontrol activity (Rosas et al., 2001) and are
• Functional Phaseomics: in collaboration with an potential candidates for mixing with rhizobia in peat
East-European partner (Slovakia) further develop- inoculants. Reduction of root-rot can lead to better
ment and implementation of Phaseolus transform- nodulation (Perdomo et al., 1995). Furthermore, cur-
ation protocols to be used in HTP gene silencing rent research indicates that allelopathic compounds
approaches (VIGS, RNAi). excreted by common tropical weeds can severely re-
• Detailed analysis of the Phaseolus/fungal/microbial duce nodulation. Further investigation in this area is
pathogen interactions using fluorescence technolo- needed to improve the bean symbiosis. Finally, in or-
gies and associated RNA profiling. der to increase the BNF capacity of beans in farmers’
fields, we will help a Haitian firm develop inoculants
Puerto Rico (UPRM – Eduardo C. Schröder) including biofertilisers for sale in the Caribbean.
Improvement of the P. vulgaris-Rhizobium symbiosis South Africa (UWC/B – Chris Gehring and Graeme
in Puerto Rico Bradley)
P. vulgaris is a major component of the diet of Carib-
bean peoples, including those of Puerto Rico. Local Stress responses – roles of natriuretic peptides
production does not satisfy the demand however, and Natriuretic peptides are a well-studied class of ver-
a large proportion of beans are imported from the tebrate molecules that are involved in the regulation
USA. Since bean production is optimum at about 21– of ion and water transport in cells and whole or-
24 ◦ C, the best yields are obtained during the winter ganisms. Natriuretic peptides thus affect osmotically
months of the northern hemisphere, or at higher el- regulated processes and are an important contributor
evations during summer. Heat-tolerant varieties have to homeostasis. A class of biologically active plant
been selected to extend the bean-producing season proteins that react with antibodies directed against
(Fernández-Toledo et al., 1997). Similarly, the estab- a vertebrate natriuretic peptide, α-hANP, have been
lishment of highly efficient, nitrogen-fixing nodules defined as novel plant natriuretic peptides that play
is limited by many factors (Buttery et al., 1997; a role in the regulation of ion and water transport
Schröder, 1992). across plant cells (Billington et al., 1977; Gehring,
The reported genetic variability in host traits that 1999). These novel immunoreactant plant natriuretic
determine the amount of nitrogen fixed should be fur- peptides have been named irPNPs. IrPNPs have been
ther exploited, particularly as new bean germplasm is shown to promote stomatal opening (Billington et
available to farmers (Schröder, 1992). We will study al., 1997), to rapidly and reversibly increase cellu-
the effect of introducting exotic genes on the nod- lar cGMP-levels (Pharmawati et al., 1998, 2001), to
ulation specificity and nitrogen fixation capacity by modulate cation transport (Pharmawati et al., 1999)
crossing P. vulgaris with P. coccineus (runner beans) and bind specifically to cell membranes in vitro and
and P. acutifolius (tepary beans). in situ (Suwastika et al., 2000). IrPNPs also signi-
Biodiversity of strains occupying nodules (even on ficantly enhance osmoticum-dependent water trans-
the same plant) has been widely demonstrated, and port in mesophyll protoplasts (Maryani et al., 2001).
competition of introduced strains with local adapted Recently, two members of the irPNP family of mo-
ones is still a practical problem. Another goal is to lecules from Arabidopsis thaliana (AtPNP-A and -B)
study the molecular differences (by DNA fingerprint- have been identified in silico and subsequently isol-
ing) between strains isolated from nodules of land ated by RT-PCR (Ludidi et al., 2002). In addition,
races as well as local cultivars and compare their sym- we have also identified irPNP homologues in EST
biotic specificity. Soil samples will also be processed databases of Medicago truncatula and Glycine max
103
(Ludidi et al., 2002). Protein sequence analyses show • Cropping systems. Monoculture and intercrop-
that irPNPs occur in two sub-families; one found ping of beans with maize has been studied under
in both monocotyledonous and dicotyledonous plants different environments in the Northwest of Spain
and the other found only in dicotyledonous plants (Lu- (Santalla et al., 1994, 1995, 1999a,b, 2001b). In
didi et al., 2002). In addition, irPNPs are closely addition, the rhizobial symbiont in the different
related to CjBAp12 (a functionally undefined pro- systems has been evaluated in local landraces and
tein from citrus that is induced in response to blight breeding lines (Santalla et al., 2001c).
infection). IrPNPs and CjBAp12 share a common • Bean evolution. An analysis of the domestica-
ancestor, primitive glucanase-like molecules that are tion process in wild Andean and antique landraces
similar to fungal β1–4 endoglucanases. Both irPNPs is being carried out by phenotypic, biochemical
and CjBA12 are related to the cell wall loosening and molecular studies (De Ron et al., 1999). Fur-
expansins, although at least CjBAp12 has no expansin- thermore, a secondary centre for diversification
like activity. Moreover, in keeping with their increased of common beans has been found in southwest-
extracellular mobility and effects on cell membranes ern Europe. Variation in allozyme patterns re-
rather than the cell wall, irPNP-like molecules do vealed forms intermediate between the Andean
not contain the wall-binding C-terminus of expansins. and Mesoamerican genetic pools (Santalla et al.,
Importantly, irPNP-like molecules are present in con- 2002).
