Euphytica DOI 10.

1007/s10681-012-0728-7

AFLP data suggest a potential role for the low genetic diversity of acid lime (Citrus aurantifolia Swingle) in Oman in 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 genotypes. 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 considerably long period of time. On the other hand, AMOVA analysis showed that only 11 % of the genetic variation exists among populations from the 18 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 limes were 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: alsadi@squ.edu.om

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Sultanate. Affected lime trees were characterized by small light green to yellow leaves, dense branching and reduced owering 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 Bridge 1978; 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 mycoplasmalike organism, now referred to as Candidatus phytoplasma aurantifolia (Garnier et al. 1991; Chung et al. 2009). WBDL has spread to most parts of Oman, especially 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 Saudi Arabia (Alhudaib et al. 2009). More than half-amillion 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 neighboring countries as well as the high susceptibility of lime 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 difcult to predict vulnerability of acid limes to future disease outbreaks. Different molecular markers have been used to characterize 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 amplied polymorphic DNA (RAPD), single sequence repeat (SSR) and amplied fragment length polymorphism (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 of different plant and fungal species (Pang et al. 2007; Al-Sadi et al. 2008a, b; Geleta et al. 2008; RoblesGonzalez et al. 2008). This study was conducted to characterize genetic diversity of C. aurantifolia in Oman. Specic objectives include: (i) to characterize genetic diversity of acid lime in Oman using AFLP ngerprinting, (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 areas will help delineate current and future disease-management 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 of acid 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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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 ne 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, SigmaAldrich) 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 amplication 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 tested using 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 ReadyTo-GoTM PCR beads (HVD Life Sciences, Vienna, Austria) and Milli-Q water up to a nal reaction mixture volume of 25 ll. The DNA was amplied by 35 cycles consisting of denaturation at 94 C for 30 s (2 min for the rst 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-amplication 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)

then transported to Plant Pathology Research Laboratory (Sultan Qaboos University) where they were stored at -80 C until used.

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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 nal 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 rst cycle), annealing for 1 min at 60 C and extension at 72 C for 1.5 min (7.5 min for cycle 35). After amplication, 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. Amplied fragment length polymorphism (AFLP) analysis DNA ngerprinting using AFLPs was conducted on 143 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 modications. 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 ngerprinting was rst performed on 8 acid lime random sub-samples using 17 primer pair combinations (seven EcoRI?2 or 3 9 7 MesI?2 or 3). 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 amplication reaction mixtures using PuReTaqTM Ready-To-GoTM PCR beads consisted of 0.65 ll of 10 lM each of EcoRI?A (50 -GACTGCGT ACCAATTCA-3) and MseI-C (50 -GATGAGTCC TGAGTAAC-3) primers, 3.7 ll of diluted restriction/ligation mix and Milli-Q water up to a volume of 25 ll. The cycling prole was as explained by Al-Sadi et al. (2012a). The pre-selective amplication product was diluted by adding 210 ll of TE0.1 to the remaining amount. The selective amplication reaction mixture and the cycling parameters were as described by Al-Sadi et al. (2012b). Fragment analysis of the PCR products from the selective amplication reactions was carried out at Macrogen Inc. (Korea) using ABI 3730XL (Applied Biosystems, Carlsbad, CA). Reproducibility of the AFLP analysis was conrmed 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 amplied 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 maximum number of alleles obtained in each population with the total number of alleles obtained for all the populations. Genotypic diversity (G) within each population (different 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 using POPGENE. A dendrogram was constructed based on Neis unbiased measures of genetic distance using UPGMA (unweighed pair group method with arithmetic mean; NTSYSpc v 2.21 m). Analysis of molecular variance (AMOVA) using the program Arlequin v.3.1 (Excofer 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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Euphytica Table 1 Evaluation of 17 different primer pair combinations for use in studying genetic diversity of C. aurantifolia populations No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Primer combinations E-AAC ? M-CG E-AAC ? M-CAG E-AAC ? M-CAT E-AGA ? M-CAG E-AGA 1 M-CTG E-AC ? M-CG E-AC ? M-CAG E-ACC ? M-CG E-ACC ? M-CAG E-AGA 1 M-CGT E-AAC 1 M-CGT E-AAG ? M-CTC E-AGT ? M-CTC E-ACA ? M-CTC E-AAG ? M-CAA E-AGT ? M-CAA E-ACA 1 M-CAA NL 128 106 81 118 166 116 46 31 44 167 135 57 9 101 103 48 193 NPA 122 103 69 118 159 116 46 31 43 166 132 57 9 101 100 46 191 PPA 95.3 97.2 85.2 100 95.8 100 100 100 97.7 99.4 97.8 100 100 100 97.1 95.8 98.9 H 0.2434 0.2115 0.1959 0.2238 0.2979 0.2249 0.1769 0.2141 0.1901 0.2575 0.2873 0.1938 0.1689 0.2234 0.2373 0.2164 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

