Isolation and characterization of fifteen polymorphic microsatellite loci

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Isolation and characterization of fifteen polymorphic microsatellite loci for the citrus mealybug, Planococcus citri (Hemiptera: Pseudococcidae), and cross-amplification in two other mealybug species RENATA F. MARTINS1 , VERA ZINA2 , ELSA BORGES DA SILVA2 , MARIA TERESA REBELO3 , ELISABETE FIGUEIREDO4 , ZVI MENDEL5 , OCTÁVIO S. PAULO1 , JOSÉ CARLOS FRANCO2 and SOFIA G. SEABRA1 ∗ 1

Computational Biology and Population Genomics Group, Centro de Biologia Ambiental, Departamento de Biologia Animal, Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal 2 Centro de Estudos Florestais, Instituto Superior de Agronomia, 1349-017 Lisboa, Portugal 3 Centro de Estudos do Ambiente e do Mar (CESAM), Departamento de Biologia Animal, Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal 4 Centro de Engenharia dos Biossistemas, Instituto Superior de Agronomia, 1349-017 Lisboa, Portugal 5 Department of Entomology, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel [Martins R. F., Zina V., Silva E. B., Rebelo M. T., Figueiredo E., Mendel Z., Paulo O. S., Franco J. C. and Seabra S. G. 2012 Isolation and characterization of fifteen polymorphic microsatellite loci for the citrus mealybug, Planococcus citri (Hemiptera: Pseudococcidae), and crossamplification in two other mealybug species. J. Genet. 91, e75–e78. Online only: http://www.ias.ac.in/jgenet/OnlineResources/91/e75.pdf]

Introduction The citrus mealybug, Planococcus citri (Risso) is a cosmopolitan and polyphagous insect pest mainly of subtropical fruit trees under Mediterranean climate conditions and ornamental plants in interior landscapes in cooler zones (Ben-Dov 1994; Franco et al. 2004). The cryptic behaviour of mealybug, its typical waxy body cover, and clumped spatial distribution pattern render the use of many insecticides ineffective. Therefore, there is a need to develop more effective, species-specific and environmentally safe approaches to control this pest. Pheromone-based control tactics, such as male attraction annihilation (mass trapping or lure and kill) or mating disruption, have been suggested as good alternatives (Franco et al. 2009). However, the success of pheromonebased control methods depends on knowledge of mating system of insect pests (Boake et al. 1996). In this respect, elucidating the existence of polyandry in a target species is a critical issue. Recently, behavioural experiments showed that mealybug females can mate several times, both on the same day and on days after the initial mating (Waterworth et al. 2011; Silva et al. 2012). Nevertheless, further studies are needed to confirm mealybug polyandry, aiming to elucidate if the progeny of multiple-mated mealybug females actually originate from more than one father. ∗ For correspondence. E-mail: [email protected].

Hypervariable molecular markers, namely microsatellites, are useful tools in establishing parentage in analysis of mating systems and have been used in a wide range of organisms, including insect pests, such as Ceratitis capitata (Wiedemann) and Bactrocera oleae (Rossi) (Bonizzoni et al. 2002; Augustinos et al. 2008). Until recently, development of even a small number of microsatellites was a time-consuming and expensive technique, but taking advantage of the next-generation sequencing technologies it is now possible to develop a large set of these markers in a very short period of time (Abdelkrim et al. 2009; Allentoft et al. 2009; Gilles et al. 2011). Here, we describe the development and characterization of 15 polymorphic microsatellites for P. citri, by applying next-generation sequencing of enriched genomic libraries, which will be used to investigate the polyandry hypothesis on mealybug species. We also tested cross-amplification in two other economically important cosmopolitan mealybug pests, the vine mealybug, P. ficus (Signoret), and the citrophilus mealybug, Pseudococcus calceolariae (Maskell) (Ben-Dov 1994; Zada et al. 2008; El-Sayed et al. 2010).

Materials and methods For the microsatellite development, we pooled DNA from 10 individuals sampled from five Portuguese populations,

Keywords. microsatellite; next-generation sequencing; polyandry; Coccoidea; scale insects; Planococcus. Journal of Genetics Vol. 91, Online Resources

