Molecular Characterization Of Male And Female Origin And Genetic Differentiation In Chinese Cashmere Goat Breeds

China has the richest cashmere goat genetic resources in the word. The origin of cashmere goats has long been considered a valuable research field in goat evolution. Many Chinese researchers have done much work on the origin of Chinese indigenoug goat breeds. But we have little knowledge on the origin, genetic differentiation, and breed genetic status of Chinese indigenous cashmere goats. In the present work, the complete control (D-loop) region and Cyt b gene of mitochondrial DNA and SRY gene coding region were sequenced in Chinese cashmere goat breeds, and were analyzed by combining the corresponding sequences of wild goats available in GenBank, in order to search for genetic markers on Y chrosome and mt DNA genome, and investigate deeply the molecular characterization of male and female origin in Chinese cashmere goat breeds, as well as, illuminate the quality characteristic of Chinese cashmere goat breeds from molecular level.These results will provide scientific basis for the genetic resource protection and utilization of Chinese cashmere goats. The conclusions are as follows in this work.(1) The fragment of SRY gene containing entire encoding region of male cashmere goat was cloned with a pair of designed primers based on the sequence of goat SRY gene sequence from GenBank. The results are as follows. The entire coding region of China cashmere goat SRY gene was 723 bp in length, encoding a peptide with 240 amino acid residues, and it contains the HMG-box with 231bp between position 190 and 420, encoding 77 amino acid residues (H64-A140). We analyzed the first structure and predicted the secondary structure and the three-dimensional structure of SRY protein, as well as, the component of SRY amino acid, characterization, structure domain and three-dimensional structure of SRY protein were also analyzed in the present work. The results showed that SRY proteinw of cashmere goats are hydrophilic without signal peptide and transmembrane region, and they have some characteristics of regulator proteins. They have three helixes and their space structure is the shape of"L", as same as the known human SRY protein. The corresponding nucleotide sequences in GenBank of goat, sheep, cattle, yak, water buffalo, forest musk deer, alpine musk deer, sika deer, hog deer, red deer, moose, white-lipped deer, elk, and pig had identities of 100%, 97.0%, 89.2%, 89.2%, 88.3%, 87.1%, 86.9%, 86.1%, 86.3%, 86.3%, 86.4%, 86.1%, 86.5% and 67.4%, and the deduced amino acid sequences had identities of 100%, 95.4%, 83.3%, 83.3%, 82.5%, 80.0%, 79.1%, 77.5%, 77.9%, 77.9%, 79.5%, 78.3%, 78.7%, and 55.7%, respectively, with the cashmere goat. These results supported well the viewpoint that SRY gene is rather conservative in evolution of species. The clustering based on the sequences of encoding region and deduced amino acid of SRY gene among species is generally in agreement with the classic taxonomic relationship. Therefore, SRY gene coding region might be useful in the construction of phylogenetic tree among different species. (2) Based on the method of purified PCR products sequencing, complete sequence of mitochondrial DNA (mtDNA) D-loop region was sequenced in 150 individuals from 11 China cashmere goat breeds. The complete sequence of mtDNA D-loop region is 1,212 or 1,213 bp in length. 144 sites were polymorphic (11.88% in 1,213) with 120 parsimony informative polymorphic sites, 24 singleton polymorphic sites and one insertion/deletion in 1075 sites. High number of nucleotide difference, nucleotide diversity and haplotype diversity were manifested in analyzed cashmere goat populations. All sequence polymorphic sites defined 94 haplotypes. Many breeds had its unique haplotypes, and the distributions and frequencies were unequilibrium either among breeds or within breeds. The average number of nucleotide differences, haplotype diversity, and nucleotide diversity of different breed range from 9.448 to 20.385, from 0.886 to 0.982, and from 0.0079 to 0.0172, respectively, indicating there is a rich genetic diversities in China cashmere goat breeds. The curve of nucleotide mismatch distributions in 11 China cashmere goat breeds took on a near unimodal, and the Fu's Fs neutrality test was significant (Fs= -23.9175, P<0.01) indicating that China cashmere goats might undergo expansions.(3) The complete sequence of Cyt b gene, which is a encoding protein gene, was sequenced in 55 individuals from 11 China cashmere goat breeds based on the method of purified PCR products sequencing. The complete sequence was 1140 bp in length that coded 379 amino acids and one termination. 21 sites were polymorphic with 15 parsimony informative polymorphic sites and 6 singleton polymorphic sites. The mutations of 6 sites including site 190, 287, 743, 728, 905 and 111 caused the amino acid changes. All sequence polymorphic sites defined 26 haplotypes. The haplotype frequencies, haplotype diversity and average number of nucleotide differences of different breeds range from 0.200 to 1.000, from 0.000 to 1.000, and from 0.000 to 3.200, respectively. Rich adenine presented in the 1st codons, while the 2nd had rich thymine. The base composition showed more bias at 3rd codon with the lowest guanine content (average only 4%). There was an obvious bias in codon usage of Cyt b gene.(4) Phylogenetic trees of NJ, ME and UPGMA method and median-joining network map were constructed based on the haplotypes defined from the sequences of mtDNA D-loop with wild goat sequences from GenBank. The results showed that there was a close genetic relationship between China cashmere goat and Capra falconeri, suggesting China cashmere goats might origin from Capra falconeri, while there no obvious divergence among China cashmere goats. The Kimura 2-parameter distances, number of nucleotide differences, number of nuc. subs. per site, and FST value between breeds range from 0.00916 to 0.01677, 10.95111 to 19.87662, 0.00904 to 0.01640, -0.02054 to 0.13538, respectively. On the whole, the result of NJ phylogenetic trees based on genetic distances of Kimura 2-parameter and Dxy of mtDNA D-loop sequences were in accordance with geographical relationship of main producing region for these cashmere breeds. But, for a few breeds, the multidimensional scaling plot of pairwise FST values constructed by SPSS software were not in accordance with geographical relationship of main producing region, indicating a weak geographical structure among breeds. The hierarchica components of mtDNA D-loop variation computed under the analysis of molecular variance (AMOVA) framework showed that smaller percentage existed among breeds (5.48%), while 94.16% within breeds with significant differences (P<0.001), indicating there were no significant divergence and weak genetic structure among breeds. The clustering and median-joining network based on the sequences of Cyt b gene were generally in agreement with that of mtDNA D-loop sequences.