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) amplication of the 16S23S rRNA gene utilizing two pairs of phytoplasma-specic 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). Presence 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 %) leaf samples 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 eld (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 the total number of alleles and polymorphic alleles for the different primer combinations ranges from 9 to 193

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 signicance 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
Fig. 3 Gel electrophoresis showing PCR amplication 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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Euphytica Table 2 Detection of phytoplasma in samples of asymptomatic lime leaves obtained from different geographical districts in Oman District Sample size (trees) 13 36 8 25 14 18 11 13 16 12 14 20 7 32 11 26 8 19 303 No. of lime samples infected with phytoplasma 1 12 5 11 4 15 6 9 1 2 6 0 3 19 1 16 3 13 127 % of lime samples infected with phytoplasma 8 33 63 44 29 83 55 69 6 17 43 0 43 59 9 62 38 68 42

Bahla Barka Boushar Dibba Ibra Ibri Madha Mahadha Mudhaibi Nizwa Qurayat Rustaq Samael Shinas Sohar Suwaiq Taqa Yanqul Overall

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 population from 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 were 0.2520.551 (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. Samples 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) showed that 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). However, 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.

and 9 to 191, respectively (Table 1). Nei (1973) gene diversity estimates for the different primer combinations range from 0.1689 to 0.2992. The primerpair 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 of C. aurantifolia AFLP analysis of 146 samples of C. aurantifolia from various parts in Oman and from Brazil and Pakistan produced 146 different AFLP genotypes (Fig. 4). Different populations of C. 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.

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Euphytica Fig. 4 UPGMA dendrogram illustrating Neis (1978) genetic distance of 146 different samples of C. aurantifolia obtained from different geographical regions in Oman and from Brazil and Pakistan based on AFLP ngerprinting analysis using 980 polymorphic alleles

Pairwise analysis of genetic differentiation among populations of C. aurantifolia obtained from 18 districts in Oman indicated the presence of low to high 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 Mahadha 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 rst 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

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Euphytica Table 3 Population genetic analysis of acid limes from different geographical origins Population (district) Bahla Barka Boushar Dibba Ibra Ibri Madha Mahadha Mudhaibi Nizwa Qurayat Rustaq Samael Shinas Sohar Suwaiq Taqa Yanqul Oman Brazil All N 10 15 3 12 9 3 4 4 11 10 8 12 4 11 10 9 3 5 143 2 145 NPL 796 908 488 860 729 423 539 278 702 779 709 771 567 765 776 831 414 589 980 152 980 PPL 81.2 92.7 49.8 87.8 74.4 43.2 55 28.4 71.6 79.5 72.4 78.7 57.9 78.1 79.2 84.8 42.2 60.1 100 15.5 100 NUA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 g 10 15 3 12 9 3 4 4 11 10 8 12 4 11 10 9 3 5 143 2 145 G 10 15 3 12 9 3 4 4 11 10 8 12 4 11 10 9 3 5 143 2 145 %G/g 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 H 0.2283 0.2139 0.1855 0.2017 0.2274 0.1534 0.1743 0.0888 0.2024 0.2183 0.1777 0.2224 0.1754 0.1779 0.1924 0.2013 0.1562 0.1723 0.2262 0.0642 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 of C. aurantifolia from different geographical regions based on AFLP ngerprinting analysis using 980 polymorphic alleles

