The Japanese ghost shrimp Neotrypaea japonica (Ortmann 1891), belongs to the group of Crustaceas (Kim et al., 2013). N. japonica is a dominant species in the macrobenthos community, which occupies important position of burrowing thalassinideans in soft-sediment environments (Mukai and Koike 1984; Posey et al. 1991; Dworschak 2000).The species quantity is facing a wide range of major threats, including environmental destruction, pollution, hydrological change and so on (Felder 2001). In addition, the species is not well managed and protected due to the lack of molecular marker research which is essential for protecting biodiversity and maintaining ecological balance. Thus, conducting genetic investigation on the population of N.japonica is an urgent need to make better use of natural resources and to protect biodiversity.
Single nucleotide polymorphisms (SNPs), as a third-generation molecular marker, are mainly used to analyze DNA sequence polymorphisms caused by mutations at the genomic nucleotide level (Kaikai et al., 2018). Moreover, SNPs have been widely used in genetics, as they can act as biological markers, helping scientists locate genes associated with certain characteristics, conditions, or diseases (Vignal et al., 2002). Here, SNPs were developed and characterized in N. japonica with the restriction-site associated DNA tags sequencing (RAD-seq), which enriched genetic marker resources for conservation genetics study (Qu et al., 2019).
Samples used in this study were collected from Yantai coast, Shandong province (37°27′N/121°30′E). Genomic DNA was extracted from tissues of 30 randomly selected N. japonica according to the manufacturer's instructions. For Illumina sequencing, RAD libraries with the paired-end short-insert of 150 bp were generated, and sample indexing, and pooling were carried out as previously described (Baird, etc. 2008). To obtain SNP marker resources, the libraries were sequenced using Illumina HiSeq4000 sequencing platform (Shanghai BIOZERON Co., Ltd.) with the total flux of one lane. Totally, 719,660 putative SNPs of N. japonica were identified in this research.
To verify the SNPs recognized by Illumina sequencing, Sanger sequencing was conducted. Primers used were designed using Primer v3.0. The PCR was performed in a total volume of 25 μL containing 12.5 μL 2 × PCR Mix (GenStar, Beijing, China), 3.5 μL cDNA template, 1 μL forward primers, 1 μL reverse primers and 7 μL ddH2O. The PCR amplification cycle was as follows: initial denaturation for 5 minutes at 94℃, followed by 40 cycles at 94℃ for 30 seconds, annealing for 30 seconds (annealing temperature of each primer pair, see table 1), 72℃ for 30 seconds, final extension of 7 minutes at 72℃. Amplified PCR products were purified by gel extraction and subjected to sequence on ABI 3730 DNA analyzer (Applied Biosystems).
Summary statistics were calculated, which include the minor allele frequency (MAF), observed heterozygosity (HO), expected heterozygosity (HE), polymorphic information content (PIC), inbreeding coefficient (FIS), and P-values representing the deviations from the Hardiy-Weinberg equilibrium (PHWE) (Kalinowski et al., 2007).123 SNP sequences were further confirmed by Sanger sequencing. The MAF ranged from 0.2500 to 0.4833. The HO, and HE ranged from 0.4333 to 0.9000 and 0.4944 to 0.6503 (Table 1), respectively. PIC was between 0.3680 and 0.5651. FIS values varied from -0.2768 to 0.1045. After Bonferroni correction, significant departures (P < 0.05) from Hardiy-Weinberg equilibrium were observed for 16 loci. The newly developed SNP markers play a significant role in population genetic analysis, conservation of natural resources, and directional breeding.