Isolation and Characterization of 25 Polymorphic Microsatellite Markers of Sepiella japonica

Samples of S. japonica were collected from the aquaculture farm of Zhoushan (China) and one of them (female) was send to BioMarker Biotech Inc. for High-throughput transcriptome. Tissue samples of eyestalk, peduncle, tentacle, gill, muscle and ovary were rapidly sampled, snap-frozen in liquid nitrogen and stored at -80°C prior to RNA extraction. And tissues samples were pooled in equal amounts for total RNA extraction, transcriptome Illumina sequencing and de novo assembly. Putative SSR markers were screened using the SSRHUNTER 1.3 software (http://www.biosoft.net/dna/SSRHunter.htm). The criteria used in SSRHUNTER to identify microsatellites were as follows: 6 repeats for di-nucleotide, 5 repeats for tri-nucleotide and tetranucleotide. Primers were designed using the PRIMER 5.0 program (http://www.premierbiosoft.com/).

Polymorphism evaluation was tested using 30 S. japonica individuals. Genomic DNA was isolated from muscle tissue by proteinase K digestion followed by a standard phenol-chloroform method. Amplification was carried out in a volume of 25µL, which contained approximately 100ng of DNA as template, 2.5µL dNTP (2.5 mM each), 2.5 µL 10×buffer,2µL MgCl2 (20 mM), 1µL primers (10 mM each), and 0.25µL of Taq DNA polymerase (5U/μL). Polymerase chain reaction were run under the following thermal cycle condition: at 94°C for 5 min, followed by 35 cycles of denaturing at 94°C for 1 min, 45s at the optimal annealing temperature of primers (Table I), and extending 72°C for 1.5 min, and an extra extension 72°C for 10 min. The products of PCR were electrophoresed on 8% denaturing polyacrylamide gels for 4-5 h at 12 W. The sizes of the alleles were estimated according to the 8 bp DNA ladder. Finally, the target bands were visualized by silver staining.

The expected and observed heterozygosity (HE and HO, respectively), allele number (NA), polymorphism information content (PIC) were analyzed using the CERVUS 3.0 (Kalinowski et al., 2007). Hardy-Weinberg equilibrium (HWE) and Tested for linkage disequilibrium (LD) were calculated by GENEPOP 4.0 (Rousset, 2008). All results for multiple tests were corrected using Bonferroni’s correction (Rice, 1989).

TA, annealing temperature; NA, observed number of alleles; HO, observed heterozygosity; HE, expected heterozygosity; PIC, polymorphic information content. *Significant deviation from HWE (P < 0.05).

A total of 13,471 simple sequence repeats were identified from 58,224 unigenes, which included 1693 di-nucleotide, 1761 tri-nucleotide, and 139 quad-nucleotide simple sequence repeats. 120 primers were designed base on repetition times and flaking sequence priority. 98 of the 120 primers were successfully amplified, but only 25 loci showed polymorphic (Table I). Number of alleles, observed and expected heterozygosity per locus ranged from 2 to 8, 0.083 to 0.922 and 0.042 to 0.866, respectively. The PIC ranged from 0.04 to 0.829. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P <0.05), but no significant linkage disequilibrium was found between all these loci.

The results of this study indicated that high throughput sequencing technology based on transcriptome was an effective method for developing microsatellite markers of S. japonica. These microsatellite markers will be helpful for further S. japonica germplasm and genetic composition evaluation.

We are grateful to Ping Hong-Ling and Zhang Tao for sample collection. This study is supported by National Natural Science Foundation of China (41676153; 41406138), the Science and Technology Project of Zhejiang province (2015F50055) and the Scientific Research Start-up Funds of Zhejiang Ocean University.

The authors declare no conflict of interest.