Molecular Ecology Notes (2004)

4

, 443–445 doi: 10.1111/j.1471-8286.2004.00699.x

© 2004 Blackwell Publishing Ltd

Blackwell Publishing, Ltd.

PRIMER NOTE

Microsatellite loci from the Caribbean Fruit Fly,

Anastrepha suspensa

(Diptera: Tephritidae)

ANN H. FRITZ* and NANCY SCHABLE†*

Department of Biological Sciences, Eastern Illinois University, Charleston, IL 61920, USA,

†

Savannah River Ecology Laboratory, P.O. Drawer E, Aiken, SC 29802, USA

Abstract

The lack of polymorphic genetic markers suitable for genotyping sperm, eggs, and all lifestages of the important agricultural pest,

Anastrepha suspensa

, have prevented detailedgenetic studies of its breeding system, reproductive dynamics, and population dynamics. Wedescribe polymerase chain reaction (PCR) primers and reaction conditions for amplifyingnine polymorphic microsatellite DNA loci isolated from this species. The PCR primers weretested on four to five individuals each collected from five geographically distant locationsin Florida. Heterozygosity values and the number of alleles per locus varied from 0.11 to 0.89,and from two to 12, respectively.

Keywords

:

Anastrepha suspensa

, microsatellite, PCR, primer, SSR

Received 14 January 2004; revision received 17 March 2004; accepted 21 April 2004

In terms of damage to agriculture, the most important Dipteranfamily is the cyclorraphan family Tephritidae. This familyincludes over 4200 species, including the MediterraneanFruit Fly, the Apple Maggot Fly, the Caribbean Fruit Fly,the Olive Fruit Fly, and over 60 other species that damageseeds, fruits, stems, etc. in a great variety of tropical, sub-tropical and temperate plants (White & Elson–Harris 1992).Because of their economic importance, many species oftephritid flies have been examined extensively throughoutthe last century, particularly in terms of taxonomy, phylo-geny, behaviour, reproduction, control, distribution, ecology,and physiology (e.g. see Robinson & Hooper 1989; Aluja

et al

. 2000). However, studies of the genetics of tephritidpopulations (e.g. for establishing origins and number ofintroductions of exotic pests), and the genetics of matingsystems (i.e. sexual selection and paternity outcomes) havebeen limited by the lack of high resolution genetic markers.

The fruit fly,

Anastrepha suspensa

, is endemic to a numberof Caribbean islands and is an introduced pest species inFlorida where it attacks a variety of tropical fruit crops(Clark & Weems 1989). We report here the first microsatel-lite loci available for examining population dynamics andmating systems in this species.

DNA was extracted from whole flies using the DNeasy®Tissue Kit (Qiagen Inc.) protocol for animal tissue. Extracted

DNA was enriched for (TG)

13

, (AG)

12

, (AAG)

8

, (ATC)

8

, (AAC)

8

,(AAT)

12

, (ACT)

12

, (AAAC)

6

, (AAAG)

6

, (AATC)

6

, (AATG)

6

,(ACCT)

6

, (ACAG)

6

, (ACTC)

6

and (ACTG)

6

following a proto-col modified from Hamilton

et al

. (1999). The detailed pro-tocol (Msat_Easy_Isolation_ 2000.rtf) is available athttp://www.uga.edu/srel/DNA_Lab/protocols.htm

.

Inbrief, the DNA was digested with

Rsa

I (New EnglandBiolabs), ligated to SuperSNX24 linkers (forward 5

′

-GTT-TAAGGCCTAGCTAGCAGAATC-3

′

, reverse 5

′

-GATTC-TGCTAGCTAGGCCTTAAACAAAA-3

′

; modified fromHamilton

et al

. 1999), hybridized to biotinylated microsat-ellite oligonucleotides, captured on streptavidin coatedmagnetic beads (Dynal), unwanted DNA was washed away,captured DNA was recovered by polymerase chain reac-tions (PCR) using SuperSNX24 forward primer. The productwas ligated into PCR 2.1-TOPO vector and used to transformOne Shot Top 10 Chemically Competent E. coli cells (TOPOTA cloning kit, Invitrogen). Colonies were screened for insertsusing

β

-galactosidase gene and positive colonies wereamplified using M13 forward and reverse primers. PCR pro-ducts of 500–1000 base pairs were sequenced using Big Dye(version 3.1, Applied Biosystems) chemistry and an ABI 377–96 sequencer. Sequences from both strands were assembledand edited in

sequencher

4.1 (Genecodes, Ann Arbor,MI) and exported to Ephemeris 1.0 (available at http://www.uga.edu/srel/DNA_Lab/programs.htm) to auto-matically search sequences for microsatellites.

