Establishment Of AFLP Markers System And Assessment Of Genetic Diversity For The Pere David's Deer

The Pere David's deer(Elaphurus davidianus) is a rare species peculiar to China and listed as the first grade of national key protected species. Since a period of great prosperity 3000 years ago, wild population was gone to decline. In the late 19th century, while it became extinct in China, only 18 individuals were collected to form a breeding herd at Woburn Abbey, which were founders for the species now. After the First World War, "ex-situ conservation" was started since the species at Woburn Abbey expanded. Thus,38 individuals were re-introduced into Beijing Naihaizi Milu Park-Beijing Milu Ecological Research Center in 1985, and the first population of the Pere David's deer in China was established after extinction.Nevertheless, since the Pere David's deer has successfully passed through the severe bottleneck of inbreeding, an issue-"how about the genetic diversity in the species?" was concerned over the academia. For that, in this paper, based on establishment of AFLP markers detection system,245 individuals in Beijing Nanhaizi Milu Park-Beijing Milu Ecological Research Center were analyzed and some parameters about genetic diversity were obtained. The major results are as follows:1. Establishment of AFLP markers system for the Pere David's deer(1) Genomic DNA with high quality and no degradation was extracted.(2) 200-400ng of genomic DNA was digested by the enzyme combination EcoRⅠ/MseⅠwith 0.5U each for 2h at 37℃, and ligation of adapters was at 20℃for 2h.(3) As detected by electrophoresis with 1% agarose gels, the products of digestion were 100-1500bp, presenting smear evenly.(4) The template should be diluted 10 times before PCR. By the same detection method, the products of pre-amplification were evenly smear at 100-800bp.(5) Assayed by electrophoresis with 6.5% denaturing polyacrylamide gel, the bands of selective amplification were at 50-700bp.2. Analysis of genetic diversity for the Pere David's deer11 pairs of AFLP primers with higher polymorphism were selected from 64 primer pairs for population analysis; and there were E-AAC/M-CAG, E-AAC/M-CAT, E-AAG/M- CTC, E-AAG/CTG, E-ACC/M-CAC, E-ACC/M-CTT, E-ACT/M-CAC, E-ACT/M-CTA, E-AGC/M-CTA, E-AGC/CTT and E-AGG/M-CTT. The results are as follows:(1) 1086 markers were obtained, including 75 polymorphic loci. The percentage of polymorphic loci (PPL) per pair of primers varied from 3.7% to 10.91%, and was 6.91% at population level of average.(2) The combined individual identity discrimination power of 75 polymorphic loci was up to 100% so that each individual could be distinguished, and each individual had its own AFLP fingerprint.(3) At population level of average, other parameters were obtained:observed number of allele (Na) was 1.0675, effective number of allele (Ne) was 1.0379, Nei's gene diversity (H) was 0.0230, and Shannon index (Ⅰ) was 0.0349. The maximum of Nei&Li genetic distance (GD) between individuals was 0.001816, and the minimum was 0.000120. The mean of coefficient of genetic similarity (GS) between individuals was 0.999057±0.0002782.The results revealed that there were extremely high genetic identity between individuals and genetic diversity of this species in Beijing was very low-close to inbred strain.(4) Based on four species as the outgroups (A. porcinus, C. nippon, M. crinifrons and M. berezovskii), dendrograms were constructed using similarity coefficient. As the coefficient of GS was 0.998810,245 individuals were classified into GroupⅠand GroupⅡ, including 181 and 64 individuals respectively. Futhermore, the genetic relationship between individuals could be easily discerned over 0.999000 of GS. In addition, the genetic relationship between Pere David's deer and outgroups was A porcinus>C. nippon>M. crinifrons>M. berezovskii; Pere David's deer was closer to the Cervus, and A. porcinus could be assigned to the Cervus. Mental test was made between dendrograms and similarity coefficient matrix, and the relative value was 0.99882; it showed that they were highly correlative, and dendrograms were of high credibility.