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AFLP data suggest a potential role for the low genetic
diversity of acid lime (Citrus aurantifolia Swingle) in Omanin the outbreak of witches broom disease of lime
A. M. Al-Sadi H. S. Al-Moqbali
R. A. Al-Yahyai F. A. Al-Said
Received: 17 September 2011 / Accepted: 22 May 2012
Springer Science+Business Media B.V. 2012
Abstract Acid lime (Citrus aurantifolia) is the
fourth largest fruit crop in terms of cultivated area
and production in Oman. However, over half a million
lime trees were lost in Oman over the past 35 years
due to witches broom disease of lime (WBDL) which
is caused by Candidatus phytoplasma aurantifolia.
This study was conducted to examine genetic diversity
of acid lime in Oman. AFLP analysis of 143 acid lime
samples from Oman, 2 from Brazil and one from
Pakistan using 4 primer pair combinations produced
980 polymorphic loci (100 %) and 146 AFLP geno-types. Despite the long history of acid lime cultivation
in Oman, populations of lime from different districts
were found to have low levels of genetic diversity
(0.08880.2284). AMOVA analysis indicated the
existence of high level of genetic differentiation
(FST = 0.271) among populations of acid lime
from Oman and Brazil, which indicates that both
populations have evolved independently for a consid-
erably long period of time. On the other hand,
AMOVA analysis showed that only 11 % of the
genetic variation exists among populations from the18 different districts in Oman. This suggests frequent
exchange of acid lime planting material across
geographical regions in Oman. Findings from this
study suggest that the low level of genetic diversity of
acid lime in Oman and frequent movement of acid
lime planting material across districts are two main
factors which contributed to the rapid spread and high
susceptibility of acid limes to WBDL in the country.
Keywords WBDL Molecular markers Key lime
Mexican lime
Introduction
Citrus is amongst the top fruit crops in production in
the world, with a total production of 116 million tons
in 2009 (FAOSTAT-Agriculture 2011). Limes and
lemons are key citrus crops in various tropical and
subtropical parts of the world with a total production
of 14 million tons in 2009 (FAOSTAT-Agriculture
2011).
Acid lime (Citrus aurantifolia Swingle) has been
grown in Oman for over four centuries. Acid limeswere brought across the sea of Oman by Arabian
sailors and then transported to Egypt and Europe
(Davies and Albrigo 1994). Acid lime is also called
Omani, Indian, Mexican or Key lime (Hodgson 1967)
and is found all over the country, with production
being concentrated in Al Batinah region.
In the early 1970s, acid lime was the leading export
commodity crop in Oman. In 1974, a new disease was
observed in lime trees in the northern part of the
A. M. Al-Sadi (&) H. S. Al-Moqbali
R. A. Al-Yahyai F. A. Al-Said
Department of Crop Sciences, College of Agricultural
and Marine Sciences, Sultan Qaboos University,
P.O. Box 34, AlKhoud123, Oman
e-mail: [email protected]
123
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Sultanate. Affected lime trees were characterized by
small light green to yellow leaves, dense branching and
reduced flowering and fruiting. Symptoms of the
disease usually appear in lime trees which are at least
two years old. Although age of healthy lime trees can
exceed 25 years, symptomatic lime trees are killed
when they are 612 years old (Waller and Bridge1978; Bove 1995; Al-Saadi et al. 2004). The disease,
which was called witches broom disease of lime
(WBDL), was found to be caused by a mycoplasma-
like organism, now referred to as Candidatus phytopl-
asma aurantifolia (Garnier et al. 1991; Chung et al.
2009).
WBDL has spread to most parts of Oman, espe-
cially in the northern part of Oman and was then
reported in the UAE in the late 1980s (Garnier et al.
1991), in Iran in the 1990s (Bove et al. 2000), in India
in 1999 (Ghosh et al. 1999), and most recently in SaudiArabia (Alhudaib et al. 2009). More than half-a-
million lime trees were lost in Oman due to WBDL,
compared to about 350,000 trees that exist today. In
addition, the area cultivated with lime and production
of lime decreased over the past two decades by 50 and
75 %, respectively (FAOSTAT-Agriculture 2011).
WBDL is known to be transmitted via cuttings
originating from infected mother plants (Chung et al.
2009).
The wide-spread of WBDL in Oman and neigh-
boring countries as well as the high susceptibility oflime to the disease raise a question concerning genetic
diversity of acid limes in Oman. Disease outbreaks in
different hosts and in different parts of the world have
been related to several factors, including the low level
of genetic diversity of the affected crops (Strange and
Scott 2005; Martinez-Castillo et al. 2008). This has
been known to make crops more vulnerable to
devastation by plant pathogens.
Previous studies have focused on the genetic
diversity of sweet orange, grapefruits, sour oranges
and other citrus species in different parts of the world(Fang et al. 1997; Corazza-Nunes et al. 2002; Abkenar
and Isshiki 2003; Yong et al. 2006; Dehesdtani et al.
2007; Jannati et al. 2009; Yang et al. 2010; EL-Mouei
et al. 2011). However, with the exception of a study
which compared genetic relatedness of 12 clones of
acid lime having varied resistance to bacterial canker
(Alpaa et al. 2010), there is a lack of knowledge
concerning genetic diversity of C. aurantifolia in
Oman and elsewhere. Such information is vital for
understanding whether the outbreak of WBDL was
partially due to low level of genetic diversity of the
acid lime germplasm in the country. In addition,
lack of knowledge in this area makes it difficult to
predict vulnerability of acid limes to future disease
outbreaks.
Different molecular markers have been used tocharacterize genetic diversity of citrus and other crop
plant species. These include the use of isozymes,
restriction fragment length polymorphisms (RFLPs),
inter-simple sequence repeat markers (ISSR), random
amplified polymorphic DNA (RAPD), single sequence
repeat (SSR) and amplified fragment length polymor-
phism (AFLP) (Corazza-Nunes et al. 2002; Abkenar
and Isshiki 2003; Dehesdtani et al. 2007; Fang et al.
1997; Geleta et al. 2008). AFLP has proven to be a
powerful technique in characterizing genetic diversity
and phylogenetic relationships in populations ofdifferent plant and fungal species (Pang et al. 2007;
Al-Sadi et al. 2008a, b; Geleta et al. 2008; Robles-
Gonzalez et al. 2008).
