The Excavation Of Key Genes And Genomic Selection For Resistance To White Gill Disease In Large Yellow Croaker(Larimichthys Crocea)

Large yellow croaker(Larimichthys crocea)is a significant economic fish for marine aquaculture in China.White gill disease(WGD)is one of the most serious diseases of large yellow croaker,causing huge economic losses to the large yellow croaker aquaculture industry.Currently,there is no effective control measure for this disease.Therefore,it is crucial to analyze the genetic mechanism of resistance to WGD from a molecular perspective to develop WGD-resistant varieties or strains of large yellow croaker.This study employed genome-wide association studies(GWAS)in combination with e QTL co-localization and other multi-omics tools to identify key candidate genes for resistance to WGD in large yellow croaker.Combined with bioinformatics analysis,the preliminary study of gene function provides an important theoretical basis for the resolution of the genetic mechanism of resistance to WGD in large yellow croaker.A preliminary exploration of the application of genomic selection(GS)in the genetic improvement of large yellow croaker was conducted.The effects of sample size,marker density,and kinship between the training and validation sets on the prediction accuracy were evaluated.The aim was to breed new varieties of large yellow croaker with resistance to WGD using the GS technique.The main research findings are as follows:1.Genome-wide association analysis of resistance to white gill disease and growth traits in large yellow croaker.Genome resequencing was performed on 590 individuals with WGD and 1,493 healthy individuals.After genotyping and quality control,10,086,856 high-quality single nucleotide polymorphisms(SNPs)were obtained.A genome-wide association study(GWAS)of the binary phenotype of WGD in 2,083 large yellow croaker was conducted using GEMMA,based on a mixed linear model.A region on Chr6 was identified as the primary effector for disease resistance.Multiple chromosomes contained variable numbers of SNP loci associated with WGD resistance.Additionally,17 immune-related genes were discovered,indicating that the resistance trait of WGD in large yellow croaker may be a complex quantitative trait controlled by micro effective polygenes.Additionally,GWAS of growth phenotypes,including body weight(BW),body length(BL),body height(BH),and body thickness(BT),were conducted on 590 individuals with WGD and 1,493 healthy individuals.The results showed that the primary loci for growth traits and WGD resistance in large yellow croaker were located in the same region of Chr6,revealing the genetic basis for the association between disease resistance and growth in this population.It has been demonstrated that we should not overlook the selection of growth traits when genetically improving disease-resistance traits in large yellow croaker.2.Genome-wide association analysis and e QTL co-localization analysis.Transcriptome sequencing of liver,muscle,spleen,intestine,and kidney samples of large yellow croaker was performed using the Illumina Nova Seq 6000 platform.e QTL analysis was conducted on each tissue,exploring 14,166,14,411,15,813,17,297,and 16,357 cis-e QTL regions,respectively.Three co-localization analyses were used to integrate the GWAS summary data with the e QTL summary data of the liver,muscle,spleen,intestine,and kidney in large yellow croaker.The analyses included the Pearson correlation coefficient,COLOC,and fast ENLOC(v1.0).The results suggest that an interval of 17 ～ 22 Mb in Chr6 might be a key candidate gene for WGD resistance and growth traits in large yellow croaker.The serpini1 gene in the region is a key candidate gene for WGD resistance and possibly for growth traits in large yellow croaker.Additionally,two SNPs,Chr14＿425884 and Chr14＿425980,are located in the 3’UTR region of the cd109 gene.This may affect the mechanism of resistance to WGD in large yellow croaker through post-transcriptional regulation.3.Mendelian randomization analysis and transcriptome-wide association analysisGene-trait association results were obtained for five tissues of large yellow croaker using SPredi Xcan and S-Multi Xcan.Transcriptome-wide association studies(TWAS)identified 100,172,108,293,and 226 genes that were significant in the liver,muscle,spleen,intestine,and kidney of large yellow croaker,with a total of 255 significant genes identified after integration.GO function and KEGG enrichment analyses of significant genes screened by TWAS showed that the Wnt signaling,NOD-like receptor,and MAPK immune signaling pathways play a role in the resistance to WGD in large yellow croaker.Using SMR and HEIDI tests,we identified 10,12,22,5,and 4genes in the liver,muscle,spleen,intestine,and kidney of large yellow croaker,respectively,that may be potentially associated with resistance to WGD in large yellow croaker.Additionally,the serpini1 gene was identified in muscle and spleen.Bioinformatics analysis and q RT-PCR