ductive tissues suggesting that they are transported, • Dry bean breeding. This work focuses on the im-
an observation that is consistent with their role as provement of specific agronomic traits and seed
systemic messenger. quality (Escribano et al., 1997; Monteagudo et
We are currently using Arabidopsis thaliana to al., 2000; Rodiño et al., 2001a; Santalla et al.,
monitor transcriptional and translational control of 1999b, 2001a), as well as on multiple resistance to
irPNP expression and these studies will be extended diseases (BCMV, Pseudomonas, Xanthomonas).
to beans. We will also test for biological activit- Selection is based on biochemical and molecular
ies of recombinant proteins and/or selected domains markers.
in vitro and in vivo. Functional testing will include • Scarlet bean breeding. P. coccineus is widely cul-
monitoring the effects on cation transport, osmoticum- tivated in Spain. Accordingly, Spanish landraces
dependent water transport, mobilisation of second have been evaluated for agronomical performance
messengers (e.g. cGMP), activity of ATPases as well and seed sensorial quality (Martínez et al., 2002).
as stomatal guard cell movement. Our major long-term Some breeding lines have been used in interspe-
programme is to first understand and then increase cific crosses.
drought and salinity tolerance in legumes. Our re- • Snap bean breeding. A new project includes eval-
search is strengthened significantly by ongoing and uation of pod quality in various landraces (e.g.
close collaboration with SANBI (South African Na- yellow pods as demanded quality by the market).
tional Bioinformatics Institute).
Switzerland (LBMPS/GE – Bill Broughton and Xavier
Spain (MBG-CSIC – Antonio M. De Ron and Marta Perret)
Santalla)
Rhizobial determinants of effective nodulation of
Our work at the Misión Biológica de Galicia (MBG- beans
CSIC, Pontevedra, Spain) is focused on: Rhizobium sp. NGR234 nodulates common beans but
• Germplasm. A large collection (1125 accessions) the nodules are often ineffective (i.e. they do not fix
of wild and cultivated beans (P. vulgaris and P. nitrogen) (see Pueppke and Broughton, 1999). Un-
coccineus) has been assembled through national doubtedly, rhizobial genes exist that help control ef-
and international missions in Europe and South fectiveness since a number of isolates (Rhizobium etli,
America (De Ron et al., 1997). This material has R. tropici and R. leguminosarum bv. phaseoli) fix large
been used in studying genetic variation, evolution, amounts of nitrogen with this plant (Michiels et al.,
breeding and cropping systems. Some accessions 1998). A simple way of identifying these genes is to
form the basis of breeding lines (Escribano et al., mass conjugate a e.g. R. etli cosmid library (cloned in
1994, 1997, 1998; Gil and De Ron, 1992; Rodiño a transmissible vector) into NGR234 and to use pools
et al., 2001b, 2002). of the transconjugants to inoculate beans. Simply
104
by screening the inoculated plants for yellow- (non- by flotation in liquid nitrogen, and the frozen tissue
fixing) or green-leaves (efficient in nitrogen-fixing) used for extraction of nucleic acids, proteins, etc.
will identify pools of transconjugants, which contain Messenger RNA can be isolated, cDNA libraries es-
R. etli genes that are able to confer the capacity to fix tablished, and selected clones sequenced. We will use
nitrogen on NGR234. Two, complementary methods these techniques to generate ≈5,000 ESTs from both
will then be used to delimit the actual transconjug- treated and untreated root–hairs as well as internodes.
ate(s) that is(are) responsible for this phenotype. One
will be to isolate the occupants from the effective nod-
Expression analysis of ESTs (transcriptomics)
ules, and from them, the cosmid in question. This
method suffers from re-arrangements that are likely Our laboratory helped pioneer studies on gene expres-
to occur amongst the different replicons in NGR234 sion of bacteria that interact with plants (Fellay et al.,
(chromosome, mega-plasmid, symbiotic plasmid, in- 1995; Freiberg et al., 1997; Perret et al., 1999). We
troduced cosmid) during nodulation. For this reason, will use this expertise, along with all the EST se-
the cosmid pool that yielded the effective nodules will quence data generated by other groups in the project
be sub-divided into smaller pools, which will then be to construct micro-arrays. RNA will be isolated from
used to inoculate and screen further bean plants. Each all the tissues listed in Table 9, labelled and hybridised
of these two types of experiments will be repeated un- against the micro-arrays to study the expression of a
til a cosmid (or a set of over-lapping cosmids) has been large collection of ESTs. These data will be essential
identified. Then, this cosmid(s) will be characterised to understanding the physiology of bean growth and
first by complete sequencing, and then by mutational how the plants respond to the environment.
analysis. Methods of this kind have successfully been
used to identify NGR234 genes that are involved in Proteomics of nodule development
nodulation of various legumes (Broughton et al., 1984,
Evidence is accumulating that the symbiotic sig-
1986; Lewin et al., 1987).
nal transduction pathway involves changes in phos-
phorylation of a number of membrane-bound proteins
Root–hair ESTs in root–hairs (Irving et al., 2000; Kelly and Irving,
Phaseolus species only fix low amounts of nitrogen 2001, 2002). We have developed ways to identify and
when compared to other legumes (see Table 8). An isolate some of these proteins (Boukli et al., 2002).
