Adaptive Evolution Of Mitochondrial DNA Atpase Synthesis Genes In Bovini Species, And Parentage Testing Of Interspecies Crossing Between Mithun And Brahman Cattle

The major functions of mitochondrion are to generate heat to maintain body temperature and ATP for action in organisms. The mitochondrial DNA (mtDNA) mutations were considered as extremely sensitive to energy-related selective pressure and available to test the adaptation of species to the environment via mtDNA genes. Cattle have played important roles in economic, cultural and religious values accompanying the migration and farming in human history. Domestic cattle were domesticated by human, while yak have always undergone hypoxia and cold temperature besides domestication in Qinghai-Tibetan Plateau. So, they had become the ideal biological models for testing domestication and adaptative evolution to the extreme inviroment. Herein, mtDNA ATPase synthesis genes were amplified to analyze the phylogeny, population expansion and adaptive evolution after domestication and in different zones in Bovini species. In addition, we investigated the genetic diversity of Yunnan mithun population and performed the interspecies crossing between mithun (Bos frontalis, frozen semen) and zebu(Bos indicus) to provide cytogenetic and molecular evidences that allowed for the potential of interspecies crossing. It will be beneficially to provide the genetic information to promote the protection and hybridization utility in mithun. The results were as follows:1Data set (including ATPase8and ATPase6genes,882bp) was concatenated as ATPase synthesis gene, and two combined data sets from Yak (Domestic yak+Wild yak) and Domestic cattle (Taurus+Zebu) were integrated into the analyzing in the context:1.1ATPase synthesis gene was combined to construct phylogenetic trees of Bovini species with five distinct clades, Bison, Yak, Zebu, Taurus and Gaur. The results were consistent with the previous phylogeny of Bovini, which clearly showed sister-groups relationships to Yak-Bison, Taurus-Zebu, with far relationship to Gaur, when Haikou buffalo used as outgroup. All tree topologies indicated that one of mithun formed its own monophyletic clade, distinct from other species of the Bovini; in addition, Yunnan mithun possessed its own58.8%genetic components except20.6%taurine and20.6%i ndicine lineages, closest to Indian mithun based on the haplotypes of ATPase synthesis gene. Haplotype diversity and nucleotide diversity in Bovini were lower, ranging from0.348-0.724and0.058-0.259%, respectively.1.2Star-burst patterns observed for the clades and differences in the shape of each mismatch distribution prompted an analysis of population demography. The presence of population expansions were supported for clades of Yak (Tajima’s D=-1.838, P<0.05; Fu and Li’s D*=-5.489, P<0.02), Domestic yak (Tajima’s D=-1.744,0.10> P>0.05; Fu and Li’s D*=-4.487, P<0.02), and Taurus (Tajima’s D=-2.198, P<0.01; Fu and Li’s D*=-3.133, P<0.05). The Zebu clade has an ambiguous signature that showed significant deviation from neutrality for the Fu and Li’s D*test (Fu and Li’s D*=-3.691, P<0.02; Tajima’s D=-1.314, P>0.10), although not for the Tajima’s D test and an uncompleted smoothly unimodal of mismatch distribution. The absences of population expansions were rejected for clades of Bison (Tajima’s D=-0.118, P>0.10) and Gaur (Tajima’s D=1.977, P>0.10), of which the scenarios were better to fit the narrow distributions and small population sizes of bison and mithun.1.3To detect variations in selective pressure along the different branches of a phylogenetic tree, all the comparisons of Ka/Ks for ATPase synthesis gene from the clades revealed Ka/Ks values were far less than1.0, indicating ATPase complex evolved globally under strong purifying selection in Bovini species. By comparing the relative rates of nonsynonymous and synonymous substitutions in ATPase synthesis gene from inter-and intra-clades, we inferred that Yak (domestics cattle and yak) had a higher excess of nonsynonymous mutations in the ATPase synthesis gene than those in other clades, indicating adaptive changes related to tolerate low temperature and hypoxia in yak than other Bovini leaning on the ATPase complex generated the energy of basal metabolism in high-altitude environment by MK test. Similarly, through MK test, domestic cattle had a higher excess of nonsynonymous mutations than those in other Bovini clades, probably indicating domestic cattle adjusted its basal metabolism and decreased in body size. Neutrality indices (NI) were higher than1.0in Bovini species showing an excess of nonsynonymous mutations and amino acid polymorphism. The counterparts from ATP6gene in the corresponding statistics were similar to those of ATPase synthesis gene, while they differentiated from ATP8gene because of short fragment, less informative and conservational functions. ND6gene was under strong purifying selection in Bovini species based on the same analyses.2Through estrus synchronization, Brahman cows were inseminated artificially with mithun’s frozen semen to gain the F1generations of interspecies crossing. Metaphase chromosome spreads were analyzed by conventional staining and fluorescence in situ hybridization with the whole chromosome1of mithun as a specific probe. And microsatellite DN A markers were scanned to reveal the genetic diversity of mithun and parentage testing of F1generation of interspecies crossing. The results were as follows:2.1Mithun (Bos frontalis) had a karyotype2n=58(N=3,3♀), but abnormal karyotype had2n=59(N=2,16(?)1♀) with a normal mithun phenotype.2.2The F1karyotypes of mithun×Brahman cow had2n=59, intermediate between their parents, consistent heterozygous carriers with a centric fusion involving rob(2;28). The female hybrids normally reproduced with whatever mated, both parents indicating their fertility when mating naturally or artificial insemination. Meanwhile, the F1sires did not produce sperm at the age of15and24months as assessed by electroejaculation to confirm that the F1bull is sterile. The female hybrids are fertile, and the male are sterile.2.3The expected heterozygosity (He) and polymorphism information content (PIC) of Yunnan mithun population were lowest amongst the beef breeds revealed by microsatellite markers,0.6332and0.5965, respectively. Yunnan mithun population hadn’t yet undergone severe bottleneck effect while suffering moderate gene introgression from local cattle (Bos taurus or Bos indicus) by Bottleneck, showing the availability of bovine microsatellite markers to facilitate the genetic background and phylogeny of mithun.2.4According to parentage testing from Cervus, two PEc of combined non-exclusion probability (first parent), combined non-exclusion probability (second parent) were99.9858%and99.9999%, respectively. These displayed the potential of interspecies crossing between mithun (Bos frontalis) and Brahman (Bos indicus) based upon molecular biological insights. As a rare genetic resource of genus Bos, it is suggested that mithun population will be under captive breeding, or to establish Mithun Preserve to protect and avoid subsequent genetic erosion from domestic cattle due to its crossing facilitation.