Molecular Ecology Resources (2009) 9, 229–232

doi: 10.1111/j.1755-0998.2008.02411.x

PERMANENT GENETIC RESOURCES NOTE Blackwell Publishing Ltd

Development of nine polymorphic microsatellite markers for the phytoparasitic nematode Xiphinema index, the vector of the grapevine fanleaf virus L . V I L L AT E ,* D . E S M E N J A U D ,† S . C O E D E L ‡ and O . P L A N TA R D *§ *INRA, Agrocampus Rennes, Univ Rennes 1, UMR BiO3P 1099 (Biology of Organisms and Populations applied to Plant Protection), F-35653 Le Rheu, France, †Equipe ‘Interactions Plantes-Nématodes’, UMR IBSV, INRA Sophia Antipolis, France, ‡Ouest-Genopole Plateforme Genotypage — INRA UMR 118, Domaine de la Motte, BP 35327, F-35000 Rennes, France

Abstract We report isolation, characterization and cross-species amplification of nine microsatellite loci from the phytoparasitic nematode Xiphinema index, the vector of grapevine fanleaf virus. Levels of polymorphism were evaluated in 62 individuals from two X. index populations. The number of alleles varies between two and 10 depending on locus and population. Observed heterozygosity on loci across both populations varied from 0.32 to 0.857 (mean 0.545). The primers were tested for cross-species amplification in three other species of phytoparasitic nematodes of the Xiphinema genus. These nine microsatellite loci constitute valuable markers for population genetics and phylogeographical studies of X. index. Keywords: cross-priming, dagger nematode, enriched library, GFLV, M13 tail, Vitis vinifera Received 2 June 2008; revision accepted 28 August 2008

Xiphinema index is a migratory ectoparasitic nematode and the vector of grapevine fanleaf virus (GFLV; Taylor & Brown 1997; Esmenjaud 2000) causing vine degeneration and considerable yield losses in vineyards. This nematode, supposedly introduced from the Middle East and now distributed worldwide (Hewitt et al. 1958), reproduces by meiotic parthenogenesis (Dalmasso & Younes 1969). A better knowledge of its genetic variability will help to trace its routes of introduction and will provide a better understanding of dispersal in this species, which is essential for sustainable management of GFLV disease. Nuclear genetic markers (i.e. microsatellites) for X. index have already been described (He et al. 2003). However, six of these seven microsatellites have a common structure composed of a unique flanking region, (GCTAT)n repeat, a common nonrepeating region, (CA)n element, and a common mammalian L1 retroposonlike element (while the seventh locus also contains a Correspondence: Laure Villate, Equipe ‘Biologie et Génétique des Nématodes Phytoparasites’, UMR BiO3P INRA-Agrocampus, Domaine de la Motte BP 35327, F- 35653 Le Rheu, France. Fax: (33) 2 23 48 51 50; E-mail: [email protected] §Present address: INRA, ENVN, UMR 1300 BioEpAR, Ecole Nationale Vétérinaire de Nantes, Atlanpôle, La Chantrerie BP 40706, F-44307 Nantes cedex 03, France © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

transposon element). The amplification results did not show any tandem repeating amplification profiles, indicating the interspersed distribution feature of the L1-like element. Thus, six of these seven markers represent the same locus that has been inserted into different parts of the genome. Those features prevent the use of those microsatellites as suitable Mendelian markers for population genetics investigations. However, it is essential to develop microsatellite loci usable to study the population structure and genetic diversity of X. index, which would certainly yield more information on the genetic diversity and history of this economically important nematode. Total genomic DNA was extracted from 500 X. index females (descendants of a single female from a French vineyard — Fréjus-) using a standard phenol–chloroform protocol. DNA was sent to SREL DNA Lab (University of Georgia) to build libraries double enriched for dinucleotide motifs as described in Glenn & Schable (2005). Positive clones (288) yielded 97 sequences with microsatellite repeats, with 58 for which primers could be designed using Primer 3 software (Rozen & Skaletsky 2000). An M13 tail (5′-CACGACGTT GTAAAACGAC-3′) was added to the 5′ end of each forward primer for amplification using a universal dye-labelling method (Schuelke 2000) (Table 1). From this set of 58

