| The yaks in the world origin from China, and now the most of the yaks live in China. During the period of 5,000-10,000 years domestication, many populations of yaks were formed through migration, segregation, natural selection, and artificial breeding, which had particularly excellent characteristics of germplasm. The number of yak breed which were recorded only in "Bovine Breeds in China" were five, and number of the well-known regional excellent breed were more than 11. As for the classification, genetic differentiation, and phylogeny of yak in China, scholar in-country or overseas had different opinions, and the current research works mainly focused on cytogenetics, blood protein polymorphism, RFLP analysis of mitochondrion et al, but a believable conclusion didn't come to as yet. And so, we decided to probe into the classification, genetic differentiation, and phylogeny of Chinese yak by the microsatellite marker and the major histo-compatibility complex (MHC) antigen, just because the former was the richest genetic marker of polymorphism information, and the latter was the gene family which contained the richest polymorphism in genome.There were few research works in yak microsatellite, and so there were lesser microsatellite marker which could be used. In order to obtain large numbers of microsatellite marker, we constructed the first of microsatellite-enriched library of yak according to the strong affinity between biotin and streptavidin. The enriched method included: digesting yak genomic DNA using flush-end restriction enzyme, extracting the 300-1,000 bp enzyme-digested fragments and then ligating with flush-end adaptors, annealing with the biotin-marked microsatellite oligoprobe (CA)12, (CCG)8, (CAG)8, (TTTC)8, capturing the microsatellite repeat using Streptavidin Magnetic Beads and so obtaining the single-stranded targeted fragments, performing PCR amplification to obtain the double-stranded targeted fragments, cloning targeted fragments into the pMD18-Tvector and then transforming to DH5a, and ultimately forming the yak genomic microsatellite-enriched library. We obtained more than 2,000 clones, and 250 of these clones were selected for sequencing. The results showed that 195 clones contained the microsatellite sequence, representing a 78% enrich ratio, whichabsolutely was higher than general small inverted-fragment genomic library of cattle, and obviously higher than the microsatellite-enriched libraries which were constructed by other researchers (45%~55%), and the data showed that our library was a high efficient library. Among the selected 298 yak microsatellite, repeated times ranged from 5~50, and the most were 5~20. Meantime, there were 143 microsatellites, which contain 9 and more than 9 continuous repeats, representing 48% of the total microsatellites, and the data showed that our library was a high-quality library. Our practice demonstrated that enriched library was the most efficient way to screen the microsatellite, and this method was simple and rapid, safe and reliable in operation, meanwhile, it had characters of strong objective, high positive clones ratio, and high efficiency.Yak genome of 6 breeds, 143 individual, are scanned by 20 fluorescence microsatellite primers selected from which with not less 9 repeat sequence. Results show, 19 microsatellites can amplify products with stable and clear bands. The average observed number of alleles is 10.9. The effective number of alleles is 4.3. The average PIC is 0.5498., and there are 57.90% microsatellite with PIC above 0.5, and 36.84% with 0.25-0.5%, and 5.26% below 0.25. These suggest that microsatellite markers we selected can provide reliable information of yaks The heterozygosity of main yak breeds in china is 0.4331-0.6053, and its mean is 0.4791. The PIC is 0.5003-0.6032, and its mean is 0.5498. These suggest that there are abundance genetic diversity in Chinese yak populations. The genetic distance between different Chinese yak breeds (population) is 0.0665-0.9994, its mean is 0.531. The genetic identity between different p... |
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