Isolation and characterization of 38 SNP markers for the black rockfish, Sebastes schlegelii by next-generation sequencing

The black rockfish (Sebastes schlegelii) is an economically important species. However, wild S. schlegelii resources have declined sharply in recent years as a result of human disturbance and habitat destruction. Thus, it is crucial to protect the current resources of S. schlegelii. In this study, 38 novel single nucleotide polymorphism (SNP) markers were developed based on restriction-site associated DNA sequencing. The results showed that the observed heterozygosity and expected heterozygosity ranged from 0.1400 to 0.6400 and 0.1487 to 0.4978, respectively. The minor allele frequency raged from 0.1429 to 0.4694. Polymorphic information content ranged from 0.174 to 0.365. Four SNPs were found to be deviated significantly from the HWE (P < 0.05). These SNP markers will serve as a useful tool for genetic studies and population evaluation aimed at the conservation of S. schlegelii.

The black rockfish (Sebastes schlegelii) is an important commercial fish distributed in Northwest Pacific enjoying high popularity in China, Korea and Japan (Yoshida et al. 2005). In recent decades, commercial exploitation and environmental changes have caused a decline in its population. Thus, demand for hypervariable molecular markers to provide a population-genetic perspective on conservation and management efforts of the species (FAO 1993) becomes urgent.
With the rapid development of next-generation sequencing technologies (NGS) (Davey et al. 2011), single nucleotide polymorphisms (SNPs) have been largely developed and widely used for genetic studies in aquaculture species such as Megalobrama terminalis (Yang et al. 2020), Coilia ectenes (Yu et al. 2019) and Ochetobius elongatus (Yang et al. 2018). For S. schlegelii resource conservation, we implemented restriction-site associated DNA (RAD) sequencing to facilitate the genetic evaluation.
In this study, a total of 50 S. schlegelii wild individuals were collected from Dandong in Liaoning province (39°86′N/124°14′E) in April 2020. Muscle tissues were sampled and stored in 95% molecular grade ethanol. Total genomic DNA was extracted from tissue samples using the TIANamp Marine Animals DNA Kit (Tiangen, Beijing, China) following the manufacturer's instructions. 20 samples were used to constructed the RAD libraries. Then, the libraries were sequenced on the Illumina HiSeq 4000 platform using 150 base pair (bp) paired-end reads. We trimmed the adapter sequences and low-quality reads (Phred score < 20) with Cutadapt (Martin 2011). Finally, 3,014,591 putative SNPs with the highest scores were generated, from which we randomly selected 90 candidate SNPs to test their applicability. The polymorphism of these candidate SNPs was further characterized in the remaining 30 samples mentioned above. Primer sequences for SNP loci were designed by Primer 5.0 software. The PCR reactions were conducted in 25 µL volume containing 50 ng of genomic DNA, 1 × PCR buffer, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 200 nM of each primer, and 1U of Taq polymerase (Takara, Dalian, China). Amplicons were checked by 1.0% agarose gel electrophoresis and sequenced on ABI 3730 DNA Analyzer (Applied Biosystems).
The observed heterozygosity (Ho), expected heterozygosity (He), minor allele frequency (MAF), and P value representing the deviations from the Hardy-Weinberg equilibrium were estimated using POPGENE 32 (Yeh et al. 2000). The polymorphism information content (PIC) was calculated These results will be useful for understanding the genetic diversity of S. schlegelii to assist in the management of this germplasm resource.
Authors contribution GXG. conceived and designed the study, contributed to the drafted the manuscript. BXB. contributed to the analysis and writing. SW participated in the data collection, purification, and management of the samples for sequencing and genotyping. LWD contributed to the design of the study and writing. LYF and LZY contributed to the collection of the samples. All authors have reviewed and approved the manuscript.

Data availability
The sequence data used for SNP discovery belongs to Liaoning Ocean and Fisheries Science Institute, and it can be available upon reasonable request.

Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval Investigations were conducted according to the guiding principles for the use and care of laboratory animals and in compliance with the Academic Ethics Committee of Liaoning Ocean and Fisheries Science Institute. Our study had been submitted to and approved by the Academic Ethics Committee of Liaoning Ocean and Fisheries Science Institute. The research permits also included the necessary ethical approval in terms of sample collection. All sample collection was undertaken in accordance with relevant Academic Ethics Committee of Liaoning Ocean and Fisheries Science Institute guidelines and regulations.