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Renata F. Martins et al. R Tissue DNA Isolation kit extracted with E.Z.N.A. (Omega, Norcross, USA) following manufacturer’s protocol. High molecular weight and final concentration were verified in a 0.5% agarose gel, stained with 20,000× Red SafeTM Nucleic Acid Staining Solution (iNtRON Biotechnology, Kyungki-do, Korea) and confirmed with NanoDropTM 1000 Spectrophotometer v3.7 (Thermo Scientific, Wathlam, USA). Isolation of microsatellites was carried out by GenoScreen (Lille, France) (http://www.genoscreen.fr/) using 454 GSR FLX technology (Roche, Branford, USA). After genomic DNA fragmentation, DNA libraries highly enriched with microsatellites were prepared using the probes with the repetitions TG, TC, AAC, AAG, AGG, ACG, ACAT and ACTC. Sequencing was performed in a quarter of a run on R plate, generating about 40 million basepair of a Titanium data, each read with an average length of 220.49 bp. A total of 19,265 good-quality sequences were obtained, of which 4156 contained microsatellite motifs. Primer pairs were validated bioinformatically for 504 of these and 24 pairs were selected for PCR amplification in seven samples of P. citri. Validation of correct amplification was performed in a 2% agarose gel and all primer pairs were amplified in most samples. From these, 16 (showing a clear band in all individuals) were selected for polymorphism testing in our laboratory and only one was revealed to be monomorphic, leaving a final set of 15 polymorphic markers. Individuals from eight different populations, five from Portugal (Silves, Mafra, Agualva, Tavira and Camarate) and three from Israel (Shilat, Iron and Yotveta), were sampled alive and kept under controlled laboratory conditions for the experimental crosses. For the genetic variability and polymorphism test, two individuals from each population were used, in a total of 16 individuals, which were kept in absolute ethanol at 4◦ C. For the paternity tests, virgin females were chosen from two Portuguese populations (Silves and Agualve) to mate with a unique male and the progeny resulting from these crosses was also sampled, in a total of seven crosses and two individuals from the F1 progeny (one male and one female). Additionally, six individuals of P. ficus, sampled from Italy (Sicily), Spain (Murcia) and Portugal (Tavira), and two individuals of P. calceolariae, sampled from a Portuguese population (Loulé) were included. DNA R Tissue DNA Isoextraction was performed with E.Z.N.A. lation kit (Omega, Norcross, USA) following manufacturer’s protocol. Amplification of microsatellite loci was performed using the M13-tailed primer protocol for fluorescence labelling of PCR fragments (Schuelke 2000). Each of the forward primers were 5’ tailed with the M13 (uni-43) tail sequence 5’AGGGTTTTCCCAGTCACGACGTT-3’ (Venkatesan et al. 2007) which hybridize with a fluorescence labelled M13 (uni-43) primer. Polymerase chain reactions (PCR) proceeded in a final volume of 10 μL, with 0.5 μL of DNA (10–70 ng/mL), 0.025 U of GoTaq DNA polymerase (Promega, Madison, USA), 1× Colorless GoTaq Flexi

Buffer, 0.2 mM of dNTPs, 2 mM of MgCl2 , 0.1 μM of the forward primer, containing the 5’ tail sequence, 0.25 μM of the reverse primer and 0.25 μM of each 5’ fluorescent primer (labelled with HEX or FAM), according to the following protocol: an initial denaturation step at 94◦ C for 5 min, followed by 10 cycles for tail binding of 94◦ C for 30 s, 60◦ C for 1 min, 72◦ C for 1 min. Primer annealing followed in 25 cycles of 94◦ C for 30 s, 55◦ C for 1 min and 72◦ C for 30 s with a final extension step of 72◦ C for 10 min. PCR products were checked to confirm correctly sized products on 0.5% agarose gels, stained with Red Safe. Microsatellites were genotyped in an ABI PRISM 310 Genetic Analyser (Applied Biosystems, Carlsbad, USA) with GeneScan Rox Size Standard (Applied Biosystems, Carlsbad, USA) as internal size standard. Microsatellite loci were scored using GeneMapper v4 (Applied Biosystems, Carlsbad, USA). Numbers of alleles, expected and observed heterozygosities and FIS were obtained with GENETIX v4.05.2 (Belkhir et al. 1996–2004) and deviations from Hardy– Weinberg equilibrium (HWE) were tested in GENEPOP 4.1.2 (Rousset 2008).

