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MULBERRY GENOMICS:
PROGRESS AND
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PROSPECT
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SMITA ROY
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SEMESTER-VI
B. SC. SERICULTURE (HONOURS)
ROLL: RGU/UG-VI/SERH/31
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NO.: 170010
SESSION: 2017-2018

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DEPARTMENT OF SERICULTURE
RAIGANJ UNIVERSITY
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RAIGANJ

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 1. Introduction

Silk, known as ‗‗Queen of textiles‘‘, originated in ancient China where its use was reserved for

the royalty and has continued to lure people through antiquity. Silk is associated with luxury,

elegance, class, and comfort. Sericulture, both an art and a science of raising silkworms for silk

production, has better prospects in developing countries as silk production is largely a cottage

industry. India has the distinct advantage of practicing sericulture throughout the year, yielding

about 4–6 crops/year as a result of its tropical climate (Gangopadhyay 2008). Trends in

international silk production indicate that India is the second largest producer of raw silk,

accounting for more than 18% of global raw silk production. India has also the unique distinction

of being the only country producing all the four kinds of silk—mulberry, eri, muga, and tasar,

but the major quantity of silk is produced by cocoons of Bombyx mori, the silkworm. The insect

being monophagus feeds only on the leaves of mulberry plants; therefore, mulberry cultivation

plays a cardinal role in the sericulture industry. Mulberry is an important component of

combined pastoral system (Talamucci et al. 2000) as its leaves are used as a forage supplement

for animal husbandry (Benavides 2000). Mulberry contains unique medicinal compounds of

great pharmaceutical worth in its leaf, fruit, stem, seed, and roots having anti-microbial, anti-

hyperglycemic, anti-hyperlipidemic, antidiabetic, chemopreventive, neuroprotective, and anti-

oxidative potential (Andallu and Varadacharyulu 2003; Chen et al. 2006; Chen and Li 2007; El-

Beshbishy et al. 2006; Kang et al. 2006; Konno et al. 2006; Singab et al. 2005). Mulberry is also

used for landscaping in Asia, Europe, and America (Tipton 1994). Global climatic changes and

scarcity of land and water in near future make it mandatory to develop varieties suitable for

different agro-climatic conditions for sustainable development of the sericulture industry. This

review illustrates in a broad sense, how recent biotechnological advances in mulberry will
generate advanced technologies for mulberry cultivation leading to economic improvement in

sericulture, impacting quality of life of those involved in sericulture practices. Figure 1 broadly

depicts the information flow in diverse aspects of genomics research in mulberry.

Fig. 1: Information flow in existing areas of research (dashes) and under-represented areas

(boxes) of genomic approaches in mulberry

2. Origin, distribution, and cytology

Mulberry (Moraceae) is a fast-growing perennial tree maintained as a shrub, occurring in wide

range of areas around the world, i.e. from the tropical, sub-tropical, to temperate climates.

Mulberry originated in the northern hemisphere, particularly in the Himalayan foothills, and later
it spread into the tropics of the southern hemisphere (Hou 1994; Benavides et al. 1994). Today, it

is present in all regions between 50°N lat. and 10°S lat. (Yokoyama 1962), from sea level to

altitudes as high as 4,000 m (Machii et al. 1999; Tutin 1996), which include Asia, Europe, North

and South America, and Africa. Although the taxonomy of mulberry is yet to be resolved fully,

according to the widely accepted classification of Koidzumi (1917), the genus Morus is divided

into two sections, the Dolichostylae (long style) and the Macromorus (short style), and each

section is again divided into two groups namely Papillosae and Pubescentae, based on the nature

of stigmatic hairs. A total of 24 species and one subspecies are recognized in this classification

(Table 1). Currently, however, four mulberry species (Morus indica, Morus alba, Morus

laevigata, and Morus serrata) have been reported from India, and more than 68 species are

widely recognized (Datta 2000). Out of which, only a few species such as Morus alba, Morus

indica, Morus bombycis, Morus latifolia, and Morus multicaulis are cultivated for leaves to feed

the silkworm while another species, Morus nigra, is cultivated for its fruits.
 Table 1: Mulberry species recognized by Koidzumi (1917)

i. M. bombycis Koidz. xiii. M. latifolia Poir.

ii. M. alba L. xiv. M. acidosa Griff.