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

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Euphytica 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 Omani districts Among populations Within populations Oman and Brazil Among populations Within populations 1 143 401.469 23301.48 60.464 162.947 27.06 72.94 0.271 \0.0001 0.67 17 125 4890.532 18334.95 17.976 146.680 10.92 89.08 0.109 \0.0001 2.04 df Sum of Squares Variance Component Percent Variation FST P Gene ow

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 diversity (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 vegetative 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 reproduction 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 populations 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. However, 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.

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Euphytica 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 Yanqul Bahla Nizwa Samael Mudhaibi Ibra Qurayat Boushar Taqa Populations Madha Dibba Sohar Rustaq Shinas Suwaiq Barka Mahadha Ibri Yanqul Bahla Nizwa Samael Mudhaibi Ibra Qurayat Boushar Taqa Madha * -0.01417 0.00493 0.2082 -0.00483 -0.00421 -0.01085 0.1652 -0.00569 -0.0154 0.10682 0.03632 -0.02305 0.19904 0.22354 0.01873 0.05795 0.16787 Yanqul 0.75676 0.63063 0.04505 0 0.31532 0.1982 0.90991 0.00901 0.93694 * 0.12663 0.06522 0.00225 0.20633 0.23577 0.04536 0.09335 0.19163 Dibba 0.86486 * 0.00173 0.1965 0.00561 0.00204 -0.00341 0.11599 -0.00572 -0.00942 0.11532 0.0184 -0.02343 0.1828 0.21014 0.00621 0.03902 0.13804 Bahla \0.00001 \0.00001 \0.00001 0.23423 \0.00001 \0.00001 \0.00001 0.04505 0.00901 \0.00001 * 0.1647 0.12527 0.00486 0.00858 0.14178 0.15919 0.01096 Sohar 0.41441 0.3964 * 0.19787 0.01558 -0.01651 0.0189 0.16382 0.02944 0.02848 0.12221 0.01503 -0.02577 0.19522 0.21842 -0.0247 0.01831 0.16486 Nizwa 0.06306 0.17117 0.10811 \0.00001 0.01802 0.02703 0.01802 \0.00001 0.04505 0.00901 \0.00001 * 0.00182 0.25096 0.26401 0.00993 0.01067 0.22442 Rustaq \0.00001 \0.00001 \0.00001 * 0.24313 0.16367 0.20775 0.18044 0.21016 0.2247 0.00946 0.25609 0.21166 -0.00109 -0.02298 0.22656 0.25074 0.00474 Samael 0.78378 0.91892 0.95495 0 0.51351 0.95495 0.53153 0.04505 0.52252 0.33333 0.00901 0.36937 * 0.20587 0.22704 0.01983 0.00207 0.15828 Shinas 0.57658 0.16216 0.09009 \0.00001 * 0.01404 -0.00189 0.16773 0.00306 0.0042 0.15783 0.03673 -0.00741 0.22891 0.25969 0.01465 0.06182 0.22182 Mudhaibi \0.00001 \0.00001 \0.00001 0.5045 \0.00001 \0.00001 \0.00001 0.01802 \0.00001 \0.00001 0.36036 \0.00001 \0.00001 * 0.00376 0.2161 0.26602 0.03007 Suwaiq 0.46847 0.36036 0.95495 \0.00001 0.09009 * 0.01943 0.12572 -0.00032 0.01984 0.10093 0.03133 -0.02537 0.15935 0.18598 0.00309 0.0322 0.11438 Ibra \0.00001 \0.00001 \0.00001 0.97297 \0.00001 \0.00001 \0.00001 0.00901 \0.00001 \0.00001 0.18018 \0.00001 \0.00001 0.36036 * 0.24614 0.26346 0.02931 Barka 0.72973 0.6036 0.05405 \0.00001 0.42342 0.03604 * 0.14313 -0.02185 -0.01714 0.1264 0.03572 -0.00416 0.1929 0.21521 0.0351 0.06203 0.16582 Qurayat 0.21622 0.25225 0.99099 \0.00001 0.14414 0.45946 \0.00001 0.00901 0.10811 0.02703 \0.00001 0.1982 0.83784 \0.00001 \0.00001 * 0.0059 0.18988 Mahadha 0.02703 \0.00001 0.00901 \0.00001 0.00901 \0.00001 \0.00001 * 0.19216 0.16449 0.09795 0.24322 0.18917 0.16298 0.21509 0.18275 0.32106 0.16914 Boushar 0.16216 0.0991 0.24324 0.02703 0.08108 0.09009 0.03604 0.05405 0.03604 0.03604 \0.00001 0.58559 0.45946 \0.00001 \0.00001 0.37838 * 0.22264 Ibri 0.7027 0.52252 0.09009 0.00901 0.38739 0.36937 0.87387 0.02703 * -0.02999 0.1136 0.06594 0.00062 0.19682 0.23245 0.04768 0.10832 0.17183 Taqa 0.03604 \0.00001 \0.00001 0.2973 \0.00001 0.00901 \0.00001 0.11712 0.15315 0.01802 0.52252 \0.00001 0.07207 0.27027 0.33333 \0.00001 0.04505 *

