Isolation and characterization of 19 microsatellite markers from the Chinese surf clam (Mactra chinensis)

Abstract

The Chinese surf clam Mactra chinensis is a commercially important bivalve, which has experienced severe population declines due to over-fishing and deterioration of coastal environment. The first set of 19 polymorphic microsatellite loci was developed and characterized for population studies. The number of alleles and expected heterozygosity ranged from 3 to 24 and from 0.419 to 0.953, respectively. The average polymorphic information content was 0.790. These microsatellite loci will facilitate future studies of population structure and conservation genetics in this species.Keywords Mactra chinensis Á Microsatellite Á FIASCO protocol Á Chinese surf clam The Chinese surf clam (Mactra chinensis), a bivalve distributed along the coastal areas of East Asia, is one of the popular fisheries resources in China. Due to over-fishing and deterioration of the coastal environment, natural populations of M. chinensis have been declining dramatically for the past decades. Demands for conservation and production improvement have resulted in growing research interests of this species (Sakurai et al. 1998;Chung et al. 2007). Assessing the genetic structure of wild populations and monitoring the genetic diversities of cultivated stocks are essential for sustainable management, genetic improvement and to avoid genetic erosion in the species. For their high variability, neutrality and codominant inheritance (Weber and May 1989), microsatellite markers provide an effective tool for investigation of genetic variation and population structure. Therefore, there is an urgent need for the development and application of microsatellites in M. chinensis by both the scientific community and the breeding industry. In this study, we reported the first set of microsatellite markers of M. chinensis, and examined the potential use of these markers for pedigree analysis.The microsatellite markers of M. chinensis were developed using the FIASCO (Fast Isolation by AFLP of Sequences Containing repeats) protocol (Zane et al. 2002) with some modifications. Briefly, genomic DNA was extracted from the adductor muscle of one live M. chinensis captured from Haiyang, Shandong province, China, using a modified phenol-chloroform protocol (Li et al. 2006). About 200 ng genomic DNA was digested with the restriction enzyme MseI, and then the fragments were ligated to MseI AFLP adaptor. The digested-ligated DNA was amplified by polymerase chain reaction (PCR) with the following reaction mixture: 2 ll buffer 19 (TaKaRa Bio Inc., Shiga, Japan), 1.5 mM MgCl 2 , 0.25 lM of primer MseI-N (5 0 -GATGAGTCCTGAGTAAN-3 0 ), 0.2 mM of each dNTP, 0.5 units of Taq DNA polymerase and 5 ll of a 1/10 dilution of digested-ligated DNA. The reaction was incubated in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA) set to 17 cycles of 94°C for 30 s, 53°C for 1 min, and 72°C for 1 min.The amplified products were hybridized to two biotinylated probes [(CA) 15 and (CT) 15 ]. The biotinylated fragments-probe complexes were captured by streptavidincoated beads (Promega...

Microsatellites reveal fine-scale genetic structure of the Chinese surf clam Mactra chinensis (Mollusca, Bivalvia, Mactridae) in Northern China

“…Individual genotypes were assessed with nine polymorphic Mactra chinensis microsatellite loci: Mc1, Mc2, Mc9, Mc10, Mc16, Mc21, Mc24, Mc28 and Mc30 (Ni et al. 2010).…”

“…The polymerase chain reactions (PCRs) were performed in 10‐μl volumes containing 0.25 U Taq DNA polymerase (Takara, Japan), 1× PCR buffer, 0.2 m m dNTP mix, 1.5 m m MgCl 2 , 1 μ m of each primer set, and 100 ng of genomic DNA. PCR was performed on a thermal cycler (GeneAmp System 9700) as follows: 3 min at 94 °C, 35 cycles of 1 min at 94 °C, 1 min at the optimal annealing temperature (Ni et al. 2010), and 1 min at 72 °C, followed by a final extension of 5 min at 72 °C.…”