iii. M. indica L. xv. M. rotundiloba Koidz.

iv. M. kagayamae Koidz. xvi. M. notabilis C. K. Schn.

v. M. boninensis Koidz. xvii. M. nigriformis Koidz.

vi. M. atropurpurea Roxb. xviii. M. serrata Roxb.

vii. M. laevigata Wall. xix. M. nigra L.

viii. M. formosensis Hotta xx. M. rubra L.

ix. M. mesozygia Stapf. xxi. M. celtidifolia Kunth

x. M. cathayana Hemsl. xxii. M. tiliaefolia Makino

xi. M. microphylla Bickl. xxiii. M. macroura Miq.

xii. M. rabica Koidz. xxiv. M. multicaulis Perr.

Cytologically, mulberry exhibits different ploidy levels. Most of the cultivating species are

diploids (2x, 2n=28). But triploids (3x, 3n=42; M. bombycis), tetraploids (4x, 4n=56; Morus

laevigata, Morus cathayana, and Morus boninensis), hexaploids (6x, 6n=84; Morus serrata and

Morus tiliaefolia), octoploids (8x, 8n=112; M. cathayana), and docosaploids (22x, 22n=308; M.

nigra) (Basavaiah et al. 1989) and even haploids (M. notabilis) with 14 chromosomes are also

available in nature (Maode et al. 1996).

Owing to the great economic importance, large numbers of mulberry germplasm accessions have

been maintained in several countries. For instance, China, India, Japan, Korea, and Bulgaria

have, respectively, more than 1,860, 1,120, 1,375, 615, and 140 germplasm accessions (FAO

2003; Machii et al. 1999; Pan 2000; Tzenov 2002; Tikader and Dandin 2006; Tikader et al.

2009). Since information on phenotypic and genotypic variability is vital for effective utilization

of germplasm accessions for long-term improvement of leaf yield, leaf quality, adaptation, and

resistance to pests and diseases, efforts have continuously been made to characterize and

evaluate germplasm accessions. Evaluations based on traits related to growth, development, leaf

yield, leaf quality, and adaptability to various agroclimatic conditions (Banerjee et al. 2007;

Bindroo et al. 1996; Machii et al. 1997, 2000; Rajan et al. 1997; Suryanarayana et al. 2002;

Tikader et al. 2003; Tikader and Kamble 2008a, 2009; Vijayan et al. 1999a, b) and screening for

tolerance to abiotic and biotic stresses (Sujathamma and Dandin 1998; Susheelamma and Jolly

1986; Susheelamma et al. 1990; Yadav et al. 1993; Vijayan et al. 2003, 2004a) revealed the

presence of considerable genetic variability among the germplasm accessions. Based on the

genetic distance estimated with different statistical methods, the germplasm accessions were

grouped into different clusters, and utilizing this information, parental selections were made for

different breeding programs (Machii et al. 1997, 2000; Rajan et al. 1997; Vijayan et al. 1999a, b;

Tikader et al. 2003; Tikader and Kamble 2008a, 2009). Highly heritable morphological

characters were documented and utilized for the development and registration of varieties (Rao

2003; Vijayan et al. 1997a, c). However, phenotypic evaluation has several limitations as most of

the agronomically important traits that are used for evaluations vary greatly depending upon the

developmental stages and environmental conditions, and collection of data is time consuming,
laborious, and expensive and is influenced by human bias. Therefore, molecular markers, which

are highly polymorphic, multiallelic, nonepistatic, neutral, and insensitive to environment

influence, have been used for genetic evaluation of mulberry germplasm resources. However,

most of these molecular markers that have been used for mulberry genetic resource assessment

are of anonymous types such as random amplification of polymorphic DNA (RAPD;

Bhattacharya and Ranade 2001; Chatterjee et al. 2004; Lou et al. 1998; Srivastava et al. 2004;

Xiang et al. 1995; Zhao and Pan 2004), amplified fragment length polymorphism (AFLP; Botton

et al. 2005; Kafkas et al. 2008; Sharma et al. 2000; Wang and Yu 2001), and intersimple

sequence repeats (ISSR; Awasthi et al. 2004; Vijayan and Chatterjee 2003; Vijayan 2004;

Vijayan et al. 2004b, c, 2005, 2006a, b; Zhao et al. 2006). Nonetheless, these studies further

confirmed the high genetic diversity among the accessions. Recently, more reliable, robust,

codominant, and better informative markers such as simple sequence repeats (SSR) markers have

been developed in mulberry (Agarwal et al. 2004; Zhao et al. 2005). A list of the 16 SSR primers

along with their nucleotide sequences and related information is available in Tikader et al.