Correspondence: Ann H. Fritz. E-mail: cfahf@eiu.edu

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Molecular Ecology

Notes

, 4, 443–445

PCR primers were developed using

oligo

6.67™(Molecular Biology Insights, http://www.oligo.net) andoptimized using 10

A. suspensa

collected from Fort Pierce,Florida. PCR amplifications were performed in a 50

µ

Lvolume using a Hybaid® PCR Express thermal cycler. Primerswere tested using touchdown thermal cycling programs(Don

et al

. 1991) encompassing a 10

°

span of annealing tem-peratures ranging between 65

°

C and 55

°

C, 60–50

°

C or55–45

°

C. Cycling parameters were: 5 cycles of 96

°

C for20 s, the highest annealing temperature for 30 s, and 72

°

Cfor 1 min; 21 cycles of 96

°

C for 30 s, the highest annealingtemperature minus 0.5

°

C per cycle for 30 s, and 72

°

C for1 min; and lastly 10 cycles of 96

°

C for 30 s, 55

°

C for 30 s,and 72

°

C for 1 min. PCR products were initially scored foramplification on 1% agarose gels and for polymorphism on3% agarose gels. PCR final concentrations for optimizingreactions were 10 m

m

Tris pH 8.4, 50 m

m

KCl, 25.0

µ

g/mLBSA, 0.4

µ

m

of each primer, 1.5 m

m

MgCl

2

, 0.15 m

m

dNTPs,0.5 units

Taq

DNA Polymerase (Promega), and approxi-mately 50 ng DNA template. One of each primer pair thatproduced an amplifiable and polymorphic microsatellitelocus was subsequently labelled with the fluorescent dyeD4-PA (Proligo®). Following optimization, three to fourflies from each of five geographically distant locations inFlorida (Bradenton, Clewiston, Ft. Pierce, La Belle, Miami)were genotyped and their amplicons sized on a Beckman

Coulter™ CEQ2000XL Sequence Analyser using the BeckmanCoulter CEQ DNA Size Standard-400 and the CEQFragment Analysis Module Program (Beckman Coulter™,Fullerton, CA).

Of 25 clones found with microsatellite arrays, nine hadsimple repeat regions that were both amplifiable andpolymorphic with the primers we developed (Table 1).Observed and expected heterozygosity values rangedfrom 0.1111 to 0.8947 and 0.3556–0.9004, respectively. Fiveloci had values that were greater than 0.7, which are par-ticularly suitable for high resolution genetic analyses.The statistical test for Hardy–Weinberg Equilibrium wascarried out in

genepop

(Raymond & Rousset 1995);however, it is important to note that the individuals in-cluded in the statistical analysis are pooled from five dif-ferent populations. Only two loci,

Asus 1–5E

and

Asus 1–6C

,deviated significantly (

P <

0.05) from Hardy–Weinbergequilibrium after sequential Bonferroni correction formultiple tests; heterozygote deficiency suggests null allelesat these loci.

Efforts to control the Caribbean fruit fly, particularly thestrategy of releasing modified flies, can only benefit from abetter knowledge of mating behaviour, sexual selectionand mating systems. The microsatellite loci reported herenow make it possible to examine the genetic details of sexualselection in an important pest.