This study was conducted to characterize genetic
diversity of C. aurantifolia in Oman. Specific objec-
tives include: (i) to characterize genetic diversity of
acid lime in Oman using AFLP fingerprinting, (ii) to
characterize genetic differentiation of acid lime from
different districts, (iii) and to characterize relatedness
of acid lime from Oman to acid lime from Brazil and
Pakistan. Information gained in these important areaswill help delineate current and future disease-man-
agement programs in acid lime cultivation regions of
Oman and across the world.
Materials and methods
Survey and collection of samples
A survey was conducted in 18 districts located in eight
geographical regions in Oman to collect samples ofacid lime leaves (Fig. 1). Leaf samples were collected
from a total of 303 lime trees from 5 to 13 farms from
each district, and 35 lime trees from each farm, except
for farms or districts which have a smaller sample size.
Only healthy lime leaves developing no disease
symptoms were collected from asymptomatic lime
trees (Fig. 2). Each sample consisted of about 20
leaves and the samples were sealed in plastic bags,
labeled, and placed in an ice box. The samples were
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then transported to Plant Pathology Research Labora-
tory (Sultan Qaboos University) where they were
stored at -80 C until used.
For comparison purposes, one leaf sample from
Pakistan and two leaf samples from Brazil were
obtained from healthy acid lime trees. The sample from
Pakistan (Faisalabad) was provided by Prof. Mumtaz
Khan (University of Agriculture, Faisalabad) and the
samples from Brazil (Vicosa) were provided by Prof.
Claudine Carvalho (Universidade Federal de Vicosa).
DNA extraction
About 5 g of leaf tissue (without midrib and petiole)
was ground into fine powder in liquid nitrogen using a
mortar and a pestle. Then 100 mg of the powder was
transferred into a 1.5 ml microcentrifuge tube. DNA
was extracted from leaf samples using GenElute Plant
Genomic DNA Extraction Kit (G2N70, Sigma-
Aldrich) according to manufacturers protocol. The
extracted DNA was maintained at -80 C until used.
Detection of phytoplasma using polymerase chain
reaction (PCR)
In order to avoid possible errors in the genetic analysis
of lime samples that may arise from the amplification
of phytoplasma DNA present in the tested lime leaves,
leaf samples which are infected with phytoplasma
were not included in the AFLP analysis. Presence of
phytoplasma in all the asymptomatic leaf samples
coming from Oman, Pakistan and Brazil was testedusing direct and nested polymerase chain reaction
(PCR). The universal primer pair P1 (50-AAGAATTT
GATCCTGGCTCAGGATT-30) (Deng and Hiruki
1991) and P7 (50-CGTCCTTCATCGGCTCTT-30)
(Schneider et al. 1995) were used for direct PCR to
amplify the 16S23S rRNA gene. The PCR reaction
mixture consisted of 1 ll of DNA preparation (approx
25 ng), 0.4 lM of each primer, PuReTaqTM
Ready-
To-GoTM PCR beads (HVD Life Sciences, Vienna,
Austria) and Milli-Q water up to a final reaction
mixture volume of 25 ll. The DNA was amplified by35 cycles consisting of denaturation at 94 C for 30 s
(2 min for the first cycle), annealing for 40 s at 60 C
and extension at 72 C for 1.5 min (7.5 min for
cycle 35).
The product of the direct PCR was diluted using
sterile deionized water (1:40) prior to re-amplification
by nested PCR using primer pair R16R2 (50-GAAA
CGACTGCTAAGACTGG-30) and R16F2n (50-TGA
CGGGTGTGTACAAACCCCG-30) as described by
Fig. 1 A map of Oman showing the main districts from which
acid lime samples were collected
Fig. 2 Typical symptoms of WBDL showing clustering of
small light green leaves (a) and a healthy lime branch (b)
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Gundersen and Lee (1996). The PCR reaction mixture
consisted of 1 ll DNA from direct PCR product
dilution, PuReTaqTM Ready-To-GoTM PCR beads,
0.4 lM of each primer and Milli-Q water up to a final
reaction mixture volume of 25 ll. The nested PCR
conditions consisted of 35 cycles: denaturation at
94 C for 1 min (2 min for the first cycle), annealingfor 1 min at 60 C and extension at 72 C for 1.5 min
(7.5 min for cycle 35). After amplification, a 5 ll
aliquot from each sample from the direct and nested
PCRs was electrophoresed on 1.5 % agarose gel
stained with ethidium bromide and visualized using
UV radiation.
Amplified fragment length polymorphism (AFLP)
analysis
DNA fingerprinting using AFLPs was conducted on143 phytoplasma-free acid lime samples obtained
from different parts of Oman as well as on one sample
from Pakistan and two samples from Brazil. The
AFLP protocol was adapted from Vose et al. (1995) as
described by Al-Sadi et al. (2008a) with slight
modifications. FAM-6-labelled EcoRI-AXX selective
primers were used in the study. Genomic DNA,
extracted in the previous step, was digested for 90 min
at 37 C using EcoRI (NEB, Frankfurt, Germany)
and MseI (NEB) enzymes (2.10 ll of 109 restriction/
ligation buffer (100 mM Tris-base; 100 mM MgAc;500 mM KAc; pH 7.5), 2 U EcoRI; 2 U MesI,
*100 ng of genomic DNA, and Mill-Q water up
to a volume of 17.5 ll). A 2.5 ll ligation mixture
consisting of 0.3 ll of 109 restriction/ligation buffer,
2.5 pmol EcoRI adaptor (50-CTCGTAGACTGCG
TACC/AATTGGTACGCAGTC-30), 25 pmol MseI
adaptor (50-TACTCAGGACTCAT/GACGATGAGT
CCTGAG-30), 0.5 U T4 DNA ligase (NEB) and
100 mM of ATP-Lithium salt (Roche Diagnostics
GmbH, Mannheim, Germany) was added to the
digested DNA and incubated for 90 min at 37 C.The restriction was checked by visualizing 5 ll of the
restriction ligation product on a thin 1.5 % agarose
gel. The remaining reaction was diluted to produce a
working restriction ligation (R/L) stock at a ratio of 3
R/L: 1 Milli-Q water.
