| Milk production traits are main economic traits in dairy goats, including milk yield, milk fat and milk protein rate and so on. At present, many scholars believe that the performance of these traits are not only controlled by minor-polygene or QTL, but also closely related to the regulation of major genes. Therefore, finding major genes of these traits or molecular markers linked with these traits and researching on the regulation mechanism of functional genes are the prerequisite of dairy goat molecular breeding work. Multigene pyramiding breeding technology centering on molecular marker-assisted selection could select genotypes of milk production traits in DNA level. It overcomes the disadvantage of low accuracy of conventional breeding technology, accelerates genetic progress and improves breeding efficiency. Making marker-assisted and conventional breeding technology to be combinated effictively and integrating innovation new breeding technology system are the trend of development of dairy goat breeding.In this study, PRLR, ELF5, MTHFR and FOLR1genes were selected as candidate genes because they played key regulatory roles in milk production traits. The study investigated the polymorphisms of PRLR, ELF5, MTHFR and FOLR1genes in Xinong Saanen (SN) and Guanzhong (GZ) dairy goat breeds by DNA sequencing and PCR-RFLP and analyzed the association of single nucleotide polymorphisms (SNPs) with milk production traits. In addition, the study detected the relative expression levels of PRLR, ELF5and FOLR1genes in10tissues of dairy goats by real time-PCR technology, and cloned the coding sequences (CDS) of these genes and analyzed the characteristics of nucleotide and amino acid sequences using bioinformatics. Finally, the research analyzed the pyramiding effect of PRLR, ELF5and MTHFR gene on milk production traits using the analysis method of quantitative genetics. The main results were as follows:1. Molecular cloning, tissue expression and association analysis of SNPs with milk production traits in PRLR geneCaprine PRLR gene coding sequence was1746bp, encoding581amino acids. Its amino acid sequence had high similarity with those of four species:Ovis aries(98%) Bos taurus (92%), Sus scrofa973%) and Homo sapiens (68%). The result of caprine PRLR amino acid sequence analysis showed that the secondary structure contains105alpha helix,114extended chains,22β-turns and340random coils. Caprine PRLR mRNA was expressed in ovary, uterus, oviduct and breast. It showed PRLR gene was involved in the reproductive physiology and lactation activities of dairy goats. Five SNPs were detected in PRLR gene. The g.40452T>C and g.40471G>A SNPs were in intron2. The g.61677G>A and g.61865G>A SNPs were in exon9, which led to p.Ser485Asn and p.Val548Met in PRLR amino acid, respectively. The g.62130C>T SNP was in3’UTR. In two dairy goat breeds, both g.40452T>C and g.40471G>A loci were closely linked (r2>0.33), in addition, the g.61677G>A and g.61865G>A loci also showed strong linkage disequilibrium (r2>0.33). In SN dairy goats, the individuals with TT genotype had higher milk yield than those with CC genotype at g.40452T>C locus for the first parity(P<0.05); at g.61677G>A locus for the first, third and average lactation, the individuals with GG genotype had higher milk yield than those with GA genotype (P<0.05); at g.61865G>A locus for the second, third and average lactation, the individuals with GG genotype had higher milk yield than those with GA genotype (P<0.05); at g.62130C>T locus for the first, third and average lactation, the individuals with CC genotype had higher milk yield than those with CT genotype (P<0.05). In GZ dairy goats, the individuals with GG genotype had higher milk yield than those with GA genotype (P<0.05) at g.61677G>A and g.61865G>A loci for the first, third and average lactation; at g.62130C>T locus for the second, third and average lactation, the individuals with CC genotype had higher milk yield than those with CT genotype (P<0.05). Association analysis of combination genotypes in g.61677G>A, g.61865G>A and g.62130C>T loci was done in two dairy goat breeds. The result showed that the three loci had significant effects on milk yield. In SN and GZ dairy goats, C2(GGGGCC) was the best combination genotype compared with other combination genotypes. PRLR gene could be used for molecular markers breeding of milk production traits in dairy goats.2. Molecular cloning, tissue expression and association analysis of SNPs with milk production traits in ELF5geneCaprine ELF5gene coding sequence was768bp, encoding255amino