Establishment And Application Of High Throughput SNP Genotyping Technology For Major Genes Of Prolific Traits In Sheep

Reproductive performance is an important economic trait of sheep.The level of fecundity directly affects the economic benefits of sheep raising and is closely related to the scale,industrialization and sustainable development of sheep raising industry.Studies have shown that FecB,FecX~G,FecX~B and FecG~Hgenes are the major genes responsible for high ovulation and lambing number of sheep.Molecular breeding of high fertility sheep can be realized by detecting the major genes,so as to promote the breeding process of multiple sheep.Objective:The purpose of this study was to establishment of high throughput SNP genotyping technology for major genes of prolific traits in sheep,improve the detection efficiency,realize accurate,rapid and high throughput typing detection of SNPs of FecB,FecX~G,FecX~B and FecG~H gene,and apply it in marker-assisted selection of multiple sheep,so as to provide technical support for the evaluation of germplasm resources of multiple sheep and the breeding of prolific traits sheep breeds.Methods:The genetic polymorphism of FecB,FecX~G,FecX~B and FecG~H genes in multiple Prolific Suffolk,Chinese Merino,Small Tailed Han sheep and Hu sheep were analyzed by PCR-RFLP.According to the gene sequences and SNPs site information of four multiple genes,four major genes wild type and mutant quality control plasmids were designed and constructed by gene cloning.Primers and TaqMan-MGB probes were designed and synthesized based on SNPs site information of four genes.Quality control plasmid was used as control group to optimize the real-time fluorescent quantitative PCR reagent,system and detection conditions of high throughput SNP typing detection technology.The sensitivity,accuracy and repeatability of the detection were analyzed.The accuracy of the typing technology was verified by PCR-RFLP and gene sequencing.To establish a high throughput SNP typing technique for four major genes.In this study,Prolific Suffolk sheep,Chinese Merino sheep,Small tail Han sheep and Hu sheep breeding groups were used to analyze the polymorphism of major genes in breeding groups by using the established high throughput SNP genotyping technique,and the least squares model was used to analyze the relationship between major genes and the phenotypic characters of sheep lambing number,and to assist the selection of multiple sheep according to genotype.Results:(1)In this study,FecB gene was detected in Prolific Suffolk,Chinese Merino,Small Tailed Han sheep and Hu sheep by PCR-RFLP,but FecX~G,FecX~B and FecG~H genes were not detected in Prolific Suffolk,Chinese Merino,Small Tailed Han sheep and Hu sheep.The mutant and wild control plasmids of FecB,FecX~G,FecX~B and FecG~H genes were successfully constructed by PCR and gene cloning.(2)By optimizing the reaction conditions and reaction system,appropriate primer and probe sequences were designed.The optimal primer concentration was 0.2μmol/L(0.4μL),probe concentration was 0.2μmol/L(0.4μL),and annealing temperature was 57℃.The established high throughput SNP genotyping method for major genes of four multi-embryo traits has the characteristics of high accuracy,good repeatability,high sensitivity and fast detection speed.(3)In this study,FecB,FecX~G,FecX~B and FecG~H genes were detected in Prolific Suffolk,Chinese Merino,Small Tailed Han sheep and Hu sheep by high throughput SNP genotyping technology.FecB gene mutations occurred in all the four breeds,but no FecX~G,FecX~B and FecG~H gene mutation were found.The results showed that BB,B+and++were found in Prolific Suffolk,Chinese Merino,Small Tailed Han sheep and Hu sheep.The frequencies in Prolific Suffolk population were 0.25,0.47 and 0.28,and the frequencies of alleles B and+were 0.49 and 0.51,respectively.The frequencies in Chinese Merino population were 0.07,0.23 and 0.70,and the frequencies of alleles B and+were 0.18 and 0.82,respectively.The frequencies in Hu sheep population were 0.94,0.06 and no++genotype,and the frequencies of alleles B and+were 0.97 and 0.03,respectively.The frequencies in Small Tailed Han sheep population were 0.19,0.57 and 0.24,and the frequencies of alleles B and+were 0.47 and 0.53,respectively.The results of least squares model analysis showed that BB and B+genotypes in Prolific Suffolk sheep and Chinese Merino sheep were significantly correlated with the number pf lambs.According to the characteristics of the breeding herd,a marker-assisted breeding scheme was developed for multiple fetal traits of sheep.Conclusion:A high throughput SNP genotyping method of FecB,FecG~H,FecX~G,and FecX~B gene for multiple fetal traits in sheep was successfully established,Which proved that FecB gene mutations in Prolific Suffolk,Chinese Merino sheep,Hu sheep and small tail Han sheep breeding herds,but no FecX~G,FecX~B and FecG~H gene mutation were found.The established high throughput SNP genotyping method has the advantages of high accuracy,good repeatability,high detection sensitivity and fast speed,and can be used to guide breeding in marker-assisted selection breeding of multi-little sheep.