AFLP fingerprinting was first performed on 8 acid
lime random sub-samples using 17 primer pair com-
binations (seven EcoRI?2 o r 3 9 7MesI?2 or3). Out
of these, four selective primer pair combinations
which produced the highest number of polymorphic
loci were chosen for analysis of the entire population
(Table 1).
Pre-selective amplification reaction mixtures using
PuReTaqTM Ready-To-GoTM PCR beads consisted of
0.65 ll of 10 lM each ofEcoRI?A (50-GACTGCGT
ACCAATTCA-3) and MseI-C (50-GATGAGTCCTGAGTAAC-3) primers, 3.7 ll of diluted restric-
tion/ligation mix and Milli-Q water up to a volume of
25 ll. The cycling profile was as explained by Al-Sadi
et al. (2012a).
The pre-selective amplification product was diluted
by adding 210 ll of TE0.1 to the remaining amount.
The selective amplification reaction mixture and the
cycling parameters were as described by Al-Sadi et al.
(2012b). Fragment analysis of the PCR products from
the selective amplification reactions was carried out at
Macrogen Inc. (Korea) using ABI 3730XL (AppliedBiosystems, Carlsbad, CA). Reproducibility of the
AFLP analysis was confirmed by repeating AFLP
analysis for all lime samples at least once.
Analysis of AFLP data
AFLP data were scored as 1 for the presence and 0 for
the absence of each amplified locus within the size
range of 50500 base pairs (bp). The number of unique
alleles within each sub-population, i.e. district, was
determined manually by comparing the maximumnumber of alleles obtained in each population with the
total number of alleles obtained for all the populations.
Genotypic diversity (G) within each population (dif-
ferent geographical locations) was determined as
described by Stoddart and Taylor (1988) followed by
scaling it by the number of genotypes (g) (Grunwald
et al. 2003). POPGENE (v 1.32) (Yeh and Boyle 1997)
was used to calculate Neis gene diversity (Nei 1973).
Genetic distance based on Neis (1978) unbiased
measures of genetic distance was also determined
between samples and populations of acid limes usingPOPGENE. A dendrogram was constructed based on
Neis unbiased measures of genetic distance using
UPGMA (unweighed pair group method with arith-
metic mean; NTSYSpc v 2.21 m).
Analysis of molecular variance (AMOVA) using
the program Arlequin v.3.1 (Excoffier et al. 2005) was
used to partition genetic variation among and within
populations of acid limes. Partition of the total genetic
variance among and within populations was based on
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geographical origins in Oman (districts) and between
populations from Oman and Brazil.
Evaluation of the level of clonality versus sexual
reproduction in C. aurantifolia was conducted using
the index of association (IA). The index and its
significance levels under the null hypothesis of
complete panmixis based on 1,000 randomizations
of the sample was determined using Multilocus
software (v.1.2).
Results
Phytoplasma in lime samples
Survey from different parts of Oman showed that
symptoms of witches broom disease of lime (WBDL)
are present in all districts with the exception of Taqa,
in the Governorate of Dhofar. Most of the surveyed
farms were found to have lime trees less than 10 years
old. Trees that exceeded 20 years of age were very few
and included one from Ibri ([25 year) and two from
Madha (4045 year). The three lime trees from Ibri
and Madha were asymptomatic and were found grown
in farms with history of the WBDL and among lime
trees which have typical WBDL symptoms (Fig. 2).A total of 303 samples of WBDL-asymptomatic
lime leaves were collected from 122 different farms
from 18 different districts in Oman. Polymerase chain
reaction (PCR) amplification of the 16S23S rRNA
gene utilizing two pairs of phytoplasma-specific
universal primers (P1/P7 and R16F2n/R2) yielded
fragments with the approximate size of 1.8 kilo base
pairs (kbp) and 1.2 kbp, respectively (Fig. 3). Pres-
ence of the two bands or at least the 1.2 kbp band
indicated infection of lime samples with phytoplasma.
PCR analysis indicated that 127 out of 303 (42 %) leafsamples from different asymptomatic lime trees were
infected with phytoplasma. Phytoplasma was detected
in all the surveyed districts in Oman, including three
samples from two farms in Taqa where WBDL
symptoms were not observed in the field (Table 2).
AFLP primer combinations
A preliminary test which evaluated 17 different
primer-pair combinations for the analysis of genetic
diversity of 8 different lime samples showed that thetotal number of alleles and polymorphic alleles for the
different primer combinations ranges from 9 to 193
Table 1 Evaluation of 17 different primer pair combinations
for use in studying genetic diversity of C. aurantifolia
populations
No. Primer combinations NL NPA PPA H
1 E-AAC ? M-CG 128 122 95.3 0.2434
2 E-AAC ? M-CAG 106 103 97.2 0.2115
3 E-AAC ? M-CAT 81 69 85.2 0.1959
4 E-AGA ? M-CAG 118 118 100 0.2238
5 E-AGA 1 M-CTG 166 159 95.8 0.2979
6 E-AC ? M-CG 116 116 100 0.2249
7 E-AC ? M-CAG 46 46 100 0.1769
8 E-ACC ? M-CG 31 31 100 0.2141
9 E-ACC ? M-CAG 44 43 97.7 0.1901
10 E-AGA 1 M-CGT 167 166 99.4 0.2575
11 E-AAC 1 M-CGT 135 132 97.8 0.2873
12 E-AAG ? M-CTC 57 57 100 0.1938
13 E-AGT ? M-CTC 9 9 100 0.168914 E-ACA ? M-CTC 101 101 100 0.2234
15 E-AAG ? M-CAA 103 100 97.1 0.2373
16 E-AGT ? M-CAA 48 46 95.8 0.2164
17 E-ACA 1 M-CAA 193 191 98.9 0.2992
These data are based on AFLP analysis of 8 randomly selected
acid lime samples from Oman
Primers highlighted in bold typeface were those selected for
further analysis of the genetic diversity of C. aurantifolia
populations
NL number of loci, NPL number of polymorphic loci, PPL
percentage of polymorphic loci, H Nei (1973) gene diversity
Fig. 3 Gel electrophoresis showing PCR amplification of the
16S23S rRNA gene using P1/P7 (a) and R16F2n/R2 (b) primer
pairs. From left to right: ladder, positive control, 11 samples
infected with phytoplasma (lanes 3, 4, 6, 7, 8, 9, 14, 16, 17, 18
and 19) and negative control (lane 20)
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and 9 to 191, respectively (Table 1). Nei (1973) gene
diversity estimates for the different primer combina-tions range from 0.1689 to 0.2992. The primer-
pair combinations E-AGA ? M-CTG, E-AGA ?