Results and discussion In a total of 16 individuals of P. citri genotyped for primer testing, 15 polymorphic loci showed variable number of alleles, ranging from 2 to 7 with a mean of 3.1 (table 1). Expected and observed heterozygosity for each locus ranged from 0.1172 to 0.8027 and from 0.1210 to 0.8286, respectively. FIS values were significantly deviated from HWE for 11 of the 15 microsatellite loci, probably due to data structuring, since individuals from two significantly distant geographic areas were used. When testing HWE within samples from each of the two regions, only Portugal showed relatively low and nonsignificant FIS values and Israel showed generally high and, in some loci, significant FIS values (table 1). Even with a smaller sample size from Israel than from Portugal, most loci (13 out of 15) had higher or equal number of alleles in Israel than in Portugal. The heterozygote deficits found may also be due to inbreeding in the populations studied. For the experimental crosses, eight loci (Pci-6, Pci-7, Pci9, Pci-14, Pci-16, Pci.17, Pci-20, and Pci-24) were chosen as they were polymorphic in the Portuguese populations. From these, four were polymorphic within each cross, being able to distinguish between female and male progenitor and thus perceiving male contribution to the progeny. Cross-amplification on P. ficus and P. calceolariae was tested and we were able to correctly amplify eight loci (Pci-2, Pci-6, Pci-8, Pci-16, Pci-17, Pci-20, Pci-22 and Pci-24) and three loci (Pci-6, Pci-8 and Pci-16), in each species respectively (table 2), with the same PCR conditions as described above. The microsatellite isolation method used here was found to be more rapid, efficient and less expensive than traditional cloning methods. Overall, these microsatellite markers show

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Journal of Genetics Vol. 91, Online Resources

JQ812727

JQ812728

JQ812729

JQ812730

JQ812731

JQ812732

JQ812733

JQ812734

JQ812735

JQ812736

JQ812737

Pci-8

Pci-9

Pci-10

Pci-12

Pci-14

Pci-16

Pci-17

Pci-20

Pci-21

Pci-22

Pci-24

F: GGAGTTTCATCATCGCGTTC R: GCCAATGAAGCTGACCTAGA F: TCAATTCGCGAGGAATTAGG R: CGAGTGCAAACAACCGGTAA F: AGGTGGAGGTACCAATGTATGTG R: CAGCAAACAAGGAGAAAACTACG F: GCCGTACGAAACCTTGTTTG R: TCGGTCTCTTGGTACTTGGTC F: CAGATTGCTTATCATCCATCCA R: CGACGACCTCTGCAAAGTATG F: AGCTGAGTTACCTACGCGAGA R: CGGCACACTTCGATACCTTT F: AAGCTGAGGTTGGACCAGAA R: TCGTATTTATGTGCCGCATC F: GCAGCTCCAGCAAAATTACC R: ATCGTATTCGCATCGCCTC F: AACGGAAGATGAAGATGATGC R: CCTGCAGATGTCATTGGTGA F: GTTTTCGATCTCCTCGATACG R: ATTTCAGCATCGTTTACGCC F: ACTGAATAGATGTGGCTCTGTGA R: TCAATATCGCAAGTCCATGA F: CGAACGCTAAGCGGGTATAA R: CGCGCTCACTTTAGTGGTTT F: GCAGGATTAAATTGCCTCCA R: CGACAACGAGAGCTTAACAGG F: CCGGCATTACTCATTCAAGT R: TGGATGTTTGCTCAGTACTTCTGT F: GCTCATTGCAGTACCAAGTACG R: GAGATCACGTGTAATGCATCG

Primer sequence (5’–3’)

(GTC)8

(ACA)8

(CGT)7

(AGG)8

(GTT)9

(ACG)9

(GAA)9

(GAC)10

(TGT)9

(CAGG)7

(ATTG)8

(AAG)8

(TCG)9

(GGA)11

(TGG)8

Repeat motif

2 2

162–170 162–170

3

2

316–319 190–196

3

4

211–223

314–344

7

197–233

2

5

187–231

253–256

3

190–196

2

3

158–164

176–179

4

2

100–124 141–177

2

Total Na

106–121

Size range (bp)

3

1

1

2

3

4

3

1

1

2

1

3

2

1

1

Na

0.4000





0.1000

0.2000

0.6842

0.6000





0.1000



0.5714

0.4684





0.1000

0.1947

0.6500

0.6842





0.1000



0.6044

0.3947





He

Portugal

0.3000





Ho

Na , number of alleles; Ho , observed heterozygosity; He , expected heterozygosity. *P value < 0.05; **P value < 0.01.

JQ812726

Pci-2

Pci-7

JQ812724

Pci-1

JQ812725

JQ812723

Locus

Pci-6

GenBank accession number

Table 1. Characterization of 15 polymorphic microsatellite loci in the citrus mealybug, Planococcus citri.

