Molecular Ecology Notes (2002),

2

, 344–345 doi:10.1046/j.1471-8278 .2002.00241.x

© 2002 Blackwell Science Ltd

Blackwell Science, Ltd

PRIMER NOTE

Microsatellite loci from the pink shrimp

Farfantenaeus notialis

(Crustacea, Decapoda)

A . ROBAINAS,* M. MONNEROT,† M. SOLIGNAC,† N. DENNEBOUY,† G. ESPINOSA‡ and E . GARCÍA-MACHADO**

Centro de Investigaciones Marinas, Universidad de la Habana, Calle 16 No. 114, entre 1ra y 3ra Miramar, C. Habana 11300, Cuba;

†

Centre de Génétique Moléculaire, CNRS, Gif-sur-Yvette, France;

‡

Facultad de Biología, Universidad de la Habana, Havana, Cuba

Abstract

Farfantepenaeus notialis

is an important resource for fisheries in Cuba. For this reason andfor a sustainable exploitation it is important to study their population structure and geneticvariability. We report and characterize microsatellites as genetic markers from this species.Fifteen microsatellite polymerase chain reaction (PCR) primers were designed and testedin some individuals from different populations. Seven pair of primers showed reliableamplification products and five were polymorphic. The allele number ranged from 4 to 33,and the observed and expected heterozygosities were relatively high with values between0.63 and 0.74 and 0.56 and 0.81, respectively. Departures from Hardy–Weinberg equilibriumwere observed for all loci.

Keywords

:

Crustacean, Farfantepenaeus notialis, microsatellites, polymorphism, shrimp, STR

Received 19 February 2002; revision received 10 April 2002; accepted 10 April 2002

Farfantepenaeus notialis

is the most abundant penaeidshrimp species around Cuba, representing an import-ant resource for fisheries. A detailed knowledge of thepopulation dynamics of this species is necessary to provideadequate elements for a sustainable exploitation. In anattempt to characterize their natural population struc-ture, a study, using allozymes and mitochondrial DNA(mtDNA) was carried out (García-Machado

et al

. 2001)with special emphasis in Ana María Gulf, the mostproductive zone in Cuba. These results have opened newquestions about the population structure and dynamics ofthis species and the study of microsatellite loci couldcomplement or help to resolve some of them.

In this study we report and characterize five microsatel-lite loci from

F. notialis

that will be used for populationgenetic analysis.

A genomic library was constructed with total DNAextracted from muscle tissue of a single pleopod (García-Machado

et al

. 1993). Following the general protocol ofEstoup & Cornuet (1994), 100

µ

g of DNA were digestedwith 150

µ

L of

Sau

3A1 and loaded in a 1.4% agarose gel.The fragments ranging between 300 and 700 bp were

recovered from gel and cloned into a

Bam

HI (Pharmacia)digested pUC18 plasmid vector. One microlitre of the ligationmixture, equivalent to 5 ng of recombinant plasmid DNA,was used to transform 40

µ

L of Top 10 F

′

cells (Invitrogen)by electroporation using a Biorad Gene Pulser equipment.

More than 1600 recombinant clones were obtained andscreened for tandem repeats using a mixture of six dig-labelled oligonucleotides: (CCT)

5

, (TG)

10

, (TC)

10

, (ATCT)

6

,(CAC)

5

and (TGTA)

6

, and detected with the Dig NucleicAcid Detection Kit (Boehringer). Two hundred and seventy-one were identified as positive and 89 were sequencedmanually using the M13 forward and reverse primers,and the Thermo Sequenase Radiolabaled Terminator cyclesequencing Kit (USB).

