| The low kidding rate is a major bottleneck restricting the development of high quality and efficient goat husbandry, and researching on the breeding methods of improving kidding rate from genetic nature is technical problems that goat breeding technology workers at home and abroad seek to overcome them. Polygene pyramiding breeding technology centering on molecular marker-assisted selection could select genotypes of litter size traits in DNA level. It overcomes the disadvantages of conventional breeding time-consuming and poor quality and rapidly improves breeding efficiency. So looking for molecular markers tightly linked with litter size traits, and researching on the regulation function and expression levels of target genes, and screening functional genes regulating litter size traits are the foundation and prerequisite for achieving organic combination of modern molecular breeding technology with conventional breeding technology and integrating innovation polygene pyramiding breeding technology system.In the study, KITLG, KIT, KISS1and NGF genes were selected as candidate genes because they played important roles in regulation of animal reproductive traits. This study investigated the polymorphisms of KITLG, KIT, KISSI and NGF genes in Xinong Saanen (SN), Guanzhong (GZ) and Boer (BG) goat breeds by DNA sequencing and PCR-RFLP and analyzed the association of single nucleotide polymorphisms (SNPs) with litter size. In addition, the study detected the relative expression levels of KITLG, KIT, KISS1and NGF genes in10tissues of goats by real time-PCR technology, and cloned the coding sequences (CDS) of KITLG, KIT and NGF genes, and analyzed the characteristics of nucleotide and amino acid sequences using bioinformatics. Finally, the research analyzed the pyramiding effect of KITLG, KIT and KISS1genes on litter size using the analysis method of quantitative genetics to provide the experimental and theoretical basis for integration innovation polygene pyramiding breeding technology system of goat litter size traits. The main results were as follows:1. Molecular cloning, tissue expression and association analysis of SNPs with litter size in KITLG geneCaprine KITLG gene coding sequence was825bp, encoding274amino acids. The amino acid sequence of caprine KITLG gene had high similarity with those of four species:Bos taurus (98%), Sus scrofa (95%), Homo sapiens (85%) and Mus musculus (81%). The result of caprine KITLG amino acid sequence analysis showed that the secondary structure contained141alpha helix,17extended chains,8β-turns and108random coils. Caprine KITLG mRNA was high expressed in ovary, breast and kidney. Six SNPs were detected in KITLG gene (g.4163G＞A, g.16474G＞T, g.17025C＞A, g.17278A＞G, g.17335T＞C and g.17453C＞T). The g.4163G＞A SNP was in intron3, and other SNPs were in3’UTR. In three goat breeds, both g.16474G＞T and g.17025C＞A loci were closely linked (r2＞0.33), in addition, the g.17278A＞G, g.17335T＞C and g.17453C＞T loci also showed strong linkage disequilibrium (r2＞0.33). Association analysis of combination genotypes in g.16474G＞T and g.17025C＞A loci was done in three goat breeds. In SN goats, the result showed that the individuals with GGCC combination genotype had higher litter size than those with TTAA in the second and average parity (P＜0.05). In GZ goats, the individuals with GGCC combination genotype had higher litter size than those with TTAA in the fouth and average parity (P＜0.05). In BG goats, the individuals with GGCC combination genotype had higher litter size than those with TTAA in the fourth parity (P＜0.05). Association analysis of combination genotypes in g.17278A＞G, g.17335T＞C and g.17453C＞T loci was done in three goat breeds. In SN goats, the result showed that the individuals with GGCCCC and GACTCC combination genotypes had higher litter size than those with GACTCT in the first parity (P＜0.05), in addition, the individuals with GACTCC combination genotype had higher litter size than those with GACTCT in average parity (P＜0.05). In GZ goats, the individuals with GGCCCC combination genotype had higher litter size than those with GACTCT in the third and average parity (P＜0.05). In BG goats, the individuals with GGCCCC combination genotype had higher litter size than those with GACTCT in the fourth and average parity (P＜0.05). These results suggest that KITLG gene could be used as molecular markers of litter size for polygene pyramiding breeding in goats2. Molecular cloning, tissue expression and association analysis of SNPs with litter size in KIT geneCaprine KIT gene coding sequence was2925bp, encoding974amino acids. The amino acid sequence of caprine KIT gene had high similarity with those of four species:Ovis aries (99%), Bos taurus (99%), Sus scrofa (94%) and Homo sapiens (90%). The result of caprine KIT amino acid sequence analysis showed that the secondary structure contained246alpha helix,223extended chains,47β-turns and458random coils. Caprine KIT mRNA was high expressed in kidney, ovary, breast and uterus. Two SNPs were detected in KIT gene (g.88430T＞A and g.120466G＞A). The g.88430T＞A SNP was in exon7, which led to Tyr>Asn at position409amino acid of KIT. The g.120466G＞A SNP was in3’UTR. At g.88430T>A locus for average parity, the individuals