0.1159





1

2

3

2

3

−0.0031 –

4

3

−0.0764 0.0388

3

2

2

2

2

4

2

2

Na









0.0317

0.2667





FIS



0.0000

0.0000

0.0000

0.3333

0.667

0.3333

0.0000

0.3333

0.1667

0.0000

0.2000

0.3333

0.5000

0.3333

Ho

Geographical location



0.3030

0.3030

0.4848

0.5455

0.8030

0.5455

0.6667

0.3030

0.5303

0.4848

0.2000

0.6667

0.5303

0.5455

He

Israel



1.2000

1.2000

1.2000*

0.5125

0.2222

0.5125

1.2000**

−0.1200

0.8229

1.2000*



0.4667*

0.0686

0.4667

FIS

Isolation of microsatellite loci in Planococcus citri

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Renata F. Martins et al. Table 2. Allele size range and number of alleles (Na ) for the microsatellite loci amplified in Planococcus ficus and Pseudococcus calceolariae. Planococcus ficus

Pseudococcus calceolariae

Locus

Na

Size range (bp)

Na

Size range (bp)

Pci-2 Pci-6 Pci-8 Pci-16 Pci-17 Pci-20 Pci-22 Pci-24

1 1 3 3 1 1 2 2

112 159 146–170 206–218 214 262 310–313 193–196

– 2 1 2 – – – –

– 159–177 170 206–215 – – – –

suitable resolution for our aims in following studies and in the near future should be able to give us insight into the genetic variability, gene flow and mating system of the citrus mealybug. Acknowledgements This work was funded by Fundação para a Ciência e a Tecnologia, Portugal (project PTDC/AGR-AAM/099560/2008).

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Interactions, CNRS UMR 5000, Université de Montpellier II, Montpellier, France. Ben-Dov Y. 1994 A systematic catalogue of the mealybugs of the world (Insecta: Homoptera: Coccoidea: Pseudoccocidae and Putoidae) with data on their geographical distribution, host plants, biology and economic importance. Intercept, Andover, UK. Boake C. R. B., Shelly T. E. and Kaneshiro K. Y. 1996 Sexual selection in relation to pest-management strategies. Annu. Rev. Entomol. 41, 211–229. Bonizzoni M., Katsoyannos B. I., Marguerie R., Guglielmino C. R., Gasperi G., Malacrida A. and Chapman T. 2002 Microsatellite analysis reveals remating by wild Mediterranean fruit fly females, Ceratitis capitata. Mol. Ecol. 11, 1915–1921. El-Sayed A., Unelius C. R., Twidle A., Mitchell V., Manning L., Cole L. et al. 2010 Chrysanthemyl 2-acetoxy-3-methylbetanoate: the sex pheromone of the citrophilous mealybug, Pseudococcus calceolariae. Tetrahedron Lett. 51, 1075–1078. Franco J. C., Suma P., Silva E. B., Blumberg D. and Mendel Z. 2004 Management strategies of mealybug pests of citrus in Mediterranean countries. Phytoparasitica 32, 507–522. Franco J. C., Zada A. and Mendel Z. 2009 Novel approaches for the management of mealybug pests. In Biorational control of arthropod pests: application and resistance management (ed. I. Ishaaya and A. R. Horowitz), pp. 233–278. Springer, Berlin, Germany. Gilles A., Meglécz E., Pech N., Ferreira S., Malausa T. and Martin J. 2011 Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing. BMC Genomics 12, 245. Rousset F. 2008 Genepop 007: a complete reimplementation of the Genepop software for Windows and Linux. Mol. Ecol. Res. 8, 103–106. Schuelke M. 2000 An economic method for the fluorescent labeling of PCR fragments. Nat. Biotechnol. 18, 233–234. Silva E. B., Branco M., Mendel Z. and Franco J. C. 2012 Mating behavior and performance in the two cosmopolitan mealybug species Planococcus citri and Pseudococcus calceolariae (Hemiptera: Pseudococcidae). J. Insect Behav. (in press; doi:10.1007/s10905-012-9344-6). Venkatesan M., Hauer M. C. and Rasgon J. L. 2007 Using fluorescently labeled M13-tailed primers to isolate 45 novel microsatellite loci from the arboviral vector Culex tarsalis. Med. Vet. Entomol. 21, 204–208. Waterworth R. A., Wright I. M. and Millar J. G. 2011 Reproductive biology o the three cosmopolitan mealybug (Hemiptera: Pseudococcidae) species, Pseudococcus longispinus, Pseudococcus viburni, and Planococcus ficus. Ann. Entomol. Soc. Am. 104, 249–260. Zada A., Dunkelblum E., Assael F., Franco J. C., Silva E. B., Protasov A. and Mendel Z. 2008 Attraction of Planococcus ficus males to racemic and chiral pheromone baits: flight activity and bait longevity. J. Appl. Entomol. 132, 480–489.

Received 14 February 2012, accepted 18 April 2012 Published on the Web: 13 July 2012

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