Below diagonal: FST values; above diagonal: Probabilities of having more extreme FST values than observed by chance alone

Findings from this study provide evidence for frequent exchange of planting material between geographically separated districts in Oman. This is evident from AMOVA analysis which showed existence of low levels (11 %) of genetic differentiation

among populations of acid lime from the different Omani districts. This nding 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

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Euphytica Table 6 Index of association (IA) values of acid lime populations from different geographical origins Wilayat Bahla Barka Boushar Dibba Ibra Ibri Madha Mahadha Mudhaibi Nizwa Qurayat Rustaq Samael Shinas Sohar Suwaiq Taqa Yanqul IA 13.519 6.8623 4.84221 7.57347 18.6564 -0.12057 2.94442 19.4395 21.6525 5.99186 8.07463 14.5749 2.92329 7.71283 7.86112 11.371 3.61594 5.61828 P value \0.0100 \0.0100 \0.0100 \0.0100 \0.0100 0.4500 \0.0100 \0.0100 \0.0100 \0.0100 \0.0100 \0.0100 \0.0100 \0.0100 \0.0100 \0.0100 \0.0100 \0.0100

IA values are not shown for populations with less than 3 individuals

from Oman and the districts from which they came from. However, a signicant and high level of genetic differentiation (FST = 0.271, P \ 0.0001) was found between the Omani population and the Brazilian population of acid lime. This may indicate that exchange of planting material (seedlings and/or seeds) between Oman and Brazil is very limited. Additionally, the large geographical distance between Taqa and most of the studied districts in Oman (7001300 km) may explain the signicant level of gentic differentiation 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 Oman have 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 certication programs in Oman, particularly for diseases transmitted via nurseries, it is possible that movement and exchange of lime seedlings could have signicantly contributed to disseminating the causal agent of WBDL among different districts. Previous studies have provided evidence 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 certication programs to planting materials in Oman will help produce seedlings free of phytoplasma 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 introduction 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. This could be related to two reasons. Firstly, it is possible that these lime trees escaped the disease. Alternatively, 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 rst 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

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acid lime in Oman has made acid lime more vulnerable 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 WBDL symptoms and are perhaps disease-tolerant. Studies are in progress to nd 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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