See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/256981351

Assessment of genetic variability in grapefruits (Citrus paradisi Macf.) and

pummelos (C. maxima (Burm.) Merr.) using RAPD and SSR markers

Article  in  Euphytica · July 2002

DOI: 10.1023/A:1016332030738

CITATIONS READS

107 671

5 authors, including:

Marcos Antonio Machado William Mario de Carvalho Nunes

Instituto Agronômico de Campinas Universidade Estadual de Maringá
368 PUBLICATIONS   12,205 CITATIONS    89 PUBLICATIONS   617 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Citrus Research View project

SWEET ORANGE GENOTYPES RESISTANCE STUDY (Citrus sinensis) THE CITRUS CANCER IN ORCHARDS IN COMMERCIAL PARANÁ STATE, BRAZIL. View project

All content following this page was uploaded by William Mario de Carvalho Nunes on 15 April 2014.

The user has requested enhancement of the downloaded file.

 Euphytica 126: 169–176, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
169

Assessment of genetic variability in grapefruits (Citrus paradisi Macf.) and

pummelos (C. maxima (Burm.) Merr.) using RAPD and SSR markers

M.J. Corazza-Nunes1,∗ , M.A. Machado2 , W.M.C. Nunes3 , M. Cristofani2 & M.L.P.N. Targon2
1 Departamento de Biologia, Universidade Estadual de Maringá, CEP 87020-900 Maringá-PR, Brazil; 2 Centro de
Citricultura Sylvio Moreira/IAC – Cordeirópolis-SP, Brazil. 3 Departamento de Agronomia, Universidade Estadual
de Maringá, Maringá-PR, Brazil; (∗ author for correspondence, e-mail: [email protected])

Received 28 February 2001; accepted 27 September 2001

Key words: Citrus paradisi, grapefruits, RAPD markers, SSR markers

Summary
The genetic variability of 38 grapefruit (Citrus paradisi Macf.) and three pummelos (C. maxima (Burm.) Merr..)
accessions was evaluated using RAPD, and single sequence repeat (SSR) analyses. Approximately 49% of the 198
RAPD were polymorphic, and 4.6 alleles per SSR loci were identified. PIC values changed from 0.093 to 0.450. A
UPGMA phenetic tree was constructed and two main grapefruit groups were identified. The grapefruit accessions
‘do Cabo’ and ‘Siamesa-Filipinas’ clustered very close to the pummelos in Group A. The Group B consisted of
three sub-groups, which comprised all of the other grapefruit accessions. The majority of grapefruit accessions
showed a narrow genetic base suggesting that the observed morphological polymorphism within the group must be
associated with somatic mutations, which were not detected by these molecular markers.

Introduction ility within the group is associated to natural muta-

tions. (Hodgson, 1967; Gmitter Jr., 1993). Brazilian
Spontaneous hybrids in the genus Citrus can be per- annual grapefruit production is around 62 thousand
petuated both by natural hybridization and assexual tons (Anon, 1998), and the reason for that is the pub-
reproduction via adventitious nucellar embryos, and lic preference for sweeter citrus fruits and, mainly,
several spontaneous hybrids are usually considered because of the high susceptibility of grapefruits to
true species (Yamamoto et al., 1993). The grapefruit, severe strains of citrus tristeza virus (CTV). Although,
Citrus paradisi Macf. (Swingle, 1943), is considered grapefruits can be considered the most tropical of cul-
a natural hybrid between a female pummelo (C. max- tivated citrus species, the Brazilian production is lim-
ima (Burm.) Merr., sin. C. grandis L. Osb.), and the ited to 62 thousand ton a year (Anon, 1998), due the
sweet orange (C. sinensis L. Osb) (Scora et al., 1982; high susceptibility to citrus tristeza virus (CTV), and
Kumamoto et al., 1987; Yamamoto et al., 1993). public preference for sweeter citrus fruits. Commer-
Grapefruits are native to the Barbados Islands in cial production has been done by using varieties with
the Caribbean, in contrast to pummelos and other cit- previous inoculation of mild and protective strains of
rus species, which are native of tropical and subtrop- CTV (Müller & Costa, 1977).
ical regions in Asia. Grapefruits became economically There are three most economically important vari-
important when introduced into Florida, from where ety groups of grapefruits: 1) the seeded fruits with
the most commercial varieties were originated (Hodg- pale-yellow pulp (‘Duncan’, ‘MacCarty’, ‘Triumph’,
son, 1967). The United States is the largest producer ‘Imperial’ and ‘Walters’), 2) pale-yellow pulp, but
of grapefruits in the world, and are responsible for al- seedless (‘Marsh Seedless’), and 3) the pigmented-
most the haft of the world production (Anon, 1998). pulp (‘Thompson’, ‘Redblush’ and ‘Star Ruby’).
Grapefruits are highly polyembryonic varieties, all of ‘Marsh Seedless’is the most cultivated variety in the
them originated from nucellar clones. Genetic variab- world, being the first grapefruit variety without seeds.
170