M-CGT, E-AAC ? M-CGT and E-ACA ? M-CAA
gave the highest number of polymorphic loci and the
highest estimates of Nei gene diversity (Table 1).
Genotypic and genetic diversity
within populations ofC. aurantifolia
AFLP analysis of 146 samples ofC. aurantifolia from
various parts in Oman and from Brazil and Pakistanproduced 146 different AFLP genotypes (Fig. 4).
Different populations ofC. aurantifolia obtained from
different geographical origins showed variations in the
percentage of polymorphic loci and gene diversity
estimates. The percentage of polymorphic loci for the
populations from various districts in Oman ranges
from 28.4 % for the population from Mahadha to
92.7 % for the population from Barka.
Gene diversity estimates based on Neis (1973)
measures of gene diversity (H) showed that the overall
gene diversity for the populations from Oman and
Brazil were 0.2262 and 0.0642, respectively (Table 3).
Gene diversity estimates for the populations from
different districts in Oman ranged from 0.0888 for the
population from Mahadha to 0.2283 for the populationfrom Bahla. No unique alleles were detected in any of
the populations (Table 3).
Genetic distance and cluster analysis
According to Neis unbiased measures of genetic
distance, genetic distance between the 143 lime
samples from different districts in Oman ranged from
0.084 to 0.726 (avg. 0.410). The level of genetic
distance between the acid lime samples from Oman
and the lime samples from Pakistan and Brazil were0.2520.551 (avg. 0.392) and 0.1690.671 (avg.
0.531), respectively (Fig. 4).
Genetic distance among populations from different
districts in Oman ranged from 0 (Sohar and Qurayat)
to 0.1194 (Boushar and Taqa) with a mean value of
0.0365. Genetic distance between the populations
from Oman and Brazil was found to range from 0.2622
to 0.3525 (avg. 0.3137) (Fig. 5).
UPGMA analysis of lime samples from different
parts of Oman and from Brazil and Pakistan showed
clustering of the samples into several clusters. Sam-ples from Brazil clustered separately from those from
Oman, while the sample from Pakistan intermixed
within the Omani cluster (Fig. 4). No relationship was
found between AFLP clustering of Omani lime
samples and the districts from which they were
obtained.
Partition of genetic variation and the index
of association
Analysis of molecular variance (AMOVA) showedthat about 11 % of the genetic variation is found
among populations of C. aurantifolia obtained from
different districts in Oman, with most of the genetic
variation being within populations (Table 4). How-
ever, about 27 % of the genetic variation was found
between the Omani and the Brazilian populations of
C. aurantifolia, indicating the existence of high levels
of genetic differentiation.
Table 2 Detection of phytoplasma in samples of asymptom-
atic lime leaves obtained from different geographical districts
in Oman
District Sample
size
(trees)
No. of lime
samples infected
with phytoplasma
% of lime samples
infected with
phytoplasma
Bahla 13 1 8
Barka 36 12 33
Boushar 8 5 63
Dibba 25 11 44
Ibra 14 4 29
Ibri 18 15 83
Madha 11 6 55
Mahadha 13 9 69
Mudhaibi 16 1 6
Nizwa 12 2 17
Qurayat 14 6 43
Rustaq 20 0 0
Samael 7 3 43
Shinas 32 19 59
Sohar 11 1 9
Suwaiq 26 16 62
Taqa 8 3 38
Yanqul 19 13 68
Overall 303 127 42
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Pairwise analysis of genetic differentiation among
populations of C. aurantifolia obtained from 18
districts in Oman indicated the presence of low tohigh levels of genetic differentiation (-0.0299 to
0.3211) (Table 5). Most of the populations from Oman
were found to have low to moderate levels of genetic
differentiation. However, the population from Maha-
dha was found to have moderate to high levels of
genetic differentiation with most of the populations
obtained from most of the districts in Oman (Table 5).
The index of association values (IA) for populations
obtained from the different districts in Oman ranged
from 2.9 to 21.6 (P\0.05), except for the population
which was obtained from Ibri (IA = -0.120567;
P = 0.45) (Table 6).
Discussion
Symptoms of witches broom disease of lime (WBDL)
have been reported for the first time in Oman in the
1970s in Shinas and Liwa (Waller and Bridge 1978).
From there, WBDL has spread to different parts of the
country, especially to districts close to the place of
Fig. 4 UPGMA
dendrogram illustrating
Neis (1978) genetic
distance of 146 different
samples ofC. aurantifolia
obtained from different
geographical regions in
Oman and from Brazil andPakistan based on AFLP
fingerprinting analysis using
980 polymorphic alleles
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origin of the disease. Findings from this study showed
that WBDL is present in all the districts of Oman that
were surveyed, from the northern Governorate of
Musandam to the southern Governorate of Dhofar.
Only 3 lime trees, of 2545 years old were found
asymptomatic and are not infected with phytoplasma
throughout Oman. These trees were found in farms
where neighboring lime trees have either been killed
Table 3 Population genetic analysis of acid limes from different geographical origins
Population (district) N NPL PPL NUA g G %G/g H
Bahla 10 796 81.2 0 10 10 100 0.2283
Barka 15 908 92.7 0 15 15 100 0.2139
Boushar 3 488 49.8 0 3 3 100 0.1855
Dibba 12 860 87.8 0 12 12 100 0.2017
Ibra 9 729 74.4 0 9 9 100 0.2274
Ibri 3 423 43.2 0 3 3 100 0.1534
Madha 4 539 55 0 4 4 100 0.1743
Mahadha 4 278 28.4 0 4 4 100 0.0888
Mudhaibi 11 702 71.6 0 11 11 100 0.2024
Nizwa 10 779 79.5 0 10 10 100 0.2183
Qurayat 8 709 72.4 0 8 8 100 0.1777
Rustaq 12 771 78.7 0 12 12 100 0.2224
Samael 4 567 57.9 0 4 4 100 0.1754
Shinas 11 765 78.1 0 11 11 100 0.1779
Sohar 10 776 79.2 0 10 10 100 0.1924
Suwaiq 9 831 84.8 0 9 9 100 0.2013
Taqa 3 414 42.2 0 3 3 100 0.1562
Yanqul 5 589 60.1 0 5 5 100 0.1723
Oman 143 980 100 0 143 143 100 0.2262
Brazil 2 152 15.5 2 2 100 0.0642
All 145 980 100 145 145 100 0.2246
N samples sizes, NPL total polymorphic loci, PPL percentage of Polymorphic loci (out of 980), NUA number of unique alleles,
g number of different genotypes recovered, G Stoddart and Taylors measure of genotypic diversity, %G/g the percentage of
maximum diversity obtained in each population, H Nei (1973) gene diversity
Fig. 5 UPGMA
dendrogram illustrating
Neis (1978) genetic
distance of 19 different
populations ofC.