acids. Its amino acid sequence had high similarity with those of four species:Bos taurus (99%), Ovis aries(99%), Sus scrofa(94%) and Homo sapiens (94%). The result of caprine ELF5amino acid sequence analysis showed that the secondary structure contained110alpha helix,29extended chains,6β-turns and110random coils. The expression level of ELF5gene in breast was significantly higher than other tissues which suggested that ELF5gene plays a key role in regulating the lactation activities. In SN and GZ dairy goats, the g.3694C>G locus was in Hardy-Weinberg disequilibrium (P<0.05), and the individuals with CC genotype had higher milk protein than those with CG genotype at g.3694C>G locus (P<0.05). In SN dairy goats, the individuals with CC genotype had higher milk yield than those with CG genotype at g.3694C>G locus for the third lactation (P<0.05). The g.3694C>G locus of ELF5gene could be used for molecular markers breeding of milk production traits in dairy goats.3. Association analysis of SNPs with milk production traits in MTHFR geneSix SNPs detected in MTHFR gene. The g.1372T>C, g.2578C>T, g.2609C>T and g.2742T>C were in5’UTR. The g.5447G>A was in intron4. The g.14635G>A was3’UTR. The g.2609C>T, g.5447G>A and g.14635G>A locus was in Hardy-Weinberg disequilibrium in SN and GZ dairy goats (P<0.05). In SN dairy goats, the individuals with CC genotype had higher milk yield than those with TT genotype at g.2578C>T locus for the third lactation (P<0.05), and the individuals with CC genotype had higher milk protein than those with TT genotype at g.2578C>T locus (P<0.05); at g.5447G>A locus, the individuals with AA genotype had higher milk protein than those with GG genotype (P<0.05); at g.14635G>A locus for the first lactation, the individuals with AA genotype had higher milk yield than those with GA genotype (P<0.05). In GZ dairy goats, the individuals with CC genotype had higher milk protein than those with TT genotype at g.2578C>T locus (P<0.05); at g.5447G>A locus, the individuals with AA and GA genotypes had higher milk protein than those with GG genotype (P<0.05); at g.14635G>A locus, the individuals with GG genotype had higher milk protein than those with GA genotype (P<0.05). Association analysis of combination genotypes in g.2578C>T and g.5447G>A loci was done in two dairy goat breeds. In SN dairy goats, the result showed that the individuals with C1(CCAA), C2(CCGA) and C3(CCGG) combination genotypes had higher milk protein than those with C7(TTGA)(P<0.05). In GZ dairy goats, the individuals with C1(CCAA) combination genotype had higher milk protein than those with C6(CTGG), C7(TTGA) and C8(TTGG)(P<0.05); the individuals with C2(CCGA) combination genotype had higher milk protein than those with C6(CTGG)(P<0.05). C1(CCAA) and C2(CCGA) were the best combination genotype compared with other combination genotypes. MTHFR gene could be used for molecular markers breeding of milk production traits in dairy goats.4. Molecular cloning and tissue expression of FOLR1geneCaprine FOLR1gene coding sequence was777bp, encoding258amino acids. Its amino acid sequence had high similarity with those of four species:Ovis aries(99%), Bos taurus (96%), Sus scrofa(80%) and Homo sapiens (80%). The result of caprine FOLR1amino acid sequence analysis showed that the secondary structure contained83alpha helix,26extended chains,5β-turns and144random coils. FOLR1mRNA had higher expression in the oviduct, lung and spleen, followed by the expression of the ovary, uterus and breast, and it was the lowest in the heart, muscle, liver and kidney. There was no polymorphism in the amplification products of different primer pairs.5. Multigene pyramiding effect of PRLR, MTHFR and ELF5genes on milk production traits in goatsIn SN dairy goats, the individuals with C1(GGGGCCCC) combination genotype had higher milk yield than those with C8(GAGACCCT) and C10(GGGACCCT) in the third lactation (P<0.05); the individuals with C1(GGGGCCCC) and C7(GAGACCCC) combination genotypes had higher milk protein than those with C6(GGGGCGTT)(P<0.05). C1(GGGGCCCC) were the best combination genotype in SN dairy goats. In GZ dairy goats, the individuals with C1(GGGGCCCC) and C2(GGGGCCCT) combination genotypes had higher milk yield than those with C3(GGGGCCTT) in the second, third and average lactation (P<0.05), in addition, the individuals with C1(GGGGCCCC) and C2(GGGGCCCT) had higher milk protein than those with C6(GGGGCGTT)(P<0.05):C1(GGGGCCCC) and C2(GGGGCCCT) were the best combination genotypes in GZ dairy goats. |
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