Moreover, it is the primary source of the germplasm Thompson (1980). The leaves were ground to a fine
used for the diversification and selection processes that powder in liquid nitrogen and resuspended in CTAB
have produced the majority of pigmented grapefruit extraction buffer (1% CTAB, 100 mM Tris HCl pH
varieties (Gmitter Jr., 1993). Since grapefruits are the 7.5, 10 mM EDTA, 0.7 M NaCl, 2% Sarcosyl and
only citrus taxon which have known origin (Scora et 140 mM 2-mercaptoethanol). The supernatant was ex-
al., 1982; Kumamoto et al., 1987; Yamamoto et al., tracted with chloroform-isoamyl alcohol (24:1), and
1993 ), the phenotypic diversity within the group may precipitated in absolute ethanol and the pellet resus-
be associated with somatic mutation, and/or hybridiz- pended in TE containing 10 g/ml RNAse. PCR was
ation with compatible species. conducted in a total volume of 13 µl (1.3 µl of 10x
Genetic variability of citrus and several other spe- 100 mM Tris-HCl pH 8.3, 500 mM KCl, 2.0 mM
cies has been accessed using DNA markers based on MgCl2 , and 0.01% gelatin), 0.2 mM of each dNTP,
PCR, such as random amplified polymorphic DNA 15 ng of primer kits Operon of 10-mer primers: AV3,
(RAPD), and single sequence repeats (SSR). Although AV5, AV8, AV13, AV15, AV16, AV17, AV20, B7,
the genetic diversity of several Citrus species have B17, C2, C5, C6, C7, C11, C14, C15, C18, C20,
been well evaluated, specially mandarins (Machado M5, U5), 7 ng of DNA and 1.5 units of DNA Taq
et al., 1996; Coletta Filho et al., 1998), it lacks polymerase (Gibco). Amplifications were performed
studies on species which origin is associated to so- in a thermocycler (MJ Research) programmed for 36
matic mutations, such as sweet oranges and grapefruit. cycles of 1 min at 92 ◦ C, followed by 1 min at 36 ◦ C, 2
The present study used RAPD and SSR markers to min at 72 ◦ C, and a final extension at 72 ◦ C for 10 min.
evaluate genetic polymorphism and similarity among Aliquots of the final amplified products were analyzed
accessions of grapefruits and pummelos in a core in 1.4% agarose gels containing 0.5 g/ml of ethidium
collection. bromide.
The primers used to amplify the microsatellite loci
regions were developed and synthesized from genomic
Material and methods DNA extracted from the sweet orange ‘Pera’ as de-
scribed by Cristofani et al. (1998). Out of 20 tested
Plant Material primers, 5 (P73, P94, P620, P1223, P1826) were se-
lected for grapefruit analysis. Sample amplification
All commercially cultivated varieties of grapefruits
was conducted in a total volume of 25 µl (2.5 µl of
and three important varieties of pummelos are rep-
the 10X buffer, 100 ng of genomic DNA, 0.2 mM
resented in this study (Table 1). The core collections
of each dNTP, 16 pM of 3’ and 5’ terminal primers
include three plants of each accession, all of them graf-
and 1.5 units of DNA Taq polymerase (Gibco)). The
ted on Cleopatra mandarin (C. reshni). A plant from
amplifications were conduced according to Kijas et
three was usually evaluated. Only from the accessions
al. (1997) (94 ◦ C /5 min, 32 cycles/94 ◦ C /1 min,
‘Marsh Seedless’, and ‘Redblush’ the three replicates
55 ◦ C /30 sec, 72 ◦ C /1 min, and a final extension
were evaluated to confirm the complete clonal identity
at 72 ◦ C / 4 min. The PCR products were denatured
among plants. The core collections belongs to active
at 95 ◦ C for 3 min and resolved in 30 cm × 30 cm
citrus germplasm collection of the Citriculture Center
denaturing gel containing 8% polyacrylamide (40:2
Sylvio Moreira/IAC, Cordeirópolis, São Paulo, Brazil.
acrylamide/bisacrylamide), 4.6 M urea and 1X TBE
The core collection include plant introduced as seeds
buffer (45 mM Tris-Boric, 1 mM EDTA (pH 8,4)).
(called new budline or new clones, in the Table 1 they
Electrophoresis was conducted at a constant 100 V or
are highlighted in italic), and as budwoods (called old
about 50 mA, for 8–10 h, after which the gels were
budline or old clones) from different part of the world.
stained with silver nitrate (Beidler et al., 1982). The
Both old and new clone of each accessions were evalu-
size of the alleles in the loci was determined using a
ated, since new clones may present genetic variations
100 bp DNA standard (Gibco). Polymorphic inform-
originated whether by seedling somatic mutations of
ation content (PIC) of SSR patterns were estimated
seedlings or by selection of zigotic hybrids.
according to Liu (1998).
Alleles RAPD (present or absent) and alleles SSR
DNA extraction and analysis
(homozigote or heterozigote) were included in a data
The total DNA was extracted from young leaves matrix (accession × marker), and used for multivari-
according to the modified method of Murray & ate analysis with the NTSYS-PC software version 1.7
 171

