Keratin5Gene Promoter Polymorphism And Their Associations With Wool Traits In Inner Mongolia Cashmere Goats

In this study, the materials are150individuals of the Inner Mongolia cashmere goats. We used the DNA sequencing and sequence analysis, bioinformatics analysis of SSCP technology for cloning and analysis of the Cashmere goat K5gene promoter region, and probing the K5gene promoter polymorphism. Application of biometrics for statistics to analyzed some wool traits. The wool traits were comprised of wool yields, length of hair, fineness of hair, length of velour, fineness of velour and thickness of velour. Account of the gene frequency, equilibrium, genetic polymorphism and statistic analysis of three SNP associations with wool traits. We obtained the corresponding molecule genetic information and found the relation wool traits with DNA marker. These would benefited for the application of DNA marker related to economic traits on marker-assist-selection, and improvement and promotion of Inner Mongolia Cashmere goat, and provided the theory accordance.EXPERIMENT1:We designed the6pairs of primers to cloned the Cashmere goat4671bp K5promoter sequence. PCR amplification used the20Cashmere goats mixed DNA as a template. Bi-directional sequencing of PCR products were purified and found that were contained three potential polymorphic loci. They were located in upstream of ATG-69,7bp,-2576bp and-2770bp, respectively. We used the bioinformatics analysis and combinet with the reports of bovine and human K5gene promoter sequence constitution and characteristic, to analyzed the Cashmere goat K5gene promoter constitution and found the Transcription start site of Cashmere goat K5gene promoter were located in upstream of translation start site ATG-101bp.Contained the two TATA boxes, and they were located in upstream of translation start site ATG-129—124bp (ATAAAA) and-178—174bp (TTAAT), respectively. Online prediction Software found (5’to3’)24species transcription factors. Among the total, the transcription factors Lyf-1(CTTGGGAGG) and CDP CR (GATTGATGGC) were specific for Cashmere goat. HNF-4, AML-1a, HSF2, AP-4, AP2, SP1, Nkx-2and GATA-1were conservation in combining site of Cashmere goat, bovine and human K5gene promoter. The two smallest enhancer in upstream of the translation initiation site ATG-138—89bp and-114—65bp. They contained a26bp overlapping region. Once again analysis the Cashmere goat K5promoter after found mutation site that found-687bp mutation site induced the occurrence of the transcription factor HSF2(AGAAGAATCT) binding site. Besides, the-2576bp mutation site caused the occurrence of the transcription factor GATA-3(GAGCGTCTC) binding site. The-2770bp mutation site did not increased any transcription factor number.EXPERIMENT2:The object of this study were150Inner Mongolia Cashmere goats, and used the SSCP method to detection the K5gene promoter polymorphism. The results were in the followings:(1) No polymorphism was detected in P1primers. P2primers amplified fragments contains-2509(G/A) and-2576(C/A) two SNP loci in K5gene promoter. The phenotype were AA, BB, CC, AB, AC, BC and CD, and genotype frequencies were49.3%,1.3%,5.3%,19.3%,15,3%,7.3%and1.3%. After the χ2test, the allele frequencies not occupy in Hardy-Weinberg equilibrium (P<0.05), polymorphic information content (PIC) was0.5039and belong to highly polymorphic (PIC>0.5).(2) P3primers amplified fragments contains a one SNP loci, and located in-2770(G/A) of ATG upstream. The phenotype were A A, AG and GG, and the size of genotype frequencies were AA>AG>GG. After the χ2test, the allele frequencies not occupy in Hardy-Weinberg equilibrium (P<0.05), polymorphic information content (PIC) was0.3333and belong to moderate polymorphic (0.25<PIC<0.5). The correlation analysis showed that:(1) The velour length of AB, BC and CD genotype individuals in P2primers was significantly higher than AA, BB, CC and AC genotype (P<0.05). The seven genotypes were not affected for wool yields, length of hair, fineness of hair, fineness of velour and thickness of velour significantly (P>0.05).(2) The three genotype in P3primers were not affected for wool traits (P>0.05). Therefore, the-2509(G/A) and-2576(C/A) two SNP may be DNA marker of fineness of velour.