Study On Candidate Genes Of Fecundity And Gene Expression In Goat

Seeking major gene influencing littler size at genetic structure or gene expression has been paid a great attention in stock farming production. To grasp the regulatory mechanism of multiplets on the level of molecular biology is the base to find the molecular mechanism of high fertility, then use it in the breeding, and select the precise location on marker assistant selection (MAS). BMPR-IB gene, BMP15 gene, GDF9 gene, FSHR gene and ESR gene as candidate genes on the fecundity of goat were studied and analyzed for their mutations and genetic effects. PCR-RFLP and PCR-SSCP were used to detect the SNP( single nucleotide polymorphism)of these gene in this study. Then sequencing, sequence analysis, ascertaining mutation type and association analysis between yean trait and polymorphism site. The mRNA levels of the ovaries in Jining Grey goat with different genotype were analyzed by semi-quantitation PCR.1. DNA samples were collected and amplified for bone morphogenetic protein receptor type IB gene (BMPR-IB) and BMP15 gene by PCR-RFLP method from 489 goats. The results showed that five markers were not polymorphic in goat and none had mutation; possibly the five markers were not the major genes for prolificacy.2. GDF9 FecGH and exon 1 as candidate genes on the fecundity of goat were studied and analyzed for their mutations and genetic effects. The results showed that there was not mutation of FecGH in GDF9 or GDF9 exon 1 of goat. It showed that the fecundity mechanism of goat was different from that of sheep. Then it was ruled out the possibility that the ovulation of goat was affected by the mutation of FecGH in GDF9 or GDF9 exon 1.3. GDF9 gene exon 2 as candidate genes on the fecundity of goat was studied and analyzed for its mutation and genetic effects. The result showed that there was a mutation in GDF9 gene exon 2 of goat. Three genotypes (AA, AB and BB) were detected in all six goat breeds. Frequency of allele A was 0.9192, frequency of allele B was 0.0808 in Jining Grey goats.The AA mutation genotype was superior in Jining Grey goat with genotype AA had 0.67 lambs more than those with genotype AB and 0.71 lambs more than those with genotype BB (P<0.05). The sequencing results indicated that there was one single nucleotide mutation (G→A) at exon 2 of GDF9 gene in goats, this mutation cause amino acid change (V→I). It could be inferred that GDF9 gene exon 2 was related with the major gene that controls the high prolificacy of goat.4. The transcriptional promoter control region 5 '-flanking of FSHR gene was studied and analyzed for its mutation and genetic effects. The result after analyzing by PCR-RFLP method showed that there was not a mutation in promoter control region of 5' terminal in gene of FSHR of goat.5. FSHR gene exon 10 as candidate genes on the fecundity of goat was studied and analyzed for its mutation and genetic effects. The result showed that there was a mutation in FSHR gene exon 10 of goat. Two genotypes (EE and FF) were detected in all six goat breeds. Frequency of allele F was 0.1692, frequency of allele E was 0.8308 in Jining Grey goats.The FF mutation genotype was superior in Jining Grey goat with genotype FF had 0.81 (P<0.05) lambs more than those with genotype EE. The sequencing results indicated that there was one single nucleotide mutation (C→T) at FSHR gene exon 10 gene in goats, this mutation did not cause amino acid change. It could be inferred that FSHR gene exon 10 was related with the major gene that controls the high prolificacy of goat.6. FSHR gene exon 10 were amplified by PCR technique and sequenced. The length of fragment was 733bp (including 3′-flanking region 37bp). Sequence comparison showed that there were 11 different sites, the two sites of 1567 and 1568 of Australian sheep were C and G, while those of Jining Grey goat were G and C. These mutations cause the code of amino acid change, CAC (histidine) became CAG (glutamine) and GTC (valine) became CTC (leucine). Other nine sites were synonymous mutations. They did not cause the amino acid change. The homology of nucleotide sequence of FSHR gene exon 10 between Jining Grey goats and Australian sheep was 98.50 percent.7. ESR gene exon 1 as candidate genes on the fecundity of goat was studied and analyzed for its mutation and genetic effects. The result showed that there was a mutation in ESR gene exon 1 of goat. Three genotypes (CC, CD and DD) were detected in all six goat breeds. Frequency of allele C was 0.6039, frequency of allele D was 0.3961 in Jining Grey goats.The CC mutation genotype in Jining Grey goat had 0.66 (P<0.05) lambs more than those with genotype CD and 0.78 (P<0.05) lambs more than those with genotype DD. The sequencing results indicated that there was one single nucleotide mutation (G→C) at exon 1 of ESR gene in goats. This mutation did not cause amino acid change. It could be inferred that ESR gene exon 1 was related with the major gene that controls the high prolificacy of goat.8. The mRNA levels of the ovaries in 9 Jining Grey goats with different GDF9 genotype were analyzed by semi-quantitation PCR. The result showed the GDF9 mRNA levels from the ovary of AA genotype ewes(0.85±0.07,0.82±0.02) were significant difference compared with AB(0.19±0.04,0.23±0.05) and BB genotype (0.31±0.09,0.27±0.04) (P<0.01). It indicated the higher mRNA levels of GDF9 from the ovary of AA genotype ewes were related to higher litter size of Jining Grey goat.9. The mRNA levels of the ovaries in 6 Jining Grey goats with different FSHR genotype were analyzed by semi-quantitation PCR.The result showed FSHR mRNA level from the right ovary of FF(1.26±0.09) ewes was higher than that of EE(0.53±0.07) ewes (P<0.01); but there were no significant difference in the left ovary among the genotypes during the oestrum. It indicated the higher mRNA levels of FSHR from the right ovary of FF ewes were related to the higher litter size of Jining Grey goat.10. The mRNA levels of the ovaries in 9 Jining Grey goats with different ESR genotype were analyzed by semi-quantitation PCR. The result showed the ESR mRNA levels from the ovary of different genotype ewes were no significant difference (P>0.05). It suggested that the high prolificacy in Jining Grey goat were no directly relative to the mRNA levels of ESR.11. The relation between different GDF9 genotype in Jining Grey goat and FSHR mRNA levels during the oestrum was studied by semi-quantity PCR. The result showed FSHR mRNA levels from the right ovary of AA (1.15±0.09) ewes were higher than those of BB(0.36±0.02) and AB(0.54±0.07) ewes (P<0.01). But there were no significant difference in the left ovary among the genotypes during the oestrum. It indicated the higher mRNA levels of FSHR from the right ovary of AA genotype ewes might be related to different GDF9 genotype in Jining Grey goat.12. The relation between different GDF9 genotype in Jining Grey goat and ESR mRNA levels during the oestrum was studied by semi-quantity PCR. The result showed ESR mRNA levels from the ovary of AA (0.93±0.11,0.82±0.04) ewes were higher than those of BB(0.26±0.07,0.28±0.03) and AB (0.56±0.05,0.53±0.04) ewes (P<0.01). It indicated the higher mRNA levels of ESR from the ovary of AA genotype ewes might be related to different GDF9 genotype in Jining Grey goat.