Table 1. Grapefruits and pummelos accessions in the core collections, and the more informative RAPD and SSR markers
associated to them

Core collection RAPD1 SSR2

p1826 p73 p94 p1223 p620

Royal C11-823∗ 457-403 384 498-430 340 442-410

Rubi AV20-750; C5-1,362 457-403 384 498-430 340 442
Redblush AV20-750; C5-1,362 457-403 384 498-430 340 442
Red Mexican AV20-750; C5-1,362 457-403 384 498-430 340 442
Triumph C11-823 457-403 384 498-430 340 442-410
Thompson Pink AV20-750; C5-1,362 457-403 384 498-430 340 442
Imperial C11-823 457-403 384 498-430 340 442-410
Marsh Seedless AV20-750; C5-1,362 457-403 384 498-430 340 442
Marsh Seedless 11656AV20-750; C5-1,362 457-403 384 498-430 340 442
Marsh Seedy AV20-750; C5-1,362 457-403 384 498-430 340 442
McCarty AV20-750; C5-1,362 457-403 384 498-430 340 442
Cannores C11-823; AV8-810 457-403 384 498-430 340 442-410
AV8-543; AV15-617
Do Cabo C6-1,285; M5-395 467-403 364 498-430 340 410
AV20-961; AV20-1,235;
C18-792; AV8-583;
C7-1,350; C7-1,310
Foster AV8-810 457-403 384 498-430 340 442
Retiro AV20-750; C5-1,362 457-403 384 498-430 340 442
Viçosa AV20-750; C5-1,362 457-403 384 498-430 340 442
Hart’s AV20-750; C5-1,362 457-403 384 498-430 340 442
Duncan AV20-750; C5-1,362 457-403 384 498-430 340 442
Duncan 1 AV20-750; C5-1,362 457-403 384 498-430 340 442
Pernambuco B7-447; C11-823; 457-420 384 498 340 442-425
AV3-1,190; AV16-1,338
Leonardy C11-823; 457-403 384 498-430 340 442-410
Marsh Seedless 1 AV20-750; C5-1,362 457-403 384 498-430 340 442
Coco Variação U5-930; AV16-1,338; 457-403 384 498-430 399-340 442-425
C5-1,662; AV20-490; AV8-828
Arananyan C11-823; C11-927; 457-403 384 498-446 340 442
AV16-1,338; C5-1,662; AV20-961;
C18-792; C7-1,350
Davis da Flórida AV20-750; C5-1,362 457-403 384 498-430 340 442
Marsh da Flórida AV20-750; C5-1,362 457-403 384 498-430 340 442
Shambar da Flórida AV20-750; C5-1,362 457-403 384 498-430 340 442
Redblush – Califórnia AV20-750; C5-1,362 457-403 384 498-430 340 442
Marsh Seedless – S. Sebastião AV20-750; C5-1,362 457-403 384 498-430 340 442
Marsh Seedless – Limeira AV20-750; C5-1,362 457-403 384 498-430 340 442
Leonardy – Flórida) C11-823; AV20-490; 457-403 384 498-430 340 442-410
De Mauritius – Mauritius C14-680; C14-1,610; 467-429 384 498-446 399-340 442-410
AV13-816; M5-1390;
AV16-1338; C5-698;
AV8-810; AV8-543;
172
Table 1. Continued

Core collection RAPD1 SSR2

p1826 p73 p94 p1223 p620

Pamplemousse – Mauritius) C14-1,610; AV13-816; 467-429 384 498-430 399-340 442-410

M5-1390; AV16-1,338;
C5-698; AV20-670;
AV8-810; AV8-543;
Siamesa – Filipinas B17-1,640; M5-2,090; 467 384-364 498-430 340 442-410
AV3-1,190; AV20-1,235;
AV20-670; C18-2,095;
AV8-828; AV8-583; C15-1,193;
Siamesa C14-1,610; AV3-1,190; 467-436 384 537-498 372-340 410
AV8-828; AV8-583
Hybrid 47389) AV20-750; C5-1,362 457-403 384 498-430 340 442
Hybrid 47449) AV20-750; C5-1,362 457-403 384 498-430 340 442
Dalandan – Filipinas AV8-810; AV8-828; 457 364 498 340 410
C7-1,350; C7-1,310;
AV15-617
Pummelos
Lau Tau M5-395; AV3-1,190; 467-388 384-364 498 340-303 442-410
AV20-961; AV20-490;
C18-2,095; AV8-828;
C7-1,350; C7-1,310
Singapura C14-680; M5-2,090; 467-403 402-364 498 340 442-410
M5-395; U5-930;
C11-927; AV3-1,190;
AV20-961; AV20-490;
AV8-828; AV8-583;
C7-1,350; C7-1,310;
C15-1,193;
Siamesa M5-395; U5-930; 467-403 384-364 498 340 425-410
AV3-1,190; AV20-961;
AV8-828; C7-1,350;
C7-1,310;

In italic the accessions of new clones or nucellar clone originated from seeds.
1 = primer number and molecular weight of the amplified fragment.
2 = molecular weight of the amplified microsatelite.

(Rohlf, 1992). Simple matching coefficients and un- bp. The more informative RAPD are included in
weighted pair-group method with arithmetic averages the Table 1. Profiles that showed bands with lower
(UPGMA) were used to build the consensus tree after or higher molecular weights and those that presen-
bootstrap test repeated hundred times . ted ambiguous interpretations were not included in
the analysis. Approximately 49% of the 198 amp-
lified fragments revealed polymorphism among the
Results and discussion accessions, with the average number of polymorphic
markers per primer being 4.6. This value was higher
Twenty-one primers were selected for the RAPD ana- than 2.2 found by Machado et al. (1996), and 1.95
lyses based upon the resolution of the amplified seg- found by Coletta Filho et al. (1998) among mandarin
ments and the number of polymorphism created. The (C. reticulata Blanco) accessions.
number of amplified fragments per primer varied from High polymorphism was observed in accessions
4 to 15, with their sizes ranging from 400 to 3,000 such as ‘Royal’, ‘Triumph’, ‘Imperial’, ‘Leonardy’,
 173

‘Cannores’, ‘do Cabo’, ‘Pernambuco’, ‘Côco Vari- ation of non-specific fragments is more evident with
ação’, ‘Arananyan’, ‘de Maurítius’, ‘Pamplemousse’, SSR based on dinucleotides than those of tri and
‘Siamesa’, ‘Siamesa-Filipinas’ and ‘Dalandan’, as tetranucleotides.
well as in the three pummelos. Accession-specific Tables 1 and 2 show that the majority of acces-
marker was also found in ‘Siamesa-Filipinas’ (OPB17- sions were heterozygous, with alleles of two different
1,640, corresponding to a fragment of 1,640bp amp- sizes, such as in loci 94 and 1826. The grapefruits
lified with primer B-17), ‘do Cabo’ (OPC6-1,285), ‘Pernambuco’, ‘Arananyan’ and ‘Dalandan’, and the
and ‘Pernambuco’ (OPB7-447). Some primers al- pummelos ‘Lau-Tau’ and ‘Singapura’ were homo-
lowed common patterns in few accessions, e.g. zygous for locus 94, while ‘Siamesa-Filipinas’ and
‘de Maurítius’ and ‘Pamplemousse’ (OPAV13-816, ‘Dalandan’ were homozygous for locus 1826. Poly-
OPC5-698, and OPM5-1,290). Reproducible band in- morphism of allele size were also detected in the
tensity among accessions was also observed, what grapefruit accessions ‘de Maurítius’, ‘Pamplemousse’,
can point out different number of copies of the target ‘Siamesa-Filipinas’ and ‘Siamesa’ for loci 94 and
fragment amplified by PCR (Wilkie et al., 1993). 1826. In the grapefruit accessions ‘do Cabo’, ‘Pernam-
RAPD markers are well known by their ability to buco’ and for the pummelos ‘Lau-Tau’, ‘Singapura’
induce misinterpretation caused by PCR conditions and ‘Siamesa’ allele size polymorphism was also ob-
(Rafalski et al., 1991; Wilkie et al., 1993; Halldén et served for locus 1826.
al., 1996). Usually the misinterpretation of the band There were 27 homozygous and 14 heterozyg-
phenotypes can affect the analysis of the genetic di- ous alleles for locus 620, and the grapefruit acces-
versity, in that, accessions can appear to be more sions ‘Royal’, ‘Triumph’, ‘Imperial’, ‘Cannores’, ‘Le-
variable than they really are. In this study, repetitions onardy’ ‘Maurítius’ and ‘Pamplemousse’ showed the
with clones showed the same RAPD profiles with all same genotype, with two alleles of the same length.
primers and PCR conditions. On the other hand, SSR Identical genotypes were found for the grapefruits
loci are more informative and can be evaluated using ‘Pernambuco’ and ‘Coco Variação’, and for the
primers developed for a species and related genera. grapefruits ‘do Cabo’ and ‘Dalandan’. The grapefruit
From the twenty primers used in this studies, all of ‘Siamesa-Filipinas’ and the pummelos ‘Lau-Tau’ and
them originally of sweet orange ‘Pera’ (C. sinensis L. ‘Singapura’ also had identical genotypes. Of the
Osb.), only five revealed polymorphism among the ac- microsatellite loci analyzed locus 73 had 4 hetero-
cessions. The number of alleles were observed in each zygous genotypes (‘Siamesa- Filipinas’, and the 3
locus changed from 4 (loci 73, 94, 620, and 1223) pummelos), while locus1223 had 5 heterozygous gen-
to 7 (locus 1826), according with Kija et al., 1995), otypes (‘Arananyan’, ‘Mauritius’, ‘Pamplemousse’,
whereas the values of PIC changed from 0,09 to 0,45 ‘Siamesa’ and ‘Lau-Tau’).
(Table 2). The average number of alleles per locus was The data obtained from RAPD (presence or ab-
4.6, which was lower than in other species as 8.4 in sence of the band) and SSR (presence or absence of
grape (Thomas & Scott, 1993), 9 in rice (Yang et al., the allele in the locus) were combined and phenetically
1994), 17.7 in barley (Saghai Maroof et al., 1994), and analyzed to determine the genetic similarity among the
18.6 in soybean (Rongwen et al., 1995). accessions of the core collection. The simple matching
According to Primmer et al. (1996), the success coefficients of genetic similarity (GS) were calculated
of amplification of SSR loci among related species using the 198 RAPD and the 820 SSR combinations
decreases as the phylogenetic distance increases. Con- for the 41 genotypes studied. Stiles et al. (1993) study-
served sequences of primers were detected in com- ing genetic similarity of papaya concluded that the
mercial and wild rice (Wu & Tanksley, 1993), grape simple matching coefficient (including common neg-
(Thomas & Scott, 1993), among genera of the Curcu- ative data) was more appropriate for the calculation
bitaceae family (Katzir et al., 1996), and among of the genetic distance between varieties that have a
conifer species (Echt et al., 1999). Close phylogen- narrow genetic base.
etic relationships were found between grapefruit and Figure 1 shows a UPGMA dendrogram produced
the its male parent, sweet orange (Scora et al., 1982; from the simple matching coefficient of similarity.
Yamamoto et al., 1993). However, the SSR primers The 41 accession fell into two main groups (A and
of sweet orange used in this study frequently pro- B). Group A contains the grapefruit accessions ‘do
duced low polymorphism and non-specific fragments. Cabo’ and ‘Siamesa-Filipinas’, which clustered with
Rongwen et al. (1995) pointed out that the amplific- the three pummelos (GS = 0.84). It therefore seems
174
Table 2. Microsatellite loci in grapefruits (Citrus paradisi) and pummelos (C. maxima)

