| The clam Meretrix petechialis is one of the most important commercial clam species farmed in China.However,in rencent years,the outbreaks of vibriosis and mass mortality in the culture process inflicted serious financial losses to the clam farming industry.This is related to factors such as the deterioration of the breeding environment and the increase of the breeding density,but the lack of resistant strains is also an important reason for the above problems.Hence,it is of great significance to carry out genetic breeding for the selection of Vibrio resistance strains of the clam M.petechialis.However,the process of resistance breeding has been hindered by the complicated genetic mechanism of disease resistance and difficulty in measuring resistance phenotypes accurately.The accurate estimation of genetic parameters is a prerequisite for developing effective breeding strategy in resistance selection breeding.And the resistance-associated markers can accelerate the improvement of resistant strains.In this study,based on the M.petechialis transcriptome database and full sibling families exhibiting stable resistance level,we identified the Vibrio resistance related gene expression and estimated the genetic parameters of gene expression traits.These results provide a reference for Vibrio resistance enhancement in the genetic breeding of clam M.petechialis.The main results were as follows.1.In this study,RNA-Seq was applied to explore global expression changes of hepatopancreas from this clam after Vibrio challenge.There were 199,318,966 clean reads obtained by Illumina sequencing,which were further assembled into 214,577 transcripts,and then 147,255 unigenes with an N50 of 1,393 bp were identified.Gene ontology(GO)analysis revealed 21 biological process subcategories,15 cellular component subcategories and 12 molecular function subcategories.A total of 8,358 unigenes were mapped onto 267 biological signaling pathways by KEGG,among which there were 16 pathways related to the immune system.In total,206 differentially expressed genes(DEGs)were identified,including 113 up-regulated unigenes and 93 down-regulated unigenes.In these DEGs,96 DEGs were annotated in at least one database,accounting for 46.60% of all significant DEGs.To validate the transcriptome dataset,15 DEGs were selected for real-time qPCR confirmation and the results showed that expression patterns of 13 genes(86.7%)agreed well with the RNA-Seq analysis.Fourteen of the 206 DEGs were annotated to be immune-related genes,and we examined the expression patterns of four immune-related DEGs(BIRC7,HSF,C1ql4 and C1ql)using clams post immersion challenge.This study enriched the M.petechialis transcriptome database and provided insight into the immune response of M.petechialis against Vibrio infection.2.Gene expression data were used to select resistance-associated markers that indicate resistance phenotypes in the clam M.petechialis.A total of 1772 clams from 23 full-sibling families were subjected to a Vibrio challenge test,and the results showed significant variation in resistance to vibriosis among different families.Three of the most resistant families and three of the most susceptible families were selected for screening of resistance-associated markers.Then,among 22 genes,12 demonstrated a significant change in expression,and their expression levels were further analysed by T-test and linear regression analyses,respectively,between resistant and susceptible families.Overall,the results showed that the basal expression levels of Big-Def,BIRC7,NFIL3,CTL9 and Bax were associated with Vibrio-resistant phenotypes and could be considered candidate resistance-associated markers.Further,two M.petechialis strains that differed in Vibrio resistance were used to examine the effectiveness of these five resistance-associated genes.The results further confirmed that the expression of BIRC7 and NFIL3 were associated with Vibrio-resistant phenotypes.Furthermore,the dynamics of bacterial load in hepatopancreas of the clams post Vibrio immersion infection were measured.The results showed that the bacterial load in hepatopancreas reached a peak at 24 h post immersion challenge,and then they dropped significantly and maintained at a lower level after 3 days.We further assessed the patterns of gene expression of resistance-related genes in high and low bacterial load groups,and that found that the gene expression levels were significantly higher in high bacterial load individuals,when compared with low bacterial load individuals.This study integrated the Vibrio-resistance phenotype,gene expression data and bacterial load for resistance evaluation and provided potential markers for the genetic breeding of high Vibrio-resistance strains in the clam M.petechialis.3.23 full sibling families which were produced using part factorial design and sire-dam model were used to estimate the genetic variation and heritability of gene transcription in the clam M.petechialis.Five Vibrio resistance-related genes(Big-Def,BIRC7,NFIL3,CTL9 and Bax)were selected for gene expression analysis in the hepatopancreas,and all the gene expression datasets exhibited normal distributions(P > 0.05).Significant additive genetic variations were observed for gene expression traits in the clam.Among those five genes,two genes(Big-Def and CTL9)showed significant sire effect,and no maternal effect variance was detected for any of the five genes.The heritabilities of the five gene expression traits varied from 0.113-0.530.And,no significant phenotypic and genetic correlations were detected between each gene transcription trait and body size.Additionally,the second generation(F2)was produced from the resistant families(No.47 and No.60).The Vibrio challenge test showed that the Vibrio resistance of the F2 was significantly improved,and the survival rate was approximately 10%-15% higher than that of their parents.This study highlights that gene expression level is heritable,and the resistance related to gene expression traits can also be improved through genetic breeding.So the discovery of gene expression markers provides references for indirect selection of M.petechialis with high Vibrio resistance. |
PDF Full Text Request