Assessment Of Genetic Diversity In The Captive Forest Musk Deer (Moschus Berezovskii) And Linkage Analysis Between The Performance Of Musk Productivity And DNA Molecular Markers

Musk deer (Moschus spp.), a rare species only distributed throughout the forest and mountainous parts of Asia, is famous for the musk secreted by the male musk deer which is economically high valuable. The distribution range and population size of musk deer have been sharply declining in the past decades due to over-hunting for musk and to loss of habitats. Therefore, all species of musk deer have been included in Category I of the State Key Protected Wildlife List in China and is also listed in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).Among all Moschus species, the Forest musk deer (Moschus berezovskii) has the widest distribution and the largest captive population. To measure genetic diversity and structure of the forest musk deer (Moschus berezovskii), we examined variations of 12 microsatellites [simple sequence repeats (SSRs)] and 908 amplified fragment length polymorphism (AFLP) fragments in 41 forest musk deer sampled from two farming populations (Baisha Farm and Jinfeng Mountain Farm) of Sichuan Musk Deer Breeding Institute. In the meantime, we analyzed the linkage of SSR and AFLP markers with musk productivity to find some linked markers. We obtained the following results:1. This study employed magnetic bead-based enrichment protocol to isolate SSR markers. Approximately 2500 clones were screened and a total of 270 recombinants that potentially contained microsatellite sequence were obtained. 100 positive clones were chosen randomly and subject to DNA sequencing. The sequencing results showed 73 clones contained eight repeats or more and had suitable flanking sequences. Thirty-seven primer pairs succeeded in PCR amplification but only 15 primer dyads were polymorphic and consistently yielded their specific PCR products for each sample.2. The 15 microsatellite markers were subjected to molecular examination of 41 musk deer and revealed that: (1) three loci showed significant deviation from Hardy-Weinberg equilibrium (P < 0.001), which were removed in the subsequent analyses; (2) the PIC values ranged from 0.631 to 0.882 with average value of 0.727; (3) the value range of single discrimination power (DP) was 85.5%-93.7%; (4) the value of cumulative discrimination power (CDP) across 11 loci was 100%. Therefore, this suite of microsatellite markers were subsequently subjected to genetic analyses of the forest musk deer. For the 2 population, a total of 108 distinct alleles were observed across 12 loci. For the Jinfengshan population, the number of alleles per locus ranged from 5 to 11 with ane average of 6.92. The average alleles richness was 6.77 per locus and the average heterozygosity across 12 loci was 0.728. For the Baisha population, the number of alleles ranged from 6 to 11 with an average of 8.08. The average alleles richness was 7.36 per locus and the average heterozygosity across 12 loci was 0.763. The T-test results showed no significant genetic variability between the 2 populations.3. A total of 22 pairs of AFLP primers, resulting in 908 AFLP polymorphic fragments, were used to assess genetic diversity of the forest musk deer. At species level, the results revealed that the average percentage of polymorphic loci (PPL) was 97.32%; the effective number of alleles per locus (N_e) was 1.643; Shannon's information index (I) was 0.528; Nei's gene diversity (H_J) was 0.386. The results reveal a high level of genetic diversity in the forest musk deer. In addition, inter-individual Nei's genetic distances (GD) ranged from 0.0110 to 0.1004 with an average of 0.0458, indicating an even distribution among individuals. To investigate population structure between these two populaltions, we analyzed the data from (1) the coefficient of genetic differentiation (F_ST), Nei's unbiased measure of genetic distance (DS) and the gene flow (Nm), which were 0.0273, 0.0131 and 22.98 respectively. These reulsts showed that there was no obvious genetic structure between the Jinfengshan and Baisha population rather than abundant gene flow instead. The comparisons of PPL and H_J value indicated the Jinfengshan population shows a higher level of genetic diversity.4. The linkage analysis between SSR or AFLP markers and musk productivity showed that (1) musk productivity significantly was associated with 2 microsatellite loci, Mb06 and Mb33. (2) The PPL of high productivity group was significantly higher than the control group (P < 0.01) and that the PPL of low productivity group was significantly lower than reference group (P < 0.01), indicating that the high productivity group had more better survival fitness than the low productivity group. (3) Homogeneity test of 908 AFLP polymorphic fragments showed that 34 loci were significantly related to the high musk productivity (P < 0.05). As a result, these 2 microsatellite and 34 AFLP markers may be linked to the genes controlling the performance of musk production, providing a pilot study for future genetic studies on musk productivity traits.