Screening of microsatellite loci and analysis of polymorphism in <i>Triplophysa hsutschouensis</i> based on RAD sequencing

To assess the genetic diversity of Triplophysa hsutschouensis population in the Shule River basin, we employed restriction site-associated DNA sequencing (RAD-Seq) to sequence T. hsutschouensis genome. Using this technology, we identified suitable microsatellite (SSR) markers specific to T. hsutschouensis population and synthesized 67 primer pairs, of which 18 were found to be polymorphic. Our analysis of 46 T. hsutschouensis individuals revealed that the number of alleles (Na) ranged from 2 to 16, with the effective number of alleles (Ne) varying between 1.293 and 8.672. Observed heterozygosity (Ho) ranged from 0.261 to 0.957, expected heterozygosity (He) from 0.227 to 0.885, and polymorphism information content (PIC) from 0.201 to 0.874. The average values obtained were Na = 7.056, Ne = 3.540, Ho = 0.609, He = 0.639, and PIC = 0.595. Hardy-Weinberg equilibrium testing showed that five of the 18 polymorphic SSR loci deviated significantly from expectations after Bonferroni correction, while the remaining 13 loci conformed to expectations, indicating no significant genetic disturbances in the population. These newly identified polymorphic SSR markers will be valuable for future genetic diversity analysis and conservation efforts regarding T. hsutschouensis populations.