PRIMERS Repetitive Alleles size Alleles Homozygous Heterozygous PIC

unit variation (bp) number

P73
F1 -CCATTGCTTACGAAGTTG (AG) 20 364-395 4 37 4 0.176
R2 -TTGACTTGCAGCAATCAG
P94
F-GATTGAATCTTCTGTAGCTC (TC) 22 430-537 4 5 36 0.214
R-ATCATCATCTAGTGTCACTG
P620
F-GACTGGATTAGAGTTCTCTG (AG)20 410-442 4 27 14 0.450
R-ATGGATGTGTTATCTCACTC
P1223
F-ATCTGTGTAAGGACTGAA (AG) 21 303-399 4 36 5 0.214
R-CCTCTATTAATGTGCCTG
P1826
F-GGACACTGTGACGGCTAA (AG) 24 388-467 7 2 39 0.093
R-AGCTACCAAGACACCACC

1 – F = Forward; 2 – R = Reverse.

Figure 1. Phenogram of 38 grapefruit and 3 pummelo accessions, derived using the UPGMA grouping method based on simple matching
similarity coefficients obtained from RAPD and microsatellite analyses.
 175

that although these two accessions were cultivated pulp, seeded and seedless fruits) such as ‘Marsh Seed-
grapefruits, they probably belong to the pummelo less’, ‘Duncan’, ‘Thompson Pink’, ‘Foster’ and ‘Red
group. The morphological characteristics of these ac- Blush’ showed complete genetic similarity. These
cessions (leaves with oval blades and serrated edges, results suggest that these accessions represent vari-
large diameter fruits with thick rinds and oval to ations of a single clone with different names or that
pear-shape fruits) support this hypothesis. The genetic they are in fact different varieties that were derived
similarity between group A and group B was 0.76, from somatic mutations that were not detected by the
an index which indicates the narrow genetic relation- molecular markers used.
ship between these grapefruits and the pummelos, the Low polymorphism has also been documented
putative maternal parents of the grapefruits. by Novelli et al. (1999), who analyzed 31 varieties
Group B includes 36 accessions, subdivided into of sweet oranges (C. sinensis) with the same SSR
three sub-groups. The accessions ‘Royal’, ‘Triumph’, primers. Studies conducted by Luro et al. (1995)
‘Leonardy’, ‘Imperial’, ‘Leonardy – Flórida’, ‘Can- showed that SSR probes could not distinguish between
nores’ and ‘Pernambuco’ formed the B1 subgroup varieties of this specie. However, a somatic variant
with a GS of 0.89. The subgroup composed of ‘Royal’, was detected by Orford et al., (1995) in hypervari-
‘Triumph’, ‘Imperial’, ‘Leonardy’ and ‘Cannores’ had able regions of an individual of sweet orange of a
a genetic similarity of approximately 100%. Accord- ‘Navel’ variety. These results support previous re-
ing to Hodgson (1967), these varieties were probably search (Cameron & Frost, 1968; Vardi, 1988) and
derived from natural hybrids with sweet oranges rep- suggest that the differences found in varieties of a few
resenting a natural group of grapefruits that have round Citrus species (e.g. C. paradisi and C sinensis) are
yellow-orange color fruits with lots of seeds and a caused mainly by somatic mutation, which explains
sweet flavor, which contrasts with the typical smell the narrow genetic base of this group.
and bitter taste of grapefruits. Selected somatic mutations that occur in buds or
The B2 subgroup has 23 accessions and is correl- branches were the only methods used to develop new
ated with the B1 subgroup with a GS of 0.88. The grapefruit varieties. These fruits showed distinct mor-
majority of the accessions of this subgroup had dif- phological characteristics such as the absence of seeds
ferent morphological characteristics, such as fruits and an increase in lycopene and carotene pigments
with pale yellow pulp and many seeds (‘Duncan’, in the peel, rind and/or pulp of the fruits (Gmitter
‘McCarthy’, ‘Marsh Seedy’, ‘Retiro’ and ‘Híbrido Jr., 1993). Cameron & Frost (1968) reported evid-
47449’), or with only a few seeds (‘Marsh Seedless’, ence that the pink-pulped varieties ‘Thompson’ and
‘Davis’, and ‘Hart’s’), as well as pigmented fruits with ‘Foster’ originated from periclinal chimeras that oc-
many seeds (‘Foster’, ‘Red Mexican’, and ‘Híbrido curred in mutant branches of the pale yellow fruited
47389’), or almost seedless fruits (‘Red Blush-Rubi’, ‘Marsh’ and ‘Walters’ varieties. Somatic mutations
‘Thompson Pink’ and ‘Shambar’). The GS of these that occurred in these varieties resulted in fruits with
clustered accessions was 0.98–1.00. The most dis- red-pigmented pulps, such as the grapefruits ‘Red-
tant accession within this subgroup was the grapefruit blush’, ‘Ruby Red’ and ‘Star Ruby’. However, the
‘Dalandan’, with a GS of 0.90. development of pink pigmentation was not observed
The B3 subgroup containing the grapefruits in the ‘Thompson’ accession held in this studied core
‘Maurítius’ and ‘Pamplemousse’ with a GS of 0.98 collections, indicating that the presence of pigments
correlated with the other B subgroups at a GS of 0.82. can also be influenced by environment factors, as was
Three grapefruit accessions were separated within the suggested by Hodgson (1967). Nucellar clones of the
B group, ‘Coco Variação’ (has a pale yellow pulp and ‘Thompson’ variety were cultivated in different ex-
a thicker and harder peel than the other varieties, GS of perimental stations in California where they produced
087) ‘Arananyan’ (with deep red pigmented pulp and fruits similar to the ‘Marsh’ variety. However, this did
rind, GS 0.84), and ‘Siamesa’. not occur with nucellar plants of the ‘Foster’ variety
RAPD amplification and SSR loci analyses re- that showed red-pigmented fruits and were identical
vealed low genetic polymorphism in the grapefruit to those from the ‘Redblush’ variety (Cameron &
accessions studied. The PIC values (Table 1) also con- Frost, 1968). It seems that, in the ‘Thompson’ vari-
firm the low polymorphism of the accessions. Varieties ety, the color mutation factor is present in histogenic
of great economic importance and distinct morpholo- layer I from which juice vesicle cells are derived,
gical characteristics (e.g. pigmented pulp, pale yellow but is absent from histogenic layer II which produces
 176

