Structure, Genetic Diversity, Phylogeny And Recombination Characteristics Of Ovine DSG4 Gene

Mitochondrial DNA (mtDNA) studies have shown that domestic sheep (Ovis aries) may possess five maternal lineages. However, the details of paternal origins are still unclear. To clarify the genetic background of domestic sheep, a genomic region of 41 kb encompassing complete DSG4 gene was directly sequenced in 17 indigenous Chinese and exotic sheep using 28 amplicons and 95 sequencing primers. NJ phylogenetic trees based on the Kimura-2 parameter model were constructed using MEGA 4.0 software. Pairwise comparison of the DSG4 haplotypes was carried out using MEGA 4.0 and DnaSP 5.0 software. A 635 bp long hypervariable region (14427-15061) of the ovine DSG4 gene was further resequenced in a panel of 450 individuals sampled from eight Chinese and four foreign breeds. Haplotype frequency, Hardy-Weinberg equilibrium (HWE) test and analysis of molecular variance (AMOVA) were estimated using Arlequin 3.1 software. Haplotype diversity value and neutrality test were assessed using DnaSP 5.0 software. Median joining network was reconstructed using Network software. The major research findings are as followed:1. Comparison of the genomic DNA and cDNA of ovine DSG4 gene, demonstrates that this gene is composed of 16 exons and 15 introns spanning approximately 41 kb, and the cDNA contains a CDS of 3123 bp. The exons and introns show large variations in sizes ranging from 36 bp to 765 bp and from 268 bp to 12118 bp, respectively.2. A total of 20 DSG4 haplotypes were reconstructed from the 17 complete genomic DNA sequences carrying a total of 648 polymorphic sites, of which 582 were SNPs with an average density of 14 SNPs per kb, and 66 were indels with eight of them larger than 10 bp and the largest one containing 69 bp. Analysis of the SNPs revealed 440 transitions and 145 transversions, giving a mutational ratio (transition/transversion) of 3.01. DSG4 gene had 34 SNPs and a TTG indel in its coding region, with 11 SNPs and the TTG indel leading to amino acid replacements, four (785, 891, 904, and 1018) of which possibly had impact on the structure and function of ovine DSG4 protein, as predicted by the Polyphen program.3. Through pairwise comparison of the 20 complete DSG4 haplotypes based on the difference of positional distribution of a large number of nucleotide variations, we observed that several homologous recombination events (Crossover) were clustered into a region of 15 kb-20 kb. Thus this region may contain recombination hotspots.4. The phylogenetic tree clustered a total of 20 DSG4 haplotypes (across the region of 0-15 kb) into eight distinct haplogroups. The average number of pairwise nucleotide differences among the eight haplogroups was 89 (indels excluded) ranging from 21 to 144, while the nucleotide variations within haplogroups were very limited as four maximal SNPs between haplotypes, suggesting that sheep may possess eight distinct genetic backgrounds.5. The 635 bp segment contained 22 SNPs defining 21 haplotypes. Of which, haplotypes h1 to h10 matched the first 10 complete DSG4 haplotypes H1 to H10. The h1 was the main haplotype with a total frequency of 0.493, which appeared more frequently in foreign sheep. The h2 haplotype showed a frequency of 0.194 in all eight Chinese breeds ranging from 0.036 to 0.397, but not present in any foreign sheep. The haplotype frequency of h6 was also exclusively present in Chinese sheep with a frequency of 0.074, much lower than the frequencies of h1 (0.424) and h2 (0.194) but higher than the frequencies of other 18 haplotypes in Chinese sheep. The frequencies of h3 to h10 ranged from 0.026 to 0.056, while the remaining 11 haplotypes of h11 to h21 was very rare and had frequencies ranging from 0.001 to 0.008 in all samples. Together, Chinese sheep had all 21 haplotypes, while foreign sheep only possessed eight haplotypes. The haplotype diversity (H_d) of Chinese sheep was higher than that of foreign sheep (0.766 vs 0.514). For the eight Chinese breeds, Tibetan sheep had the highest H_d (0.846) while Hu sheep showed the lowest H_d (0.451); this estimate was similar among Duolang (0.767), Tan (0.748) and Small-tailed Han sheep (0.747).6. For the 635 bp segment, all 22 SNPs conformed to the HWE in 12 breeds but three out of the 22 SNPs significantly deviated from the HWE in Tibetan sheep. The AMOVA results showed that most variation was found among individuals within populations. Tests of neutrality showed that polymorphisms of this segment in domestic sheep were possibly influenced by selection. The phylogenetic tree constructed for the 21 haplotypes indicated that the h12 haplotype was possibly originated from a distinct genetic background. Combined with the results of network analysis, we demonstrate that some rare haplotypes were possibly derived from recombination.The ovine DSG4 gene has eight different haplogroups, suggesting that domestic sheep may contain additional paternal origins different from the documented maternal lineages. The 611 bp segment of the sex determining region Y (SRY) gene was directly resequenced in a panel of 152 male sheep from nine Chinese and two Iranian breeds and two SNPs (460G>T as a novel transversion mutation identified in this study) were further scanned in additional 226 male sheep, leading to the following main results: None of the 152 SRY sequences conformed to the documented SRY sequences of Argali (Ovis ammon) and Urial (Ovis vignei) sheep; For 88A>G, the G allele was fixed in Suffolk, Texel and Polled Dorset but present only in two of the 322 Chinese sheep; For 460G>T, the T allele was observed in Tan (n = 45) and Hetian (n = 20) sheep with frequencies of 0.16 and 0.10 but not present in the remaining 22 breeds. Considering several Argali subspecies are found today in northwestern China, we postulate that Tan and Hetian sheep may contain a distinct paternal origin, thus was possibly originated from the introgression of unknown Argali species.