【作者】 武玉珍；

【导师】 张峰；

【作者基本信息】 山西大学 ， 生态学， 2008， 博士

【摘要】 褐马鸡是我国特有的濒危鸟类,国家一级重点保护动物。目前其分布区被严重分隔成了3个地理种群,即山西吕梁山脉的中部种群、河北与北京地区的东部种群、陕西黄龙山的西部种群。本文以山西省庞泉沟国家自然保护区、芦芽山国家自然保护区、太原市动物园的褐马鸡种群为研究对象,采用分子生物学和光谱学的研究手段,进行了线粒体DNA控制区序列变异分析、ISSR多样性分析、褐马鸡的分子性别鉴定以及褐马鸡矿质元素含量的测定分析,初步探讨了褐马鸡种群的遗传多样性和遗传结构、以及它们的系统发生关系,为褐马鸡的保护与管理提供科学依据。主要研究结果如下:利用雉类线粒体控制区通用引物获得褐马鸡线粒体控制区(D-loop)序列长度为1236bp～1237bp,序列测定与分析20个个体,共检测出26个突变位点,13个单倍型,其中11个个体具有独特的单倍型,2个共享单倍型。太原市动物园种群与庞泉沟自然保护区种群的遗传多样性参数分别为核苷酸多样性平均值(π)为0.0024,单倍型多样性平均值(h)为0.916。两个种群具有较高的单倍型多样性和较低的核苷酸多样性,因而褐马鸡种群的遗传变异较低,个体间差异较大。太原市动物园种群与庞泉沟自然保护区种群内单倍型间的遗传距离均值分别为0.003和0.002,核苷酸多样性分别为0.003和0.002,单倍型多样性分别为0.933和0.911。结果表明:两个种群单倍型间的遗传距离和遗传多样性较接近,统计分析无显著差异,两个种群尚未表现出明显的遗传分化,且两个种群间有基因流存在。从合成的20条ISSR引物中筛选出扩增产物稳定、条带清晰、重复性好的引物10条。35个样本通过10条引物共扩增出65条清晰稳定的条带,其中有50条具有多态性,多态带百分率(PPB)为76.9%。在种群水平上,平均期望杂合度(He)、Shannon指数(I)和多态带百分率(PPB)分别为0.1904、0.2842和54.10%。在物种水平上,平均期望杂合度(He)、Shannon指数(I)和多态带百分率(PPB)分别为0.2281、0.3367和62.30%。结果表明:物种水平的遗传多样性高于种群水平的遗传多样性,而两个种群的的遗传多样性指数比较接近,与线粒体控制区(D-loop)序列分析结果一致。两个种群间的遗传分化系数(Gst)以及种群间的基因流估计值(Nm)分别为0.1640和2.5493,相似性系数为0.9079,遗传距离为0.0966,因而两个种群的遗传多样性水平较一致,种群间的遗传分化较小。太原动物园和庞泉沟自然保护区种群个体之间遗传距离最大为0.3279和0.3607,遗传距离最小值一致均为0.0328。表明两个种群个体之间的亲缘关系较近,近交程度较高。采用鸟类CHD基因的引物2550F/2718R对褐马鸡进行了性别鉴定,经过扩增、序列测定和Clustal X分析,雌性可以扩增出CHD1-Z和CHD1-W基因两条主带而雄性仅扩增到CHD1-Z基因一条主带。对18个已知个体和18个未知性别的个体进行了测定,18个已知性别个体测定结果与实际性别完全吻合。太原市动物园饲养的褐马鸡羽毛中除K和Cu外,其余8种元素的含量均比芦芽山和庞泉沟国家自然保护区野生的褐马鸡羽毛中的含量低,特别是Fe,仅占野生含量的大约1/3。各个组织器官中元素的含量差异极显著(P＜0.01)。大部分组织器官中含量较高的元素有:Fe、K、Zn、Cu、Mn,而Cr、Pb、Cd、Ni四种元素含量较低。褐马鸡羽毛、卵壳与机体大部分组织器官矿物元素呈正相关关系,而且绝大部分呈显著(P＜0.05)及极显著(P＜0.01)正相关关系。分析了褐马鸡的濒危原因,提出了褐马鸡自然保护区的科学管理和保护措施,加强各保护区之间卵、幼鸟、成年鸟的交换,促进种群间的基因交流,防止种群遗传衰退。对于圈养种群应进行野放试验,同时为了提高褐马鸡种群的遗传多样性水平,遗传管理十分重要。更多还原

【Abstract】 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.更多还原