(2009). Thus, a number of proven marker systems are now available for mulberry germplasm

assessment. Using these marker systems, effective assessments of mulberry germplasm to

identify genetically distant accessions with desirable traits can be made to assist the breeders for

selection of parents. In the meantime, efforts can also be made to develop single-nucleotide

polymorphism (SNP) markers in mulberry, as these are the abundant markers present in any

genome (Collins et al. 1997). Hence, SNP can make genotyping more effective and faster than

any other marker systems. Owing to the current impetus on mulberry genomic research, large

numbers of expressed sequence tags (ESTs) from mulberry have been started to be deposited in

GenBank (Lal et al. 2009; Zhao 2008; Sajeevan et al. unpublished). Presently, nearly 1,366 ESTs
from healthy and stress subjected plants of M. alba and M. indica are available. Using these

ESTs, new markers such as SNPs and SSRs can be developed. Likewise, attempts can also be

devoted to develop SNPs from ISSR and RAPD markers that are associated with valuable

phenotypic traits through locusspecific amplification and comparative resequencing from

multiple individuals (Rieder et al. 1998;). Recently, chloroplast genome of mulberry (M. indica

cv. K2) has been completely sequenced (Ravi et al. 2006). This 158,484-bp circular DNA

contains two identical inverted repeats of 25,678 bp each, separating a large single-copy region

of 87,386 bp and a small single-copy region of 19,742 bp. The sequence information on the

cpDNA can be of much use to develop suitable polymerase chain reaction (PCR) primers for

phylogenetic assessments, evolutionary investigations (Cruzen et al. 1993), and bar coding of

mulberry species (CBOL plant working group 2009). Furthermore, information on the complete

sequence of chloroplast DNA has great potential for manipulating the photosynthetic efficiency

of mulberry through genetic engineering because a large number genes involved in the process of

photosynthesis are located in the chloroplast.

4. Mulberry breeding

Palatability of mulberry leaves to silkworms and production of appreciable number of good

quality cocoons are the main targets in mulberry breeding which depend on several quantitative

traits such as leaf retention capacity, leaf size and weight, total biomass, resistance to pest and

diseases, tolerance to abiotic stresses like drought, salinity, and cold stress. The horizontal

expansion of sericulture has succeeded as traditional methods of plant breeding have made

significant contributions to mulberry improvement by developing several varieties with desirable

agronomic traits which have been released for commercial exploitation. The importance of

evolving a new mulberry variety is to bring all desirable characteristics in a single genotype to
feed the silkworm (Bombyx mori) to obtain high-quality cocoons. Although considerable efforts

have been directed for mulberry breeding for salinity resistance (Vijayan et al. 2009), it is

necessary to develop mulberry varieties specific to different agro-climatic zones for vertical

development of sericulture, and at present suitable mulberry varieties are not available for

alkaline, saline, acidic soil conditions as well as for inundated conditions (Tikader and Kamble

2007). Traditional breeding approaches have been slow due to obstacles like inherent genetic

limitations, heterozygous and perennial nature of the plant system, non-availability of

information about inheritance of various planta traits and genetic markers. Moreover, abiotic

stresses like salt and drought which are inimical to mulberry‘s productivity are complex

quantitative traits and thus difficult to manipulate through conventional breeding and phenotypic

selection. Another factor which fends off genetic improvement of mulberry is the lack of

sufficient variability, a pre-requisite to initiate the breeding programs. In spite of sufficient

genetic variability in mulberry germplasm, the cultivated forms of mulberry belong to M. alba

and M. indica and are used in existing breeding programs (Tikader and Kamble 2008).

Integration of wild/exotic species like M. laevigata and M. serrata in the existing breeding

programs can provide sufficient genetic diversity as they may harbor novel genes regulating

important traits like abiotic and biotic responses (Tikader and Dandin 2007). Mulberry being a

perennial woody plant has long juvenile periods, and it takes approximately 15–20 years to

develop a new variety. Therefore, efforts need to be directed towards understanding genetic

control of major agronomic traits of the domesticated species. Parallely, intervention of advances

in genomics and molecular biology techniques have the potential to overcome problems

associated with conventional breeding and open new vistas for amelioration of mulberry

productivity under adverse environments to revolutionize the sericulture practices (Vijayan

in Fig. 2.

