Development and Characterization of 35 SNP Markers in the Fat Greenling Hexagrammos otakii ()
1. Introduction
Hexagrammos otakii (Fat greenling), belonging to the family Hexagrammidae and genus Hexagrammos, is mainly distributed in the North Korea, Japan and China offshore [1]. H. otakii is an important kind of economic fish species in East Asia. H. otakii has high protein content (18.50%) and essential amino acid (7.25%), especially since the protein content is higher than most other fish [2]. Featuring meat quality, good taste, and high nutritional value, H. otakii is challenged by continuously growing market requirements and reduction of fishery resources [3]. Thus, the demand for hypervariable molecular markers to provide a population-genetic perspective on conservation and management efforts of the spices becomes urgent [4] [5].
With the rapid development of next-generation sequencing technologies (NGS) [6], single nucleotide polymorphisms (SNPs) have been largely developed and widely used for genetic studies in aquaculture species such as Sebastes schlegelii, Megalobrama terminalis and Coilia ectenes [7] [8] [9]. For H. otakii resource conservation, we implemented restriction-site associated DNA (RAD) sequencing to facilitate the genetic evaluation which could provide a reference for the development of SNP markers.
2. Materials and Methods
2.1. Materials
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.
2.2. Methods
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 [10]. 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 MgCl2, 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).
3. Results
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. The polymorphism information content (PIC) was calculated using Cervus 3.0 [11]. 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 ranged 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 significantly from the HWE (P < 0.05) (see Table 2 for details). These results will be useful for understanding the genetic diversity of H. otakii to assist in the management of this germplasm resource.
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Table 1. Summary information for the 35 SNP markers developed for the H. otakii.
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Table 2. The detailed information about the six SNPs found to be deviated significantly from the HWE (P < 0.05).
4. Conclusion
Wild H. otakii resources have declined sharply in recent years as a result of human disturbance and habitat destruction. In this study, 35 novel SNP markers were developed based on restriction-site associated DNA sequencing. The results showed that the Ho and He ranged from 0.1875 to 0.6562 and 0.2679 to 0.5079, respectively. The minor allele frequency ranged from 0.1875 to 0.4375. Polymorphic information content ranged from 0.229 to 0.375. Six 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 H. otakii.
Acknowledgements
This study was funded by the Agricultural Major Project of Liaoning Province (2020JH1/10200002) and China Agriculture Research System of MOF and MARA (CARS-47).
NOTES
*Corresponding author.