5A-04-Euphytica 2012 Lime Genetic Diversity (2)

7/29/2019 5A-04-Euphytica 2012 Lime Genetic Diversity (2)

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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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DOI 10.1007/s10681-012-0728-7


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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).

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