important objective of ‘Phaseomics’ therefore is to So far these methods have focused on individual pro-
ameliorate nitrogen fixation in beans. Detailed ana- teins, but in collaboration with the Australian Pro-
lysis of nodulation and nitrogen fixation in beans is teome Analysis Facility (see Australia APAF, p. 79)
thus essential to increase production of beans. Gen- full, proteome scale analysis of especially membrane
erally, root–hairs emerge at the apical end of some proteins will be studied. Approaches of this kind have
epidermal cells, and elongate by polar growth of the already been applied to thylakoid membranes (Hippler
tip. Young, elongating root–hairs are extensively col- et al., 2001) and should thus also be applicable to
onised by soil-borne micro-organisms. Rhizobia enter root–hairs. Knowledge of how nodulation is regulated
the roots (and occasionally adventitious-roots on the along with access to the controlling genes should per-
stems) of legumes, and induce the formation of highly mit the development of more efficient nodulation and
specialised organs called nodules. Rhizobia present in nitrogen-fixing plants.
the root nodules convert to an endo-symbiotic form,
the bacteroids, in which dinitrogen is reduced to
ammonia. Bacteroids within nodules contribute the Coordination of the global project
majority of fixed nitrogen to the global pool. In addition to the research listed above, LBMPS
In other words, symbiotic bacteria first contact the will coordinate the ‘Phaseomics’ project. At the sci-
root–hairs of legumes. Obviously it is within this inter- entific level this will involve collecting all the dispar-
face that early recognition events occur which largely ate information from the research laboratories, ana-
control further development of the symbiosis. In our lysing it, and making it available on our web-site
laboratory, we have developed methods for analysing [www.phaseolus.net]. LBMPS will also be responsible
the molecular changes that occur in root–hairs fol- for co-ordinating the raising of funds to support the
lowing inoculation with rhizobia (Krause et al., 1992, international collaboration that will be necessary for
1994; see Irving et al., 2000). Root–hairs are isolated the success of this project.
105
Figure 6. A. Chlorophyll a fluorescence imaging of diuron transport in Arabidopsis thaliana leaves. Diuron (a herbicide, inhibits photosynthetic
electron transport. As a result, light energy is dissipated as heat and fluorescence) was topically applied to one leaf that is discernible in panels
b–e by its uniformly high chlorophyll a fluorescence. Diuron is first transported to the upper leaves. (a) The rosette before treatment; (b) 2 h
after treatment (c), 4 h after application of diuron, the herbicide has spread to the petioles of lower leaves in the rosette; (d) 6 h after diuron
application. The increase in chlorophyll a fluorescence has expanded into the upper leaves of the rosette and along the main veins of the lower
leaves; (e) 10 h after application, diuron has spread to the side veins; (f) greyscale reflectance image of the treated Arabidopsis rosette. Further
spreading of diuron along the side veins is evident. B. O-J-I-P-induction kinetics of chlorophyll a fluorescence in leaves of Vigna unguiculata
plants grown in a nutrient solution containing 0.5; 1.0; 5; 10; 20 mM KNO3 (Ft from top to bottom) along with the corresponding differences
in kinetics of each treatment compared with plants grown in 20 mM KNO3 (Ft ). Kinetic data are presented on a logarithmic time scale from
50 µs to 2 s, and this period has been sub-divided into the O-J-I-P steps. The fluorescence traces were normalised between 50 µs and 100 ms
(measuring time 2 s per sample). A PEA fluorimeter (Plant Efficiency Analyser – Hansatech Instruments, Narborough Road, Pentney, King’s
Lynn, Norfolk PE32 1JL, UK) was used. The increase in fluorescence intensity at 300 µs is typical of nitrogen starvation.
106
Figure 7. Evolutionary divergence among key taxa (labelled 1–4) in the Phaseolus genus. Each taxon is followed by a description of its
phenotype for the APA locus (PHA: phytohaemagglutinin; αAI: alpha-amylase inhibitor; ARL: arcelin) and the PHS (phaseolin) locus. For
further explanations, see text. For each of the four taxa, a BAC library is being developed.
Switzerland (BIOEN/GE – Reto Strasser) 3. The instrument that measures fluorescence is light-
weight, portable and independent of external
Our goal is to establish functional behavioral patterns power sources for more than a working day.
of living plants. As an index of plant health, we 4. Fluorescence data are digitised in real time and
use the poly-phasic chlorophyll a fluorescence tran- stored in the instrument’s core memory.
sient O-J-I-P (Srivastava et al., 1995; Strasser et al.,
1995; Tsimilli-Michael et al., 2000). Fast fluorescence
United States of America (ARS/UC – Paul Gepts)
kinetics (10 µs to 15 min) emitted by chlorophyll
containing tissues are analysed using the so-called JIP- Evolutionary genomics of beans
test. Typically, data are collected with a measuring
Knowledge of evolutionary patterns and processes is
time of ≈1 s, a 10 µs time of resolution and 12-bit
essential to understand how organisms, in general,
digitalisation of the signal (Figure 6). As the JIP-test
and plants, in particular, develop new traits, espe-
can easily be used to analyse plants under stress, it
cially those of agronomic interest. Common bean is
is a valuable tool for screening different phenotypes.
an excellent model in this respect (see Introduction),
JIP-test technology has been used in international pro-
because of the extensive knowledge developed on the
jects dealing with pulses (in India), establishment
phylogeny of the genus and the genealogy of the
of banks of stress-data (Australia), sugar cane and
species.
