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Isolation and characterization of fifteen polymorphic microsatellite locifor 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∗

1Computational Biology and Population Genomics Group, Centro de Biologia Ambiental, Departamento de BiologiaAnimal, Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal

2Centro de Estudos Florestais, Instituto Superior de Agronomia, 1349-017 Lisboa, Portugal3Centro de Estudos do Ambiente e do Mar (CESAM), Departamento de Biologia Animal, Faculdade de Ciências da

Universidade de Lisboa, 1749-016 Lisboa, Portugal4Centro de Engenharia dos Biossistemas, Instituto Superior de Agronomia, 1349-017 Lisboa, Portugal

5Department 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 andcharacterization of fifteen polymorphic microsatellite loci for the citrus mealybug, Planococcus citri (Hemiptera: Pseudococcidae), and cross-amplification 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 cos-mopolitan and polyphagous insect pest mainly of subtrop-ical fruit trees under Mediterranean climate conditions andornamental plants in interior landscapes in cooler zones(Ben-Dov 1994; Franco et al. 2004). The cryptic behaviourof mealybug, its typical waxy body cover, and clumped spa-tial distribution pattern render the use of many insecticidesineffective. Therefore, there is a need to develop more effec-tive, species-specific and environmentally safe approaches tocontrol this pest. Pheromone-based control tactics, such asmale attraction annihilation (mass trapping or lure and kill) ormating disruption, have been suggested as good alternatives(Franco et al. 2009). However, the success of pheromone-based control methods depends on knowledge of matingsystem of insect pests (Boake et al. 1996). In this respect,elucidating the existence of polyandry in a target species is acritical issue. Recently, behavioural experiments showed thatmealybug females can mate several times, both on the sameday and on days after the initial mating (Waterworth et al.2011; Silva et al. 2012). Nevertheless, further studies areneeded to confirm mealybug polyandry, aiming to elucidateif the progeny of multiple-mated mealybug females actuallyoriginate from more than one father.

∗For correspondence. E-mail: sgseabra@fc.ul.pt.

Hypervariable molecular markers, namely microsatellites,are useful tools in establishing parentage in analysis ofmating systems and have been used in a wide range oforganisms, including insect pests, such as Ceratitis capi-tata (Wiedemann) and Bactrocera oleae (Rossi) (Bonizzoniet al. 2002; Augustinos et al. 2008). Until recently, devel-opment of even a small number of microsatellites was atime-consuming and expensive technique, but taking advan-tage of the next-generation sequencing technologies it isnow possible to develop a large set of these markers in avery short period of time (Abdelkrim et al. 2009; Allentoftet al. 2009; Gilles et al. 2011). Here, we describe the devel-opment and characterization of 15 polymorphic microsatel-lites for P. citri, by applying next-generation sequencing ofenriched genomic libraries, which will be used to inves-tigate the polyandry hypothesis on mealybug species. Wealso tested cross-amplification in two other economicallyimportant cosmopolitan mealybug pests, the vine mealybug,P. ficus (Signoret), and the citrophilus mealybug, Pseudococ-cus calceolariae (Maskell) (Ben-Dov 1994; Zada et al. 2008;El-Sayed et al. 2010).

Materials and methods

For the microsatellite development, we pooled DNA from10 individuals sampled from five Portuguese populations,

Keywords. microsatellite; next-generation sequencing; polyandry; Coccoidea; scale insects; Planococcus.

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Renata F. Martins et al.

extracted with E.Z.N.A. R© Tissue DNA Isolation kit(Omega, Norcross, USA) following manufacturer’s pro-tocol. High molecular weight and final concentrationwere verified in a 0.5% agarose gel, stained with20,000× Red SafeTM Nucleic Acid Staining Solution(iNtRON Biotechnology, Kyungki-do, Korea) and confirmedwith NanoDropTM 1000 Spectrophotometer v3.7 (ThermoScientific, Wathlam, USA).

Isolation of microsatellites was carried out by GenoScreen(Lille, France) (http://www.genoscreen.fr/) using 454 GS-FLX R© technology (Roche, Branford, USA). After genomicDNA fragmentation, DNA libraries highly enriched withmicrosatellites were prepared using the probes with therepetitions TG, TC, AAC, AAG, AGG, ACG, ACAT andACTC. Sequencing was performed in a quarter of a run ona Titanium R© plate, generating about 40 million basepair ofdata, each read with an average length of 220.49 bp. A totalof 19,265 good-quality sequences were obtained, of which4156 contained microsatellite motifs. Primer pairs were val-idated bioinformatically for 504 of these and 24 pairs wereselected 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 sam-ples. From these, 16 (showing a clear band in all individuals)were selected for polymorphism testing in our laboratory andonly one was revealed to be monomorphic, leaving a final setof 15 polymorphic markers.