aurantifolia from different
geographical regions based
on AFLP fingerprinting
analysis using 980
polymorphic alleles
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or are affected by WBDL but none of these has
exceeded 12 years old. Although this may indicate
that the three lime trees have resistance or tolerance to
WBDL, future studies are needed to evaluate the
mechanisms controlling lime resistance or tolerance to
WBDL.
Analysis of genetic diversity within populations of
acid lime from different parts of Oman showed that all
Omani populations have low levels of genetic diver-
sity (0.08880.2283). Furthermore, the levels of
genetic diversity are low compared to the previously
reported levels for sweet orange (0.20450.4044),
mandarin (0.5124), lemon (0.4543) and other citrus
species (Yong et al. 2006; Dehesdtani et al. 2007;
Jannati et al. 2009; Yang et al. 2010; EL-Mouei et al.
2011).
Although acid lime has been known in Oman for
over 400 years (Davies and Albrigo 1994), the low
levels of genetic diversity could be related to two main
factors. Firstly, it is possible that all cultivated acid
lime has been introduced into Oman from a common
source. Previous reports indicated that acid lime has
been moved to the west from India via Oman (Davies
and Albrigo 1994), which makes it possible that a
single acid lime cultivar was introduced and cultivated
in Oman in the past. Using UPGMA analysis, the
sample of acid lime from Pakistan, part of the Indian
Subcontinent, was found within clusters of acid lime
from Oman. This supports the hypothesis that acid
lime in Oman has been introduced from countries in
the northern part of the Indian Ocean. However, due to
the small sample size from Pakistan, future studies
may consider evaluating the relationship between acid
limes from Oman and other parts of the world,
especially India, using larger sample sizes.
Another factor that may have contributed to the low
levels of genetic diversity of acid lime in Oman is the
method of propagating lime which is mainly vegeta-
tive by layering. This traditional and the most common
way for propagating citrus in Oman, may have led to
the low levels of genetic diversity in acid limes in the
country. The index of association values provided
evidence that limes are propagated asexually or that
outcrossing between different lime genotypes, which
results from sexual reproduction, is not common in
lime growing areas in Oman. These modes of repro-
duction are known to affect diversity in citrus species
(Novelli et al. 2006; Culley and Wolfe 2001), which
can result in low levels of genetic diversity as
compared to species or cultivars reproducing by
outcrossing between different genotypes.
The low levels of genetic diversity within popula-
tions of acid lime in Oman could be one of the main
reasons for the rapid decline and high susceptibility of
acid lime to WBDL. Since WBDL was reported in the
1970s, the disease wiped out over half a million lime
trees throughout the country. Previous studies have
shown that crops with low levels of genetic diversity
are more vulnerable to diseases than crops with high
levels of genetic diversity (Strange and Scott 2005).
Since Candidatus phytoplasma aurantifolia is very
selective for acid limes compared to other citrus
species and cultivars in the country (Moghal et al.
1993; Chung et al. 2009), widening the genetic base of
acid lime in Oman may help in the management of
WBDL. This can be achieved through introduction of
new acid lime cultivars from places with high levels of
genetic diversity, which may help in overcoming
future disease outbreaks to which crops with a low
level of genetic diversity are more vulnerable. How-
ever, whether widening the genetic base of acid lime in
Oman could help in the management of WBDL is a
question which deserves further investigation in the
future.
Table 4 Variation as measured using AFLPs among and within populations of acid limes from different geographical regions based
on hierachiacl analysis of molecular variance (AMOVA)
Source of Variation df Sum of Squares Variance Component Percent Variation FST P Gene flow
Omani districts
Among populations 17 4890.532 17.976 10.92 0.109 \0.0001 2.04
Within populations 125 18334.95 146.680 89.08Oman and Brazil
Among populations 1 401.469 60.464 27.06 0.271 \0.0001 0.67
Within populations 143 23301.48 162.947 72.94
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Findings from this study provide evidence for
frequent exchange of planting material between geo-
graphically separated districts in Oman. This is
evident from AMOVA analysis which showed exis-
tence of low levels (11 %) of genetic differentiation
among populations of acid lime from the different
Omani districts. This finding is also supported by the
lack of unique alleles in any of the populations which
were obtained from the different parts of Oman and the
lack of relationship between clustering of acid limes
Table 5 Pairwise genetic differentiation (FST) among populations of acid limes obtained from different districts
Populations Madha Dibba Sohar Rustaq Shinas Suwaiq Barka Mahadha Ibri
Madha * 0.86486 0.41441 \0.00001 0.57658 0.46847 0.72973 0.02703 0.7027
Dibba -0.01417 * 0.3964 \0.00001 0.16216 0.36036 0.6036 \0.00001 0.52252
Sohar 0.00493 0.00173 * \0.00001 0.09009 0.95495 0.05405 0.00901 0.09009
Rustaq 0.2082 0.1965 0.19787 * \0.00001 \0.00001 \0.00001 \0.00001 0.00901
Shinas -0.00483 0.00561 0.01558 0.24313 * 0.09009 0.42342 0.00901 0.38739
Suwaiq -0.00421 0.00204 -0.01651 0.16367 0.01404 * 0.03604 \0.00001 0.36937
