Study On High Prolificacy Candidate Genes In Small Tail Han Sheep

The method of PCR-SSCP to detect sheep inhibin a (INHA) gene, bone morphogenetic protein 4(BMP4) gene and bone morphogenetic protein 7 (BMP7) gene mutations were established in this study.The inhibin a (INHA) gene, the bone morphogenetic protein 4 (BMP4) gene and the bone morphogenetic protein 7 (BMP7) gene were studied as candidate genes on the prolificacy of Small Tail Han sheep. Single nucleotide polymorphisms of 5 ' regulatory region, exon 1 of INHA gene;exon 2, exon 3 and exon 4 of BMP4 gene and exon 2, exon 3 of BMP7 were detected in high fecundity breed (Small Tail Han sheep) and low fecundity breeds (Chinese Merino sheep, Corriedale sheep and South African Mutton Merino sheep) by PCR-SSCP. The results indicated that there was a mutation (316C→T) in 5' region of INHA gene in Small Tail Han sheep, Chinese Merino sheep and Corriedale sheep, the same mutation did not exist in South African Mutton Merino sheep. In Small Tail Han sheep, Chinese Merino sheep, Corriedale sheep and South African Mutton Merino sheep, frequency of AA genotype was 0.58, 0.98, 0.96, 1.00, frequency of AB genotype was 0.40, 0.02, 0.04, 0.00, frequency of BB genotype was 0.02, 0.00, 0.00, 0.00, respectively. The ewes with mutation homozygous genotype BB or mutation heterozygous genotype AB had 1.45 (P<0.01) or 0.90 (P<0.01) lambs more than those with wild type AA in Small Tail Han sheep, respectively. The sequencing results showed that there was a mutation (T→C) at 877 bp of exon 1 of INHA gene in Small Tail Han sheep and Corriedale sheep, the same mutation did not exist in Chinese Merino sheep and South African Mutton Merino sheep. In Small Tail Han sheep, Chinese Merino sheep, Corriedale sheep and South African Mutton Merino sheep, frequency of CC genotype was 0.74, 1.00, 0.89, 1.00, frequency of CD genotype was 0.25, 0.00, 0.11, 0.00, frequency of DD genotype was 0.01, 0.00, 0.00, 0.00, respectively. The ewes with mutation homozygous genotype DD or mutation heterozygous genotype CD had 1.32 (PO.01) or 0.77 (P<0.01) lambs more than those with wild type CC in Small Tail Han sheep,respectively. For BMP 4 Primer 1 was used to amplify the exon 2. Primer 2 was used to amplify the exon 3. Other five pairs of primer were used to amplify the exon 4. No single nucleotide polymorphism was detected for exon 2 and exon 4 of BMP A gene in four sheep breeds. One polymorphism was detected for exon 3 of BMP4 gene in four sheep breeds. For primer 2, three genotypes (AA, AB and BB) were detected in four sheep breeds. In Small Tail Han sheep, Chinese Merino sheep, Corriedale sheep and South African Mutton Merino sheep, frequency of AA genotype was 0.017, 0.216, 0.115, 0.429, frequency of AB genotype was 0.102, 0.317, 0.269, 0.500, frequency of BB genotype was 0.881, 0.467, 0.616, 0.071, respectively. Sequencing revealed one single nucleotide mutation (C—>A) at 305 bp of exon 3 of BMP4 gene in the BB genotype in comparison to the AA genotype, and this mutation resulted in an amino acid change: alanine—?aspartic acid. The ewes with genotype BB had 0.61 (P<0.05) or 1.01 (P<0.05) lambs more than those with genotype AB or AA in Small Tail Han sheep. These results preliminarily showed that the INHA gene and the BMP4 gene is either a major gene that influences the prolificacy of Small Tail Han sheep or a molecular genetic marker in close linkage with such a gene.For BMP7 Primer 1 was used to amplify the exon 2. Primer 2 was used to amplify the exon 3. No single nucleotide polymorphism was detected in four sheep breeds. This result indicated that the detected loci of BMP7 gene had no significant effect on prolificacy of Small Tail Han sheep.A new PCR method— AuNPs-PCR was used for amplifying exon 3 of BMP4, which can enhancing the specelity of PCR.