Table 1 Characterization of nine primer pairs that amplify microsatellites from Anastrepha suspensa. Touchdown temperature refers to theinitial annealing temperature of the Touchdown protocol used; N refers to sample size; repeat sequence is of the cloned microsatellite DNA;HO and HE are observed and expected heterozygosity. Size range refers to the observed distribution of alleles at each locus; clone size refersto size of the PCR product amplified from the clone used to develop each locus, which was identical to the size predicted from the DNAsequence. The first primer listed for each pair was fluorescently labelled

Locus Primer Sequence (5′–3′)

GenBank Accession Number

Touchdown Temp

Repeat Sequence N

No. Alleles

Size Range

Clone Size HO HE

Asus 1–1HF TGG TAG TCA GGC ATC AA AY520434 60 (AG)12 19 8 208–230 220 0.8947 0.8816Asus 1–1HR CAG TAT ATC TTG GGC AAT AAAsus 1–2BF CAG CAG CGT ATG TAT GT AY520435 60 (AGC)5 20 2 129–132 132 0.3000 0.3845Asus 1–2BR CTT CGG TGT TGT TTA CTT AAsus 1–2FF GCC ACT GGT TTA TTA CTC T AY520436 60 (CA)10 20 8 262–286 278 0.8000 0.7757Asus 1–2FR ACG CCA GAC ATT TTA GTTAsus 1–3BF CGT TCA GCA TTA CTT TGA AY520437 60 (ACTC)6 20 3 107–123 123 0.4500 0.5218Asus 1–3BR CTT ATT TGG AAG TGA CTGAAsus 1–4HF TGC CAT GTC TTG CTA GT AY520438 60 (GT)7 20 6 158–204 162 0.5500 0.5256Asus 1–4HR TTA CCC TGA CTG ATT GTT ATAsus 1–5EF CAA CCC GAT TCA GAT TA AY520439 60 (AAAC)11 18 8 235–275 269 0.6111 0.8190Asus 1–5ER CGA AAA ATC CAA ATA TCT TAAsus 1–6CF AAA TCG TGG TAA ATA AAG TAA C AY520440 60 (AAAT)4 18 2 333–377 333 0.1111 0.3556Asus 1–6CR CGC TGC TCA ATT TAA TAC TAsus 1–8DF GTT AAG CCA TTC CTG TTC AY520441 55 (AG)9 18 8 214–254 238 0.7778 0.8222Asus 1–8DR CTG ACA GGG CAA AGT TACAsus 1–9AF AAA CCA TAC TTG AGA AAA AC AY520442 60 (AAG)25 19 12 278–329 320 0.8947 0.9004Asus 1–9AR TTG GAA CGA GAA TAA AAC

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Molecular Ecology Notes

, 4, 443–445

Acknowledgements

We sincerely thank Travis Glenn for helping make this projectsuccessful and the assistance of Kena Dow, Jessica Dailey, LauraNovak, Allison Kenney, for helping screen primers. This projectwas supported in part by contract DE-FC09–96SR18546 betweenthe U.S. Department of Energy and the University of Georgia’sSavannah River Ecology Laboratory. This material is also basedupon work supported by the Material Science Foundation underGrant No. 0221297.

References

Aluja M, Piñero J, Jácome I, Díaz-Fleischer F, Sivinski J (2000) Beha-vior of flies in the Genus Anastrepha (Trypetinae: Toxotrypanini).In: Fruit Flies (Tephritidae): Phylogeny, Evolution of Behavior (eds.Aluja M, Norrbom AL) pp. 375–406. CRC Press, Boca Raton,USA.

Clark RA, Weems HV Jr (1989) Detection, quarantine, and eradica-tion of fruit flies invading Florida. Proceedings of the Florida StateHorticulture Society, 102, 159–164.

Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991)‘Touchdown’ PCR to circumvent spurious priming during geneamplification. Nucleic Acids Research, 19, 4008.

Hamilton MB, Pincus EL, Di Fiore A, Flesher RC (1999) Universallinker and ligation procedures for construction of genomic DNAlibraries enriched for microsatellites. Biotechniques, 27, 500–507.

Raymond M, Rousset F (1995) genepop Version 1.2.: population-genetics software for exact tests and ecumenicism. Journal ofHeredity, 86, 248–249.

Robinson AS, Hooper G (1989) Fruit Flies Their Biology, NaturalEnemies and Control, Vol. 3A 3b. Elsevier, New York.

White IM, Elson–Harris MM (1992) Fruit Flies of Economic Significance:Their Identification and Bionomics. CAB International, Walling-ford, UK.