Individuals from eight different populations, five fromPortugal (Silves, Mafra, Agualva, Tavira and Camarate) andthree from Israel (Shilat, Iron and Yotveta), were sampledalive and kept under controlled laboratory conditions for theexperimental crosses. For the genetic variability and poly-morphism test, two individuals from each population wereused, in a total of 16 individuals, which were kept in abso-lute ethanol at 4◦C. For the paternity tests, virgin femaleswere chosen from two Portuguese populations (Silves andAgualve) to mate with a unique male and the progeny result-ing from these crosses was also sampled, in a total of sevencrosses and two individuals from the F1 progeny (one maleand one female). Additionally, six individuals of P. ficus,sampled from Italy (Sicily), Spain (Murcia) and Portugal(Tavira), and two individuals of P. calceolariae, sampledfrom a Portuguese population (Loulé) were included. DNAextraction was performed with E.Z.N.A. R© Tissue DNA Iso-lation kit (Omega, Norcross, USA) following manufacturer’sprotocol.

Amplification of microsatellite loci was performed usingthe M13-tailed primer protocol for fluorescence labelling ofPCR fragments (Schuelke 2000). Each of the forward pri-mers 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) pro-ceeded in a final volume of 10 μL, with 0.5 μL ofDNA (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 theforward primer, containing the 5’ tail sequence, 0.25 μM ofthe reverse primer and 0.25 μM of each 5’ fluorescent primer(labelled with HEX or FAM), according to the following pro-tocol: an initial denaturation step at 94◦C for 5 min, followedby 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 of94◦C for 30 s, 55◦C for 1 min and 72◦C for 30 s with afinal extension step of 72◦C for 10 min. PCR products werechecked to confirm correctly sized products on 0.5% agarosegels, stained with Red Safe.

Microsatellites were genotyped in an ABI PRISM 310Genetic Analyser (Applied Biosystems, Carlsbad, USA) withGeneScan Rox Size Standard (Applied Biosystems, Carls-bad, USA) as internal size standard. Microsatellite loci werescored using GeneMapper v4 (Applied Biosystems, Carls-bad, USA). Numbers of alleles, expected and observed het-erozygosities and FIS were obtained with GENETIX v4.05.2(Belkhir et al. 1996–2004) and deviations from Hardy–Weinberg equilibrium (HWE) were tested in GENEPOP4.1.2 (Rousset 2008).

Results and discussion

In a total of 16 individuals of P. citri genotyped for primertesting, 15 polymorphic loci showed variable number of alle-les, ranging from 2 to 7 with a mean of 3.1 (table 1). Expectedand observed heterozygosity for each locus ranged from0.1172 to 0.8027 and from 0.1210 to 0.8286, respectively. FISvalues were significantly deviated from HWE for 11 of the15 microsatellite loci, probably due to data structuring, sinceindividuals from two significantly distant geographic areaswere used. When testing HWE within samples from eachof the two regions, only Portugal showed relatively low andnonsignificant FIS values and Israel showed generally highand, in some loci, significant FIS values (table 1). Even witha smaller sample size from Israel than from Portugal, mostloci (13 out of 15) had higher or equal number of alleles inIsrael than in Portugal. The heterozygote deficits found mayalso be due to inbreeding in the populations studied.

For the experimental crosses, eight loci (Pci-6, Pci-7, Pci-9, Pci-14, Pci-16, Pci.17, Pci-20, and Pci-24) were chosen asthey were polymorphic in the Portuguese populations. Fromthese, four were polymorphic within each cross, being ableto distinguish between female and male progenitor and thusperceiving male contribution to the progeny.

Cross-amplification on P. ficus and P. calceolariae wastested 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) andthree loci (Pci-6, Pci-8 and Pci-16), in each species respec-tively (table 2), with the same PCR conditions as describedabove.

The microsatellite isolation method used here was foundto be more rapid, efficient and less expensive than traditionalcloning methods. Overall, these microsatellite markers show

Journal of Genetics Vol. 91, Online Resources e76

Isolation of microsatellite loci in Planococcus citri

Tabl

e1.

Cha

ract

eriz

atio

nof

15po

lym

orph

icm

icro

sate

llite

loci

inth

eci

trusm

ealy

bug,

Plan

ococ

cusc

itri.

Gen

Ban

kSi

ze

Geo

grap

hica

lloc

atio

n

acce

ssio

nR

epea

tra

nge

Tota

lPo

rtuga

lIs

rael

Locu

snu

mbe

rPr

imer

sequ

ence

(5’–

3’)

mot

if(b

p)N

aN

aH

oH

eF I

SN

aH

oH

eF I

S

Pci-1

JQ81

2723

F:G

GA

GTT

TCAT

CAT

CG

CG

TTC

(TG

G) 8

106–

121

21

––

–2

0.33

330.

5455

0.46

67R

:GC

CA

ATG

AA

GC

TGA

CC

TAG

APc

i-2JQ

8127

24F:

TCA

ATTC

GC

GA

GG

AAT

TAG

G(G

GA

) 11

100–

124

21

––

–2

0.50

000.

