Studies On The Cytogenetics And Microsatellite Markers Of Forest Musk Deer (Moschus Berezovskii)

Musk deer (Moschus spp.) is the most important resource animal of China, and over 70% of the musk deer and the musk of the world come from China. Due to poaching for musk pods and the disturbance of habitat, musk deer populations are reduced sharply, and are becoming seriously endangered animals. Because of the serious conservation status, musk deer became the first class of "key" species of wildlife protected by Chinese legislation on the Nov. 4 of 2002. Owing to its rarity, researches have interested in the study of ecology, taxology, histology, physiology of musk deer and pharmacology and clinical applications of musk for many years. However, little data is about the genetics of musk deer. In this research, the karyotype, G-banded chromosomes, the isolation and polymorphism of microsatellites of forest musk deer (Moschus berezovskii) was studied. The genetic diversity of the wild and captive forest musk deer populations was also analyzed.Using peripheral blood lymphocytes of forest muck deer (M berezovskii) as materials, a new culture system has been developed to induce more peripheral blood lymphocytes of forest muck deer into cell cycle by adding an equal quantity of phytohemagglutinin (PHA) and concanavalin A (ConA) at 38℃. The chromosomes were prepared from the cultured cells, and the results showed that the karyotype of M. berezovskii is 2N=58, and all of the chromosomes are telocentric ones. Usingchromosome G-banding technique, the chromosomes of forest musk deer were studied for the first time. The results showed that the longest chromosome is X and the shortest one is Y in the karyotype of forest musk deer.Because of the reliability of microsatellite markers, three microsatellite enriched libraries of forest musk deer (M berezovskii) were constructed. They were (AC)n-enriched, (AAG)n-enriched and (GATA)n-enriched library, respectively. Each library had more than ten thousands transformants. The results showed that about 75% of clones in the (AC)n library were positive ones. Sequencing of the positive clones showed that about 98% of positive clones were (AC)n-containing clones, of which 10% of clones occurred twice or more in the libraries. The average size of inserts of the library was 534±128bp. In the (AC)n library, microsatellite with perfect repeat sequences was about 44.4%, with imperfect repeat sequences about 53.3% and compound repeat sequences 2.2%. The highest array contained 59 repeats, and the lowest 9 repeats, with an average of 25 repeats in the library. All these results showed that the library constructed here had a high quality.The polymorphism and the application of microsatellite loci isolated from the library were studied in this research. At first, the follicle DNA of forest musk deer was extracted with proteinase K/phenol/chloroform method. The yield of DNA isolated from follicles is about 4~ 9 ng/hair, which can be used for digestion of restriction enzyme and amplification of gene and microsatellite loci. Then the microsatellites were screened and the PCR condition was optimized. The genetic diversity of the wild forest musk deer population in Miyalo, and the captive population in Dujiang Jinfeng Mountain and Maerkan was studied at last. The polymorphic study showed that 8 reliable and highly polymorphic microsatellite loci had been isolated in this research. The number of alleles per locus ranged from 6 to 14, with an average of 9.25. The average number of effect alleles of each locus was 6.83, and the observed and expected heterozygosities ranged from 0.41 to 1.0 and 0.8 to 0.9, respectively. The polymorphic information content (PIC) value for these markers was from 0.76 to 0.89, with an average of 0.82. Compared with the wild population in Miyalo, the two captive forest musk deer populations had a low geneticdiversity. The research also showed that the rare allele of captive population was 23.3% and 22%, which was higher than that of the wild population in Miyalo (only 13.5%). The homozygosity of the two forest musk deer populations was 61.25% and 46.32, which is also higher than that of the wild population, and both were departure from Hardy-Weinberg balance. The results also showed that both wild and captive forest musk deer had a more abundant genetic diversity than that of the endangered animals such as tiger, deer and alligator. This means it is not late to carry out the protection project for forest musk deer. Cross amplification results showed it failed to amplify product from related species such as pig, own, ovine, dog, exception that only locus Mber14H succeed both in alpine musk deer (M.chrysogaster) and sika deer (Cervus nippon), and locus Mber 86B succeed only in alpine musk deer. Sequences of locus Mber14H from the 3 species showed the relationship of forest musk deer and alpine musk deer is closer than that of forest muks deer and sika deer. The hybridization result of digoxigenin labled DNA fragments of the 8 microsatellites with the genome of forest musk deer showed that they came from the genome of forest musk deer. Undoubtedly, these results provide the most direct practical and theory values for forest musk deer conservation. Therefore the management measures for protecting captive forest musk deer population were proposed in this study.