with TT genotype had higher litter size than those with AA genotype in BG, SN and GZ goat breeds (P＜0.05). At g.120466G>A locus for the third parity, the individuals with AA genotype had higher litter size than those with GG genotype in SN goats (P＜0.05). In GZ goats, the individuals with A A genotype had higher litter size than those with GG genotype at g.120466G＞A locus for the first parity (P＜0.05). In BG goats, the individuals with AA genotype had higher litter size than those with GG genotype at g.120466G＞A locus for the fourth parity (P＜0.05). These results suggest that KIT gene could be used as molecular markers of litter size for goat breeding.3. Tissue expression of KISS1gene and association analysis of SNPs with litter sizeCaprine KISS1mRNA was high expressed in ovary and muscle. Six SNPs were detected in KISS1gene (g.384G＞A, g.2124T＞A, g.2270C＞T, g.2489T＞C, g.2510G＞A and g.2540C＞T). The g.384G＞A SNP was in5’UTR, and other SNPs were in intron1. In three goat breeds, both g.2124T＞A and g.2270C＞T loci were closely linked (r2＞0.33), in addition, the g.2510G＞A and g.2540C＞T loci also showed strong linkage disequilibrium(r2＞0.33). At g.384G＞A locus for the second and average parity, the individuals with A A genotype had higher litter size than those with GG genotype in SN goats (P＜0.05). In GZ goats, the individuals with AA genotype had higher litter size than those with GA and GG genotypes at g.384G＞A locus for the third parity (P＜0.05). Association analysis of combination genotypes in g.2124T>A and g.2270C＞T loci was done in three goat breeds. In SN goats, the result showed that the individuals with C5(TTTC) and C6(TTTT) combination genotypes had higher litter size than those with C1(AACC) in the fourth and average parity(P＜0.05). In GZ goats, the individuals with C6(TTTT) combination genotype had higher litter size than those with C1(AACC) in the second and average parity (P＜0.05). In BG goats, the individuals with C3(TATC) and C6(TTTT) combination genotypes had higher litter size than those with C1(AACC) in average parity (P＜0.05). Association analysis of combination genotypes in g.2510G＞A and g.2540C＞T loci was done in three goat breeds. In SN goats, the result showed that the individuals with C1(AACT) combination genotype had higher litter size than those with C2(AATT) and C5(GATT) in the third parity (P＜0.05). In GZ goats, the individuals with C1(AACT) combination genotype had higher litter size than those with C6(GGCC) and C7(GGCT) in the third parity (P＜0.05). In BG goats, the individuals with C3(AATT) combination genotype had higher litter size than those with C1(AACC), C4(GACT) and C6(GGCC) in average parity (P＜0.05). These results suggest that KISSl gene could be used as a candidate gene for goat breeding.4. Molecular cloning, tissue expression and association analysis of SNP with litter size in NGF gene Caprine NGF gene coding sequence was726bp, encoding241amino acids. The amino acid sequence of caprine NGF gene had high similarity with those of five species:Bos taurus (99%), Sus scrofa(95%) Canis lupus(92%), Homo sapiens (92%) and Mus musculus(83%). The result of caprine NGF amino acid sequence analysis showed that the secondary structure contained44alpha helix,50extended chains,15β-turns and132random coils. Caprine NGF mRNA was high expressed in ovary, uterus and lung. One SNP was detected in NGF gene (g.705A＞G in exon1). At g.705A＞G locus for the second and average parity, the individuals with GG genotype had higher litter size than those with AA genotype in three goat breeds (P＜0.05). In SN and GZ goats, the individuals with GG genotype had higher litter size than those with AA genotype in the second and fourth parity (P＜0.05). These results suggest that the g.705A＞G locus of NGF gene could be used as a molecular marker of litter size for goat breeding.5. Polygene pyramiding effect of KITLG, KIT and KISS1genes on litter size in goatsIn SN goats, the individuals with C1(GGCCTTTTTT) combination genotype had higher litter size than those with C5(GTCATTAACC) and C22(GTCATATACC) in the first and average parity (P＜0.05); the individuals with C1(GGCCTTTTTT), C2(GTCATTTTTT), C3(TTAATTTTTT) combination genotypes had higher litter size than those with C11(GTCAAATTTT), C15(GTCAAAAACC) and C17(TTAAAAAACC) in the third parity (P＜0.05). In GZ goats, the individuals with C16(GGCCTATTTT) combination genotype had higher litter size than those with C4(GTCATTAACC) and C5(GGCCTTTATC) in the second and average parity (P＜0.05); the individuals with C5(GGCCTTTATC) combination genotype had lower litter size than those with C6(TTAATATATC), C9(TTAATTTATC) and C16(GGCCTATTTT) in the fourth parity (P＜0.05). In BG goats, the individuals with C1(GGCCTTTTTT), C3(TTAATTTTTT) and C6(GGCCTTTATC) combination genotypes had higher litter size than those with C5(GTCATTAACC) and C8(GTCATTTATC) in average parity (P＜0.05). In SN goats, C1(GGCCTTTTTT) was the best combination genotype compared with other combination genotypes. In GZ goats, C16(GGCCTATTTT) was the best combination genotype. In BG goats, C1(GGCCTTTTTT) was the best combination genotype and other excellent combination genotypes included C3(TTAATTTTTT) and C6(GGCCTTTATC)... |
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