France Locus with GenBank Accession no. Xi04 EU678745

Xi08 EU678746

Xi13 EU678747

Xi16 EU678748

Xi22 EU678749

Xi24 EU678751

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

Xi27 EU678752

Xi29 EU678753

Xi32 EU678754

All loci

Primer sequence (5′–3′) F-m13: CACGACGTTGTAAAACGAC GTGAGCAAACGCAGAAGAGA R: CAAGAAACCGATTGAAATTATGG F-m13: CACGACGTTGTAAAACGAC AGACGAAGATGACGATGCTG R: TACGAATACCAAACGCCAAG F-m13: CACGACGTTGTAAAACGAC AGGACGTCACTGCTTTTGGT R: TGCCTAAAATGGAGGGCTTA F-m13: CACGACGTTGTAAAACGAC CGACAGGTGGCAGTTATTGA R: CGCAACGAATAAGGGAAGAG F-m13: CACGACGTTGTAAAACGAC CAAAGTGTTTTGGGCGAGAT R: TGTTCTGTAAGGTCGGCACA F-m13: CACGACGTTGTAAAACGAC GAGAATCGAGCGTTTTCCTG R: CGCGAGAATCATCTGCCTA F-m13: CACGACGTTGTAAAACGAC CGGTGCACTGGTATAGTTGC R: TCGCTGTGGTGATGTTCTTC F-m13: CACGACGTTGTAAAACGAC GTGGCAGAACCCAATTCACT R: TTAGTTACACTGGCCCATCC F-m13: CACGACGTTGTAAAACGAC ATGACCACCCAATGACGAA R: CCGCCGGTATTTCCAGTAT

Crete

Fluorescent tag

Ta (°C)

Repeat in sequenced allele

6-FAM

55

(TG)10–11

2

195–197

35

2

0.357

0.299

NS

27

1

—

—

NA

PET

55

(GAT)8

3

149–164

35

3

0.185

0.175

NS

27

3

0.778

0.511

**

PET

55

(CA)11

10

215–259

34

3

0.267

0.391

NS

27

5

1.000

0.702

***

VIC

55

(TC)11

6

131–155

35

3

0.3

0.269

NS

27

3

0.37

0.319

NS

VIC

50

(AAAC)8

4

144–168

35

3

0.897

0.598

***

26

2

1.000

0.510

***

6-FAM

55

(AC)10

4

227–237

35

2

0.862

0.499

***

27

3

0.704

0.519

*

NED

55

(GT)8

3

243–249

35

2

0.867

0.506

***

27

2

0.296

0.257

NS

VIC

62

(GA)9

4

124–136

35

3

1.000

0.554

***

27

2

0.296

0.307

NS

NED

55

(GTT)7

5

147–165

35

4

1.000

0.600

***

27

3

0.704

0.498

*

0.637

0.432

***

27

0.572

0.403

***

NA

Size range alleles (bp)

n

NA

HO

HE

HWE test

n

NA

HO

HE

HWE test

35

F-m13 is the forward primer with M13 tail in 5′ (underlined and in italics). 6-FAM, NED, VIC and PET are the fluorescent tag for the M13 primer. Ta, optimal annealing temperature. NA, number of alleles observed among the 62 individuals genotyped. NA, sample sizes (n), observed (HO) and expected (HE) heterozygosities, and probability of the Hardy–Weinberg test for a heterozygote excess (HWE test) are given, locus by locus and for all loci, for two populations, France and Crete: ***P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05, NS P > 0.05.

230 P E R M A N E N T G E N E T I C R E S O U R C E S N O T E

Table 1 Characterization of nine microsatellite loci developed for Xiphinema index

P E R M A N E N T G E N E T I C R E S O U R C E S N O T E 231

N Xiphinema americanum 10 Xiphinema diversicaudatum 24 Xiphinema vuittenezi 13

XI04

XI08

XI13

XI16

XI22

XI24

XI27

XI29 XI32

— — 1

— — 1

3 2 3

2 2 3

— — 1

— — 1

— 4 3

— — 2

Table 2 Number of alleles observed for each locus in cross-species amplification tests

— 2 2

N, number of individuals tested; —, no amplification.

