| Reproductive traits of swine, including puberty, litter size, weight at birth, and so on, are important economic traits in modern pig production, which highly influence productive efficiency and economic profits. And also they are quantitative traits of low heredity, which are easily influenced by environmental factors. Identification of genes associated with reproductive traits could give an insight into the genetic mechanism of pig reproductive performance and be helpful for pig breeding with high reproductive performance. Candidate gene approach is one of the strategies in mapping quantitative traits. Some major genes have been mapped successfully by this method, e.g. sex-linkage dwarf gene of chicken, stress syndrome gene of swine and estrogen receptor gene of swine, and so on.To investigate the genes associated with swine reproductive traits, we selected FSHβ, FSHR, LHR, PGR, KISS-1and GPR54gene as candidate genes to analyze the relationship of those genes with reproductive performance in Xiaomeishan pig population. Mixed genome DNA pool and direct sequencing methods were used to screen the SNPs of those above six genes, PCR-SSCP, PCR-RFLP, mismatched PCR-RFLP, and direct PCR methods to detect at each SNP locus in a large scale, and then their relationship with reproductive traits in Xiaomeishan pig population were analyzed. The developmental patterns of FSHβ, FSHR, LHR, PGR, KISS-1and GPR54mRNA expression in the hypothalamic-pituitary-gonadal (HPG) axis in Xiaomeishan pig population and serum FSH, LH, E2and P4were determined by relative quantitative RT-PCR and ECLIA methods, respectively. The main results were as follows:1. All exons in FSHβ gene were screened, and seven SNPs in total were detected. No mutations were detected in exon1, but one SNP in intron1, that is, C5807A; one SNP in exon2, that is, C6699T; five SNPs in exon3, that is, A8753G, C8766T, T8788C, A8880G and8943delllbp. Only transition C6699T in those above SNPs was located in CDS region, but not causes the change of amino acid, and was a synonymous mutation. In Xiaomeishan pig, Fengjing pig and Large White pig populations, there were4,4and3loci displayed polymorphisms in FSHP gene, respectively, and all were highly informative loci. Those above seven SNPs were in intense state of linkage disequilibrium. It could be found that genotype BB at FSHβ-1locus, genotype DD at FSHβ-2locus, genotype FF at FSHβ-3.1, genotype HH at FSHβ-3.2and haplotype combination H5H5(BDFH/BDFH) had significant effect or highly significant effect on total number born (TNB) or number born alive (NBA) traits in Xiaomeishan sows after the second parity or sows in all parities, the heredity of which is mainly influenced by gene additive effect at each locus. So haplotype combination H5H5is a predominant genotype, and can be regarded as a molecular marker in pig breeding of reproductive traits.2. Six SNPs were discovered in swine FSHR gene. Amongst them, three SNPs were in exonl, that is, C70T, C74G and C81T; three SNPs in exon10, that is, C1166T, T1491C and T1977C, in which transversion C74G and transition C1166T caused the change of amino acid, that is, Thrl3Ser and Thr377Ile, respectively. In Xiaomeishan pig, Fengjing pig and Large White pig populations, there were3,3and4loci displayed polymorphisms in FSHR gene, respectively; FSHR-1locus in Xiaomeishan pig was highly informative locus, the other were reasonably informative loci; three loci in Fengjing pig were all reasonably informative loci; FSHR-1locus in Large white pig was highly informative locus, the other were slightly informative loci. It could be found that genotype AC at FSHR-1locus, genotype BB at FSHR-10.4locus, genotype DD at FSHR-10.5and haplotype combination H3H5had significant effect or highly significant effect on TNB or NBA traits in Xiaomeishan sows after the second parity or sows in all parities, the heredity of which is mainly influenced by gene dominant effect at FSHR-1locus and by gene additive effect at FSHR-10.4and FSHR-10.5loci.3. Only one SNP was found in exonll of swine LHR gene, that is, C1351T, which not caused the change of amino acid, and was a synonymous mutation. And it only existed in Xiaomeishan pig population, not in Fengjing pig and Large White pig populations. The results suggested that genotype MN at LHR-11.4locus had significant effect or highly significant effect on TNB or NBA traits in Xiaomeishan sows after the second parity or sows in all parities, the heredity of which is mainly influenced by gene dominant effect at LHR-11.4locus.4. Only one SNP was detected in exonl of swine