| This study was intended to investigate the relationship between ZP3 genes’ mutations and altitude and litter size,investigate the ZP3 gene’s evolution in different specises and the selection types in sheep populations, find the SNP sites in ZP3 gene that associated with high fertility, which will provide a theoretical reference in sheep breeding work.Nine populations living in different altitudes(Plateau-type Tibetan Sheep, Oura-type Tibetan sheep, Valley-type Tibetan sheep, Ujimqin sheep, Sunite Sheep, Tan sheep, Guangling big-tailed sheep, Small-tailed Han sheep and Hu sheep) were used as the experiment material. We detected the SNP sites of sheep ZP3 gene with mixed samples, separated by genotypes through direct sequencing, analyzed the association between allele frequencies, genetype frequencies and altitude. ZP3 genes of 15 species were chosen for phylogenetic relationships, similarity, and evolution analysis, and we determined the intraspecific evolution in ZP3 genes’ SNP sites among nine sheep populations. We collected 158 Hu sheep lambing data, and analyzed the relationship between SNP genotypes, haplotypes and diplotype of linkage area and litter size.The results are as follows:1.The intra-species evolution analysis of ZP3 gene among 15 species showed that ZP3 was under strong positive selection.In the Naive Empirical Bayes(NEB) of model M2 a,absence of positive selection sites was found, but in Bayes Empirical Bayes(BEB), three positive selection sites(364V, 371 S, 372T) were identified. In NEB of model M8, 83 positive selection sites were identified, including seven sites were under significant positive selection(344Q, 364 V, 371 S, 372 T, 374 A, 399 T, 401 L, P>0.95); in BEB, eight positive selection sites(344Q, 364 V, 371 S, 372 T, 374 A, 399 T, 401 L, 402P) were found. Most of these amino acids are located in the sperm specific recognition binding area.2. 20 SNP sites were detected by the mixed sample sequencing in sheep ZP3 gene. There are 10 sites in introns,10 in exons. g.78T>C, g.195G>A, g.2293C>T, g.7576C>T are synonymous mutations, g.259A>C and g.7610G>A are non-synonymous mutations.3. Association analysis results of allele frequency and altitude in nine sheep populations’ SNP loci were displayed: at the site of g.7202A>G, allele A has a significant positive correlation with altitude, and G has the negative relationship; at g.9438G>C, allele G was significantly positive correlated with altitude, and C was negatively correlated. The association results of genotypes with altitude showed: in g.7179G>T locus,genotype GG has significant positive correlation with altitude,and GT has the negative correlation; in g.7202A>G locus, genotype AA was positively correlated with altitude, and AG was negatively correlated; in g.9438G>C locus, GG genotype was under significant positive correlation with altitude, GC genotype was under negative correlation.4. Intra-breed evolution analysis of ZP3 SNP sites was detected by Lositan among 9 sheep populations, the average differentiation coefficient(Fst) was 0.038, indicating that only 3.8% of the variation among populations was generated, 96.2% of the variation within each group was generated, and only two sites(g.2046G>A and g.7576C>T) were under strong positive selection, the remaining sites were under neutral selection.5.The association between SNP genotypes, three chain haplotype blocks and double haplotype and first and second parity litter size in Hu sheep were analyzed and the results showed that: the g.328G>A site is significantly correlated(P<0.05) with the first litter size.indivisuals of genotype GG had more litter size than GA in the first parity. The g.7342A>G site was significantly correlated(P<0.05) with litter size. AG genotype had more litter size than AA genotype. The results of second parity showed no significant association with litter size(P>0.05). The results of association analysis between SNP haplotype, diplotype and the first and second parity litter size were not significant(P>0.05). |
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