Barka -0.01085 -0.00341 0.0189 0.20775 -0.00189 0.01943 * \0.00001 0.87387
Mahadha 0.1652 0.11599 0.16382 0.18044 0.16773 0.12572 0.14313 * 0.02703
Ibri -0.00569 -0.00572 0.02944 0.21016 0.00306 -0.00032 -0.02185 0.19216 *
Yanqul -0.0154 -0.00942 0.02848 0.2247 0.0042 0.01984 -0.01714 0.16449 -0.02999
Bahla 0.10682 0.11532 0.12221 0.00946 0.15783 0.10093 0.1264 0.09795 0.1136
Nizwa 0.03632 0.0184 0.01503 0.25609 0.03673 0.03133 0.03572 0.24322 0.06594
Samael -0.02305 -0.02343 -0.02577 0.21166 -0.00741 -0.02537 -0.00416 0.18917 0.00062
Mudhaibi 0.19904 0.1828 0.19522 -0.00109 0.22891 0.15935 0.1929 0.16298 0.19682
Ibra 0.22354 0.21014 0.21842 -0.02298 0.25969 0.18598 0.21521 0.21509 0.23245
Qurayat 0.01873 0.00621 -0.0247 0.22656 0.01465 0.00309 0.0351 0.18275 0.04768
Boushar 0.05795 0.03902 0.01831 0.25074 0.06182 0.0322 0.06203 0.32106 0.10832
Taqa 0.16787 0.13804 0.16486 0.00474 0.22182 0.11438 0.16582 0.16914 0.17183
Populations Yanqul Bahla Nizwa Samael Mudhaibi Ibra Qurayat Boushar Taqa
Madha 0.75676 \0.00001 0.06306 0.78378 \0.00001 \0.00001 0.21622 0.16216 0.03604
Dibba 0.63063 \0.00001 0.17117 0.91892 \0.00001 \0.00001 0.25225 0.0991 \0.00001
Sohar 0.04505 \0.00001 0.10811 0.95495 \0.00001 \0.00001 0.99099 0.24324 \0.00001
Rustaq 0 0.23423 \0.00001 0 0.5045 0.97297 \0.00001 0.02703 0.2973
Shinas 0.31532 \0.00001 0.01802 0.51351 \0.00001 \0.00001 0.14414 0.08108 \0.00001
Suwaiq 0.1982 \0.00001 0.02703 0.95495 \0.00001 \0.00001 0.45946 0.09009 0.00901
Barka 0.90991 \0.00001 0.01802 0.53153 \0.00001 \0.00001 \0.00001 0.03604 \0.00001
Mahadha 0.00901 0.04505 \0.00001 0.04505 0.01802 0.00901 0.00901 0.05405 0.11712
Ibri 0.93694 0.00901 0.04505 0.52252 \0.00001 \0.00001 0.10811 0.03604 0.15315
Yanqul * \0.00001 0.00901 0.33333 \0.00001 \0.00001 0.02703 0.03604 0.01802
Bahla 0.12663 * \0.00001 0.00901 0.36036 0.18018 \0.00001 \0.00001 0.52252
Nizwa 0.06522 0.1647 * 0.36937 \0.00001 \0.00001 0.1982 0.58559 \0.00001
Samael 0.00225 0.12527 0.00182 * \0.00001 \0.00001 0.83784 0.45946 0.07207
Mudhaibi 0.20633 0.00486 0.25096 0.20587 * 0.36036 \0.00001 \0.00001 0.27027
Ibra 0.23577 0.00858 0.26401 0.22704 0.00376 * \0.00001 \0.00001 0.33333
Qurayat 0.04536 0.14178 0.00993 0.01983 0.2161 0.24614 * 0.37838\
0.00001Boushar 0.09335 0.15919 0.01067 0.00207 0.26602 0.26346 0.0059 * 0.04505
Taqa 0.19163 0.01096 0.22442 0.15828 0.03007 0.02931 0.18988 0.22264 *
Below diagonal: FST values; above diagonal: Probabilities of having more extreme FST values than observed by chance alone
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from Oman and the districts from which they came
from. However, a significant and high level of genetic
differentiation (FST = 0.271, P\ 0.0001) was found
between the Omani population and the Brazilianpopulation of acid lime. This may indicate that
exchange of planting material (seedlings and/or seeds)
between Oman and Brazil is very limited. Addition-
ally, the large geographical distance between Taqa and
most of the studied districts in Oman (7001300 km)
may explain the significant level of gentic differenti-
ation between lime populations in this district and lime
populations in other distcrics in Oman (Yang et al.
2010).
Distribution and exchange of planting material
between geographically separate regions in Omanhave largely been done by government-owned or
commercial nurseries. This resulted in the country
wide distribution of lime seedlings to growers from the
same stock plants; thus the cultivation of genetically
identical acid limes in different districts.
Data generated from AMOVA analysis which
indicated frequent exchange of planting material
between geographically separate districts in Oman
may explain the rapid spread of WBDL from the place
of origin to other citrus growing districts in Oman. Due
to the lack of nursery budwood certification programs
in Oman, particularly for diseases transmitted via
nurseries, it is possible that movement and exchange
of lime seedlings could have significantly contributed
to disseminating the causal agent of WBDL among
different districts. Previous studies have providedevidence that exchange of planting material between
countries or regions in the same country could help
transmit pathogens/diseases among these regions
(Al-Sadi et al. 2008a, b; Al-Sadi et al. 2012a).
Applying certification programs to planting materials
in Oman will help produce seedlings free of phytopl-
asma and other serious diseases of citrus, including
severe viruses and viroids (Bove 1995; Al-Sadi et al.
2012a). This should be coupled with identifying
WBDL-free areas in Oman and at the same time
applying strict quarantine measures to prevent intro-duction of the causal agent of WBDL into these areas.
In addition, research aiming at management of
Hishimonus phycitis, the potential vector of ca.
phytoplasma aurantifolia in acid lime (Chung et al.
2009; Salehi et al. 2007), is required to keep disease
levels under economically acceptable levels.
Although three lime trees which are grown among
WBDL-affected lime trees were found to be free of
WBDL symptoms, no relationship was found between
the apparent tolerance of these lime trees to WBDL
and AFLP-based clustering of the lime samples. Thiscould be related to two reasons. Firstly, it is possible
that these lime trees escaped the disease. Alterna-
tively, clustering of the lime samples was based on
multiple genes that control several characteristics
other than resistance. Therefore, clustering will be
based more on the genes which are common between
these lime samples, rather than depending only on
genes controlling resistance to a particular disease.
