Genetic Variations Analysis Of Goat LHX3 Gene And MYF5 Gene CDS Cloning

In this study, the single nucleotide polymorphisms (SNPs) of LHX3 gene were detected in 1073 goats belong to four Chinese indigenous breeds (GZ, XS, INWC and HNBG) by DNA pools, sequencing and PCR-RFLP methods, and their genetic population structures were analyzed in the four breeds. To assess whether LHX3 gene could be efficient molecular markers for marker-assisted selection (MAS), the associations between these SNPs or their combinations and economic traits were evaluated in dairy goat, which will provide some genetic information for the efficient selection program in goat breeding. Furthermore, the coding sequence (CDS) of goat Myf5 gene was cloned by RT-PCR method, and the amino acid sequence and their specific domains were predicted through bioinformatics. The results were as follows:1. Polymorphisms detection and population genetic analysis of goat LHX3 geneFour novel SNPs (AY923832: g.7778 A>T, g.8035 T>C, g.10592 T>C and g.11260 T>C) of goat LHX3 gene were identified, which were located at intron 2, intron 2, exon 6 and 3'UTR, respectively. Among the above mentioned four SNPs, the SNP in exon 6 revealed a synonymous mutation. The g.11260 T>C mutation was not detected in INWC and HNBG goat populations. The four SNPs can be genotyped by the method of DraI,HinfI,MspI and EcoRII-PCR-RFLP, so named as P3-DraI,P3-HinfI,P6-MspI and P7-EcoRII, respectively.By Chi-square fit testing, four goat breeds (GZ, XS, INWC and HNBG) were in Hardy-Weinberg disequilibrium (P>0.05) at these four loci except for the milk utility (GZ and XS) at two loci (P3-DraI and P7-EcoRII) and the meat utility (HNBG) at P6-MspI locus. In addition, Chi-square independent testing showed that the genotype distribution of the four loci was significantly different among species except for P3-DraI locus in GZ and XS breeds.Through linkage disequilibrium analysis, the results demonstrated that P3-HinfI and P6-MspI loci were in strong linkage disequilibrium in all breeds (r2>0.33), while P7-EcoRII and the other three loci were linked weakly in XS breed, respectively.2. Association of polymorphisms in LHX3 gene and economic traits in goatAssociation between single locus and economic traits revealed that different genotypes of P6-MspI locus had significant (P<0.05) effects on Body Weight and Cannon Circumference in HNBG breed. The associations were also analyzed between combinations of genotype and economic traits. The results showed that the combined genotypes indeed had more profound impacts than in separation. When combined the two P3-DraI and P3-HinfI, the different diplotypes had significant (P<0.05) effects on protein (A) and milk yield, and the diplotype TTTC had higher protein than TTCC; diplotype AATT had significantly higher milk yield compared with diplotypes ATTT, TTTT, ATTC and AACC in XS breed, respectively. The diplotype AACC had significantly higher Body Length than ATTC in HNBG breed. While in the two P6-MspI and P7-EcoRII combination, the different diplotypes had significant (P<0.05) effects on protein in GZ and milk yield in XS breeds. Diplotype TCGG had higher protein than CCTT and TTTT in GZ, while CCTT had higher milk yield than CCTG in XS.3. Cloning of goat Myf5 geneThe whole coding sequence (768 bp) of goat Myf5 gene was cloned by RT-PCR method, and was recombined to PGM-T and pEGFP-C1 vector. The homologies of cDNA nucleotide sequence of Myf5 gene with the counterparts of Bovine (NM₁74116.1), Homo (NM₀05593.2), Pan (XM₅22481.2) and Mus (NM₀08656.4) were 99%, 90%, 90% and 88%. The prediction of structure of goat Myf5 protein revealed that it had a Myogenic Basic domain, Helix-loop-helix DNA binding domain and Myf5 domain.