Development of SNP molecular markers for Russian sturgeon（Acipenser gueldenstaedti） based on RAD-seq technology

【Objective】 To develop potential single nucleotide polymorphism（SNP） molecular markers for the Russian sturgeon（Acipenser gueldenstaedti） using restriction site-associated DNA sequencing（RAD-seq） and evaluate the genetic diversity of artificially farmed populations, which could provide technical support for in-depth research on the conservation genetics of sturgeon species. 【Method】 DNA was extracted from fins of Acipenser gueldenstaedti using the CTAB method. DNA libraries were constructed, and RAD-seq was performed on the Illumina NovaSeq 6000 platform. Potential SNP loci were identified through a series of steps, including quality control, filter, cluster assembly and mapping analysis. The identified SNPs were validated using SNaPshot and Sanger sequencing. 【Result】 From 5 Acipenser gueldenstaedti samples, a total of 14.24 Gb of raw base was generated via RAD-seq. Each sample had an average clean base of 2.84 Gb, with a clean base rate of 99.54% and Q20 values exceeding 96.00%. The GC content ranged from 40.52% to 41.36%. The total length of assembled data was 53211245 bp, with an average contig length of 343 bp and N50 of 404 bp. The alignment rate of reads to the assembled genome ranged from 59.89% to 62.43%, with an average sequencing depth of 15.75 to 23.84 times. RAD-seq identified 299416-367709 SNP loci across the 5 samples, with SNP transition loci number of 183070-224458 and SNP transversion loci of 116346-143251. The average transition/transversion ratio（Ts/Tv） was 1.57. The heterozygosity rate of SNP loci ranged from 82.81% to 97.43%, with mutation types classified into 6 categories（T/C, T/A, T/G, C/G, C/A, A/G）, among which T/C or A/G occurred most frequently. Based on Sanger sequencing, 36 polymorphic SNPs loci were screened from 100 randomly selected potential SNPs loci. Among the mutation types, A/G had the highest frequency（44.4%）, followed by T/C（36.1%）. The frequencies of A/C, C/G and G/T were relatively low, at 8.3%, 8.3% and 2.7% respectively. A total of 72 alleles were detected at the 36 SNPs loci, with the minimum allele frequency（MAF） ranging from 0.0147 to 0.5000, observed heterozygosity（H_O） ranged from 0.029 to 0.971, and expected heterozygosity（H_E） ranged from 0.029 to 0.523. Except for 4 SNPs loci（Ag18, Ag33, Ag35 and Ag36）, which exhibited low polymorphism（PIC≤0.25）, the remaining 32 SNPs loci were moderately polymorphic（0.25＜PIC≤0.50）, indicating that the genetic diversity of artificially farmed Acipenser gueldenstaedti in southwestern China was relatively low. Hardy-Weinberg equilibrium testing revealed that 19 SNPs loci were in equilibrium(P_HWE＞0.05). 【Conclusion】 Developing SNP molecular markers for Acipenser gueldenstaedti using RAD-seq is a practical and feasible approach. These markers exhibit high universality, large quantity, and wide coverage, providing valuable technical support for population genetics studies, genetic linkage map construction and molecular-assisted breeding programs for Acipenser gueldenstaedti.