Previous studies have shown that samples belonging to
the same genotype do not have to share the same
physiological characteristics (Al-Sadi et al. 2008a, b;Al-Sadi et al. 2010, b).
Conclusion
This study is the first to examine genetic diversity of
acid lime. The study provides evidences that two
factors have contributed to devastation of the acid lime
industry in Oman. The low level of genetic diversity of
Table 6 Index of association (IA) values of acid lime popu-
lations from different geographical origins
Wilayat IA P value
Bahla 13.519 \0.0100
Barka 6.8623 \0.0100
Boushar 4.84221 \0.0100Dibba 7.57347 \0.0100
Ibra 18.6564 \0.0100
Ibri -0.12057 0.4500
Madha 2.94442 \0.0100
Mahadha 19.4395 \0.0100
Mudhaibi 21.6525 \0.0100
Nizwa 5.99186 \0.0100
Qurayat 8.07463 \0.0100
Rustaq 14.5749 \0.0100
Samael 2.92329 \0.0100
Shinas 7.71283 \0.0100
Sohar 7.86112 \0.0100
Suwaiq 11.371 \0.0100
Taqa 3.61594 \0.0100
Yanqul 5.61828 \0.0100
IA values are not shown for populations with less than 3
individuals
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acid lime in Oman has made acid lime more vulner-
able to infection by phytoplasma. The problem was
made worse by frequent exchange of planting material
between districts which helped in the spread of WBDL
to different areas in Oman. Surveys of over 9000 lime
trees from different areas in Oman have shown that
only 3 lime trees, aged 2545 years old, lack WBDLsymptoms and are perhaps disease-tolerant. Studies
are in progress to find out mechanisms controlling the
apparent resistance or tolerance in these lime trees.
Acknowledgments Authors would like to acknowledge Prof.
Claudine Carvalho (Brazil) and Prof. Mumtaz Khan (Pakistan)
for providing the acid lime sample. Thanks are due to lime
growers for their help in sample collections and to research
assistants and Issa Al-Mahmooli for help in technical work. We
acknowledge Sultan Qaboos University for funding this study
through the Strategic Research Project: Rejuvenating lime
production in Oman: resolving current challenges (SR/AGR/
CROP/08/01).
References
Abkenar AA, Isshiki S (2003) Molecular characterization and
genetic diversity among Japanese acid citrus (Citrus spp.)
based on RAPD markers. J Hortic Sci Biotechnol 78:
108112
Alhudaib K, Arocha Y, Wilson M, Jones P (2009) Molecular
identification, potential vectors and alternative hosts of the
phytoplasma associated with a lime decline disease in
Saudi Arabia. Crop Protection 28:1318Alpaa K, Gopal K, Gopi V, Aliya S, Sreenivasulu B, Purusho-
tham K (2010) Fingerprinting of acid lime varieties and
clones having varied resistance to bacterial canker, using
RAPD marker. Arch Phytopathol Plant Prot 43:624633
Al-Saadi AM, Khan IA, Deadman ML (2004) Economic losses
caused by witches broom disease of lime and some man-
agement aspects in Shinas area of Oman. In: 10th inter-
national citrus congress, Morocco, 1520 February 2004,
pp 817818
Al-Sadi AM, Drenth A, Deadman ML, Aitken EAB (2008a)
Genetic diversity, aggressiveness and metalaxyl sensitivity
of Pythium aphanidermatum populations infecting
cucumber in Oman. Plant Pathol 57:4556
Al-Sadi AM, Drenth A, Deadman ML, de Cock AWAM, Al-
Said FA, Aitken EAB (2008b) Genetic diversity, aggres-
siveness and metalaxyl sensitivity of Pythium spinosum
infecting cucumber in Oman. J Phytopathol 156:2935
Al-Sadi AM, Al-Masoudi RS, Al-Habsi N, Al-Said FA, Al-
Rawahy SA, Ahmed M, Deadman ML (2010) Effect of
salinity on Pythium damping-off of cucumber and on the
tolerance of Pythium aphanidermatum. Plant Pathol
59:112120
Al-Sadi A, Al-Hilali S, Al-Yahyai R, Al-Said F, Deadman M,
Al-Mahmooli I, Nolasco G (2012a) Molecular character-
ization and potential sources of Citrus Tristeza Virus in
Oman. Plant Pathol. doi:10.1111/j.1365-3059.2011.02
553.x
Al-Sadi AM, Al-Ghaithi AG, Al-Balushi ZM, Al-Jabri AH
(2012b) Analysis of diversity in Pythium aphanidermatum
populationsfrom a singlegreenhousereveals phenotypic and
genotypic changes over 2006 to 2011. Plant Dis 96:852858
Bove JM (1995) Virus and virus-like disease of citrus in the near
east region. FAO, Rome
Bove JM, Danet JL, Bananej K, Hassanzadeh N, Taghizadeh M,
Salehi M, Garnier M (2000) Witches broom disease of
lime (WBDL) in Iran. In: Paper presented at the proceed-
ings of the fourteenth conference of IOCV
Chung KR, Khan IA, Brlansky RH (2009) Citrus diseases exotic
to Florida: witches broom disease of lime (WBDL). EDIS
Publications, University of Florida, Gainesville, PP228,
pp 1-3
Corazza-Nunes MJ, Machado MA, Nunes WMC, Cristofani
MN, Targon MLP (2002) Assesment of genetic variability
in grapefruits (Citrus paradise Macf.) and pummelos (C.
maxima (Burm) Merr.) using RAPD and SSR markers.