soybeans (South Africa), and drought stress of peas
In this respect ARS/UC studies two main themes:
(Spain). Advantages of the JIP test (Strasser et al.,
(1) evolution of small multi-gene families involved
2000) include:
in seed protein production; and (2) evolution of do-
1. The short measuring time (a few seconds per mestication traits. The former include the gene family
sample) permits the analyses of large numbers of coding for phaseolin, the major seed storage protein of
plants. This is important in screening programmes common bean, and the APA family, i.e., the arcelin–
and to ensure statistical significance. phytohaemagglutinin–alpha-amylase inhibitor family
2. Samples can vary enormously and include leaves, that is involved in defence against animal predators,
micro-plants, tissue- or suspension cultures, sus- especially seed weevils. Domestication traits include
pensions of cells or chloroplasts, clones of algae those that distinguish various bean cultivars from one
on agar plates, etc. another. As examples, the determinacy gene controls
107
growth habit and is often found in domesticated bush In a segregating population such as an F2 , BC1 , or
beans, especially in snap-bean cultivars, where it as- RI population, correlation between the segregations of
sures both earliness and single-pass harvest of pods of genes is generally interpreted as resulting from phys-
more or less the same age. The pod-shattering gene ical linkage on a DNA molecule (or chromosome).
is essential to wild beans to assure seed dissemination An advantage of this approach is that all individuals
and reproduction of the plant. In domesticated beans, in these populations are genetically related because
this is obviously a deleterious trait. they stem from the same two parents. This approach
Genomics offers the potential to isolate the genes therefore circumvents genetic drift and can lead to cor-
responsible for these traits and, in turn, improve them relations among genes, regardless of whether they are
in superior cultivars. Some of the results to be ex- linked or not. A disadvantage is the limited number
pected from genomics activities in association with of segregating generations during which effective re-
classical breeding are increased protein levels, resist- combination (i.e., between double heterozygotes) can
ance to seed-boring insects, reduced pod shattering, take place. Thus, the placement of a gene is typic-
and improved plant type (Table 8). For the isolation ally imprecise. This is especially true for genes that
of the phaseolin and APA gene families, both coded have small phenotypic effects and/or environmental
by a single, complex locus, we are developing four regulation.
bacterial artificial chromosome (BAC) libraries. These Linkage disequilibrium (LD) analysis is an altern-
are being constructed from genotypes that were care- ative measure of association, which relies on existing
fully chosen to represent successive stages in the APA populations of unrelated individuals rather than on se-
and phaseolin multi-gene families (see Figure 7). Gen- gregating populations resulting from a cross. The use
otype 1 is a Phaseolus lunatus cv. Henderson (lima of genomics promises to instill a new life into this
bean) line that is representative of the legume fam- concept, principally as a way to locate genes on a
ily in that it only has the PHA (phytohaemagglutinin) linkage map, by allowing the development of markers
component of APA. Its phaseolin is characterised by that: (A) can be used to develop a fine map around the
post-translational cleavage, which is unusual in Phase- locus of interest; and (B) analyse the structure of ge-
olus species. Genotype 2 is P. vulgaris DGD 1962, netic diversity. Indeed, one of the drawbacks is that LD
which is representative of the presumed ancestor of P. can be caused by factors other than physical linkage,
vulgaris from Ecuador and northern Peru and there- such as genetic drift, gene flow (admixture), and selec-
fore a key to the understanding the genetic diversity tion. Many of these disadvantages can be overcome if
of P. vulgaris. Genotype 3 is P. vulgaris cv. BAT93, a large number of well-chosen molecular markers are
a multiple disease resistant genotype that is also one used on a genome-wide basis. Most of the recent pub-
of the parents of the core mapping population of com- lished studies on LD on a genome-wide analyses have
mon bean (Freyre et al., 1998; Kami and Gepts, 2000). been conducted in a limited number of species, includ-
Genotype 4 is P. vulgaris G02771, a carrier of arcelin ing humans (Daly et al., 2001; Jeffreys et al., 2001),
and strong resistance against seed weevils. Ordered Drosophila pseudoobscura (Schaeffer et al., 2001),
groups of overlapping clones (contigs) each compris- and Zea mays (Remington et al., 2001; Thornsberry
ing about 180 kbp will be constructed around the APA et al., 2001). Irrespective of the inherent variability
locus and the organisation of APA genes determined. among loci, the lowest levels of LD were found in D.
Sequencing of the APA locus in the four genotypes pseudoobscura and maize (about 1 kbp), followed by
will then be initiated. Similar techniques will be used humans (several kbp). Towards these ends, the fin and
on the phaseolin locus (Phs) that encompasses some st genes have been mapped on linkage groups 1 and
190 kbp. 2, respectively (Koinange et al., 1996 – see Figure 7).
In addition, we will focus on the determinacy (fin) RFLP markers have been or are being transformed into
and pod string (st) loci. Unlike the seed protein loci, Sequence Tagged Sites (STS) to facilitate their use as
the genes for these loci have not yet been isolated, PCR-based markers, with or without subsequent di-
although candidate genes may be found amongst the gestion with restriction enzymes. These markers will
ESTs of meristems and pods, respectively. An addi- serve in preliminary experiments (on a genome-wide
tional tool, which will have great repercussions in crop basis) to measure LD in common beans around loci of
biodiversity, characterisation and utilisation, is link- interest. Target loci will include not only the fin and
age disequilibrium (LD) analysis. Traditionally, genes st loci but also the Phs and APA loci to pinpoint the
have been located on genetic maps by linkage analysis.