Fig. 2: Integration of various fields of biotechnology for mulberry improvement

5. Trait improvement through conventional breeding

In mulberry, leaf is the primary product and, therefore, leaf productivity was the principal trait

targeted by most of the breeding schemes. Leaf productivity is a multifactorial trait that depends

on a number of quantitative traits such as plant height, number of branches, leaf retention

capacity, nodal length, leaf size and weight, total biomass, etc. (Bindroo et al. 1990; Sahu et al.

1995; Tikader and Kamble 2008a, 2009; Vijayan et al. 1997b). The other important traits that

have been targeted by the mulberry breeders are the adaptability, resistance to pests and diseases,

tolerance to abiotic stresses like drought, salinity, and cold, higher vegetative propagation ability,
better leaf quality, and better coppicing ability. Coppicing ability is important in tropical

sericulture zones where silkworms are reared four to five times per year, and to feed the

silkworms with fresh leaves, mulberry is pruned regularly for four to five times per year to

obtain juvenile shoots. Since mulberry is a crosspollinated perennial plant with high

heterozygosity and long juvenile period, traditional breeding methodologies mostly relied on the

production of F1 hybrids (Das 1984). F1 hybrids are obtained either through controlled crossing

or by collecting open-pollinated seeds from selected female parents. Intensive within-family

selection and clonal propagation were adopted to multiply superior hybrids. Once a hybrid with

desirable traits is identified, it is mass multiplied for further assessment. Based on its

performance in multilocation trials, varieties suitable for a particular agroclimatic zone is

selected and released for filed cultivations (Fig. 2). Mulberry varieties are multiplied clonally

(planting stem cuttings) to capture the additive and nonadditive variations. Using these breeding

methods, leaf productivity has been increased significantly. Major silk-producing countries like

China and India have released a number of mulberry cultivars for commercial exploitation

(Tikader et al. 2009; Vijayan 2009). In spite of the large germplasm collections, breeders so far

have exploited only a few accessions mostly from M. alba and a few other species closely related

to it (Tikader and Kamble 2008b), leaving huge genetic resources unutilized. This reduced

genetic base makes them susceptible to disease epidemics and reduces the chance of their further

improvement using new combinations of genes (Tikader and Dandin 2007). This limited use of

germplasm resources is mainly due to the paucity of information on the genetic control of most

of the agronomically important traits. Thus, it is difficult to transfer desirable traits from the

unadapted genotypes to the elite lines. Introgression of characters from wild relatives to the elite

breeding line is, often, accompanied by undesirable traits that are difficult to be removed because
of their close physical linkage with the desired traits, a phenomenon called linkage drag. In order

to get rid of these undesired traits, back crossings are to be made for several generations.

Mulberry being a tree crop with long juvenile period, back crossing for many generations is

highly time consuming and laborious and is almost prohibitive. Similarly, no inbred lines or

doubled haploid lines are available to study the genetic controls on important agronomic traits.

Therefore, except a few crossability, heritability, and combining ability studies (Dandin et al.

1987; Dwivedi et al. 1989; Tikader and Dandin 2001; Vijayan et al. 1997a, 2008), no effort was

made to understand the genetic control of major growth and adaptability traits. The major

challenges, thus, faced by the mulberry breeders, such as (1) understanding the basis of heterosis

and prediction of hybrid performance, (2) identification of useful genetic factors in divergent

populations or lines, (3) introgression of desired traits with minimal linkage drag, (4)

understanding the genotype by environment interaction, remain unresolved. In order to resolve

these problems, proper understanding on (a) the number of genetic factors (loci) influencing the

expression of the traits,(b) the chromosomal location of these loci, (c) the relative size of the

contribution of individual loci to trait expression, (d) pleiotropic effects, (e) epistatic interactions

among genetic factors, and (f) variation of expression of individual factors in different

environments are required. This necessitates the integration of recent advances in genomic

research with conventional breeding techniques to dissect the complexity of most of these traits

to increase the productivity and nutritional quality of the leaves and the adaptability of the

mulberry for sustaining the sericulture industry.