Euphytica 126:169176
Culley T, Wolfe A (2001) Population genetic structure of theclestogamous plant species Viola pubescens Aiton (Viola-
ceae), as indicated by allozyme and ISSR molecular
markers. Heredity 86:545556
Davies FS, Albrigo LG (1994) Citrus. CAB International,
United Kingdom
Dehesdtani A, Kazemitabar SK, Rahimian H (2007) Assessment
of genetic diversity of Navel sweet orange cultivars grown
in Mazandaran Province using RAPD markers. Asian J
Plant Sci 6:11191124
Deng SJ, Hiruki C (1991) Genetic relatedness between two non-
culturable mycoplasmalike organisms revealed by nucleic
acid hybridization and polymerase chain reaction. Phyto-
pathology 81:14751479
EL-Mouei R, Choumane W, Dway F (2011) Molecular char-acterization and genetic diversity in Genus Citrus from
Syria. Int J Agric Biol 13:351356
Excoffier L, Laval G, Schneider S (2005)Arlequin (version 3.0):
an integrated software package for population genetics data
analysis. Evol Bioinf 1:4750
Fang DQ, Roos ML, Krueger RR, Federic CT (1997) Finger-
printiing trifoliate orange germplasm accessions with iso-
zymes RFLPs and inter-simple sequence repeat markers.
Theor Appl Genetics 95:211219
FAOSTAT-Agriculture (2011) http://faostat.fao.org/site/567/
default.aspx#ancor
Garnier M, Zreik L, Bove JM (1991) Witches broom, a lethal
mycoplasmal disease of lime trees in theSultanate of Oman
and the United Arab Emirates. Plant Dis 75:546551
Geleta M, Bryngelsson T, Bekele E (2008) Assessment of
genetic diversity of Guizotia abyssinica (L.f.) Cass. (As-
teraceae) from Ethiopia using amplified fragment length
polymorphism. Plant Genetic Resour Character Util 6
(1):4151
Ghosh DK, Das AK, Singh S, Singh SJ, Ahlawat YA (1999)
Occurrence of witches broom, a new phytoplasma disease
of acid lime (Citrus aurantifolia) in India. Plant Dis 83:302
Grunwald NJ, Goodwin SB, Milgroom MG, Fry WE (2003)
Anlaysis of genotypic diversity data for populations of
microorganisms. Phytopathology 93:738746
Euphytica
123
http://dx.doi.org/10.1111/j.1365-3059.2011.02553.xhttp://dx.doi.org/10.1111/j.1365-3059.2011.02553.xhttp://faostat.fao.org/site/567/default.aspx#ancorhttp://faostat.fao.org/site/567/default.aspx#ancorhttp://faostat.fao.org/site/567/default.aspx#ancorhttp://faostat.fao.org/site/567/default.aspx#ancorhttp://dx.doi.org/10.1111/j.1365-3059.2011.02553.xhttp://dx.doi.org/10.1111/j.1365-3059.2011.02553.x -
7/29/2019 5A-04-Euphytica 2012 Lime Genetic Diversity (2)
13/13
Gundersen DE, Lee IM (1996) Ultrasensitive detection of
phytoplasmas by nested PCR assay using two universal
primer pairs. Phytopathol Mediterr 35:144151
Hodgson RW (1967) Horticultural varieties of citrus. In: Reu-
ther W, Batchelor LD, Webber HD (eds) The citrus
Industry. Univeristy of California Press, Berkeley,
pp 431591
Jannati M, Fotouhi R, Abad AP, Salehi Z (2009) Genetic
diversity analysis of Iranian citrus varieties using micro
satellite (SSR) based markers. J Hortic For 1:120125
Martinez-Castillo J, Colunga-GraciaMarin P, Zizumbo-Villar-
real D (2008) Genetic erosion and in situ conservation of
lima bean (Phaseolus lunatus L.) landraces in its Meso-
american diversity center. Genet Resour Crop Evol 55:
10651077
Moghal SM, Shivanathan P, Mani A, Al-Zidjali AD, Al-Zidjali
TS, Al-Raeesy YM (1993) Status of pests and diseases in
Oman: series 1: plant diseases in the Batinah. Ministry of
Agriculture and Fisheries, Muscat
Nei M (1973) Analysis of gene diversity in subdivided popu-
lations. Proc Nat Acad Sci 70:33213323
Nei M (1978) Estimation of average heterozygosity and geneticdistance from a small number of individuals. Genetics
89:583590
Novelli VM, Cristofani M, Souza AA, Machado MA (2006)
Development and charaterization of polymorphic micro-
satellite markers for the sweet orange (Citrus sinensis L.
Osbeck). Genetics Mol Biol 29(1):9096
Pang X-M, Hu C-G, Deng XX (2007) Phylogentic relation-
ships within Citrus and its related genera as inferred from
AFLP markers. Genet Resour Crop Evol 54:429436
Robles-Gonzalez MM, Medina-Urrutia VM, Velazquez-Mon-
real JJ, Simpson J (2008) Field performance and molecular
profiles of Mexican lime selection. Euphytica 161:401411
Salehi M, Izadpanah K, Siampour M, Bagheri A, Faghihi SM
(2007) Transmission of Candidatus phytoplasma auran-
tifolia to Bakraee (Citrus reticulata Hybrid) by Feral Hi-
shimonus phycitis leafhoppers in Iran. Plant Dis 91:466
Schneider B, Seemuller E, Smart CD, Kirkpatrick BC (1995)
Phylogenetic classification of plant pathogenic myco-
plasmalike organisms or phytoplasmas. In: Raszin S,
Tully JG (eds) Molecular and diagnostic procedures in
mycoplasmology, vol 2. Academic Press, New York,
pp 369380
Stoddart JA, Taylor JF (1988) Genotypic diversity: estimation
and prediction in samples. Genetics 118:705711
Strange RN, Scott PR (2005) Plant disease: a threat to global
food security. Annu Rev Phytopathol 43:83116
Vose P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes
M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M
(1995) AFLP: a new technique for DNA fingerprinting.
Nucleic Acids Res 23:44074414
Waller JM, Bridge J (1978) Plant diseases and nematodes in the
Sultanate of Oman. Proc Am Nat Sci 24:313326
Yang Y, Pan Y, Gong X, Fan M (2010) Genetic variation in the
endangered Rutaceae species Citrus hongheensis based onISSR fingerprinting. Genet Resour Crop Evol 57:1239
1248
Yeh RC, Boyle TJB (1997) Population genetic analysis of co-
dominant and dominant markers and quantitative traits.
Belg J Bot 129:157
Yong L, De-Chung L, Bo W, Zhong-Hai S (2006) Genetic
diversity of pummelo (Citrus grandis Osbeck) and its rel-
atives based on simple sequence repeat markers. Chin J
Agric Biotechnol 3:119126
Euphytica
123