Isolation and Characterization of New 24 Microsatellite DNA Markers for Golden Cuttlefish (Sepia esculenta)

Twenty-four microsatellite DNA markers were isolated and characterized for golden cuttlefish (Sepia esculenta) from a (GT)13—enriched genomic library. Loci were tested in 48 individuals from Jiaozhou bay of China. The numbers of alleles per locus ranged from two to 25 with an average of 10.3. The observed and expected heterozygosities ranged from 0.063 to 0.896 and from 0.137 to 0.953, with averages of 0.519 and 0.633, respectively. Six loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni’s correction and no significant linkage disequilibrium between loci pairs was detected. These microsatellite markers would be useful for analyzing the population genetic structure to make conservation and management decisions for S. esculenta.


Introduction
Golden cuttlefish (Sepia esculenta) is a cephalopod that lives in neritic and abyssal zones, and sometimes in sandy habitats. It is widely distributed, from the island of Honshu in Japan to Vietnam and the Philippines. The golden cuttlefish is an economically important target species in China, South Korea and Japan [1][2][3]. Unfortunately, since the 1980s, S. esculenta resources have been gradually OPEN ACCESS declining possibly due to over-exploitation and ocean environmental change, especially deterioration of the spawning grounds [3]. With respect to the four traditional main spawning grounds of S. esculenta along the coast of China, only one remains [4].
Estimating population genetic structure may lead to a better understanding of the effects of over-exploitation and environmental change on S. esculenta stocks, and the result would provide valid reference for the management and conservation of fishery resources [5][6][7][8]. Among molecular markers, microsatellite markers have been shown to be an extremely valuable tool for the study of population structure because of their variability, abundance, neutrality, co-dominance and unambiguous scoring of alleles [9,10]. Until now, 31 microsatellite markers have been reported for S. esculenta [6,8]. However, only 25 of these conformed to the Hardy-Weinberg equilibrium (HWE). Therefore, additional highly informative microsatellite markers are needed for investigating the population genetic structure.

Results and Discussion
The number of alleles per locus ranged from two to 25, with an average of 10.3. The observed and expected heterozygosities ranged from 0.0625 to 0.8958 and from 0.1366 to 0.9533, with an average of 0.519 and 0.633, respectively (Table 1). Six loci (J5/J8/J17/J61/J63/J74) deviated from HWE in the tested population after Bonferroni's correction (adjusted P-value < 0.002), which might be caused by the heterozygotes deficit. There are three possible explanations for the heterozygotes deficit. Firstly, it might be due to allelic "dropouts", which are artifacts in the PCR amplification process. Secondly, the deficit might be caused by the limited sample size or size homoplasy. Thirdly, it could result from the presence of null alleles [11,12]. Twenty four microsatellite loci were tested for the presence of null alleles and the results showed that they might be present in seven loci (J5/J8/J10/J17/J60/J63/J74), five of which deviated from HWE. According to Zheng' report, a significant deviation from HWE was observed in one locus, and null allele existed in the same locus [6]. Wang also reported that, significant occurrences of null alleles were found for four of five loci that deviated from HWE [8]. No significant linkage disequilibrium (LD) was found between all pairs of these 24 loci after Bonferroni's correction (P-value > 0.002).

DNA Extraction
Forty-eight individuals of S. esculenta were collected from Jiaozhou bay and preserved in alcohol until DNA extraction. DNA was extracted from muscle tissue using the phenol-chloroform procedure [13].

Microsatellite-Enriched Library Construction
Genomic DNA was simultaneously digested with Mse I for three hours (New England Biolabs, USA), and the digested DNA (10 µL) was ligated to Mse I adaptors (100 pmol) (5'-TACTCAGGAACTCAT-3'/5'-GACGATGAGTCCTGAG-3'). Linker-ligated DNA was amplified in a 25 µL reaction mix using the adapter-specific primer (5'-GATGAGTCCTGAGTAA-3'). Polymerase chain reaction (PCR) conditions were as follows: 20 cycles at 94 °C for 30 s, 53 °C for 1 min, 72 °C for 1 min. The PCR products were purified using DNAmate (TaKaRa, Japan) and hybridized to a biotin labeled (GT) 13 probe. The mixture was denatured at 94 °C for 5 min, then at 53 °C for 15 min. The hybrids were captured with streptavidin-coated magnetic beads (Promega, USA). Unhybridized DNA was washed away, and the remaining DNA was eluted from the magnetic beads and amplified using the adaptor-specific primer and the above PCR program. Following purification, DNA fragments ranging from 500 base pair to 1000 bp were selected by separation on 1.5% agarose gels. The fragments were ligated to pMD18-T vectors (TaKaRa), and transformed into Escherichia coli DH5α competent cells to construct an enriched microsatellite library. After amplifying with (GT) 10 and M13 primers,180 positive clones were obtained. The positive clones were sequenced on an ABI 3730 automated DNA sequencer (Applied Biosystems, USA).

PCR Amplification and Genotyping
Eighty-five pairs of primers were designed using PRIMER PREMIER5 (Premier Biosoft International, USA) and tested for polymorphism with six S. esculenta. After preliminary screening, only 24 polymorphic microsatellite loci were tested on a sample of 48 individuals. PCR for all loci was performed separately in a 25 µL reaction volume containing 0.4 µM of each primer, 0.2 mM dNTPs, 2 mM MgCl 2 , 1× PCR buffer, 1 U Taq polymerase (Fermentas, Canada) and 50-100 ng DNA. Amplification was carried out with the following thermal profile: 94 °C for 5 min, followed by 35 cycles of 94 °C for 45 s, optimal annealing temperature ( Table 1) for 45 s, and 72 °C for 45 s, and a final extension step at 72 °C for 10 min. PCR products were separated on 6% denaturing polyacrylamide gels and visualized by silver-staining.

Genetic Data Analysis
Allele sizes were estimated according to the pBR322/Msp I marker. The variability at each locus was measured in terms of number of alleles, expected heterozygosity and observed heterozygosity, and Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium were tested using GENEPOP 4.0 [14].
Null allele frequencies were calculated using Micro-Checker 2.2.3 [15]. The significant value for all diversity tests of significance was corrected by the sequential Bonferroni's procedure [16].

Conclusions
In the present study, we isolated and characterized 24 polymorphic microsatellite loci for S. esculenta. These new high variable microsatellite markers will enrich S. esculenta microsatellite marker resources and be useful for various population genetic analyses of S. esculenta. In our results, there are some highly heterozygous microsatellite loci (i.e., J13/J14/J35/J77), which we will use for designing conservation strategies for the management of S. esculenta stocks.