Selection of functional EPHB2 genotypes from ENU mutated grass carp treated with GCRV

Meher Un Nissa,Zhu-Xiang Jiang,Shu-Ming Zou,Guo-Dong Zheng

doi:10.1186/s12864-021-07858-x

Abstract

BackgroundN-ethyl-N-nitrosourea (ENU) mutagenesis is a useful method for the genetic engineering of plants, and the production of functional mutants in animal models including mice and zebrafish. Grass carp reovirus (GCRV) is a haemorrhagic disease of grass carp which has caused noteworthy losses in fingerlings over the last few years. To overcome this problem, we used ENU mutant grass carp in an attempt to identify functional resistance genes for future hereditary rearing projects in grass carp.ResultsThis study used ENU-mutated grass carp to identify genetic markers associated with resistance to the haemorrhagic disease caused by GCRV. Bulked segregant analysis (BSA) was performed on two homozygous gynogenetic ENU grass carp groups who were susceptible or resistant to GCRV. This analysis identified 466,162 SNPs and 197,644 InDels within the genomes of these mixed pools with a total of 170 genes annotated in the associated region, including 49 genes with non-synonymous mutations at SNP sites and 25 genes with frame shift mutations at InDel sites. Of these 170 mutated genes, 5 randomly selected immune-related genes were shown to be more strongly expressed in the resistant group as compared to the susceptible animals. In addition, we found that one immune-related gene, EPHB2, presented with two heterozygous SNP mutations which altered the animal’s responded to GCRV disease. These SNPs were found in the intron region of EPHB2 at positions 5859 (5859G > A) and 5968 (5968G > A) and were significantly (p = 0.002, 0.003) associated with resistance to GCRV. These SNP sites were also shown to correlate with the GCRV-resistant phenotype in these ENU grass carp. We also evaluated the mortality of the different ENU fish genotypes in response to GCRV and the SNPs in EPHB2. The outcomes of these evaluations will be useful in future selections of GCRV-resistant genes for genetic breeding in grass carp.ConclusionOur results provide a proof of concept for the application of BSA-sequence analysis in detecting genes responsible for specific functional genotypes and may help to develop better methods for marker-assisted selection, especially for disease resistance in response to GCRV.

Highlights

N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful method for the genetic engineering of plants, and the production of functional mutants in animal models including mice and zebrafish
We selected 5 genes with genetic variations and examined their mRNA expression in liver, kidney and gill tissues. The results of these analyses showed that mRNA expression levels of Sterile alpha motif domain containing 9 like (SAMD9L) and EPH receptor B2 (EPHB2) were increased in all three tissues from the resistant groups in response to Grass carp reovirus (GCRV), while BCL2 interacting protein 3 like (BNIP3L) and Adaptor protein (APPL2) expression was increased in the liver and kidney tissues of resistant fish when compared to the gill tissues of the same animal
Our results demonstrate the utility of applying BSAsequence analysis to the detection of genes responsible for interesting phenotypes and will help in developing new protocols for completing marker-assisted selection for GCRV disease resistance in farmed fish

Summary

Introduction

N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful method for the genetic engineering of plants, and the production of functional mutants in animal models including mice and zebrafish. Grass carp reovirus (GCRV) is a haemorrhagic disease of grass carp which has caused noteworthy losses in fingerlings over the last few years. To overcome this problem, we used ENU mutant grass carp in an attempt to identify functional resistance genes for future hereditary rearing projects in grass carp. Genetic breeding of aquaculture fish species primarily depends on the identification of naturally occurring mutants with high performance, and subsequent hybridisation or marker-assisted breeding to produce betterquality strains [1]. GCRV infection results in haemorrhagic disease in grass carp and causes noteworthy losses in fingerlings, with recent outbreaks exhibiting a significant economic impact

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