Genetic Mining And Functional Study Of Sheep Reproduction Traits Related Genes Based On Whole Genome Data

Fecundity is one of the crucial economic traits in sheep,directly influencing the breeding efficiency of meat sheep in the sheep industry.With the gradual development of the meat sheep industry in China towards scale and intensification,the demand for high-prolific sheep breeds is becoming increasingly urgent.The reproductive performance of ewes is influenced by factors such as the litter size and seasonal estrus.Therefore,the identification of candidate genes and molecular markers affecting traits like prolificacy and year-round estrus is essential for establishing modern meat sheep production and breeding systems.In this study,genomic data from 2409 sheep worldwide,including whole-genome resequencing,and Pac Bio Hi Fi long-read sequencing data from 15 sheep,were collected from public databases.Additionally,120 Australian Merino sheep with lambing data available were newly sequenced for the whole genome.The study focused on three known major fecundity genes:bone morphogenetic protein receptor 1B（BMPR1B）,bone morphogenetic protein 15（BMP15）,and growth differentiation factor 9（GDF9）,as well as two important candidate genes,melanocortin 1 receptor（MC1R）and melanocortin 4receptor（MC4R）.Various types of genetic variations,including single nucleotide polymorphisms（SNPs）,insertions/deletions（In Dels）,and structural variations（SVs）,were comprehensively explored.The frequency distribution patterns of these variations were studied,and candidate mutations related to lambing were identified using methods such as signal detection.Finally,association analyses were conducted by combining the collected lambing data and genome data of Australian Merino sheep.The study aimed to uncover new variant sites influencing the litter size.Additionally,eight Romanov sheep（a high-prolificacy European breed）were sequenced for the whole genome to identify new genes affecting lambing.Selection signal analysis was performed in conjunction with published data,and functional studies were conducted on candidate mutations identified in the MC1R gene.In order to discover new genes influencing year-round estrus,genomic data from 6 breeds with year-round estrus and 10 breeds with seasonal estrus were analyzed through selection signal detection and GWAS.Functional studies were conducted on candidate mutations identified in the MC4R gene.The ultimate goal of this research is to provide scientific data for the development of modern meat sheep production and breeding.The study has yielded the following important results:1.Exploration and validation of genetic variations in the major sheep fecundity genes BMPR1B,BMP15 and GDF9Based on the compiled WGS data of 75 global sheep breeds（or populations）,totaling2409 sheep,as well as Pac Bio Hi Fi sequencing data from 15 sheep,this study systematically explored the SNP,In Del,and SV variations of three key reproductive genes in sheep-BMPR1B,GDF9 and BMP15.A plethora of known and novel variant sites were identified,and the distribution patterns of these sites across the 75 global sheep populations were comprehensively reported.To validate the correlation between significant mutations and lambing numbers,this research additionally collected samples from 800 Australian Merino sheep,along with their lambing data.By combining this information with the whole-genome sequencing data（10×）from 120 sheep,the study conducted an association analysis between relevant variations and lambing numbers.（1）Sheep BMPR1B Gene:A total of 11,297 SNPs were identified,including 7 coding mutations,with one being novel.Additionally,508 In Dels were identified,of which 3 were located in coding regions or untranslated regions.Nineteen SVs were identified,ranging in length from 51 to 5,144 bp.Using a selection signal detection method,a synonymous mutation,g.30050773C>T,was screened in the Duolang sheep.This mutation was under selection in Duolang sheep,forming a long haplotype,suggesting its potential impact on litter size.Using Kompetitive allele-specific PCR（KASP）technology,we found that this site exhibited polymorphism in the Australian Merino sheep and was significantly associated with lambing numbers（P<0.05）.This indicates that the g.30050773C>T mutation can serve as an effective molecular marker for early selection of lambing numbers.（2）Sheep BMP15 Gene:A total of 139 SNPs were identified,including 10 coding mutations,with 9 being novel.Seven In Dels were identified,and no SV were found.This study focused on a missense mutation site g.54284258A>G（L251P）,which showed a relatively high frequency in diverse sheep breeds.Using KASP technology,the frequency of this mutation was examined in the Australian Merino sheep,and its correlation with lambing numbers was analyzed.Results revealed polymorphism at this site in the Australian Merino sheep population,showing a significant correlation with lambing numbers（P<0.05）.This suggests that the g.54284258A>G mutation can be an effective molecular marker for early selection of lambing numbers.