108
specific areas that may be involved in e.g. the amount tide for transport into the chloroplast. In wheat, maize
of protein produced. and rice, the cytoplasmic and chloroplast isozymes
differ in sequence at many places, but one residue
USA (DMGCB/UC – Bob Haselkorn) in particular, an ile/leu residue, determines sensitivity
or resistance to herbicides of the aryloxyphenoxypro-
Acetyl CoA carboxylase in Phaseolus vulgaris pionate class (Joachimiak et al., 1997; Zagnitko et
Acetyl CoA carboxylase (ACCase) catalyses the first al., 2001). Monocotyledonous chloroplast isozymes
committed step in fatty acid biosynthesis, the addi- are sensitive while those of the Dicotyledons, with
tion of CO2 to acetyl CoA to make malonyl CoA. their prokaryote-type of chloroplast ACCase, are all
This reaction occurs in two steps: the ATP-dependent resistant.
carboxylation of biotin followed by the transfer of In green tissues of wheat, chloroplast ACCase
the carboxy group to acetyl CoA. In prokaryotes, accounts for 95% of the total ACCase mRNA and AC-
this synthesis involves the activation of CO2 by bi- Case activity. In roots, most of the ACCase mRNA
otin carboxylase (BC, a homodimer) and addition of and activity are cytoplasmic. The malonyl CoA pro-
the CO2 to biotin covalently linked to a lysine side- duced in chloroplasts is used for fatty acid syn-
chain on the biotin carboxyl carrier protein (BCCP, a thesis. In the cytoplasm, malonyl CoA is required
homodimer). Carboxytransferase, an a2b2 heterotet- for malonylation reactions, synthesis of the nuclear
ramer, then transfers the carboxy group from biotin to envelope, and secondary metabolite synthesis, includ-
acetyl CoA. In most eukaryotes, all four domains of ing flavonoids. In wheat, the levels of all classes of
ACCase are located on single, large polypeptides of at ACCase mRNA are developmentally regulated. This
least 2600 amino acids. These function as dimers or has been studied using sectioned seedlings, measur-
higher order polymers. Many levels of phosphoryla- ing the RNA levels using Northern gels, RT-PCR,
tion as well as feedback using downstream metabolites and promoter-GUS fusions in transient expression as-
control ACCases. says, both callus and intact embryos. Virtually nothing
In plants, fatty acids are synthesised in chloro- is known about ACCase in beans. Based on experi-
plasts. The chloroplast ACCase in grasses is a typical ence with other plants such as soybean and various
eukaryotic multi-domain ACCase, whose transport Brassicae, however, it should be possible to identify
into the chloroplast is facilitated by a transit peptide both cDNA and genomic clones of ACCase genes in
at the N-terminus. In dicotyledonous plants, however, libraries of Phaseolus DNA. We expect to find genes
the chloroplast ACCase is like that of prokaryotes, encoding the multi-domain cytoplasmic ACCase and
comprised of four separate polypeptides. In the cases the multi-component chloroplast enzyme. Sequencing
already studied (Arabidopsis, spinach, tobacco) one of these genes and cDNAs will provide the necessary
these polypeptides, the beta subunit of the carboxy- background information for developmental studies of
transferase, is encoded in chloroplast DNA while the ACCase gene expression including the response to
other three are encoded in the nucleus. The situation nodulation and Nod-factors. As the complete pathway
in P. vulgaris is unknown. for fatty acid biosynthesis has been described in Ar-
We have studied the organisation, evolution and abidopsis, it should be possible to use Arabidopsis
function of the ACCase gene family in hexaploid information to clone the Phaseolus counterparts. In
wheat (Gornicki et al., 1993a,b; 1994). The cytoplas- this way, we expect to assemble a set of probes to
mic enzyme is encoded in a small gene family consist- follow expression, using limited micro-arrays, of the
ing essentially of two tandemly repeated genes on each entire fatty acid pathway during nodulation of beans.
of the ancestral chromosome sets (Podkowinski et al.,
1996). These genes each contain 32 introns, are about USA (EL/MSU – James Kelly)
20 kb long, undergo alternative splicing, and have two
promoters each yielding leaders with complex splicing Bean breeding is a multifaceted challenge requiring
and translational control sequences. A single gene on an optimum balance of the ‘tried and true’ traditional
each ancestral chromosome set encodes the chloro- breeding approaches with the need to incorporate new
plast enzyme (Gornicki et al., 1997). The chloroplast methods that could improve efficiency or permit the
isozyme is similar to the cytoplasmic enzyme in size exploitation of genetic variability for traits of eco-
and organisation, differing principally by the inclusion nomic value. Traditional breeding methods need to
of an N-terminal extension serving as a transit pep- be varied depending on germ-plasm, objectives, traits,
109
and resources and should be periodically evaluated or locus. In addition, we are developing SSR and
changed, as no single method is suitable for all situ- AFLP markers on B8.
ations (Kelly et al., 1998). Finding ways to incorporate 2. Constructing a BAC library of the genotype SEL
the new biotechnology tools and ‘traits’ will be chal- 1308. One essential requirement for molecular
lenging as the technologies demand increased costs cloning of genes is the generation a clone library
and facilities associated with an increased level of un- with large DNA inserts. The most commonly used
certainty regarding outcome and usefulness (Kelly and system for constructing a large insert genomic lib-
Miklas, 1998). The potential divisiveness of intellec- rary is the bacterial artificial chromosome (BAC)
tual property considerations, and consumer concerns system. A BAC library of the Co-42 locus con-
currently cloud the future of biotechnology, plant taining genotype SEL 1308 will be constructed
transformation and the potential impact of genomics in (Vanhouten and MacKenzie, 1999).
bean breeding in the XXI century. The bean breeding 3. Selecting BAC clones that contain the Co-42 locus.
and genetics programme at Michigan State University A PCR based strategy will be used to select BAC
utilizes an integrated approach to bean improvement clones that contain the Co-42 locus. BACs will
that employs methodologies that identify and exploit be separated into pools and markers linked to the
novel genetic variability in the wild species; that incor- gene will be used to select pools that contain the
porate marker technologies to enhance quality traits locus. Pools that are positive will be screened for
as well as protect against biotic stresses; and that in- individual BACs that contain the gene by PCR.
tegrate genetic maps to assist in gene discovery using 4. Chromosome walking to sequence the Co-42
map based cloning and plant transformation systems locus. Chromosome walking to sequence the re-
to achieve specific objectives. A gene that imparts res- gion will be performed using the Universal Gen-
istance to a major disease pathogen of common bean ome WalkerTM Kit (Clonetech). The selected
is a current target. clones will be digested and a Genomewalker ad-
We have discovered a unique anthracnose resist- apter will be ligated to the blunt ends. PCR amp-
ance gene, Co-42 that conditions resistance to 97% lification using primers designed for the adaptor
of races of Colletotrichum lindemuthianum present in and a marker linked to the gene will be carried out.
North and South America (Balardin and Kelly, 1998). Hopefully, the result will be a single PCR product
Tests indicate that the Co-4 locus is multi-allelic and that has a known 5 end sequence and extends
encodes a protein kinase (Melotto and Kelly, 2001). into the unknown adjacent genomic DNA. This
Understanding the molecular organisation of resist- product will be cloned and sequenced. Sequences
ance genes will shed light on their evolution and are aligned in a contig based on overlapping re-
facilitate studies on plant–pathogen interactions. We gions.
plan to use a map-based cloning strategy to isolate 5. Identification of promoters, ORFs, splice-sites,
the Co-4 locus in the black bean genotype SEL 1308. etc. Using the sequence information, candid-
Map-based cloning includes: (1) saturating the region ate genes will be tested for function and race
with molecular markers and identifying tightly linked specificity using a plant transformation biolistic
flanking markers; (2) chromosome walking to the gene method developed by Aragão et al. (1998, 2002)
of interest using a genomic library; and (3) confirming for use in common bean.
the function of the isolated gene. Many disease resist-
ance genes have been successfully cloned using this USA (NDSU/F – Phil McClean)
approach. The experimental procedure will consist
of: Beans are an important international source of
1. Saturating the Co-42 gene locus with tightly linked protein as reflected by the fact that the dry
markers. To facilitate the cloning of the Co-42 gene bean export market alone (exclusive of canning
we need to further saturate that region with addi- beans) has a value to the US economy of $1.8
tional markers. To date, we have identified four billion (http://www.ers.usda.gov/briefing/drybeans/).
SCAR markers tightly linked to the Co-42 gene The cash value of the crop at the farm gate is $1
and with the recent mapping of Co-42 to linkage billion. Many agronomic factors contribute to their im-
group B8 on the core Phaseolus map (see Figure portance, particularly because they provide a valuable
4), we have access to other genetic linkage maps crop to the farmer even though production has been
providing >20 tentative markers adjacent to this pushed to marginal quality soils by other crops. Farm-
110
ers utilize these marginal soils from sea level to as high lished data). R. tropici pho mutants can be effective
as 3000 m. Furthermore, although common bean is tools for dissecting host-bacteroid P relations. We
a tropical legume, its cultivated range extends from plan to use these mutants in experiments to invest-
the tropics to 45◦ latitude. This agronomic plasticity igate host nodule gene expression in response to ex-
is a direct result of the genetic diversity of the species. perimentally manipulated bacteroid P acquisition and
As an example, the United States Department of Agri- metabolic activities. We will pair these mutants and
culture (USDA) recognises ten common bean market their wild-type parental strain with common bean gen-
classes, each distinguished by their size, seed coat otypes (two cultivars) that differ in P-use efficiency
colour and pattern. Although the species is diverse, and are available from our collaborations with Dr. J.-J.
breeding efforts have only made marginal improve- Drevon.
ments in yield, especially in comparison with other The experimental approach that we will use in the
crops such as cereals. An important objective for the first instance will be based on micro-arrays. Each of
Phaseolus research community in the new millennium the two bean P-use efficiency cultivars will be grown
is to describe research approaches that best charac- in combination with the wild-type R. tropici strain
terise the important agronomic diversity necessary to CIAT899. Nodule-specific mRNA from each symbi-
improve this crop. osis will be isolated using oligo-dT affinity columns,
and cDNA libraries prepared. For hybridisation ex-
Genomics, especially ESTs periments, mRNA from appropriate pair-wise com-
ESTs are important in developing new classes of mo- binations between the bacterial pho mutants and the
lecular markers needed for applied genetics research, bean P-use cultivars will be isolated, and labeled
and will lead to the discovery of simple sequence by reverse transcrition in the presence of the fluor-
repeat (SSR) variation in common beans. This in- escent dyes Cy3-dUTP, or Cy5-dUTP. Differentially
formation is necessary to develop user-friendly SSR labeled mRNA populations will be co-hybridised to
markers that can be applied, for example, in crop im- each respective array, and the arrays will be scanned to
provement programmes. To do this, cDNA libraries assess Cy3/Cy5 ratios from each spot. Differentially
will be created from two different genotypes for each expressed (both up- and down-regulated) sequences
tissue source. Comparable genes identified in the two will be identified by relative differences in fluorescent
parental genotypes will be scanned for SSR variation. color emitted. Replicate arrays will be made, allow-
The use of BAT93 and Jalo EEP558, two genotypes ing for statistical analysis (ANOVA) on each candidate
used extensively for mapping purposes, will allow us differentially expressed gene. Confirmation of candid-
to place these newly discovered ESTs on a common ate differentially expressed genes will be made using
linkage map. The map can be used for both basic Northern blot analysis. We will also prepare labelled
and applied genetics research. Steps in this project cDNAs from leaf and root tissues from these same
will include: (1) cDNA library development; (2) high pairings, but will provide these to other interested
throughput sequencing; (3) search for novel genes; (4) labs within the collaborative group for examination of
SSR discovery; (5) data analysis (see also UW/M – system-wide effects of altered nodule P metabolism on
Eric Triplett). general host metabolism and gene expression.
