南方农业学报2025，Vol.56Issue(6)：1691-1703,13.DOI:10.3969/j.issn.2095-1191.2025.06.001
基于RAD-seq技术的俄罗斯鲟SNP分子标记开发
Development of SNP molecular markers for Russian sturgeon(Acipenser gueldenstaedti)based on RAD-seq technology
摘要
Abstract
[Objective]To develop potential single nucleotide polymorphism(SNP)molecular markers for the Rus-sian 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 map-ping analysis.The identified SNPs were validated using SNaPshot and Sanger sequencing.[Result]From 5 Acipenser guel-denstaedti 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 num-ber 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 al-lele frequency(MAF)ranging from 0.0147 to 0.5000,observed heterozygosity(HO)ranged from 0.029 to 0.971,and ex-pected heterozygosity(HE)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(PHWE>0.05).[Conclusion]De-veloping 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.关键词
俄罗斯鲟/SNP分子标记/遗传多样性/RAD-seqKey words
Acipenser gueldenstaedti/SNP marker/genetic diversity/RAD-seq分类
农业科技引用本文复制引用
陈叶雨,赖见生,罗晓含,吴晓雲,宋明江,龚全..基于RAD-seq技术的俄罗斯鲟SNP分子标记开发[J].南方农业学报,2025,56(6):1691-1703,13.基金项目
四川省科技计划项目(2025ZNSFSC0276) （2025ZNSFSC0276）
国家现代农业产业技术体系四川淡水鱼创新团队项目(SCCXTD-2025-15) （SCCXTD-2025-15）
雅安市创建国家现代农业产业科技创新中心"揭榜挂帅"项目(kczx2023-2025-24,kczx2023-2025-25) Sichuan Science and Technology Plan Project(2025ZNSFSC0276) （kczx2023-2025-24,kczx2023-2025-25）
Sichuan Fresh Water Fish In-novation Team Project of China Agriculture Research System(SCCXTD-2025-15) （SCCXTD-2025-15）
"Leading the Charge with Open Com-petition"Project for Ya'an to Establish National Modern Agricultural Industry Science and Technology Innovation Center(kczx2023-2025-24,kczx2023-2025-25) （kczx2023-2025-24,kczx2023-2025-25）