PCR amplification reactions were carried out in a finalvolume of 20

µ

L, containing 21

µ

L of DNA from a Chelexextraction solution (Walsh

et al

. 1991), 0.2

m

of each primer,1 U of

Taq

DNA polymerase (Promega), 200

m

of dNTPs,2.0

µ

L of 10

×

PCR reaction buffer [10 m

m

Tris

−

HCl(pH 9.0), 50 m

m

KCl, 1% Triton X-100], and the optimalMgCl

2

concentration (see Table 1). The amplification pro-gramme followed the profile of: 94

°

C for 3 min as initialdenaturing, 94

°

C for 45 s, experimental annealing temper-ature for 45 s, 72

°

C for 2 min and 30 s and a final extensionstep of 72

°

C for 10 min (see Table 1). The polymerase chain

Correspondence: Aymée Robainas Barcia. Fax: 537-321321; E-mail:abarcia@fbio.uh.cu

men_241.fm Page 344 Friday, August 16, 2002 10:23 AM

P R I M E R N O T E

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© 2002 Blackwell Science Ltd,

Molecular Ecology Notes

, 2, 344–345

reaction (PCR) products were denatured for 5 min at95

°

C, loaded in 5 or 6% denaturing polyacrylamide gelsand stained using the Silver Sequence Staining ReagentsKit (Promega).

PCR primers for 15 microsatellites were designed(EMBL Accession nos: AJ270744 (PnS11), AJ270746(PnS04), AJ270748 (PnS09), AJ270749 (PnS01), AJ270750(PnS08), AJ270751 (PnS13), AJ270752 (PnS02), AJ270753(PnS03), AJ270757 (PnS12), AJ270758 (PnS10), AJ270759(PnS14), AJ270760 (PnS06), AJ250376 (PnS07), AJ427483(PnS16), AJ427857 (PnS15). Some of them amplified non-specific bands (i.e. PnS07, PnS10, PnS15), while in othercases the selected loci did not amplifiy at all (i.e. PnS02,PnS08, PnS11, PnS12, PnS14).

Seven loci (

PnS01

,

PnS03

,

PnS04

,

PnS06

,

PnS09

,

PnS13

and

PnS16

) were consistently amplified, and all but the loci

PnS09

and

PnS13

were polymorphic (Table 1). The allelenumbers ranged from 4 to 33 with sizes between 112 and319. The estimation of deviations from Hardy–Weinberggenetic equilibrium (

F

IS

estimated according to Weir &Cockerham 1984, and significance of departures from zerodetermined by permutation procedure

genetix

4.01; Belkir

et al

. 2000), using between 20 and 50, individuals per popu-lations (8 populations) showed significant departuresfrom equilibrium (Table 1). However, loci

PnS01

,

PnS04

and

PnS16

were at equilibrium in most of the populationsanalysed. Departures from genetic equilibrium were alsoreported for many loci from different penaeid shrimps(Moore

et al

. 1999; Benzie 2000), that led some authors tosuggest that larger sample sizes (> 50) could be necessaryin most cases for population level analysis (Pongsomboon

et al

. 2000). The observed and expected heterozygositiesestimated for the five loci ranged between 0.63 and 0.74and 0.56 and 0.81, respectively. Although we did notobserve particularly low levels of diversity with allozyme(García-Machado

et al

. 2001) and microsatellites, temporal

replicates would be necessary to assess trends in geneticvariability over time.

Acknowledgements

We thank P.A. Morin for valuable suggestions. This research waspartially supported by grants from the International Foundationfor Science, Stockholm, Sweden and from Havana UniversityAlma Mater Concurs to EGM.

References

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Table 1 Optimal amplification conditions and variability for five of the microsatellite loci characterized from Farfantepenaeus notialis

Locus

Optimum annealing temp. (°C)

MgCl2 conc. (mm)

No. cycles

No. ind.

No. alleles

Allele size (bp)

Global FIS

Ave HO

Ave HE

PnS01 (CA)20 48 1.5 35 357 12 146–168 0.089b ± 0.025 0.74 0.81PnS03 (GA)34 51 1.5 35 240 33 237–319 0.320b ± 0.020 0.63 0.94PnS04 (CT)12 52 1.0 40 340 16 112–131 0.149b ± 0.023 0.69 0.79PnS06 (GA)41 56 2.0 40 24 22 342a nac 0.63 0.73PnS16 (GA)43 56 2.0 40 40 5 151–163 0.155* ± 0.115 0.64 0.56

a. Size of the cloned allele. Alleles of the locus PnS06 were identified by their relative migration on gel, because of the large size of the amplification products.b. Significant departure from 0 after 1000 permutations.c. As the number of individuals tested for locus PnS06 was small no calculation was made for this locus.

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