Thus, it is evident that growth-related traits play a significant role in leaf productivity and

adaptations in mulberry. Understanding on these traits through genomic approach is essential to

tackle them effectively and efficiently. Although much advancement has been made in silkworm

genomic research, the same could not be achieved in mulberry. However, in many tree species

like Eucalyptus (Gratapaglia and Kirst 2008), Pinus (Pot et al. 2006), and Populus (Rae et al.

2006) both forward and reverse genomic approaches were proven their worth in elucidating

genes involved in the expression of different traits and chemical pathways. Generally, in forward

genomics, existing phenotypic traits are analyzed to identify the underlying genetic variations

whereas in reverse genomic manipulation of specific gene through transgenic technologies such

as insertional mutagenesis, overexpression of genes, and RNA interference (RNAi) and miRNA

approaches is used to study the gene–trait relationship. Reverse genomic methods are, however,

mostly species specific, expensive, and transgenic or only transiently disrupt gene functions.

Forward genomic approaches, thus, seem to be well suited for mulberry as considerable

phenotypic variation is present in the natural populations for the important traits like leaf yield,

leaf quality, and adaptability. The commonly used technologies in forward genomics are

genotyping, genetic mapping, discovery of quantitative trait loci (QTL), association mapping,

positional cloning and sequencing, functional genomics, and the recently introduced

metabolomics.

6.1. Genetic linkage mapping

A genetic map constructed from a population segregating for a trait of interest is required for

QTL identification (Hong et al. 2010); therefore, genetic maps are an important component of
mulberry research, underpinning the improvement programs. Nevertheless, mulberry research

still lacks adequate tools and resources for genetic, genomic research, and breeding, and

therefore, expansion of thebasic knowledge of the genetic control of complex traits is required.

First, genetic linkage map of mulberry was generated by Venkateswarlu et al. (2006) with 50 F1

full-sib progeny using RAPD, ISSR, and SSR markers and pseudotest cross mapping strategy to

provide reference information for future molecular breeding work on Morus and its relatives.

They selected 100 RAPD, 42 ISSR, and 9 SSR primers that amplified 517 markers. Two separate

male and female maps were constructed using 94 each of female and male-specific test cross

markers, containing 12 female linkage groups and 14 male linkage groups. However, the female

map covered an average distance of 15.75 cM and maximum map distance of 37.9 cM between

two loci, while average distance of male map was 18.78 cM, maximum distance between two

loci being 34.7 cM. Though the markers were randomly distributed between the linkage groups,

the information generated has limited utility. A detailed linkage map integrating information

from multiple mapping populations with sufficient markers is necessary for QTL identification

and marker-assisted selection. These maps need to be constructed with the aim of determining

the relative position of transferable markers, increasing the number of available DNA markers,

obtaining saturated maps, and comparing the locations of quantitative trait loci (QTL) and

candidate genes of interest across germplasm. The construction of a consensus linkage map for

mulberry should enable us to determine the stability of locus positions across mulberry

germplasm, as well as increase the number of loci in the linkage map.

6.2. Expression quantitative trait locus mapping

A QTL identified through the aforementioned approaches may have genomic regions with

several hundred genes (Salvi and Tuberosa 2005). Thus, it is difficult to determine which gene(s)
is responsible for the variation in the trait, for which extensive cloning and sequencing are

required (Mackay 2001). System biology approaches such as transcriptomics has shown that

modifications in gene regulation cause wide phenotypic variations in natural populations (Gilad

et al. 2008; Hansen et al. 2008). Thus, mapping of QTLs that control the transcript level of gene

expression, called expression QTL (eQTL) mapping or systems genetics, helps us understand the

relationship between the genome and the transcriptome. Generally, two types of eQTLs are

recognized, cis-QTLs and trans-QTLs. cis-QTLs are due to polypmorphism in or closer to the

gene that codes for the mRNA product and, thus, are likely to represent structural genes, while

trans- QTLs are due to polymorphism located elsewhere in the genome but control the

transcription of the interested gene and, thus, are likely to be transcription regulator(s). The

major difference of eQTL mapping from traditional QTL mapping is the ability to analyze

thousands of traits at a time to find the QTL responsible for the variations (Chesler 2007). In

order to achieve the full potential of eQTL mapping, it is necessary to develop highly saturated

maps like those with SNP markers (Gilad et al. 2008). Several software packages are freely

available, with which eQTLs can be identified from repository genomic data. EQTL

EXPLORER is one such soft ware available fromhttp://web.bioinformatics.ic.ac.uk/eqtlexplorer.