5303

0.06

86R

:CG

AG

TGC

AA

AC

AA

CC

GG

TAA

Pci-6

JQ81

2725

F:A

GG

TGG

AG

GTA

CC

AAT

GTA

TGTG

(TC

G) 9

141–

177

42

0.30

000.

3947

0.26

674

0.33

330.

6667

0.46

67*

R:C

AG

CA

AA

CA

AG

GA

GA

AA

AC

TAC

GPc

i-7JQ

8127

26F:

GC

CG

TAC

GA

AA

CC

TTG

TTTG

(AA

G) 8

158–

164

33

0.57

140.

6044

0.03

172

0.20

000.

2000

–R

:TC

GG

TCTC

TTG

GTA

CTT

GG

TCPc

i-8JQ

8127

27F:

CA

GAT

TGC

TTAT

CAT

CC

ATC

CA

(ATT

G) 8

162–

170

21

––

–2

0.00

000.

4848

1.20

00*

R:C

GA

CG

AC

CTC

TGC

AA

AG

TATG

Pci-9

JQ81

2728

F:A

GC

TGA

GTT

AC

CTA

CG

CG

AG

A(C

AG

G) 7

162–

170

22

0.10

000.

1000

–2

0.16

670.

5303

0.82

29R

:CG

GC

AC

AC

TTC

GAT

AC

CTT

TPc

i-10

JQ81

2729

F:A

AG

CTG

AG

GTT

GG

AC

CA

GA

A(T

GT)

917

6–17

92

1–

––

20.

3333

0.30

30−0

.120

0R

:TC

GTA

TTTA

TGTG

CC

GC

ATC

Pci-1

2JQ

8127

30F:

GC

AG

CTC

CA

GC

AA

AAT

TAC

C(G

AC

) 10

190–

196

31

––

–3

0.00

000.

6667

1.20

00**

R:A

TCG

TATT

CG

CAT

CG

CC

TCPc

i-14

JQ81

2731

F:A

AC

GG

AA

GAT

GA

AG

ATG

ATG

C(G

AA

) 918

7–23

15

30.

6000

0.68

42−0

.076

43

0.33

330.

5455

0.51

25R

:CC

TGC

AG

ATG

TCAT

TGG

TGA

Pci-1

6JQ

8127

32F:

GTT

TTC

GAT

CTC

CTC

GAT

AC

G(A

CG

) 919

7–23

37

40.

6842

0.65

000.

0388

40.

667

0.80

300.

2222

R:A

TTTC

AG

CAT

CG

TTTA

CG

CC

Pci-1

7JQ

8127

33F:

AC

TGA

ATA

GAT

GTG

GC

TCTG

TGA

(GTT

) 921

1–22

34

30.

2000

0.19

47−0

.003

13

0.33

330.

5455

0.51

25R

:TC

AAT

ATC

GC

AA

GTC

CAT

GA

Pci-2

0JQ

8127

34F:

CG

AA

CG

CTA

AG

CG

GG

TATA

A(A

GG

) 825

3–25

62

20.

1000

0.10

00–

20.

0000

0.48

481.

2000

*R

:CG

CG

CTC

AC

TTTA

GTG

GTT

TPc

i-21

JQ81

2735

F:G

CA

GG

ATTA

AAT

TGC

CTC

CA

(CG

T)7

314–

344

31

––

–3

0.00

000.

3030

1.20

00R

:CG

AC

AA

CG

AG

AG

CTT

AA

CA

GG

Pci-2

2JQ

8127

36F:

CC

GG

CAT

TAC

TCAT

TCA

AG

T(A

CA

) 831

6–31

92

1–

––

20.

0000

0.30

301.

2000

R:T

GG

ATG

TTTG

CTC

AG

TAC

TTC

TGT

Pci-2

4JQ

8127

37F:

GC

TCAT

TGC

AG

TAC

CA

AG

TAC

G(G

TC) 8

190–

196

33

0.40

000.

4684

0.11

591

––

–R

:GA

GAT

CA

CG

TGTA

ATG

CAT

CG

Na,

num

bero

falle

les;

Ho,

obse

rved

hete

rozy

gosi

ty;H

e,ex

pect

edhe

tero

zygo

sity

.*P

valu

e<

0.05

;**P

valu

e<

0.01

.

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Table 2. Allele size range and number of alleles (Na) for themicrosatellite loci amplified in Planococcus ficus and Pseudococcuscalceolariae.

Planococcus Pseudococcusficus calceolariae

Locus Na Size range (bp) Na Size range (bp)

Pci-2 1 112 – –Pci-6 1 159 2 159–177Pci-8 3 146–170 1 170Pci-16 3 206–218 2 206–215Pci-17 1 214 – –Pci-20 1 262 – –Pci-22 2 310–313 – –Pci-24 2 193–196 – –

suitable resolution for our aims in following studies and inthe near future should be able to give us insight into thegenetic variability, gene flow and mating system of the citrusmealybug.

Acknowledgements

This work was funded by Fundação para a Ciência e a Tecnologia,Portugal (project PTDC/AGR-AAM/099560/2008).

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

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