| The Pere David's deer(Elaphurus davidianus)is a rare and peculiar species in China and was listed as the first grade of national key protected species.It had been extinct in the wild,and had been reintroduced to China in 1985.With the bottleneck effect and founder effect,the genetic diversity of the Pere David's deer is very low,and the species is still critically endangered.In this study,the noninvasive genetic sampling was used,and 9 microsatellite loci were applied to study genetic diversity of the wild population of the Pere David's deer,which live in third core areas of Jiangsu Dafeng Milu National Nature Reserve(i.e.,Dafeng Milu wild population).Also,the genetic differentiation and the probable cause were discussed with the comparisons to the Beijing Milu population.The main results were as follows:1.When used the noninvasive genetic sampling to conduct the research on the wild Pere David's deer,the nucleic acid extracted from feces which collected in winter was better than that in summer.The proportion was about 61.57%for the winter samples which the OD 260/280 ratio was between 1.60 and 1.90,and about 43.51%for the summer samples.The success rate of PCR amplification for gender identification and microsatellite genotyping were 84.98%and 96.25%for winter samples,and 93.13%and 95.25%for summer samples respectively.There was no significant difference between the different seasons.The weather such as rain and snow influenced the DNA quality significantly.The proportion of OD 260/280 ratio which between 1.60 and 1.90 was about 61.57%for the sunny winter samples,about 23.68%and 23.88%for the samples after raining and snowing respectively.The rain would wash out the intestinal tissue,mucosa or blood on the surface of fecal samples,and made the degradation of the DNA.The results suggest that the noninvasive genetic sampling for David's Deer should be done in sunny winter,as it can improve the DNA quality effectively.2.For the Dafeng Milu wild population,total of 472 fecal samples was collected and the genome DNA was extracted according the standard procedure of the QIAamp DNA Stool Mini Kit.Among them,the mitochondrial cyt-b gene of 444 samples could be amplified successfully,and 118 unique genotypes(or individuals)were identified,including 63 females,38 males,and 17 individuals which the sex unknown.The sex ratio of male to female was 1:1.66.The effective population size was about 94.81 for the wild population.The average allele number was 2.667 for the all loci.The PIC was between 0.177 and 0.612 for the nine loci,the average was 0.362;the expected heterozygosity was between 0.197 and 0.678,and the average was 0.449;the observed heterozygosity was between 0.220 and 0.746,the average was 0.542.There were 7 loci(P2G6,F65,FCB48,BM4107,URB021B,Elda-16 and T172)significantly deviate from the Hardy-Weinberg equilibrium(P<0.05).FiS was-0.209,and Fis<0,which indicated the genetic heterozygosity excess in the population.The Dafeng Milu wild population exhibited a low genetic diversity,which suggested this population could have experienced severe bottleneck effect in history.3.The genetic diversity of Dafeng Milu wild population was higher than that of Beijing Milu population.The average number of alleles for Dafeng Milu wild population was 2.67 ± 1,and 2.11 ± 0.60 for Beijing Milu population.The expected heterozygosity and observed heterozygosity of Dafeng Milu population was 0.450 ±0.047 and 0.539 ± 0.015,while for Beijing Milu population was 0.403 ± 0.060 and 0.385 ± 0.042 respectively.Among 9 microsatellite loci,there were 7 loci significantly deviated from the Hardy-Weinberg equilibrium in Dafeng Milu population,while all the loci were under the Hardy-Weinberg equilibrium for Beijing Milu population.Through the FAC analysis,the Dafeng Milu wild population was clustered into one branch,and the Beijing Milu population was clustered into another branch.The results indicated that they were significant population differentiation between Dafeng Milu wild population and Beijing Milu population. |
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