The success of eQTL mapping in Eucalyptus and Populus (Krist et al. 2004) suggests that eQTL

is much suited to mulberry for identification of genes responsible for adaptability, pathogen

resistance, and early leaf senescence and leaf nutritional qualities.

6.3. Metabolomic QTL mapping

The nutritional status of mulberry leaf has a high impact on the cocoon quality as mature

silkworms use more than 70% of the leaf protein for synthesizing the silk proteins, fibroin, and

sericin. (Fukuda et al. 1959). Nutritional status of mulberry leaf can be improved by integrating
genetic and metabolic approaches. Metabolic profiling facilitates understanding of plant

metabolic networks (Fernie and Schauer 2008). Pathway-based approaches have been found

useful in identifying the genetic determinants of crop compositional quality. These approaches

have identified several mQTLs in tomato (Schauer et al. 2006, 2008), Cucumis (Alba et al.

2005), and sesame (Laurentin et al. 2008). The most commonly used technologies for metabolic

profiling are mass spectrometry and nuclear magnetic resonance (NMR). Different types of mass

spectrometry are available. Of which, gas chromatography–mass spectrometry, gas

chromatography–time-of-flight mass spectrometry, liquid chromatography–mass spectrometry,

capillary electrophoresis– mass spectrometry, and Fourier transform–ion cyclotron resonance–

mass spectrometry are most widely used. NMR uses magnetic nuclei of atoms after application

of a constant magnetic field. NMR has the advantage of being used for assessing living cells; it

provides subcellular information, and it is easier to drive atomic information for flux modeling

from NMR than from mass-spectrometrybased approaches (Fernie and Schauer 2008).

Considering the great potentials of mQTL mapping, it can be adopted for improving the leaf

quality in mulberry to enhance the silk productivity per unit area, which will have a great impact

on the sustainability of sericulture.

6.4. Association mapping

Association mapping or linkage disequilibrium (LD) mapping is an emerging technique in

plants, though it was well established in human. Association mapping differs from traditional

linkage mapping in many aspects (Table 4); it exploits the historical and evolutionary

recombination events rather than the limited recombinations available in biparental mapping

populations (Nordborg and Tavaré, 2002). LD mapping uses the genetic diversity present in

natural populations to identify molecular markers that are tightly linked to complex phenotypic
traits on the basis of significant allele frequency differences between individuals with the

phenotype of interest (―cases‖) and a set of unrelated control individuals (Pritchard et al. 2000).

Based on the nature of the study, LD mapping can be categorized into candidate gene association

mapping and genome-wide association mapping (Zhu et al. 2008). Candidate gene association

mapping relates polymorphisms in selected candidate genes with phenotypic variation for

specific traits whereas genome-wide association mapping or gene scan surveys genetic variation

in the whole genome to locate genes or narrow regions that have significant statistical

connections to various complex traits (Zhu et al. 2008). Many computer softwares can be used

for visualizing linkage disequilibrium (Table 5). Association mapping has already proven its

efficiency in many tree plants (Table 3). Detection of gene–trait associations for important

growth traits like phenology, disease resistance, drought resistance, and wood properties in those

trees suggest the suitability of association mapping in mulberry, though it has not been attempted

in mulberry.

6.5. Marker-assisted selection breeding

Variety development in mulberry based on phenotypic characters needs large-scale

multienvironmental testing (Fig. 3). In addition, mulberry needs at least 3–4 years to become

well established to be able to express important agronomic traits properly. For appropriate

statistical analysis, data have to be recorded in all the seasons for a minimum of 3 years. Thus,

field testing of a large number of progenies of mulberry requires huge space, time, labor, and

money. Therefore, assistance from environmentally insensitive, selectively neutral, and less

expensive and more time-saving methods like molecular marker-assisted selection (MAS)

techniques can be used for accelerating mulberry breeding as it is easier, faster, nondestructive,

and cheaper than the phenotypic assessment, especially for complex traits which are expensive to
assess (Koebner and Summers 2003). The most important requirement for MAS is molecular

markers that are tightly linked to traits of interest (Collard et al. 2005). If there is a marker linked

tightly to a particular phenotype, then one can select the phenotype indirectly based on the

presence or absence of the marker on a gel or on autoradiogram, depending on the marker system

