Development and characterization of EST-derived microsatellite makers for Manila clam (Ruditapes philippinarum)

Abstract

Twenty-five polymorphic microsatellite makers were developed and characterized from expressed sequence tag sequence of the Manila clam, Ruditapes philippinarum. The number of alleles at each locus ranged from 3 to 20 with an average of seven alleles per locus. The observed and expected heterozygosity varied from 0.081 to 0.730 and from 0.127 to 0.926, respectively. Thirteen loci were found deviate significantly from Hardy–Weinberg equilibrium. These microsatellite loci will be useful for further studies on the population structure and genetic variation of this species.

The Manila clam, Ruditapes philippinarum, which is widely distributed on tidal flats in the West Pacific coasts from Russia to the Philippines, is one of the important commercial resources for the coastal fisheries. However, the wild stocks of R. philippinarum have been declining dramatically for last decades due to over-exploitation and the deterioration of environmental conditions in China. In recent years, recovery efforts such as artificial breeding program and stock enhancement are in progress (Zhang and Yan 2009). But, the genetic effects of hatchery individuals on wild populations of R. philippinarum have not yet been fully evaluated. Therefore, reasonable stock management and genetic improvement are required for sustainable development of R. philippinarum aquaculture industry.

Microsatellites or simple sequence repeats (SSRs) have become one of most commonly used DNA markers in population genetics and evolutionary biology research, and they have been widely applied in studies of biological breeding, and genetic linkage maps. Although some microsatellite markers have been developed in R. philippinarum (Yasuda et al. 2007; An et al. 2009), more polymorphic microsatellites are still required in R. philippinarum to enable parentage, population genetics and genome mapping studies. In this study, we report 25 novel polymorphic microsatellite markers developed from expressed sequence tags (ESTs) of the R. philippinarum that will be useful for genetic research of this species.

A total of 5,844 R. philippinarum ESTs obtained from GenBank (Sep 20, 2012) were scanned and assembled using SeqMan II sequence assembly software (DNASTAR Inc., Madison, WI) and 4,549 potential unigenes that contain contigs and singletons were generated. SSRHUNTER program (http://www.biosoft.net/dna/SSRHunter.htm) was used to find regions containing microsatellites. Parameters were set for the detection of di-, tri-, tetra-, penta-, and hexa- nucleotide motifs with a minimum of five repeats. Primers flanking microsatellites were designed using the PRIMER 5.0 program (http://www.premierbiosoft.com/).

Polymorphism evaluation was tested by 38 wild individuals of R. philippinarum collected from Dalian, Liaoning province, China. Genomic DNA of each specimen was extracted from adductor muscle tissue by standard proteinase K digestion, phenol–chloroform extraction, and DNA precipitation. Polymerase chain reaction (PCR) was performed in 10-μl volumes containing 0.5 U easy Taq DNA polymerase (TransGen, Beijing), 1 × PCR buffer, 0.2 mM dNTP, 0.4 μM of each primer set, 1.5 mM MgCl₂, and about 25 ng template DNA. The reactions were performed using the following parameters: 3 min at 94 °C, followed by 35 cycles of 45 s at 94 °C, 45 s at the annealing temperature listed in Table 1 and 45 s at 72 °C, then a final extension of 5 min at 72 °C. Amplification products were resolved on a 8 % polyacrylamide gel and visualized by silver staining.

Table 1 Characterization of 25 EST-SSR markers in R. philippinarum

A total of 228 microsatellite-containing EST sequences were identified from 5,844 ESTs in the R. philippinarum EST database. Of the 228 sequences, 57 were selected for microsatellite marker optimization because of repetition times and flaking sequence priority. Of the 57 potential microsatellite markers, 18 were not easily amplified, 14 were monomorphic, and 25 were found to be polymorphic among 38 individuals of R. philippinarum. Of the 57 primer pairs developed, 25 microsatellite loci (43.9 %) showed polymorphism in the population of R. philippinarum (Table 1).

The number of alleles, and observed (H _O) and expected (H _E) heterozygosities were estimated by MICROSATELLITE ANALYSER software (Dieringer and Schlötterer 2003). Tests for linkage disequilibrium (LD) and deviations from Hardy–Weinberg equilibrium (HWE) were performed by GENEPOP 4.0 (Rousset 2008). Sequential Bonferroni corrections (Rice 1989) were applied for all multiple tests (P < 0.05).

The number of alleles per locus ranged from 3 to 20 with an average of 7.76, and the observed and expected heterozygosities ranged from 0.081 to 0.730 and from 0.127 to 0.926, with an average of 0.324 and 0.571, respectively (Table 1). Tests for linkage disequilibrium showed a nonrandom association (P < 0.01) between four pairs of loci (RpN13/RpN14, RpN10/RpN13, RpN37/RpN38, RpN36/RpN56). Thirteen loci (RpN04, RpN10, RpN13, RpN14, RpN27, RpN30, RpN35, RpN36, RpN44, RpN46, RpN48, RpN50 and RpN53) deviated significantly from HWE after correction for multiple tests, which may be due to the presence of null alleles and sampling effect. The results obtained in this study indicated that these SSRs developed from EST in the Manila clam will be a useful tool for the genetic research such as population variation, parentage analysis, stock enhancement evaluation, and the establishment of effective conservation strategy of R. philippinarum.