nucellar embryos. In the ‘Foster’ variety, this same Machado, M.A., H.D. Coletta Filho, M.L.P.N. Targon & J. Pom-
mutation factor must be present in layer II and in all peu Jr, 1996. Genetic relationship of Mediterranean mandarins
(Citrus deliciosa Tenore) using RAPD markers. Euphytica 92:
the histogenic layers of the varieties that produce red- 321–326.
pigmented fruits, and it seems that somatic mutations Müller, G.W. & Costa, A.S. 1977. Tristeza control in Brazil by
from ‘Thompson’ to ‘Redblush’ implies an exchange preimmunization with mild strains. In: Congress International
of cells from histogenic layer I to histogenic layers II Society of Citriculture, Orlando, USA, 1977. Proc Int Soc for
Citriculture 3: 868–872.
and III. Murray, M.G. & Thompson, W.F., 1980. Rapid isolation of high
molecular weight plant DNA. Nucl Acds Res 8: 4321–4325.
Novelli, V.M., M.A. Machado & M. Cristofani, 1999. Marcadores
References microssatélites (SSR- Simple Sequence Repeats) na identificação
de cultivares de laranja doce. Genet Mol Biol 22: 724.
Orford, S.J., N.S. Scot & J.M. Timmis, 1995. A hypervariable
Anon., 1998. Production Yearbook – 1998, FAO, Rome. middle repetitive DNA sequence from citrus. Theor Appl Genet
Beidler, L.L., P.R. Hilliard & R.L. Rill, 1982 Ultrasensitive staining 91: 1248–1252.
of nucleic acids with silver. Analyt Biochem 126: 374–380. Rafalski, J.A., S.V. Tingey & J.G.K. Williams, 1991. RAPD mark-
Cameron, S.W. & H.B. Frost, 1968.Genetics, breeding, and nucel- ers – a new technology for genetic mapping and plant breeding.
lar embryony. In: W. Reuther, L.D. Batchelor & H.J. Webber Ag Biotech News Info 3: 645–648.
(Eds.), The Citrus Industry, pp. 325–389, vol. 2. University of Rohlf, F.J., 1992. Numerical Taxonomy and Multivariate Analysis
California, USA. System. Version 1.70. Exeter Software, Seatauket, N.Y.
Coletta Filho, H.D., M.A. Machado, M.L.P.N. Targon, Rongwen, J., M.S. Akkaya, A.A. Bhagwat, U. Lavi & P.B. Cregan,
M.C.P.Q.D.G. Moreira & J. Pompeu Jr, 1998. Analysis of 1995. The use of microsatellite DNA markers for soybean
the genetic diversity among mandarins (Citrus spp.) using genotype identification. Theor Appl Genet 90: 43–48.
RAPD markers. Euphytica 102: 133–139. Saghai Maroof, M.A., R.B. Biyashev, G.P. Yang, Q. Zang &
Cristofani, M., R.V. Brondani, V.M. Novelli & M.A. Machado, R.W. Allard, 1994. Extraordinarily polymorphic microsatellite
1998. Desenvolvimento de marcadores microssatélites para an- DNA in barley: species diversity, chromosomal locations, and
álise genética em citros. Genet Mol Biol 21: 222. population dynamics. Proc Natl Acad Sci USA 91: 5466–5470.
Echt, C.S., G.G. Vendramin, C.D. Nelson & P. Marquardt, 1999. Scora, R.W., J. Kumamoto, R.K. Soost & E.M. Nauer, 1982. Con-