（3）Sheep GDF9 Gene:A total of 49 SNPs were identified,including 22 coding mutations,with 15 being novel.Additionally,20 In Dels and 2 SVs were identified.This study focused on a missense mutation site g.42114493C>T（V332I）and used KASP technology to examine its frequency in the Australian Merino sheep and its correlation with lambing numbers.The site was found to be polymorphic in the Australian Merino sheep but showed no significant correlation with reproductive traits such as lambing numbers（P>0.05）.However,the possibility of a correlation between this mutation site and other traits cannot be ruled out.2.Exploration of candidate genes for high fecundity in romanov sheep and functional study of MC1R gene mutationsRomanov sheep is a globally renowned representative breed known for high prolificacy.In this study,the population structure of Romanov sheep was assessed using whole-genome data from 17 individuals（including 8 newly sequenced in this study）and compared with data from other 11 European breeds.Candidate genes associated with high prolificacy in Romanov sheep were identified,and functional studies were conducted.Selective sweeps in Romanov sheep genome were identified using population differentiation indices(F_ST)and population-specific extended haplotype homozygosity（XP-EHH）methods.A strong selection signature was detected on chromosome 14（Chr14:14.2 Mb-14.3 Mb）,encompassing a long haplotype containing genes such as MC1R,SPIRE2,TCF25 and DEF8.This suggests a potential association between this haplotype and reproductive and cold resistance traits.Considering the well-established methods for functional mutation studies in G protein-coupled receptor（GPCRs）family genes like MC1R,and the potential"one gene,multiple effects"phenomenon observed in MC1R in this study,further analysis focused on the global distribution of MC1R gene mutations and their impact on gene function.Based on WGS data from 75 global sheep populations,139 SNPs were identified in the MC1R gene,including 22 missense mutations,11 synonymous mutations,and 2 In Dels.For the missense mutation g.14251947T>A（M73K）,compositional activity testing was conducted using a dual-luciferase reporter system.The study found that the mutant composition activity at the g.14251947T>A（M73K）site in the MC1R gene significantly increased（P<0.05）.When cells expressing wild-type and mutant recombinant vectors were stimulated withα-MSH as an agonist,the EC50 of the mutant MC1R K73 receptor binding to theα-MSH ligand increased threefold,indicating that this mutation significantly enhances receptor-ligand binding.This suggests that the missense mutation may affect the function of the MC1R gene,laying the groundwork for future in-depth research on the impact of this mutation on reproductive traits.3.Exploration of candidate genes for year-round estrus in sheep and functional study of MC4R gene mutationsUtilizing WGS data from 392 sheep,including 6 breeds with year-round estrus and 10breeds with seasonal estrus,which are widely recognized,this study employed selection signal detection methods and genome-wide association analysis（GWAS）to identify key genes associated with year-round estrus in sheep.The study identified strong selection in the MC4R gene in breeds with year-round estrus,and GWAS revealed the association of this gene with year-round estrus.As MC4R is also a G protein-coupled receptor（GPCR）with mature methods for functional mutation studies,the study further analyzed the global distribution of MC4R mutations and their impact on gene function.Based on whole-genome data from 75 global sheep populations,48 SNPs were identified in the MC4R gene,including 4 missense mutations,4 synonymous mutations,2 In Dels,and 13SVs with lengths ranging from 54 to 594 bp.In-depth analysis revealed a significant missense mutation,g.59480440G>C（I173M）enriched in breeds with year-round estrus.Functional studies on this enriched missense mutation revealed that it did not affect protein expression levels.However,a dual-luciferase reporter assay found a significant increase in receptor compositional activity after the mutation.Additionally,the mutant receptor showed a 20-fold increase in EC50 when binding to theα-MSH ligand,indicating a substantial enhancement in c AMP signal transduction activation.This suggests that the mutation is a crucial functional site affecting sheep estrus.In summary,this study systematically explored the SNP,In Del,and SV variations in three key reproductive genes（BMPR1B,GDF9 and BMP15）using a large panel of genome sequence data.A series of new variant sites were identified,and two new molecular markers for sheep prolificacy were discovered.In the globally renowned Romanov sheep,new candidate genes MC1R were identified,and functional studies revealed that the missense mutation M73K affects the gene function at the cellular level.Leveraging resequencing data from breeds with year-round and seasonal estrus,this study identified,for the first time,candidate gene MC4R associated with year-round estrus and found that the missense mutation I173M affects the gene function at the cellular level.These findings provide a basis for exploring critical genes regulating sheep prolificacy,deciphering their regulatory mechanisms,and conducting marker-assisted selection for sheep reproductive traits,ultimately contributing to the theoretical foundation for improving sheep prolificacy in China.