Isolation and characterization of polymorphic microsatellite loci from transcriptome sequence of Ruditapes philippinarum

In this study, 38 novel polymorphic microsatellite markers derived from transcriptome sequence of Ruditapes philippinarum were reported. The polymorphisms of these markers were detected in a natural population of R. philippinarum. The number of alleles per locus ranged from 2 to 10 with an average of 4.8. The observed and expected heterozygosity per locus ranged from 0.000 to 0.939 and 0.086 to 0.832, with an average of 0.255 and 0.542, respectively. Eighteen loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0013) and two pairwise combinations of four loci (RpT178/RpT146 and RpT200/RpT249) were significant after Bonferroni correction. These loci will provide useful information for the studies on genetic diversity and structure, construction of genetic linkage maps and the effective management of R. philippinarum.


INTRODUCTION
The Manila clam, Ruditapes philippinarum, is an economically-important marine bivalve species of the China aquaculture industry and is widely distributed in the coasts of China.The world production of this species was 3.9 million metric tons in 2013.China is the first largest country in the world in terms of production of the Manila clam, producing about 3.0 million metric tons annually, which accounts for about 90% of global production (Zhang and Yan, 2010).In aquaculture, genetic diversity is the fundamental resource on which stock improvements rely.However, many aquaculture practices, such as producing large numbers of offspring from a few parents, inbreeding and using broodstocks derived from hatchery seed, are likely to reduce genetic diversity and thereby diminish disease resistance and reduce the population's ability to adapt to new environments (Allendorf and Phelps, 1980).In recent years, the wild resources of R. philippinarum have experienced dramatic population declines due to over-exploitation and the deterioration of coastal environment.*Corresponding author.Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License The significant decline of wild R. philippinarum stocks makes people to pay close attention to its genetic variation and population structure which will provide essential information on maintenance and management of the clam resources (An et al., 2012;Mura et al., 2012;Xing et al., 2014).
Microsatellite or simple sequence repeat (SSR) markers, which are inherited in a Mendelian fashion as codominant markers, have been increasingly popular in genetic studies because of their high levels of allelic variability, wide dispersal and abundance throughout the genome (Chistiakov et al., 2006).Until recently, microsatellite markers have been developed in the R. philippinarum derived from both expressed sequence tag (EST) and anonymous genomic sequence (Yasuda et al., 2007;An et al., 2009;Nie et al., 2014).Molecular markers can be divided into type I (coding) markers which are associated with genes of known functions and type II (noncoding) markers which are associated with anonymous genomic sequences (O'Brien, 1991).As Type I markers that are associated with genes of known function, the EST-SSRs are superior to anonymous genomic SSR in functional diversity assessment and interspecific transferability (Pashley et al., 2006), but genomic SSRs usually are more polymorphic than EST-SSRs (Ellis and Burke, 2007).
About 60 microsatellite markers were developed in R. philippinarum until now (Yasuda et al., 2007;An et al., 2009;Hu et al., 2014), including 36 genomic SSRs and 25 expressed sequence tag derived SSRs (EST-SSRs).These markers provide sufficient information to evaluate wild and cultured genetic resources, but are still deficient for the development of genetic linkage map and markerassisted selection (MAS).Therefore, the objective of the present study was to develop more polymorphic microsatellite markers using the transcriptome data derived from 454 pyrosequencing.

Samples collection and DNA extraction
Thirty five individuals of R. philippinarum in total were collected from Jinzhou, Dalian, Liaoning province China.Genomic DNA of each specimen was isolated from muscle tissues following the standard phenol-chloroform method (Li et al., 2006) with some modifications.The adductor muscle was removed from fresh specimens and preserved in 100% ethanol until DNA preparation.Tissue was homogenized in 500 μL of extraction lyses buffer together with 0.5 μg/mL proteinase K and incubated at 55°C.Following phenol: chloroform: isoamyl alcohol (25:24:1) extractions, the supernatants were precipitated by the addition of 2 volumes of absolute ethanol.DNA was washed with 70% ethanol, dissolved in TE and stored at -20°C.

Microsatellite primer design and PCR
Microsatellite sequences were screened from transcriptome sequences derived from R. philippinarum in our laboratory.
Microsatellite sequences were screened from a total of 9450 ESTs in the 454 database using the software SSRHUNTER 1.3 (Li and Wan, 2005).Microsatellite primers were designed using Primer Premier 5.0 software (http: //www.premierbiosoft.com/primerdesign/).From 9450 sequences, 324 were identified with microsatellite motifs, and a set of 105 microsatellite primer pairs were designed and synthesized.The major parameters for primer design were set as follows: primer length from 19 to 25 nucleotides, the size of PCR product from 100 to 350 bp, and annealing temperature at 50-65°C.The primers were synthesized by Sangon Company (Shanghai).ESTs containing SSRs were then annotated using BLAST software as described by Maneeruttanarungroj et al. (2006).The BLAST results were classified into 3 groups: known gene products, hypothetical proteins and unknown genes.

Polymorphism assessment for primers
The polymorphisms of microsatellite primers were tested in 35 individuals of R. philippinarum.Of the 105 potential microsatellite markers, 39 were not easily amplified, and 38 were found to be polymorphic among 8 individuals of R. philippinarum.Then, thirty eight microsatellite markers were selected to test polymorphic and genetic diversity of natural population of R. philippinarum in Jin Zhou, Dalian, China.
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.

Data analysis
The number of alleles, and observed (HO) and expected (HE) 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).MICRO-CHECKER (Van Oosterhout et al., 2004) was employed to infer the most probable technical cause of HWE departures, including null alleles, mis-scoring due to stuttering and allelic dropout due to short allele dominance.

RESULTS AND DISCUSSION
Microsatellites, which are inherited in a Mendelian fashion as codominant markers, have been increasingly popular in genetic studies because of their high level of heterozygosity, wide dispersal and abundance throughout the genome and transferability across different strains.For R. philippinarum, numerous microsatellites have been developed recently (Yasuda et al., 2007;An et al., 2009;Hu et al., 2014).But, the pace of development has been limited by the time-consuming and labor intensive requirement to construct, enrich and sequence genomic libraries (Edwards et al., 1996).Recently, identification of microsatellites from expressed sequences has been

Table 1 .
Characterization of thirty eight microsatellite markers in the Manila clam, Ruditapes philippinarum.