PGR gene, that is, C1319T, which not caused the change of amino acid, and was a synonymous mutation. Polymorphism was detected in all three pig populations. The result showed that AB genotypic sows of Xiaomeishan pigs produced more TNB and NBA than AA genotypic sows after the second parity or in all parities.5. Six SNPs were found in swine KISS-1gene. Amongst them, one SNP was in5’-UTR, that is, A7G; one SNP in exon1, that is, G41T; three SNP2in intron1, that is, A859G, C1026A and633addl9bp; one SNP in exon2, that is, G2343C. In Xiaomeishan pig, Fengjing pig and Large White pig populations, there were4,5and5loci displayed polymorphisms in KISS-1gene, respectively. All four loci in Xiaomeishan pig were highly informative loci; KISS1-4locus in Fengjing pig and KISS1-5locus in Large White pig were reasonably informative loci, the other slightly informative loci. SNPs A7G and G41T, A7G and C1026A, G41T and C1026A were all in complete state of linkage disequilibrium. The results indicated that genotype AB at KISS1-3locus, genotype KL at KISS1-4locus, genotype MN at KISS1-6, genotype PQ at KISS1-7and haplotype combination H1H5(ALNQ/BKMP) had significant effect or highly significant effect on TNB or NBA traits in Xiaomeishan sows after the second parity or sows in all parities, the heredity of which is mainly influenced by gene additive effect at each locus.6. Three SNPs were detected in swine GPR54gene. Amongst them, one SNP was in exon1, that is, T245C; one SNP in intron1, that is,1984addlbp; one SNP in exon5, that is, T3295C, in which transition T245C caused the change of amino acid, that is, Leu35Pro. In Xiaomeishan pig, Fengjing pig and Large White pig populations, there were3,2and3loci displayed polymorphisms in KISS-1gene, respectively. GPR54-1.1locus in Large White pig was slightly informative locus, the other in three pigs were all reasonably informative loci. SNPs1984addlbp and T3295C were in complete state of linkage disequilibrium. The results indicated that genotype BB at GPR54-1.1locus, genotype CD at GPR54-3locus, genotype EF at GPR54-5.1and haplotype combination H1H1(BDE/BDE) had significant effect or highly significant effect on TNB or NBA traits in Xiaomeishan sows after the second parity or sows in all parities, the heredity of which is mainly influenced by gene additive effect at GPR54-1.1and by gene dominant effect at GPR54-3and GPR54-5.1loci.7. The developmental patterns of FSHp, FSHR, LHR, PGR, KISS-1and GPR54mRNA expression in the hypothalamic-pituitary-gonadal (HPG) axis in Xiaomeishan pig population were studied. The results showed that FSHp, FSHR, LHR, PGR, KISS-1and GPR54mRNA were all tested in different tissues in HPG axis from birth to5-month-old in Xiaomeishan pig. FSHβ gene presented high expression in pituitary and low expression in oviduct and ovary. FSHβ gene expression in pituitary was54.557times higher than in oviduct, and it increased with the age, attained the peak at4-month old, and then declined. FSHR gene performed high expression in hypothalamus before4-month-old, and high in ovary and hypothalamus after4-month-old, and low in uterus during the sexual development phases. LHR gene showed high expression in hypothalamus in all phrases, and after4-month-old also high in ovary. PGR gene showed high expression in uterus in all phrases, after4-month-old high in hypothalamus and ovary, but low in pituitary in all phrases, and it increased with the age, attained the peak at4-month old, and then declined in all tissues, especially in ovary. KISS-1and GPR54genes presented high expression in hypothalamus after4-month-old. But there were big difference between them before4-month-old, KISS-1gene high in uterus and GPR54gene different. We also found that the tendency of KISS-1and GPR54gene expression in hypothalamus, pituitary, ovary and uterus were the same as FSHp gene in pituitary, FSHR gene in ovary and uterus, LHR gene in ovary and hypothalamus, and PGR gene in all tissue.8. The patterns of serum FSH, LH, E2and P4during sexual development phrases in Xiaomeishan pig population were also studied. The results showed that at birth serum FSH, LH, E2and P4were all in high levels, there was a slight fluctuation from1-4month old, but presented a increasing trend, and attained a peak at4-month-old, had significant or highly significant difference, at5-month-old they all rapidly decreased. Based on the changes of those four above hormones in association with the practice, it could be speculated that the puberty time of Xiaomeishan pig was between3.5-month-old and4.5-month-old. |
PDF Full Text Request