potential loci, 36 microsatellites that harboured at least seven repeats were screened for polymorphism. DNA was extracted from single individuals (natural populations from France and Crete) by a simplified protocol. Each individual was hand-picked, placed in 20 μL of NaOH 0.25 m in a 250 μL Eppendorf tube and stored one night at room temperature (Stanton et al. 1998). Then, the Eppendorf tube was placed at 99 °C for 2 min. 20 μL of a mix (10 μL of HCl 0.25 m, 5 μL of Tris-HCl 0.5 m pH 8and 5 μL of Triton X-100 2%) was added, and the Eppendorf tube was placed once more at 99 °C for 2 min (Floyd et al. 2002). Finally, we added 10 μL of lysis buffer (Tris-HCl 50 mm pH 8, EDTA 5 mm, Tergitol 5% and Proteinase K 500 μg/mL) and each tube was incubated at 65 °C for 1 h and 95 °C for 10 min (Ibrahim et al. 1994). Extracted DNA can be stored at –20 °C until polymerase chain reaction (PCR) amplification. PCRs were carried out in 10-μL simplex reaction containing 0.5 μL of DNA extract, 1× Taq buffer (Elmer), 0.8 μL of MgCl2 at 25 mm (Elmer), 0.4 μL of each primer (1 μm for forward M13 primer and 10 μm for reverse primer), 0.36 μL of M13 10 μm fluoro-labelled with a FAM, PET, NED or VIC dye at the 5′ end (see Table 1), 0.8 μL of 4 × 2.5 mm dNTPs and 0.08 μL of 5 U/μL Taq DNA polymerase (AmpliTaq, Applied Biosystems). Reactions were carried out in a PTC-100 thermocycler (MJ Research) with the following amplification conditions: 5 min at 95 °C; 20 cycles of 95 °C for 1 min, primer specific annealing temperature (see Table 1) for 1 min, 72 °C for 1.5 min; 20 cycles of 95 °C for 1 min, 53 °C for 1 min (annealing temperature of the fluorescent dye-labelled M13 primer), 72 °C for 1.5 min, and 10 min at 72 °C. Nine loci were chosen according to amplicon size, variability and amplification success. A total of 62 X. index individuals, sampled in vineyards from France (N = 35) and Crete (N = 27), were genotyped in 10 μL of the mix described previously. The species identification was based on morphology and confirmed using PCR markers designed from internal transcribed spacer sequences (Wang et al. 2003). Amplification conditions were the same as described before. PCR products were stored at –20 °C until genotyping. All PCR products were run on an ABI PRISM 3130xl Genetic Analyser 16 Capillary system (Applied Biosystems) and sized with internal lane standard (500 LIZ; Applied Biosystems) using the program GeneMapper version 3.7 (Applied Biosystems). © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

All primer pairs tested in simplex reactions amplified one polymorphic locus (see Table 1). The fragments amplified ranged from 124 to 259 bp. The number of alleles varied between two and 10 depending on locus and population (Table 1). fstat 2.9.3 (Goudet 2001) was used to analyse genetic data, in particular to test for departures from Hardy-Weinberg equilibrium and linkage disequilibrium of loci. Observed heterozygosity calculated for each locus across all populations varied from 0.32 to 0.857 (mean 0.545) and expected heterozygosityranged from 0.25 to 0.701 (mean 0.464). A significant global excess of heterozygosity was detected in the two populations France and Crete (Table 1) and all pairs of loci showed a significant genotypic disequilibrium. In both populations, all loci do not exhibit an excess of heterozygosity and some of them are in deficit (Table 1). This pattern is consistent with the meiotic parthenogenesis of X. index (Balloux et al. 2003; De Meeus et al. 2006) and will be the object of a forthcoming study. Testing of those loci was conducted in three other Xiphinema species after morphological and molecular identification (Wang et al. 2002). The cross-priming test revealed from two, four and nine loci amplifying for Xiphinema americanum, X. diversicaudatum and X. vuittenezi, respectively, among the 10, 24 and 13 individuals tested for each species, respectively (Table 2). Two, four and five of these loci were polymorphic for X. americanum, X. diversicaudatum and X. vuittenezi, respectively. The availability of these polymorphic microsatellite loci will allow the investigation of the genetic structure of those dagger nematodes, a poorly known aspect of their biology.

Acknowledgements The authors would like to thank the estates and their vineyard managers that have funded this research. Many thanks to Travis Glenn, Mandy Schable and Cris Hagen of the SREL DNA Lab for the building of the library. We thank INRA of Sophia Antipolis, particularly Roger Voisin, for providing us with Xiphinema nematodes, and Robert Robinns (University of Arkansas) for providing us with X. americanum samples.

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© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd