Identification of SNPs associated with disease resistance in juveniles of Sinonovacula constricta using RNA-seq and high-resolution melting analysis

Abstract

Sinonovacula constricta is one of the important economic bivalves in the world with high output value. However, vibriosis is a serious threat to the razor clam aquaculture industries, especially Vibrio parahaemolyticus. Molecular breeding such as marker-assisted selection or genomic selection is an efficient method for genetic improvement of disease resistance. In this study, the resistant and susceptible juvenile clams were evaluated according to the survival time after V. parahaemolyticus challenge and the two groups were genotyped by RNA sequencing technology to develop disease-resistant single nucleotide polymorphism (SNP) markers. The reference transcriptome was de novo assembled by Trinity, containing a total of 146,651 unigenes and 40,574 unigenes annotated. Almost 2 million SNPs were called from the two groups, of which, 23,970 SNPs showed significant allelic imbalance between groups (P valve <0.05). As shown by functional annotation, 396 SNPs were selected as significantly immune-related SNPs, which presented in immune-related genes participating in pathogen infection. It is interesting that significant SNPs in ABC transporters pathways were enriched, and some of SNPs located in ABC transporter genes were exclusive in the susceptible group indicating they maybe deleterious, which can be used as biomarker to distinguish disease-susceptible individuals. Six SNPs were further validated by High-resolution melting analysis in wild population, and five SNPs were verified successfully, which were correlated significantly with disease. These single nucleotide polymorphisms located in coding regions can be applied in the further studies elucidating the molecular mechanisms of immune responses. And the SNPs identified in this study could be used as molecular markers for molecular breeding of disease resistance in S. constricta.