Development and characterization of 56 SNP markers in Misgurnus anguillicaudatus

The dojo loach Misgurnus anguillicaudatus is an endemic freshwater species in Asia. The effective conservation and molecular-aided selection of M. anguillicaudatus were limited without sufficient molecular markers. In this study, 112 novel single nucleotide polymorphism (SNP) markers were screened based on 2b-RAD sequencing database, and 56 SNPs of them were developed and characterized by genotyping 40 individuals using SNaPshot method. The observed heterozygosity (Ho) ranged from 0.025 to 0.675, while the expected heterozygosity (He) varied from 0.025 to 0.500. The minor allele frequency (MAF) ranged from 0.013 to 0.500. Among these SNPs, 18 loci were found to deviate significantly from the Hardy–Weinberg equilibrium after Bonferroni correction (P < 0.05). The first set of SNP markers developed from M. anguillicaudatus will provide valuable information in further population genetic analysis and natural resource conservation.

The dojo loach Misgurnus anguillicaudatus, a member of the family Cobitidae, is an endemic freshwater species in Asia and widely distributed along the eastern coasts of the Asian continent (Li et al. 2008;Zeng et al. 2012). For delicious meat and traditional Chinese medicine values, M. anguillicaudatus has become one of the most important economic fish in several East Asian countries. In addition to their edible value, M. anguillicaudatus was regarded as an excellent animal model to explain the biological origin and evolutionary significance of polyploidization because of its complex ploidy composition (Zhou et al. 2014;Zhong et al. 2019). Despite its economic importance, M. anguillicaudatus for human consumption were mostly obtained through the exploitation of wild populations. In recent years, natural populations have declined dramatically because of over-exploitation and habitat destruction. Therefore, it is urgent to perform population genetic investigation on M. anguillicaudatus to protect the natural resources.
The research of genetic markers in M. anguillicaudatus has mainly concentrated on mitochondrial DNA and microsatellite markers at present Fujimoto et al. 2017;Lee et al. 2018). Owing to their wide genomic distribution and high level of polymorphism, single nucleotide polymorphism (SNP) markers have gradually become one of the most important DNA markers for genetic studies (Vignal et al. 2002;Seeb et al. 2011). Unfortunately, limited reports were available for M. anguillicaudatus on SNP. To better understand the genetic background and enrich the molecular marker, we firstly developed a set of SNP markers by SNaPshot technique in M. anguillicaudatus.
In this study, the constructed RAD libraries were sequenced on the Illumina Hiseq Xten platform using 350 bp paired-end reads (unpublished data). After quality filtering, 36,916 putative SNPs were identified, and 112 novel SNP loci were randomly selected for primer design using Primer 5.0 to test polymorphic. Forty caudal fin samples of diploid M. anguillicaudatus were randomly collected from wild populations in Qingyuan City, Guangdong Province, China (24°35′1.31″N, 113°15′86.66″E). Genomic DNA was extracted from tissue samples using the HiPure Mollusc DNA Mini Kit (Magen, China) according to the   Of the 112 potential SNP markers, 56 loci were found to be polymorphic and bi-allelic. The Ho and He varied from 0.025 to 0.675 (mean 0.271) and 0.025 to 0.500 (mean 0.345), respectively. The minor allele frequency ranged from 0.013 to 0.500 (mean 0.261). The PIC ranged from 0.024 to 0.375 (mean 0.274) (Table1). Among these SNPs, 18 loci showed significant deviations from the Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.05). These HWE deviations may be caused by natural selection, gene mutation, genetic drift, and presence of null alleles. To our knowledge, this is the first development of SNP markers in M. anguillicaudatus, and these novel markers offer a valuable tool for future studies of population conservation and natural resource conservation in this species.
Authors' contribution Conceived and designed the experiments: GC ML. Performed the experiments, analyzed the genetic data: GH. Contributed reagents/materials/analysis tools: FG ZL JC. Wrote the paper: GH ML. All authors read, revised, and approved the manuscript.
Funding This research was funded by China Agriculture Research System of MOF and MARA (CARS-46) and the National Key R&D Program of China (No. 2018YFD0901201). We thank Yaosen Qian who provided loach tissue samples for this research.

Data Availability
The data used to support the findings of this study are available from the article and its supplementary information.

Conflicts of interest
The authors declare no competing financial interests.
Ethical approval All experimental procedures for loach handling were approved by the Animal Care and Ethics Committee of the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences.