Development and Characterization of 35 SNP Markers in the Fat Greenling Hexagrammos Otakii

Xianggang Gao Liaoning Ocean and Fisheries Science Institute Weidong Liu Liaoning Ocean and Fisheries Science Institute Zhongying Liu Liaoning Ocean and Fisheries Science Institute Wei Sun Liaoning Ocean Fisheries Science Institute Yulong Li Liaoning Ocean and Fisheries Science Institute Yunfeng Li Liaoning Ocean and Fisheries Science Institute Xiangbo Bao (  bobo_1325@126.com ) Liaoning Ocean and Fisheries Science Institute


Abstract
Hexagrammos otakii is an important kind of economic sh species in East Asia. However, wild H. otakii 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 H. otakii. In this study, 35 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 0.1875 to 0.6562 and 0.2679 to 0.5079, respectively. The minor allele frequency raged from 0.1875 to 0.4375. Polymorphic information content ranged from 0.229 to 0.375. Six SNPs were found to be deviated signi cantly 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 H. otakii.

Full Text
Hexagrammos otakii (Fat greenling), belonging to family Hexagrammidae and genus Hexagrammos, mainly distributed in the North Korean, Japan and China offshore (Nishikawa 1982). H. otakii is an important kind of economic sh species in East Asia. H. otakii has high protein content (18.50%) and essential amino-acid (7.25%), especially the protein content is higher than most other sh (Kang and Wu 1999). Featuring meat quality, good taste, high nutritional value, H. otakii is challenged by continuously growing market requirement and reduction of shery resources (Shen et al. 2019). Thus, demand for hypervariable molecular markers to provide a population-genetic perspective on conservation and management efforts of the spices (FAO 1993) becomes urgent.
With the rapid development of next-generation sequencing technologies (NGS) ( In this study, a total of 32 H. otakii wild individuals were collected from northern Yellow Sea in China. 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. All samples were used to construct 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, 707,105 putative SNPs with the highest scores were generated, from which we randomly selected 100 candidate SNPs to test their applicability. The polymorphism of these candidate SNPs was further characterized in the 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, 250 nM of each primer, and 1U of Taq polymerase (Takara, Dalian, China). The 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 using Cervus 3.0 (Kalinowski et al. 2007). Among the test SNP markers in H. otakii, 35 polymorphic SNP markers were characterized in Table 1. The Ho and He were ranged from 0.1875 to 0.6562 and 0.2679 to 0.5079, respectively. The MAF raged from 0.1875 to 0.4375. The PIC varied from 0.229 to 0.375, with an average of 0.3293. Six SNPs were found to be deviated signi cantly from the HWE (P < 0.05). These results will be useful for understanding the genetic diversity of H. otakii to assist in the management of this germplasm resource. Availability of data The sequence data used for SNP discovery belongs to Liaoning Ocean and Fisheries Science Institute, and it can be available upon reasonable request.
Con ict of interest The authors declare that they have no con ict 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.