(Hospital 2009), because the banding patterns indirectly reveal the presence or absence of a

specific chromosomal segment which carries the desired alleles (Varshney et al. 2004). Although

tightly linked markers for phenotypic traits through genetic mapping have not been identified in

mulberry, using multiple regression analysis on germplasm accessions, several molecular

markers associated with many important agronomic and biochemical characters have been

identified (Vijayan and Chatterjee 2003; Vijayan et al. 2006a, 2009; Kar et al. 2007). After

appropriate validations, these markers can initially be employed for MAS breeding in mulberry.

6.6. Genetic transformation system

Reverse genomic tools such as insertional mutagenesis, overexpression of genes, and RNAi are

increasingly being used for dissection of complex traits in trees. Insertional mutants with

suppressed or activated target genes are used for identifying phenotypes (Busov et al. 2005).

Overexpression of dominant genes in transgenic plants leads to visible phenotypic changes and it

has been shown in trees (Grattapaglia et al. 2009). RNAi is another powerful tool that has been

developed recently for validating gene function in the context of plant developments. The

essence of RNAi is the delivery of double stranded RNA into the plant to induce a sequence-

specific RNA degradation mechanism that effectively silences targeted gene (Waterhouse and

Helliwell 2003). For elucidating genes involved in many biochemical pathways, this methods

appear to be very promising (Capell and Christou 2004). In order to apply these technologies in

mulberry, an efficient transformation system is a prerequisite. Bhatnagar et al. (2002, 2003) have
developed an efficient Agrobacterium-mediated transformation system for the mulberry. Using

this transformation system, a glycinin gene AlaBlb, an oryzacystatin gene OC (Wang et al.

2003), and a barley HVA1 gene (Lal et al. 2008) were successfully incorporated into the

mulberry genome. Thus, an efficient genetic transformation system in mulberry is ready for the

breeders to take advantage of. Overexpression of genes and reverse genetics are also promising

options to study the trait–gene relationships in mulberry. However, transgenesis of a highly

cross-pollinated plant like mulberry should be done with great caution. In this regard, it is

important to note that chloroplast genetic engineering has been viewed as a safer method of

transgenic plant development as, unlike nuclear genes, transgenes in the chloroplast DNA have

little chances of dissemination through cross-pollination.

7. Conclusion and perspectives

Mulberry is one of the most economically important plants with significant contributions to the

Indian economy. Abiotic stresses are major factor affecting the productivity of mulberry

worldwide. Comprehensive evaluations of genotypic variations suggested new perspectives for

efficient use of its genetic resources. Genetic markers have been employed in mulberry to study

its unique genotypic variation. However, only limited progress has been made in development of

linkage maps in mulberry, and therefore, efforts are needed for development of mulberry

genomic toolbox which would provide valuable basic information for functional genomics

accelerating the creation of improved mulberry varieties. Biotechnology has energized research

and fostered a surge of new ideas for mulberry improvement. Tissue culture studies have been

standardized and exploited for raising stable transgenic plants for different characters. Morus

genomic resources provided numerous novel candidate genes and fueled endeavors of

developing improved stress-tolerant and disease-resistant plants by genetic engineering. The

paved the way for evolutionary studies in mulberry. Redundant sequences found in EST

databases can be a useful resource for mining SNPs or developing DNA markers, for mapping

expressed genes to a linkage map, thus making the map more useful for OTL analysis and

marker-assisted selection. The potential of proteomics and metabolomics has yet to be exploited;

however, an integrated analysis of genomic, transcriptomic, proteomic, and metabolomic

analysis is expected to yield desirable results for understanding mechanisms of enhancing the

productivity of mulberry. We have now excellent chances to translate our knowledge into major

gains with the recent sequencing of the model dicot angiosperms—Arabidopsis thaliana, Populus

tricocarpa. Using reference genome systems, the potential of comparative genomics should be

exploited to transfer the information on gene function, genetic markers and for a detailed

knowledge of conserved genome structures (synteny). The mulberry community needs an

International mulberry consortium, presence of which renders the data to be universally useful.