Study On The Genetic Diversity And Conservation Of Endangered Species, Crossoptilon Mantchuricum

Brown-eared pheasant(Crossoptilon mantchuricum) is one of the endangered species that is endemic to north China,and one of the state first-protection animals.Since its population size is believed to be declining because of habitat loss and damaged,hunting and other human activity interventions,geographical barrier(Yellow River) and natural vegetation destruction(Taihang Mountain),there are three isolating populations of Brown-eared pheasant,including western Shanxi,north-western Hebei, western Beijing and northern central Shaanxi,moreover,it is resulted in that the gene flows of the populations is obstructed and the frequency of inbreeding within the populations is more and more.In this study,the genetic diversity and genetic structure were investigated among Pangquangou Natural Reserve and Taiyuan Zoo in Shanxi using mtDNA and ISSR DNA. Furthermore,the molecular method for sex determination of Brown-eared pheasant and the determination of 10 elements in the feather,eggshells and the organisms of Brown-eared pheasant from Luya Moutain Natural Reserve, Pangquangou Natural Reserve and Taiyuan Zoo by ICP and AAS were reported in order to provide scientific basis for the protection of the endangered bird.The main results and implication for the conservation of the Brown-eared pheasant species were as follows:The mitochondrial control region with 1236～1237bp were sequenced from 20 individuals from the two populations of Pangquangou Natural Reserve and Taiyuan Zoo.26 mutaion loci were found.13 haplotypes were detected by defining of 1～7 variation loci.11 of which occurred in a single individual and two of which is shared.The average number of haplotype diversity(h) was 0.916,the mean value of nucleotide diversity(π) was 0.0024,it was indicated that the bird has the distinct high haplotype diversity and the distinct low nucleotide diversity. It was suggested(The above data indicated) that the genetic diversity of the current Brown-eared pheasant population is relatively low. The average genetic distance within each population,Taiyuan Zoo was 0.003 and Pangquangou Natural Reserve 0.002,and the haplotype diversities were 0.933 and 0.911,and the nucleotide diversities were 0.003 and 0.002 respectively.The differences between the two populations in the two genetic diversity parameters were not statistically significant,which mean that there was no significant genetic differentiation occurred the two populations.The phylogenetic tree for those populations constructed with UPGMA and NJ methods further showed that there was no significant genetic differentiation among two groups,and there was gene flow among two populations.Twenty ISSR primers were screened and 10 primers were selected.A total of 65 DNA bands was amplified,50 of which were polymorphic and the percent of polymorphic bands(PPB) was 76.9%.A relatively high level of intraspecific genetic diversity was revealed:He=0.1904,I=0.2842, PPB=54.10%at population level;He=0.2281,I=0.3367,PPB=62.30%at species level.It was demonstrated that the genetic polymorphism at species level was higher than at population level and the genetic diversity index were near almost among two populations.The genetic heterogeneity(Gst),genetic flow(Nm),genetic similaritive index and genetic distance were 0.1640,2.5493,0.9079 and 0.0966 respectively.The results showed that the genetic diversity level was identical, and the heterogeneity was less between the two populations.There was no significant genetic differentiation among the groups.The largest genetic distance was 0.3279 in Taiyuan Zoo and 0.3607 in Pangquangou Natural Reserve,and the least was identical that were 0.0328 among the two populations.The result of genetic distance analysis revealed that the origin among the individuals was near,and there was high degree of inbreeding.The relatively low genetic diversity were proved using mtDNA and ISSR DNA markers,and two molecular marker were two useful molecular markers in detecting the genetic diversity of Brown-eared pheasant population.The results suggested that the genetic resource of Brown-eared pheasant in China is lack. A simple,fast and reliable method for sex identification was developed using CHD intron amplified by one pair of 2550F/2718R primers from the DNA samples extracted from blood and feather samples of Brown-eared pheasant.The males showed one main band,whereas the females were with two clear main bands,and both CHD-Z(594 bp) and the CHD-W(452 bp) fragments could be separated on 2%agarose gel.In order to test 2550F/2718R sexing methods in Brown-eared pheasant,the DNA samples from the bloods and feathers of eighteen known-sex individuals and eighteen unknown-sex individuals were used.Our results demonstrated that eighteen known-sex individuals were agreement with the corresponding fact samples.The results for the elements of two populations showed that the contents of the eight elements of the feather in the Taiyuan Zoo were lower than that of Luya Mountain Natural Reserve and Pangquangou Natural Reserve,and the contents of Fe is only one third.Moreover,Fe is the highest among those ten elements,Cd was not found,Mo and Cr were much lower than the others.The differentiation was significant of element contents among organisms (P＜0.01).There were high contents of Fe,K,Zn,Cu,Mn in mostly the organisms,Cr,Pb,Cd,Ni were low,and Pb,Cd were the lowest.The results showed that the living environment was slight pollution of Brown-eared pheasant.There were positive correlation between the feather,eggshell,and organisms,and mostly were significant(P＜0.05),or higher significant(P＜0.01).Therefore,through detected the contents of the feather and eggshell, the body supply were understood.It provides valuable information for captive breeding and reproduction of Brown-eared pheasant.The endangered causes of Brown-eared pheasant were analyzed,the strategies for the protection of this bird were discussed,including enhancing the exchange between the eggs,poults and matures from different regions so that improve the gene exchange between populations in order to prevent from genetic decline.In addition,the population from the raised bird should be reintroduction in the field so that the distribution of the birds was increasing gradually.