Identification Of Candidate Genes Underlying Chicken Breast Muscle Traits Based On Whole-genome Association Analysis

Chicken meat is considered to be the most significant meat product globally due to its high nutritional value,affordability,and vital role in the national economy.Therefore,research aimed at improving and enhancing the growth performance and meat production of domestic chickens has become a current area of great interest.Recently,the gradual maturation of molecular breeding techniques has provided a new opportunity to enhance the meat production capabilities of domestic chickens.In this study,we intercrossed Yunnan Daweishan Mini Chickens and Cobb Broilers to establish an F₂ resource population and measure phenotypic indicators related to breast muscle traits in the F₂ generation.We then performed whole-genome resequencing on this population and integrated these indicators to conduct a genome-wide association study（GWAS）.By doing so,we identified key candidate SNPs and genes associated with breast muscle traits.Additionally,we carried out functional validation of the candidate genes in vitro,providing a comprehensive genetic analysis of the chicken breast muscle from a genetic perspective.The results of this study are shown below:Firstly,we established 478 F₂ resource populations by crossing Cobb broilers and Daweishan mini-chickens.The populations were divided into K21（246 individuals）and K22（232 individuals）groups based on the direction of hybridization.We measured 11 trait indicators related to breast muscle traits in the F₂ populations,with heritability ranging from 0.01 to 0.62.High-heritability traits included body weight,breast muscle weight,breast width,keel length,and full evisceration weight,while low-heritability traits included breast muscle fiber density,single muscle fiber diameter,and cross-sectional area.Breast muscle weight was significantly correlated with all other traits（p<0.05）.It showed a strong positively correlation with body weight,keel bone length,breast width,breast muscle percent,and full evisceration weight;a moderate positively correlation with breast depth and sternum weight;and a weak positively correlation with the diameter and cross-sectional area of single muscle fibers in the breast muscle.In contrast,it was negatively correlated with muscle fiber density.When comparing the K21 and K22 populations,breast depth was the only trait that showed a significant difference（p<0.01）,with K22（87.25 mm）being larger than K21（84.32 mm）.Next,we used whole-genome resequencing technology（10×）to genotype 478 F₂-generation individuals and 40 parental individuals,generating 5.6 TB of data for the F₂ resource population with an average of 11.72 GB per individual,which identified 4,828,637 SNPs.The parental population generated 0.6 TB of data,averaging 15 GB per individual,and detected 17,164,853 SNPs.Referring to the parental genotype data,we phased the F₂ resource population genotyping and performed data quality control,obtaining 12,378,481 SNPs.We set P-value thresholds of 4.04×10^-8 and 4.04×10^-10 for significant SNPs in GWAS for different traits,identifying 213 SNPs significantly correlated with breast muscle traits.These SNPs were distributed across chicken chromosomes 1,3,4,14,15,and Z,and based on their genomic distribution,we identified 45candidate genes.After GO annotation and KEGG pathway analysis,only seven genes were successfully annotated,including UFM1,FNDC3A,ALOX5AP,PPP1CB,CALM2,LDB2,and TBX3,involved in biological functions related to protein synthesis and binding,such as myosin binding,calcium ion binding,and protein phosphatase activation.All candidate genes were expressed in the breast muscle tissue of the F₂ resource population,with CALM2 showing the highest expression level and LDB2 exhibiting a relatively lower expression level.The F₂ resource population was divided into different genotypic groups based on the candidate SNPs.The SNP（g.G176412883C）divided the F₂ resource population into three groups（GG,GC,CC）,with GG being the original genotype.Individuals with this genotype had significantly smaller breast muscle weights than those with the other two genotypes（p<0.01）,and the corresponding candidate gene ALOX5AP exhibited significantly lower expression levels in the GG genotype individuals compared to those with CC+GC genotypes（p<0.0001）.Finally,the functional validation of ALOX5AP showed that it is highly expressed in myogenic cells.Real-time quantitative PCR revealed that the expression of ALOX5AP m RNA was 1.6 times higher in mixed cells with a significant difference（p<0.001）.An ALOX5AP overexpression cell line was established,and both the m RNA and protein levels were significantly increased,with m RNA levels at 157%and protein levels at 180%of the control cell line（p<0.001）.Furthermore,an ALOX5AP knockdown cell line was also established,and both the m RNA and protein levels were significantly decreased,with m RNA levels at 24%and protein levels at 12%of the control cell line（p<0.001）.The alteration of ALOX5AP expression levels in myogenic cells has an impact on their proliferative capacity.Specifically,the ALOX5AP overexpression cell line showed significantly higher proliferation vitality compared to the control cell line 24 hours after inoculation（p<0.001）.Additionally,the m RNA expression levels of cell proliferation-related genes,including IGF1,Myo D,and Myo G,were significantly increased compared to the control cell line（p<0.05）.On the other hand,the ALOX5AP knockdown cell line exhibited significantly lower proliferation vitality than the control cell line 48 hours after inoculation（p<0.05）.Furthermore,the m RNA expression levels of cell proliferation and differentiation related genes,including PAX7,Myo G,and Myo D,were significantly downregulated compared to the control cell line（p<0.05）.The study investigated cell cycle changes in ALOX5AP differentially expressed cell lines.The ALOX5AP overexpression cell line showed a significant decrease in the number of cells in G0/G1 and S phases（p<0.05）and a significant increase in the number of cells in the G2 phase（p<0.001）,indicating enhanced cell proliferation capacity.Furthermore,the expression levels of cell cycle-related genes CDKN1A and CDK1 were significantly upregulated compared to the control cell line（p<0.05）.In contrast,the ALOX5AP knockdown cell line showed a significant increase in the number of cells in the S phase（p<0.05）,a significant decrease in the number of cells in the G2 phase（p<0.001）,and no significant difference in the number of cells in the G0/G1 phase（p>0.05）,suggesting impaired DNA synthesis capacity in myogenic cells.The expression level of the cell cycle-related gene CCNA2 was significantly upregulated compared to the control cell line（p<0.05）,while CDK1 and CDK2 were significantly downregulated（p<0.05）.Overall,this study has successfully established a resource population and identified significant genetic markers and potential candidate genes associated with chicken breast muscle trait.The study’s focus on ALOX5AP has confirmed its influence on myogenic cell development,providing insights into the genetic patterns underlying chicken breast muscle traits.These findings can contribute to further research in understanding the genetic mechanisms behind chicken breast muscle trait and potentially aid in improving poultry breeding and meat production.