Polymorphism Analysis Of Multi-vertebrae Regulatory Genes Btg2 And NR6A1 In Hebei Small Tail Han Sheep

Multi-vertebra trait is a positive mutation existing in the Hebei Small Tail Han Sheep, most of which can result in lengthening of the spine, which increases the body size and mutton production significantly. PCR-RFLP, dCAPS, PCR-SSCP and sequencing were used to study the correlation between the polymorphism of Btg2 and NR6A1 gene, and the Multi-vertebra trait. The associated molecular markers were expected to be found, which will be used in Marker-Assisted Selection in order to improve meat performance of Hebei Small Tail Han Sheep, and will lay the foundation for the breeding of meat sheep.The 1226bp fragment containing partial exon3 and partial 3′UTR was amplified using a pair of PCR primers designed based on the available nucleotide sequence of Btg2 gene from cattle and pig, and was sequenced in some representative individuals with distinct phenotypes. According to the homologous alignment of the sequenced results, 4 SNPs were detected (A150G, C243T, A607G and T704C), one of which was silent mutation existing in the exon.The dCAPS primers were designed according to A150G, C243T and A607G, respectively, followed by population analysis via PCR-RFLP. The results of independence Chi-square test showed that genotype frequencies at Site 150 had no significant difference among different vertebral patterning sheep (P>0.05). At Site 243, the genotype frequencies had extremely significant difference among different vertebral patterning sheep (P<0.05), and the resegmentation of Chi-square test showed that the genotype frequencies were extremely significantly different (P<0.01) between T14L6 and T14L7, and between T14L6 and T14L5, and were significantly different (P<0.05) between T14L6 and T13L7. At site 607, the genotype frequencies were significantly different (P<0.05), and according to the resegmentation of Chi-square test, the genotype frequencies between T14L6 and T14L7 were significantly different. The results showed that Site 243 and Site 607 might be associated with multi-vertebra trait. According to independence Chi-square test, haplotypes showed significant difference among different phenotypic individuals, and the haplotype GTA might be related to multi-vertebra trait.8 pairs of primers have been designed based on the available DNA and mRNA sequences of NR6A1 gene from cattle and pig, followed by amplification of the complete coding sequence (CDS) in Hebei Small Tail Han Sheep. Mutation screening has been carried out via PCR-SSCP and direct sequencing, which showed there was one silent mutation (T25G) at the 25th base in exon5. With respect to this site, the population genotyping and independence Chi-square test showed that the genotype frequencies were significantly different (P<0.05) among the five kinds of multi- vertebra phenotypes, and according to the resegmentation of Chi-square test, the genotype frequencies were significantly different (P<0.05) between T14L5 and T14L6, and between T14L5 and T14L7, showing this site might be related to the multi-vertebrae trait.