Molecular Evolution And Molecular Clock Hypothesis Testing Based On Structural Gene Of Domestic Species Of Bovinae

Blood samples of six domestic species of Bovinae were collected from Bos taurus, Bos indicus, Bos grunniens, Bos frontalis, Swamp buffalo(Bubalus bubalis) and Water buffalo(Bubalus bubalis) using simple random sampling method in typical colony which avoiding the same bloodline into the samples. Single nucleotide polymorphisms and molecular evolution characters were analyzed basing on complete coding sequence of growth hormone gene, myostatin gene and mt DNA cytochrome b gene from six bovine species combinating PCR technology and direct DNA sequencing technology. Non-parameter test was used to testing relative evolution rate of nucleotide sequences and amino acid sequences of Cyt b gene together with cited sequences of other bovine species which provide partial evidences for molecular clock hypothesis. The conclusions indicated as follows: 1. Sixteen and twenty-four SNP sites were determined from coding region of GH gene and MSTN gene of six bovine species respectively. These SNP sites all showed two-allele model whether within species or between species. Two hundred and twenty SNP sites were determined from coding region of Cyt b gene, most sites showed two-allele model, 5% sites showed three-allele model and only 0.55% sites showed four-allele model.2. There was no complete accordance between haplotype polymorphism and nucleotide polymorphism of three structure genes in different bovine species which represented the variation track of genetic diversity extent of different gene. There was accordant variation between totall haplotype polymorphism and nucleotide polymorphism of three structure genes of six bovine species, which reflected the decreased polymorphisms gradually of Cyt b gene, MSTN gene and GH gene of domestic species of Bovinae.3. There was little difference in base composition of coding region sequences of structure gene whether within species or between species. Most base substitution arised in the third codon, few arised in the first codon and the second codon was the most conservative. Transition biased was observed and the ratio of transition to transversion were 4.6, 2.8 and 5.4 in GH gene, MSTNgene and Cyt b gene respectively.4. Codon usage biased arised in the coding region of GH gene, MSTN gene and Cyt b gene. There were twenty-five, thirty and twenty-seven biased codons in GH gene, MSTN gene and Cyt b gene respectively. Eleven biased codons were found in GH gene and MSTN gene. Five biased codons were shared between exo-nucleus mtDNA Cyt b gene and nucleus gene, which coding the same amino acid.5. Most substitutions were synonymous and nonsynonymous (missense mutation) was greatly less than synonymous in the coding region of GH gene, MSTN gene and Cyt b gene. The ratio of synonymous and nonsynonymous substitution was all less or equal to one, which manifesting that three structure genes of domestic species of Bovinae were not affected by Darwin positive selection in the process of evolution and few site was affected by negative selection which was the result of purifying selection. These results further demonstrated that three structure genes still retain important biological functions.6. Molecular tree basing on GH gene coding sequence showed that swamp buffalo and water buffalo clustered and there was obvious differentiation between these two buffalo together with very obvious differentiation between these two buffalos and Bos taurus, Bos indicus, Bos grunniens, Bos frontalis. These four bovine species shared an identical sequence, which maybe an ancestral sequence of them and other sequences could be regarded as the random mutation type deriving from it (mainly affected by genetic drift).7. Molecular tree based on coding sequence of MSTN gene showed that there was not only obvious differentiation between swamp buffalo and water buffalo but also more obvious differentiation between these two buffalos and Bos taurus, Bos indicus, Bos grunniens, Bos frontalis. No complete differentiation appeared among Bos taurus, Bos indicus, Bos grunniens, Bos frontalis and no shared nucleotide sequence among them. Except few shared sequence between few bovine species, the rest haplotypes were specific types of different bovine species. 8. The phylogenetic relationship revealed by Cyt b gene showed that domestic species of Bovinae could be divided into four genus with obvious differentiation which including Bos, Bibos, Poephagus and Bubalus. The phylogenetic relationship between Bos and Bibos, Poephagus, Bubalus decreased gradually.9. There was obvious differentiation of gayal population in China from maternal origin and it maybe possess a special origin. Although part haplotypes of Bos frontalis clustered with haplotypes of Bos taurus and Bos indicus, it couldn't origin from Bos taurus or Bos indicus. This phenomenon could be regarded as that Bos frontalis, Bos taurus and Bos indicus once shared identical ancestral sequences. The basic characters of these ancestral sequences were still retained in Bos frontalis in the process of long evolution. There was very close relationship between Bos frontalis and Bos gaurus, which showed that they might share the same maternal origin in an anterior time and maybe the progeny of a kind of extinct wild bovine species.10. It was sure that Leiqiong cattle origined from Bos indicus from maternal genetics. Leiqiong cattle was also few cattle breed of zebu type possessing single origin. Network relationships of Bos indicus showed that Leiqiong cattle and other Asian zebu might be domesticated independently from wild Bos nanadicus in different locality. It was deduced that there was an independent domesticated center of Bos nanadicus in antiquity in the south of China.11. The result from molecular clock testing showed that there was no obvious relevance between accepting or refusing molecular clock hypothesis and phylogenetic relationship of tested species. Molecular clock hypothesis existed in some extent of species. It was easy to refusing molecular clock hypothesis basing on the tested result from nucleotide sequence compared with the result from amino acid sequence.12. There was no gene or the product of gene variing in a stable rate absolutely in long evolution. Searching of popular molecular clock of gene or protein was in vain, that was to say, molecular clock only existed in some extent and possessed no generality.