Isolation and Characterization of 19 Polymorphic Microsatellite Markers of Sepia esculenta

Twenty-four individuals of S. esculenta were collected in October from the coastal waters near Jiaonan, China. The samples were preserved at -20°C until DNA extraction. One of them was send to Novogene Biotech Inc., for high-throughput DNA sequencing using 454 Genome Sequencer FLX platform (Roche). Thousands of DNA sequences which contained the short tandem repeat fragments were subsequently obtained and PRIMER 5 software was used to analyze these fragments. Eventually, 80 perfect microsatellite repeats were picked up to test polymorphism among S. esculenta individuals.

The designed primers were evaluated using 24 individuals of S. esculenta. PCR was performed on a Veriti Thermal Cycler (Applied Biosystems, USA) in a total volume of 25 μl containing 0.4 μM of eachprimer, 0.2 mM of each dNTP, 1×PCR buffer, 2 mM of MgCl2, 1 unit Taq polymerase (TaKaRa, Japan) and 10-100 ng DNA. The amplification profile included an initial denaturing at 94°C for 5 min, 35 cycles of 45 s at 94°C, at the locus-specific annealing temperature for 1 min, and 72°C for 45 s, and final period at 72°C for 10 min. The PCR products were separated on 6% denaturing polyacrylamide gel, and visualized by silver staining.

The observed and expected heteozygosities together with tests for Hardy-Weinberg disequilibrium were calculated by GENEPOP 4.0 (Rousset, 2007).

Ta, optimized annealing temperature; NA, number of alleles; HO, observed heterozygosity; HE, expected heterozygosity. *locus may harbor null alleles (null allele frequency > 5%). †locus deviated from Hardy-Weinberg proportions (adjusted P-value < 0.0021).

Null allele frequencies (Brookfield, 1996) were calculated by MICRO-CHECKER 2.2.3 (van Oosterhout et al., 2004). All results for multiple tests were corrected using Bonferroni’s correction (Rice, 1989).

Among 80 primer pairs tested, 68 amplified single PCR products of expected size, and the others had either no products or smear only. Only 19 microsatellite markers showed polymorphism. The number of alleles, observed and expected heterozygosity per locus ranged from 6 to 10, from 0.292 to 0.861 and from 0.260 to 0.904, with an average of 7.300, 0.670 and 0.741, respectively. The polymorphism information content (PIC) ranged from 0.566 to 0.823. Four loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P <0.0021), which may be due to the small sample size or the presence of null alleles confirmed by MICRO-CHECKER (Table I), but no evidence for stuttering and allelic dropout were found in all loci. Three loci showed evidences of null alleles (null allele frequency > 0.05). No significant genotypic linkage disequilibrium (LD) was found among all pairs of the 19 loci after Bonferroni’s correction (P>0.0021).

We are very grateful to Mr. Binbin Shan for collecting samples. This study was supported by National Natural Science Foundation of China (41676153; 41406138), National Infrastructure of Fishery Germplasm Resource and Key Laboratory for Exploitation & Utilization of Marine Fisheries Resource in South China Sea, Ministry of Agriculture (No. LSF2014-02).

Authors have declared no conflict of interest.