Isolation and characterization of 45 SNP markers in Triplophysa tenuis

Triplophysa tenuis is an endemic species of ray finned fish native to China, which is mainly distributed in Xinjiang and Gansu Province. Effective conservation and management of this species is limited by insufficient availability of molecular markers. In the present study, we report the isolation and characterization of 45 SNP markers in T. tenuis. The minor allele frequency ranged from 0.046 to 0.500, and the observed and expected heterozygosity ranged from 0.061 to 0.667 and 0.088 to 0.508, respectively. Polymorphic information content ranged from 0.083 to 0.375. Among these SNPs, three loci showed significant departures from the Hardy–Weinberg equilibrium. The novel polymorphic SNPs will be helpful for the future study on genetic management and population conservation for this species.

Triplophysa tenuis is distributed both in the Bosten lake and Tarim river systems in the south of Xinjiang and in the Shule river and Ruoshui river in the Hexi corridor of Gansu Province (Zhu 1989). Although T. tenuis has not yet been classified as an endangered species, the fish species in Gansu Province, especially the endemic fish species, show an obvious decreasing trend (Wang et al. 2015b). At present, the reports on T. tenuis mainly focus on biological characteristics (Chen et al. 2017;Yao et al. 2018). There are very few reports about molecular markers in this species. Genetic markers that are currently available for T. tenuis are mostly based on mitochondrial DNA (Wang et al. 2015b;Wang et al. 2016). Single-nucleotide polymorphism (SNP) markers are useful in population genetic studies because of their co-dominance, high levels of polymorphism, low cost and wide distribution (Vignal et al. 2002;Wang et al. 2015a;Blanc-Jolivet et al. 2017). In the present study, we developed and characterized 45 SNP markers in T. tenuis. It is expected that these results will contribute to population conservation, genetic management and the construction of genetic linkage maps for this species.
In this study, we collected six T. tenuis from Shule river and used their fin chips for restriction-site associated DNA sequencing (RAD-seq) to isolate and characterize SNP markers. 146,960 SNP loci were generated by RAD-seq. A total of eighty potential SNP loci were selected for primer design using Primer 5.0 (Lalitha 2000). Fin clips of T. tenuis were collected from 33 individuals of the Shule river (Gansu, China). Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method (Doyle 1987) and was stored at − 20 °C.
PCR amplifications were performed in 30 µL volumes containing 1 µL of genomic DNA, 14 µL of Premix Taq (2 × Taq Plus MasterMix, CWBIO), 1 µL of each gene primer and 13 µL of PCR-grade water. The PCR programme was 94 °C for 5 min, then 32 cycles at 94 °C for 30 s, annealing for 30 s (for annealing temperatures of each primer pair, Table 1), 72 °C for 45 s, and one cycle of 72 °C for 7 min for the final extension. Amplification products were sequenced using Sanger technology. The genotypes per locus were determined by BioEdit (Alzohairy 2011). The minor allele frequency (MAF), observed heterozygosities (Ho), expected heterozygosities (He), polymorphism information content (PIC) and P value representing the deviations from Hardy-Weinberg equilibrium (HWE) were calculated using Cervus 3.0 (Kalinowski et al. 2007).
A total of 45 loci were found to be polymorphic and bi-allelic in the 33 individuals of T. tenuis (Table 1

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Supplemental Table 1). The minor allele frequency ranged from 0.046 to 0.500. The observed heterozygosity varied from 0.061 to 0.667, while the expected heterozygosity ranged from 0.088 to 0.508. Polymorphic information content ranged from 0.083 to 0.375. Only three loci showed significant deviations from the HWE. As far as we know, this is the first report of SNP identification in T. tenuis, which will be a valuable tool for population conservation in this species.