As development of the EST database pro-
USA (MSU/B – Tim McDermott and Dan Bergey) gresses, comprehensive and relatively rapid analysis
of changes in host gene expression during nodulation
Over the last decade, the McDermott laboratory has will become increasingly feasible using serial analysis
focused on phosphorous (P) metabolism in Rhizobium of gene expression (SAGE). We have practical experi-
tropici-bean symbiosis (Al-Niemi et al., 1997, 1998; ence making SAGE libraries and using SAGE-specific
Botero et al. 2000). Based on this work we sug- analytical software.
gest that the bean plant provides its bacteroids with
very low levels of inorganic P. We also know from USA (UA/T – Richard Musser)
labeling work (Al-Niemmi et al., 1998), and P fer-
tiliser placement experiments (unpublished data) that Functional genomic analyses of bean defense
bean nodules are a strong sink for root-acquired P, and responses to insect attack
that proper nodule P nutrition appears to be critical Herbivory triggers plant responses that are qual-
to the overall performance of the symbiosis (unpub- itatively and quantitatively different from artificial
111
wounding or mechanical damage. Recently, oral secre- Butterfly) which feeds on maize, beans, and Ara-
tions from insects have been shown to elicit profound bidopsis, respectively. We expect that the patterns of
effects on the responses of plants. Oral secretions of gene expression in the host-plants will depend on the
herbivorous insects can stimulate anti-herbivore de- specific herbivore and their salivary elicitor.
fences, plant growth, and phytopathogen resistance. Hopefully, these experiments will provide a gen-
The elictiors β-glucosidase and volicitin stimulate ome wide indication of how plants respond to salivary
anti-herbivore defences. However, we found that gluc- elicitors and insect herbivory. Knowledge of how a
ose oxidase, a salivary component of Helicoverpa plant detects and responds to herbivory is severely lim-
zea can suppress induced anti-herbivore defences in ited especially in comparison to our understanding of
tobacco plants. Glucose oxidase also appears to be how a plant detects and responds to plant pathogens.
an elicitor of plant pathogen defences. Ribonuclease
activity is found, at high levels, in Mexican bean USA (UN/L – Sally Mackenzie)
beetle regurgitant. We found that Phaseolus vulgaris
cv. Pinto bean leaves that were wounded and treated Genomics and transcriptomics
with RNase had increased virus resistance. This evid- We have constructed a BAC library of total gen-
ence suggests that RNase activity in the regurgitant omic DNA of the bean cultivar Sprite (Vanhouten
of the Mexican bean beetle functions as an elicitor of and Mackenzie, 1999). Sequencing of the ends of
plant pathogen defences. individual BACs will be initiated so that a complete
The specific objectives of our proposal are to: physical map of the bean genome can be developed.
1. Identify salivary factors responsible for plant de- BAC contigs will be anchored to molecular mark-
fence responses. ers from beans, soybeans, Medicago spp. and Vigna
2. Determine how insect herbivory alters plant de- unguiculata (long-beans, cowpeas) for comparative
fence genes via DNA microarray analysis. genome analysis. In addition, mRNA will be isolated
3. Correlate plant protein expression with gene ex- from developing ovules and roots of P. vulgaris cv.
pression in response to herbivory. Sprite and these ESTs contributed to the Phaseomics
4. Correlate metabolic data with gene expression in database.
response to herbivory.
5. Correlate whole plant effects with susceptibility to USA (MU/M – Dale Noel)
herbivory, and to disease.
6. Compare gene expression in plants with insect age, Determinants of infection and bean nodule
and location of herbivory. development
7. Compare gene expression of plants with tolerance Insufficient nitrogen fixation by beans in agriculture
to herbivores. often derives from poor infection and nodule devel-
8. Examine the possible relationships between gene opment. At Marquette University we study bacterial
for gene interaction between herbivores and plants. factors that affect infection of the plant after Nod-
We will compare the genome wide response to factors trigger nodule initiation. These studies have
herbivory by insects with different feeding modes and shown that progress of the infection thread determ-
salivary components that are thought either to stimu- ines whether later events in nodule development occur.
late or suppress plant defences on two economically Unless the infection thread crosses five cell layers of
important host plants, maize and bean. In addition, the developing nodule, the primordium develops into a
we will utilise Arabidopsis thaliana as a comparat- pseudo-nodule that resembles a lateral root rather than
ive model genomic system. As insects we will use a true nodule (Newman et al., 1992; Noel, unpub-
two generalist herbivores, Helicoverpa zea (Corn Ear- lished). If however infection stops after crossing ten
worm/Tomato Fruitworm) that feeds on maize, beans, cell layers, development proceeds to a later endpoint
and A. thaliana as well as Whiteflies which feed on that has true nodule anatomy with a central zone of
beans, and Arabidopsis. Gene expression of plants two types of cells; a normal peripheral vasculature,
suffering from herbivory will be compared to those and other tissue layers characteristic of a true nod-
of plants that have been fed upon by insect special- ule, even though bacteroids are absent (Newman et al.,
ist herbivores such as Diatraea grandiosella (South- 1992; Noel, unpublished).
western Corn Borer), Epilachna varivestis Mulsant These generalisations apply to determinate nod-
(Mexican Bean Beetle), Pieris rapae (Lesser Cabbage ule development, at least as it occurs in the tribe
112
Phaseoleae (that includes soybeans as well as com- or for ESTs induced early in infection by the wild-type
mon beans). Other ‘model’ legumes (L. japonicus and bacteria but not by mutants defective in LPS.
M. truncatula) are not suited to studying this type of
development. Nodulation of M. truncatula is inde- USA (UW/M – Eric Triplett)
terminate; while that of L. japonicus is determinate
but deviates in many important details from that of Genomic Interspecies Micro-array Hybridisation
the Phaseoleae. Aside from its agronomic importance, (GIMH) will be performed in collaboration with
P. vulgaris is an excellent biological model for study- NDSU/F – Phil McClean (see above) to rapidly
ing nodule development. It nodulates prolifically with identify genes in P. vulgaris (see Dong et al., 2001). To
appropriate rhizobial strains, nodule initiation is syn- do this, micro-arrays will be spotted with a unique set
chronous (a burst of 50 or more nodules in a cluster of soybean ESTs. Then, genomic DNA of both beans
appear within about 1 day of one another), and nodules and soybeans that has been digested with restriction
are large, thereby facilitating biochemical analyses. enzymes and fluorescently labelled will be hybridised
The main limitation to full utilisation of this system to the arrays. This will be followed by experiments
to understand determinate nodule development is the where digested, fluorescently labelled genomic DNA
lack of tools for genomic and transcriptomic analysis from soybeans and Medicago truncatula will be hy-
of this plant. bridised to the arrays. This will identify genes that are
We use purine auxotrophs that are completely expressed in P. vulgaris, and provide a quantitative
blocked in infection unless the purine intermediate relationship assessment of the relationships between
AICA riboside is supplied (Newman et al., 1992) to beans, soybeans and M. truncatula. We expect that
study nodule development in beans. Here we will con- beans and soybeans will have more common homo-
struct cDNA libraries from nodules in which infection logues than in comparison with M. truncatula, and that
stops after penetrating ten cell layers and will use sev- these will be enriched in determinants of grain yield.
eral approaches to eliminate those sequences that are Based on the results of these experiments we will con-
expressed in nodule primordia in which infection stops struct new micro-arrays using selected soybean ESTs.
before penetrating five cell layers. The existence of a Gene expression in all tissues will be studied this way.
reasonably complete genome sequence or a more com- Each array experiment will be replicated at least six
plete bank of ESTs would obviously greatly accelerate times and be performed before ESTs from P. vulgaris
this effort. become widely available.
Another bacterial determinant that is critical for
infection of beans and many other legumes is the
polysaccharide portion of the surface lipopolysacchar- Common names of Phaseolus spp.
ide (LPS). It is a dynamic structure that is altered
by the bacteria in response to conditions that ex- − P. acutifolius A. Gray=tepary bean.
ist in the rhizosphere and inside the nodule. In the − P. coccineus L.=(scarlet) runner bean.
R. etli-bean symbiosis, certain structural features of − P. lunatus L.=Burma or butter or Lima bean.
this polysaccharide appear to be required for normal − P. vulgaris L.=baked or canellini or common or
infection (Noel et al., 2000). One possible explan- dwarf or flageolet or frijoles or French or kidney
ation for this structural specificity is that the LPS or navy or pinto or snap or string or wax or haricot
acts as a ligand for receptors, which when bound to or Nuñas bean.
LPS trigger responses that are required for infection
thread development. We are looking for a protein re-
ceptor that binds the wild-type LPS but less well to Acknowledgements
mutant LPSs that cause slower infection. Again, the
availability of genome sequences and EST databases We wish to thank Dora Gerber for her unstinting help
would be of great help. N-terminal sequencing would with all aspects of this work. We gratefully acknow-
immediately identify candidate gene(s) for putative ledge the financial support of the IIIe Cycle Romand
receptors and we could immediately design tests of en Sciences Biologiques (C. Penel), le Décanat of
specific expression of members of the gene family. the Faculté des Sciences (J. Weber), as well as la
With micro-arrays, we could look for expression of Section de Biologie (P. Spierer) of l’Université de
genes uniquely induced by appropriate LPS structures Genève and Applied Biosystems Rotkreuz Branch
113
(A.L. Monsutti), during the second Phaseomics con- Aragão F J L, Vianna G R, Albino M M C and Rech E L 2002 Trans-
ference (Geneva, May 16 to 19, 2002). Research in genic dry bean tolerant to the herbicide glufosinate ammonium.
Crop Sci. 42, 1298–1302.
LBMPS is supported by the Fonds National Suisse de Ariyarathne H M, Coyne D P, Jung G, Skroch P W, Vidaver J
la Recherche Scientifique (Project 31-63893.00). R, Steadman A K, Miklas P N, Bassett M J 1999 Molecular
mapping of disease resistance genes for halo blight, common
bacterial blight, bean common mosaic virus in a segregating pop-
ulation of common bean. J. Amer. Soc. Hort. Sci. 124, 654–662.
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CIAT – International Center for Tropical Agriculture,
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Dr. Jorge A. Acosta-Gallegos, INIFAP
jamk@prodigy.net.mx Dr. Matthew W. Blair, CIAT
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124
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