Genetic Variation Identification Of MMAF-related Genes And Their Associations With Litter Size In Shaanbei White Cashmere Goat

Shaanbei white cashmere goat is a kind of goat with fine cashmere and meat,but its reproductive performances need to be improved.Therefore,the use of molecular marker assisted selection is of great significance for the genetic improvement of its reproductive traits.As the candidate genes related to goat reproduction traits.There are seven genes have been proved to associate with multiple morphological abnormalities of the sperm flagella(MMAF),including DNAH1,QRICH2,CFAP43,CFAP44,CFAP69,CCDC39,and AKAP4gene.In this study,the INDEL loci of MMAF-related genes were firstly identified and the genetic effects of the INDEL loci and litter size traits were analyzed.The CNV loci of significantly related genes were analyzed to explore their genetic effects on litter size traits.Finally,MMAF-related genes that both INDEL and CNV loci can affect goat litter size traits were determined to further explore the relationship between SNP mutations of these genes and litter size traits.This study expected to provide effective DNA markers and scientific data for improving its reproductive traits through MAS1.INDEL mutation identification of MMAF-related genes and correlation analysis with litter size in goatConsidering the rapidity,convenience and operability of INDEL detection technology,and the lack of relevant studies on the INDEL polymorphism of goat MMAF-related genes,this study firstly explored the INDEL mutation of MMAF-related genes and analyzed its correlation with the first litter size traits in 1025 Shaanbei white cashmere goats(SBWC).This experiment was conducted by Ensembl database,agarose gel electrophoresis and Sanger sequencing method.Result found DNAH1 gene exist P1(rs636295440)and P3(rs662697370)polymorphic loci,QRICH2 gene exist one P4 polymorphism locus(rs651821102),CFAP43 gene exist P20 polymorphism locus(rs644941577),CFAP69 gene had P4(rs646423399),P6(rs654691768)and P7(rs658907780)polymorphism loci.Different genotypes of mutations associated with litter size traits of SBWC found that DNAH1-P1(rs636295440)and CFAP43-P20(rs644941577)loci were significantly associated with litter size traits(P<0.05),and linkage disequilibrium analysis results show that the two loci had strongly linked relationship(r~2<0.33),the above results show that both the two mutations could be used as goat litter size trait markers of genetic breeding.2.Analysis of genetic effects of CNV mutation in DNAH1 gene and correlation analysis with litter size in goatAccording to the Animal Omics Database,only DNAH1 gene has three exon CNV mutations,while CFAP43 gene has no exon CNV mutations,so this study fu-rther explored the three DNAH1 exon CNVs in 318 goats.In this study,q PCR me-thod was used to identify three CNV mutations of DNAH1 gene,and it was found that polymorphisms existed in these three mutation sites,namely,Loss genotype,Medium genotype,and Gain genotype.In CNV1(NC?030829.1:g:48593201-48595200),CNV2(NC?030829.1:g:48603601-48605200)and CNV3(NC?030829.1:g:48617201-48618800)mutation,Gain genotype had the highest litter size(P<0.01).The res-ults showed that all three exons CNV of DNAH1 gene could be used as molecular markers of goat litter size traits.3.Analysis of genetic effect of SNP mutation in DNAH1 gene and correlation analysis with litter size in goatBased on the significance results of INDEL and CNV mutations,and considering the complexity and long time of SNP detection methods,this study finally detected the SNP mutations of DNAH1 gene,and analyzed the correlation between these SNP mutations and the lambing traits of 327 goats.According to the result of prediction of transcription factors of DNAH1,this study found ten SNP mutations can differently combined with C/EBP alpha,the TBP,PR,and many other transcription factors,so this research using DNA sequencing method,identifying ten SNP mutations of DNAH1 gene upsteream region and its correlation with litter size traits of goats.The results showed that SNP1(rs665366227),SNP3(rs638339874),SNP4(rs653533484),SNP5(rs646237158),SNP7(rs640739292),SNP9(rs656018411)and SNP10(rs644545438)existed mutations.The SNP1(rs665366227),SNP5(rs646237158)and SNP7(rs640739292)loci were significantly associated with goat litter size traits,and the wild genotypes had best litter size traits,results also found that SNP1(rs665366227),SNP5(rs646237158),SNP7(rs640739292)and SNP10(rs644545438)sites belong to strong linkage disequilibrium relationship(r~2>0.33).The above results indicated that DNAH1 had rich genetic polymorphism and may play a role in influencing litter size traits through linkage relationship,which can be used in the genetic breeding of litter size traits in goats.4.Correlation analysis between combination genotypes of key mutations of goat MMAF-related genes and litter sizeLitter size trait is a complex quantitative trait involving multiple genes and loci,which may be due to the co-regulation of multiple sites,so analyzing the linkage disequilibrium and connection effect of multiple loci on litter size traits is necessary.In this study,the relationship between molecular polymorphisms of MMAF-related genes and goat litter size traits was explored.Results found a total of eight polym-orphism sites were correlated with goat litter size traits.DNAH1-SNP1(rs665366227),DNAH1-SNP5(rs646237158),DNAH1-SNP7(rs640739292),DNAH1-P1(rs636295440),DNAH1-CNV1(NC?030829.1:g:48593201-48595200),DNAH1-CNV2(NC?030829.1:g:48603601-48605200)and DNAH1-CNV3(NC?030829.1:g:48617201-48618800)loci located in DNAH1 gene could significantly affect goat litter size traits,and the CFAP43-P20(rs644941577)locus in CFAP43 gene was significantly associate with litter size traits of goats.The combined genotypes analysis indicated that in the combined effects of CNV and SNP mutation sites,there were 18 combined genotypes that could be used for association analysis,and the combined genotypes 11(Medium-Medium-Medium-AA-TT-CC),16(Medium-Gain-Gain-AA-TT-CC)and 17(Gain-Gain-Medium-AA-TT-CC)had the highest litter size,which were significantly different from the combined genotype 1(Loss-Loss-Loss-AA-TT-CC)and 2(Loss-Loss-Loss-AG-TA-CT)(P<0.01).Linkage disequilibrium analysis showed that only DNAH1-SNP1,DNAH1-SNP5,DNAH1-SNP7loci were in a strongly linkage disequilibrium state(r~2>0.33).The results showed that the above 8 mutations could be used for the effective genetic breeding of Shaanbei white cashmere goats.