Development and association analysis of SNP markers of nrlc3 gene in grass carp against Ichthyophthirius multifiliis based on HRM technology

Abstract:

To investigate the relationship between the polymorphism of the nlrc3 gene in grass carp （Ctenopharyngodon idella） and resistance to Ichthyophthirius multifiliis， susceptible and resistant groups were selected from grass carp populations infected with I. multifiliis. The single nucleotide polymorphism （SNP） sites of the nlrc3 gene in the two groups were analyzed and compared. High-resolution melting （HRM） and Sanger sequencing techniques were used to validate the genotypes of the SNP sites， and the obtained SNP sites were analyzed for their association with the disease resistance traits in grass carp. The results showed that four SNPs sites were identified from the nlrc3 gene in grass carp， and three SNP sites （SNP1， SNP3， and SNP4） were successfully genotyped. Among them， SNP1 and SNP3 exhibited two genotypes each （AA and AG for SNP1， AA and GG for SNP3） and were significantly associated with resistance to I. multifiliis. The analysis of haplotype combinations and their correlation with disease resistance revealed that the genotype composed of SNP1 AG and SNP3 AA （D1） showed 100% disease resistance， while the genotype composed of SNP1 AG and SNP3 GG （D2） showed 95% disease resistance， both representing advantageous genotypes for disease resistance. The study demonstrated the existence of nlrc3 gene polymorphism in the entire population of I. multifiliis resistant grass carp， and different genotypes were significantly correlated with the ability to resist I. multifiliis infection. Among them， the D1 genotype with SNP1 AG and SNP3 AA combination exhibited the strongest resistance. These findings provide important theoretical and technical support for further breeding and prevention of I. multifiliis disease in grass carp.