Microsatellite DNA as shared genetic markers among conifer tribution to the origin of grapefruit, Citrus paradisi (Rutaceae).
species. Can J Res 29: 365–371. Syst Bot 7: 170–177.
Gmitter, F.G., Jr. 1993. ‘Marsh’ Grapefruit. Fruit Var J 47: 130–133. Swingle, W.T., 1943. The botany of citrus and its wild relatives
Halldén, C., M. Hansen, N.O. Nilsson, A. Hjerdin & T. Säll, 1996. of the orange subfamily. In: W. Reuther, H.J. Webber & L.D.
Competition as a source of errors in RAPD analysis. Theor Appl Batchelor (Eds.), The Citrus Industry, pp. 129–474, vol.1 (1st
Genet 93: 1185–1192. edn), University of California, USA.
Hodgson, R.W., 1967. Horticultural varieties of Citrus. In: W. Thomas, M.R. & N.S. Scott, 1993. Microsatellite repeats in grapev-
Reuther, H.J. Webber and L.D. Batchelor (Eds.), The Citrus ine reveal DNA polymorphisms when analysed as sequence-
Industry, pp. 431–591, vol. 1. University of California, USA. tagged sites (STSs). Theor Appl Genet 86: 985–990.
Katzir, N., Y. Danin-Poleg, G. Tzuri, Z. Karchi, U. Lavi & P.B. Vardi, A., 1988. Application of recent taxonomical approaches and
Cregan, 1996. Length polymorphism and homologies of mi- new techniques to citrus breeding. Proc Int Soc Citriculture 1:
crosatellites in several Cucurbitaceae species. Theor Appl Genet 303–308.
93: 1282–1290. Wilkie, S.E., P.G. Isaac & R.J. Slater, 1993. Random amplified poli-
Kijas, J.M.H., M.R. Thomas, J.C.S. Fowler & M.L. Roose, 1997. morphic DNA (RAPD) markers for genetic analysis in Allium.
Introduction of trinucleotide microsatellites into a linkage map Theor Appl Genet 86: 497–504.
of Citrus. Theor Appl Genet 94: 701–706. Wu, K. & S.D. Tanksley, 1993. Abundances, polymorphisms and
Kumamoto, J., R.W. Scora, H.W. Lawton & W.A. Clerx, 1987. genetic mapping of microsatellites in rice. Mol Gen Genet 241:
Mystery of the forbidden fruit: historical epilogue on the origin 225–235.
of grapefruit, Citrus paradisi (Rutacea). Econ Bot 41: 97–107. Yamamoto, M., S. Kobayashi, Y. Nakamura & Y. Yamada, 1993.
Liu, B., 1998. Statistical Genomics: Linkage, Mapping, and QTL Phylogenic relationships of Citrus revealed by RFLP analysis of
Analysis, pp. 156–162. CRC Press LLC, Boca Raton. mitochondrial and chloroplast DNA. Japan J Breed 43: 355–365.
Luro, F., F. Laigrent, J.M. Bové & P. Ollitrault, 1995. DNA Yang, G.P., M.A. Saghai Maroof, C.G. Xu & Q. Zhang, 1994.
Amplified fingerprinting, a useful tool for determination of ge- Comparative analysis of microsatellite DNA polymorphism in
netic origin and diversity analysis in Citrus. HortScience 30: landraces and cultivars of rice. Mol Gen Genet 245: 187–194.
1063–1067.

View publication stats