validation of the serpini1 gene in spleen tissues revealed that the serpini1 gene was significantly highly expressed in individuals infected with WGD.It was hypothesized that the serpini1 gene is a key candidate gene for resistance to WGD in large yellow croaker.4.Excavation of a rare variant of resistance to WGD in large yellow croakerThe variant annotation was conducted using snp Eff(v4.3),resulting in the retention of 35,093(MAF < 0.01)and 49,982(MAF < 0.05)variants following the application of bcftools filtering(with F＿MISSING ≤ 50% as the criterion).A total of 1,778 coding variants and 33,315 noncoding variants were screened with MAF < 0.01,while 2,580 coding variants and 47,402 noncoding variants were screened with MAF < 0.05.Filtering with F＿MISSING ≤ 20 %,all 4,483 loci with MAF < 0.01 were identified as non-coding variants.Furthermore,6,165 loci with MAF< 0.05 contained 203 coding variants and 5,962 non-coding variants.With F＿MISSING ≤ 20 %,and F＿MISSING ≤ 50 % & MAF < 0.01,variants were not identified at both the variant level and at the gene level,and no significant loci were unearthed.At F＿MISSING ≤ 50 % & MAF < 0.05,single variant association analysis revealed 3 rare variant loci significantly associated with resistance to WGD in large yellow croaker,screening for mutations in the intergenic region of the gene that may regulate the associated immune response by affecting the tc1 a gene,and thus have an impact on WGD in large yellow croaker.A total of 10,9,10,and 12 genes were identified as potentially associated with resistance to WGD in large yellow croaker,with the SKAT＿Binary,SKATO＿Binary,SKAT＿Robust,and SKATO＿Robust methods,respectively.The SKAT＿Binary and SKATO＿Binary methods consistently identified 11,11,10,and 13 genes from 2,580 coding variants,respectively.Additionally,they identified 7,7,6,and 9 genes potentially related to the disease from 47,402 non-coding variants,respectively.Important immune genes,such as the ampd3 gene and the nf1 gene,which have been screened for their close association with a variety of human diseases,may play an important role in the development of WGD in large yellow croaker.5.Genomic selection for resistance to white gill disease in large yellow croakerA mixed linear model was employed to estimate the heritability of resistance to WGD in large yellow croaker,which was found to be 0.33 based on the genomic relationship matrix.GBLUP was used to calculate genomic estimated breeding values.The effects of sample size,marker density,and kinship between the training and validation sets on prediction accuracy were assessed using 5-fold cross-validation.The results indicate a significant difference in prediction accuracy between the related group(GP1)and the(near)independent group(GP3),with accuracies of 0.53 and 0.17,respectively,representing a three-fold difference.This suggests that the kinship between the training and validation sets has a significant effect on prediction accuracy,and should be kept as as high as possible in practice.Additionally,heritability has a significant impact on prediction accuracy.For instance,when the number of QTL was 500,increasing the heritability from 0.1 to0.5 resulted in an increase in the prediction accuracy of GP1 from 0.36 to 0.55 and an increase in the prediction accuracy of GP3 from 0.12 to 0.22.The accuracy of predictions increased slightly with an increasing number of QTLs,but there was little difference in accuracy with different numbers of QTLs.Increasing the sample size from 100 to 2,083 resulted in a 1.5-fold increase in prediction accuracy.When the marker density was increased from 0.5 K to 30 K,the prediction accuracy increased significantly from 0.10 to 0.55,which represents more than a five-fold increase in accuracy.After the marker density exceeded a certain threshold of 30 K,the prediction accuracy stabilized and was comparable to the accuracy achieved using genome-wide variants.This study demonstrated the feasibility of using low-depth sequencing combined with a genotype-imputation strategy for the genetic improvement of WGD in large yellow croaker.In summary,this study identified serpini1 as a key gene for WGD in large yellow croaker through GWAS and e QTL co-localization strategy.serpini1 is also a key gene that leads to a genetic association between resistance to WGD and growth traits in large yellow croaker.The study also assessed the feasibility of GS for genetic improvement of WGD in large yellow croaker,providing an important reference for the application of genomic selection in breeding large yellow croaker for WGD resistance.This study is a valuable reference for future applications of genomic selection in breeding WGD-resistant large yellow croaker.It also establishes a foundation for breeding new varieties of large yellow croaker with WGD resistance.The study contributes to the theoretical research on WGD resistance in large yellow croaker and has reference value for breeding disease-resistant traits in other aquatic animals.