Large-scale SNP screenings identify markers linked with GCRV resistant traits through transcriptomes of individuals and cell lines in Ctenopharyngodon idella

Zhiwei Liao,Jianguo Su,Xueying Shang,Quanyuan Wan

doi:10.1038/s41598-017-01338-7

Abstract

Grass carp (Ctenopharyngodon idella) is an important economic species in freshwater aquaculture and its industry has been confined due to variety degeneration and frequent diseases. Marker-assisted selection is a feasible method for selective breeding of new varieties. Transcriptome data have greatly facilitated high-throughput single nucleotide polymorphism (SNP) marker discovery and phenotype association study. In this study, we gained a total of 25,981 and 5,775 high quality SNPs in two transcriptomes from individuals and cell lines, respectively. Comparative transcriptome analysis identified 413 and 832 grass carp reovirus (GCRV)-resistant-association SNPs as well as 1,381 and 1,606 GCRV-susceptible-association SNPs in individuals and cell lines, respectively. Integrated analysis indicated 22 genes with single SNP share common resistant/susceptible traits in two transcriptomes. Furthermore, we infected grass carp with GCRV, genotyping and association analyses were performed, and 9 in 22 SNPs were confirmed by PCR-RFLP. Meanwhile, mRNA expression profiles of 6 genes containing confirmed SNPs were examined by qRT-PCR. The results demonstrated that mRNA expressions were significant differences in resistant/susceptible individuals and cell lines. The present study develops an important strategy for high throughput screening of phenotype association genetic markers and the results will serve in grass carp breeding for GCRV resistance.

Highlights

Grass carp (Ctenopharyngodon idella) is an important economic freshwater fish in China and accounted for 15.6% of global freshwater aquaculture production in 20111
Two transcriptomes were integrated to look for their communal variation locus to improve the single nucleotide polymorphism (SNP) accuracy, the candidate SNPs were confirmed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) experiments
Taking into consideration the identical SNPs in C1, R2 and S3 libraries, it showed a total of 5,775 SNPs in cell lines (Fig. 1E and F)

Summary

Introduction

Grass carp (Ctenopharyngodon idella) is an important economic freshwater fish in China and accounted for 15.6% of global freshwater aquaculture production in 20111. Massive SNPs have been identified by transcriptome sequencing in aquaculture species such as catfish (Clarias gariepinus), Atlantic cod (Gadus morhua), oyster (Crassostrea gigas), shrimp (Litopenaeus vannamei), half-smooth tongue sole (Cynoglossus semilaevis), Atlantic herring (Clupea harengus), Atlantic salmon (Salmo salar), silver carp (Hypophthalmichthys molitrix) and C. carpio[16] These data supply a large amount of genetic information related to development and disease resistance in aquaculture species. MRNA expression profiles of genes corresponding to confirmed SNPs were examined by quantitative real-time RT-PCR (qRT-PCR) as well as transcriptome data analysis in resistant and susceptible groups These results provide precious resources for molecular and genetic breeding as well as immune researches

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