Characterization of multi-copy C3 genes and association analysis of C3.1 polymorphism with GCRV resistance traits in Ctenopharyngodon idella

Abstract

Molecular-assisted breeding plays an essential role in the selection of high disease -resistant fish in grass carp (Ctenopharyngodon idella) aquaculture. Complement 3 (C3) is a central molecule in the complement cascade and plays a polyfunctional role in immune defense, complement activation, and the inflammatory response. In this study, 9 copies of the C3 gene (named C3.1-C3.9) were identified in C. idella, and their structure, genomic location, phylogeny, expression patterns, and genetic characteristics were systematically analyzed. Phylogenetic analysis indicates that nine copies of grass carp C3 (hereafter called CiC3) cluster in the branch of Osteichthyes together with that of other fish, forming a different evolutionary branch distinct from other vertebrates. CiC3 are constitutively expressed in all examined tissues, among which CiC3.1 is the dominant C3 molecule. At 14 days of grass carp reovirus (GCRV) challenge, the CiC3s expression patterns share a similar expression trend. Furthermore, the comparison of the level of C3 protein, the mRNA expression level of dominant CiC3 (C3.1), and the mortality after GCRV challenge between the Xiangjiang River and Yangtze River grass carp indicate that the C3 protein exhibits distribution of polymorphism in a population, and the expression level of C3 is heritable and positively correlated with GCRV resistance. Furthermore, we identified 10 SNPs (named SNP1–10) of the C3.1 gene between GCRV-resistant and GCRV-susceptible grass carp groups. Inspiringly, SNP7 (g.76773C/T), SNP8 (g.76945 T/C), SNP1 (g.7065C/T), SNP9 (g.77153 A/C) and SNP10 (g.77252 T/C) have been verified to be significantly associated with the GCRV-resistant trait. Collectively, our findings provide potential molecular markers and relevant SNP sites for future grass carp breeding with respect to GCRV resistance.