基于KASP技术的大口黑鲈抗蛙虹彩病毒SNP分子标记开发与验证OA

Largemouth bass ranavirus(LMBV)has emerged as a devastating pathogen that poses a severe threat to the global largemouth bass(Micropterus salmoides)aquaculture industry.In this context,identifying key genetic loci underlying disease resistance and developing highly resistant selective breeding populations have become critical strategies to ensure the sustainable development of the industry.To screen for molecular markers significantly associated with LMBV resistance in largemouth bass,a total of 3600 individuals from the elite"Youlu No.3"strain were subjected to standardized LMBV challenge via intraperitoneal injection,from which distinct disease-resistant and susceptible subpopulations were successfully established.Fifty representative samples were randomly selected from each of the two subpopulations for whole-genome resequencing.Using the reference genome assembly NW_024044237.1,a genome-wide association study(GWAS)was systematically performed,and a total of 2348 variant loci that showed statistically significant associations with the LMBV resistance trait were identified.On the basis of these GWAS results,the most significantly associated single nucleotide polymorphism(SNP)locus,designated G38928188(G/T),was selected to develop a low-cost,high-throughput genotyping assay using kompetitive allele-specific PCR(KASP)technology.This newly developed assay can accurately distinguish three genotypes at the target locus:GG(resistant homozygote),GT(heterozygote),and TT(susceptible homozygote),with a verified genotyping accuracy of 100%.Furthermore,this KASP method was applied to genotype largemouth bass populations across three successive selective breeding generations(F1,F2,and F3).The results showed that the frequency of the resistant GG genotype increased progressively across generations,reaching 22.7%,54.1%,and 100%in F1,F2,and F3,respectively.The KASP genotyping assay established in this study is highly accurate,robust,and suitable for large-scale population screening,providing a reliable and effective tool for marker-assisted selection(MAS)of LMBV-resistant largemouth bass.This research carries important theoretical significance and practical application value for accelerating the selective breeding process of disease-resistant largemouth bass varieties and promoting the healthy and high-quality development of the largemouth bass aquaculture industry.