Isolation, Characterization, Genetic Effect And Functional Analysis Of Ten Candidate Genes Affecting Porcine Reproduction Traits

Litter size is one of the import factors which impact the benefit of porcine industry. In this study, the candidate gene approach has been emploied to discover new molecular markers associated with reproductive traits. According to physiological or biochemical functions of genes, QTL mapping results and pig-human comparative map, ten candidate genes were selected. The objective of this study was to isolate and character these candidate genes, to analyze the expression of these genes in different tissues, to identify mutations in these genes sequence and establish suitable method to detect polymorphisims, to determine associations between the polymorphisms and the reproductive performance of sows in Qingping, New Qingping line, Chinese lean type new line DIV and Large White×Meishan F₂ resource populations. The main results are as following:1. The analysis results of genetic effect of RNF4 gene(1) An 1154bp cDNA sequence of porcine RNF4 gene spanning complete coding sequences(CDS) was isolated by RT-PCR. A 4441bp genomic sequence encompassing complete intron 3, 5 and 6 was amplified from total porcine genomic DNA.(2) A PCR-Sacâ…¡-RFLP assay was established to detect the SNP-T358C in intron 5; a PCR-AluI-RFLP assay was established to detect the cSNP-C487T in exon 8 of RNF4 gene.(3) Statistical analysis demonstrated that: Sacâ…¡polymorphism is associated with TNB, NBA and LWB in the second and subsequent litters of Qingping sows, an additive effect was detected(p<0.05 or p<0.01); this SNP is associated with NBA in the second and subsequent litters of Line DIV sows(p<0.05) as well. AluI polymorphism is associated with NBA, GL and LWB in the second and subsequent litters of Qingping sows(p<0.05), an additive effect was detected(p<0.05); this cSNP is associated with TNB in the econd and subsequent litters of DIV sows(p<0.1) as well.2. The analysis results of genetic effect of HSD17B8 and HSD17B7 gene(1) A 961bp cDNA sequence of porcne HSD17B8 gene spanning the complet CDS was isolated by RT-PCR. A 2873bp genomic sequence encompassing all the introns and 729bp promoter sequence of porcne HSD17B8 gene were amplified from genomic DNA. Partial genomic sequence of porcine HSD17B7 gene was isolated as well.(2) PCR-HhaI-RFLP assay was established to detect the cSNP-A140C and cSNP-A199C in exon 2 of HSD17B8 gene.(3) Statistical analysis demonstrated that: cSNP-A140C is associated with GL in the second and subsequent litters of Qingping sows(p<0.05); this SNP is associated with LWB in the first litters of DIV sows(p<0.05) as well.3. The analysis results of genetic effect of MMP9,MMP2,MMP23 gene(1) An 8436bp genomic sequence of porcne MMP9 which encompass all the introns and 1452bp promoter sequence gene were amplified from porcine genomic DNA. A 18973bp genomic sequence of porcne MMP2 gene which encompass encompass all the introns except intron 10 were amplified from genomic DNA. A 1552bp genomic sequence of porcne MMP23 which encompass all the introns except intron 1 gene were amplified from genomic DNA.(2) For MMP9 gene: a PCR-RsaI-RFLP assay was established to detect the SNP-G-1257A in up-stream sequence; a PCR-Accâ…¡-RFLP assay was established to detect the SNP-T1284C in intron 12; a PCR-MspI-RFLP assay was established to detect the cSNP-A170G in exon7; a PCR-SmaI-RFLP assay was established to detect the SNP-T311C in intron 10. For MMP2 gene: a PCR-ForkI-RFLP assay was established to detect the SNP-G637A in intron 1.(3) Statistical analysis demonstrated that: RsaI polymorphism is associated with TNB, NBA, LWB and WW in the second and subsequent litters of Qingping sows(p<0.05), an additive effect was detected(p<0.05); this SNP is associated with LWB in the second and subsequent litters of New Qingping line(p<0.05), an additive effect was detected(p<0.05). Accâ…¡polymorphism is associated with NBA in the first litters of Line DIV sows(p<0.05), a dominent effect was detected(p<0.05); this SNP is associated with TNB in the second and subsequent litters of Line DIV sows(p<0.05), a dominent effect was detected(p<0.05) as well. MspI polymorphism is associated with TNB, NBA and NSP in the second and subsequent litters of Qingping sows(p<0.05), an additive effect was detected(p<0.05); this SNP is associated with NBA in the second and subsequent litters of Line DIV sows(p<0.01), an additive effect was detected(p<0.05) as well. ForkI polymorphism is associated with TNB in the second and subsequent litters of Line DIV sows(p<0.05).4. The analysis results of genetic effect of OVGP1 gene(1) Partial genomic sequence of porcne OVGP1 gene which encompass intron 6 and 9 were amplified from genomic DNA.(2) A PCR-.PstI-RFLP assay was established to detect the SNP-G215A in intron 6; a PCR-EcoRI-RFLP assay was established to detect the SNP-T934A in intron 9 of OVGP1 gene.(3) Statistical analysis demonstrated that: EcoRI polymorphism is associated with NBA in the second and subsequent litters of Qingping sows, additive and dominent effect were detected(p<0.05 or p<0.01); this polymorphism is associated with OW(p<0.05) in LargeWhitexMeishan F₂ resource populations as well.5. The analysis results of genetic effect of PREI3 gene(1) A 697bp cDNA sequence of porcine PREI3 gene spanning partial CDS was isolated by RT-PCR. A 2086bp genomic sequence encompassing complete intron 4, 6 and 7 was amplified from total porcine genomic DNA.(2) A PCR-MspI-RFLP assay was established to detect the SNP-T802G in intron 6 of PREI3 gene.(3) Statistical analysis demonstrated that: MspI polymorphism is associated with NBA in the sEcond and subsequent litters of New Qingping sows(p<0.05); this SNP is associated with TNB, NBA in the first litters and NAB in the sEcond and subsequent litters of DIV sows(p<0.05), an additive effect was detected(p<0.05). 6. The analysis results of genetic effect of SPAG1 gene(1) A 1715bp cDNA sequence of porcine SPAG1 gene spanning partial CDS was isolated by RT-PCR. A 2985bp genomic sequence encompassing complete intron 24 and 25 was amplified from total porcine genomic DNA.(2) A PCR-Sau3AI-RFLP assay was established to detect the cSNP-T48C in intron 6 of SPAG1 gene.(3) Statistical analysis demonstrated that: Sau3AI polymorphism is associated with UW(p<0.01) and OW(p<0.05) in Large White×Meishan F₂ resource populations, a dominent effect was detected(p<0.05).7. The analysis results of genetic effect of BYSL gene(1) A 1620bp cDNA sequence of porcine BYSL gene spanning complete CDS was isolated by RT-PCR. A 2892bp genomic sequence encompassing all the introns except intron 1, 2 was amplified from total porcine genomic DNA.(2) A PCR-MspI-RFLP assay was established to detect the cSNP-C96G in exon 4 of BYSL gene.These results indicate that the SNP-T358C in inron 5 and cSNP-T487C in exon 8 of porcine RNF4 gene; SNP-G-1257A in promoter region and cSNP-A170G in exon 7 of porcine MMP9 gene could be useful to accelerate the genetic improvement of reproductive traits. Further investigations in more pig populations with large sample size are needed to confirm these. In addition, the identification, polymorphism and expression analysis of these ten candidate genes would be useful in discoving their biological function as well as the molecular mechanism of litter size trait.