Potential SSR marker detection and validation

The MIcroSAtellite identification tool (MISA, version 1.0) was applied to identify the simple sequence repeat (SSR) sequences in the assembled transcriptome. The minimum repeat number for the various unit types were as follows: twelve for mono-nucleotide, six for dinucleotide, five for tri-nucleotide and tetra-nucleotide, and four for penta-nucleotide and hexa-nucleotide microsatellites. Then, primers for every SSR locus were designed on flanking sequences by using Primer 3³¹.

In order to assess the polymorphism of the SSR markers, we used a wild P. edita population sampled in the Beibu Gulf, genomic DNA was isolated from 24 randomly selected P. edita specimens using a marine animal DNA kit (Tiangen, Beijing, China). Then, 1% agarose gel electrophoresis was used to access the integrity of the DNA samples. Fifty pairs of primers were initially selected and tested. The annealing temperatures of the primers were initially tested for amplification using pooled DNA samples. PCR amplifications were carried out using a Mastercycler gradient thermal cycler (Eppendorf) in a final volume of 25 μl, and the mixture and conditions of PCR followed the methods of Shan et al³². Subsequently, genomic DNA from 24 samples were amplificated under appropriate annealing temperatures, and the products were resolved in 6% denaturing polyacrylamide gels and visualized by silver staining.