| There is a rich variety of buffalo resources,both B.bubalus arnee and B.bubalus carabanesis,mainly used for arable land and meat production in China.In recent years,with the utilization of buffalo milk performance and the economic value of milk production performance produced,the buffalo industry has developed rapidly.Buffalo milk is a high-quality milk that is rich in nutrients and has a high digestion and absorption rate.It has a wide market prospect for its high-quality dairy products,such as "milk tofu","milk cake" and "double skin milk" with good taste.Although the amount of buffalo in our country is large,the performance of buffalo milk yield and reproduction are low.In addition,there are no specialized breeds of dairy type buffalo so far,which has led to the the difficulty of the buffalo industry development.Both reproduction and milk production traits are important economic traits for buffalo,and belong to typical quantitative traits.Because of the low heritability and many influential factors,the traditional method of breeding for high reproduction and high milk yield buffalo is progressed slowly.Genome wide association analysis and transcriptional sequencing provide a new direction for the study of quantitative traits.Therefore,we used genome wide association analysis and transcriptome sequencing to screen and identify candidate genes that affect buffalo reproductive traits and milk production traits.The purpose of this study was to reveal the molecular genetic basis of milking and reproductive performance of buffalo,so as to provide a theoretical basis for improving the milk production and reproductive performance.1.Identification of candidate genes for buffalo reproductive traits:This experiment is divided into three parts.First of all,a genome-wide association analysis using commercial buffalo 90 K gene chip for 6 reproductive traits of Italy Mediterranean buffalo,including first calving age,second calving age,third calving age,calving interval,the number of artificial insemination,and open days.The SNP,genomic region and candidate genes that affect the reproductive traits of buffalo will be identified by GWAS.Next,the next generation sequencing technology was used to carry out the transcriptome sequencing of granulosa cells from four development periods of antral follicle in buffalo(the follicle diameter was <5mm,5~8mm,8~12mm,>12mm),and the sequencing results were qualitatively and quantitatively analyzed.The candidate genes that affect the development of follicle were identified by the enrichment analysis of GO and KEGG.Finally,we identified the key candidate genes affecting reproductive performance of buffalo by integrating GWAS results and transcriptome analysis results.Moreover,one of the key candidate genes,IGFBP7 was experimentally confirmed by RNAi technique in buffalo granulosa cells.The main results are as follows:(1)Total 40 SNPs and 28 candidate genes related to reproductive traits were identified by genome wide association analysis.(2)Haplotype analysis revealed that there was a QTL related to reproductive traits of buffalo in chromosome 2.3-2.7 Mb region of chromosome 5,in which multiple SNPs were mainly located within and near the TRHDE gene.(3)The transcript analysis of buffalo granulosa cells identified 17700 genes,of which 1076 genes were significantly differentially expressed at different stages of follicular development,and 30 genes were differentially expressed in all stages of follicular development.The largest number of differentially expressed genes was observed in the last two stages of follicular development.(4)The differentially expressed genes were mainly enriched in immune related signaling pathways at the end of follicle development,suggesting that follicles are preparing for oocyte release at the last stage.(5)Integrating GWAS analysis and transcriptome sequencing,we found that 20 of 28 candidate genes related to reproductive traits were expressed at different stages of follicle development,suggesting that these genes may directly participate in the development of buffalo follicle and affect the reproductive performance of buffalo.(6)IGFBP7 has high expression in all four stages of follicle development.Using RNAi to knock down the expression of IGFBP7 in buffalo granulosa cells,the granulosa cell proliferation,apoptosis and estrogen and progesterone secretion were significantly changed after IGFBP7 knocking down.2 Analysis of candidate genes affecting buffalo milk production traitsThis experiment is divided into two parts.First of all,1002 buffalo's 16 body size index and milk yield were analyzed by regression analysis.The body size index which significantly related to milk yield was screened out,and the index was verified by the Mediterranean buffalo group.Secondly,according to the index of body conformation obtained,using 90 K Affymetrix Axiom SNP Buffalo SNP Array for genotyping 176 Italy Mediterranean buffaloes.After quality control,the genome wide analysis was carried out with the key body conformation index to find the SNPs,candidate genes and QTLs related to the body conformation index.The main results are as follows:(1)According to the analysis of body conformation index and milk yield of buffalo,we found that breast traits were significantly correlated with milk yield,and breast traits could be regarded as direct or indirect indicators of buffalo milk production.(2)A total of 10 SNPs and 5 candidate genes related to breast traits were obtained from the genome wide association analysis of 5 breast traits in buffalo.The candidate genes include PTPN11,ZNF385 B,LRP2,COX18 and DTHD1.(3)Haplotype analysis identified a QTL associated with breast traits,which located in the candidate gene LRP2.Expression profile analysis of LRP2 revealed that LRP2 was highly expressed in breast tissues.We speculated that the gene may participate in the reabsorption of milk protein and milk fat during lactation. |
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