Establishment Of High Throughput Genotyping Methods For FecB Mutation In Sheep And Screening For Candidate Genes Of Prolificacy In Bamei Mutton Sheep

The mutation of bone morphogenetic protein receptor 1B(BMPR1B)gene present in prolific Booroola Merino sheep can increase the ovulation rate and litter size of ewes significantly.It is the first major gene in sheep for high fecundity and was assigned as FecB(Fecundity Booroola)by Sheep and Goat Genetic Nomenclature Committee.The traditional FecB genotyping methods include PCR-RFLP(polymerase chain reaction-restriction fragment length polymorphism)and PCR-SSCP(polymerase chain reaction-single strand conformation polymorphism).Unfortunately,the flux of these methods was low,and the program was complicate and difficult to achieve high-throughput automatic measurement.Two high-throughout methods for FecB detection(Taqman and SNaPshot)had been developed in this study.33987 sheep of 28 sheep breeds including Small Tail Han,Hu,and Bamei Mutton Sheep et al.were used for FecB detection using genomic DNA.The genotyping results of 33987 sheep showed that the allele B frequencies of Hu and Small Tail Han sheep are much higher than other breeds.The allele B frequency in Bamei Mutton Sheep was 0,which indicated that FecB was not the major gene of prolificacy in Bamei Mutton Sheep.These results showed that these two high-throughout methods can save the time and increase efficiency dramatically.Those methods have potential utilization in large-scale molecular breeding.Additionally,blood samples from 5 polytocous Bamei ewes and 5 monotocous Bamei ewes were collected.The genomic DNA was extracted and FecB genotype of the ten Bamei ewes were detected.All the genotypes of ten Bamei ewes were ++ suggesting that there were other genes which may affect the litter size of Bamei Mutton Sheep.Moreover,whole-genome sequencing was performed on these ten ewes,and 150 G raw data were obtained,the average depth of coverage of each individual had reached 5�.Finally,we acquired more than 21 million SNP variants.XP-EHH and FST selection signal methods were used to screen the selected regions for litter size in Bamei Mutton sheep.87 unique genomic regions were identified according to FST selection.One top signal of selective sweep was identified at an intergenic region upstream of KDM4B(Lysine(K)-Specific Demethylase 4B).After protein interaction analysis,we found that KDM4B and ESR1(Estrogen Receptor 1)had interaction,and ESR1 played an important role in prolificay and seasonal estrus.198 unique genomic regions were identified by XP-EHH method.We overlapped these regions identified by FST and XP-EHH respectively,and 12 unique regions were selected.We analyzed these 12 unique regions by bioinformatics tools,and some candidate genes were found such as CPS1,HERC5,NSF,which involved in the digestive tract development,ketone body metabolism,and the process of cell cycle.After GO enrichment analysis,we found a group of genes which may influence reproduction,such as ITIH1,ITIH3,ITIH4 genes involved in the process of hyaluronic acid metabolism,CPS1 associated with the urea metabolism of ruminants,and GNA12 and LMBR1 associated with embryonic digit morphogenesis.