| PITX1and PITX2are paried-like homeodomain transcription factors, also known as pituitary transcription factors. They play crucial roles in pituitary development and are important regulator of hypothalamic-pituitary-adrenal (HPA) axis related hormones, directly or indirectly regulate the expression of GH and PRL, which are internationally recognized to influence mammals’lactation performance. Therefore, the genetic and epigenetic changes of PITX1and PITX2genes will directly or indirectly influence the milk performance of dairy goats. This study mainly explored the genetic variation and DNA methylation as well as their effects on milk performance using Xinong Saanen and Guanzhong dairy goats, from the perspective of genetics and epigenetics, expecting to screen out the specific SNPs and DNA methylation loci which have significant impact on milk performance, and to decipher the possible influences of SNP and DNA methylation on mRNA expression, in order to provide theoretical support and practical information for molecular breeding of dairy goats.1. Genetic variations of PITX1, PITX2genes and their effects on milk performanceThis study detected the genetic variations of PITX1and PITX2genes in925Xinong Saanen and Guanzhong dairy goats using DNA pool sequencing and PCR-RFLP, firstly discovered one SNP (IVS1+4G>A) in intronl of PITX1, two SNPs (Cys28Arg, Pro42Pro) in exon1and two SNPs (IVS1+79C>A, IVS1+110T>C) in intronl of PITX2. Population genetic analysis demonstrated that one SNP in PITX1possessed moderate polymorphism, while other4SNPs in PITX2were all in intermediate polymorphism. Association analysis showed that different genotypes in the PITX1(IVS1+41G>A) and PITX2(IVS1+110T>C, Pro42Pro) were significantly associated with milk performance (fat content, total solid content, milk density ect.) in dairy goats (P<0.05or P<0.01), suggesting that these3SNPs could be used as DNA markers in improving milk performance through marker-assisted selection (MAS) in dairy goats.2. Influence of genetic variations within PITX1and PITX2gene on mRNA expression This study identified the most stably expressed housekeeping genes in each tissue of adult dairy goats and detected the relative mRNA expression of PITX1and PITX2in each tissue, also identified the relationship between genotype of SNPs and mRNA expression using DNA and tissue RNA sampls of18Xinong Saanen dairy goats. Detection in muscle, heart and small intestine tissues all showed that3SNPs (Cys28Arg, Pro42Pro, IVS1+110T>C) in exon1and intronl of PITX2gene significantly affected mRNA expression level (P<0.05or P<0.01).3. DNA methylation profile of PITX2gene and its effect on mRNA expressionUsing various dairy goat tissues, this study detected the DNA methylation profile of promoter CpG island in PITX2gene by bisulfite sequencing PCR (BSP) method, also detected the relative mRNA expression of PITX2gene by real-time PCR. Owing to the extremely low DNA methylation rate of promoter CpG island in PITX2, no significant relationship was found between DNA methylation and mRNA expression.4. Influence of DNA methylation of PITX1and PITX2genes on milk performanceAccording to plenty of DNA samples of Xinong Saanen dairy goats and detailed lactation data, this study identified the relationship between DNA methylation profile of CpG islands in PITX1/PITX2genes and milk performance. Results showed that the methylation level of promoter CpG island was significantly lower than other region, and that the overall methylation level of3’CpG island in PITX1,5’promoter CpG island and exon CpG island in PITX2as well as methylation rates of10specific CpG-dinucleotide loci presented significant differences in low and high milk yield groups (P<0.05or P<0.01), demonstrating that DNA methylation might have influence on milk performance of dairy goats and may become epigenetic marker in improving milk performance through MAS in dairy goats. |
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