【作者】 郑书星；

【导师】 张建国；

【作者基本信息】 中国林业科学研究院 ， 森林培育， 2009， 博士

【摘要】 新疆额尔齐斯河流域分布着大面积的杨树天然林,包括有四大派系（白杨派、黑杨派、青杨派和胡杨派）,其中,欧洲山杨、银白杨、银灰杨、欧洲黑杨、苦杨等杨属植物在我国仅天然分布于该地区。额尔齐斯河流域拥有一个丰富的天然杨树基因资源库,具有独特的生态和遗传学研究价值。本论文通过实地调查和实验分析,初步弄清了额尔齐斯河流域杨属天然林的分布状况、繁殖方式、遗传多样性和种间亲缘关系,为这一天然基因资源库的保存利用提供科学依据;取得了以下主要研究结果:1.白杨派树种克隆结构及多样性分析:（1）额尔齐斯河流域分布的白杨派天然林分多呈雌雄独立斑块状分布,从独立斑块小尺度上研究表明斑块内所有单株是同一个克隆系,即独立分布斑块是单克隆结构。白杨派树种天然林分以无性克隆繁殖方式（根孽）来产生新个体,维持种群的延续。（2）流域大尺度分析结果显示欧洲山杨和银白杨天然居群的Simpson指数（D）分别为0.987和0.983,表明欧洲山杨和银白杨天然居群具有较高水平的克隆多样性。其原因是:最初白杨派树种建群过程中,多个基因型不同的基株占据不同地点生境,通过根孽方式繁殖后代,形成克隆斑块;现在的植株是最初基株的克隆后代或者克隆后代的后代。（3）雌雄独立斑块是单克隆结构,而沿流域分布的多个斑块间具有遗传变异。通过对额尔齐斯河流域白杨派居群小尺度（独立斑块）和大尺度（流域几百平方公里）的研究,发现由于人为活动和环境恶化的影响,使得生境碎片化,大的斑块形成了相邻的多个小斑块,相邻的不同斑块植株是属于同一个大斑块的同一克隆系。同一居群内空间上不相邻的斑块也聚为一类,是一个克隆系,表明欧洲山杨和银白杨的根蘖繁殖距离长,克隆系空间分布的跨度很大。（4）分析欧洲山杨和银白杨居群的遗传多样性,结果表明欧洲山杨的Shannon信息指数（I）平均为1.0689, Nei多样性指数（h）平均为0.5056,以检测到的期望杂合度（He）为标准,将3个居群按照遗传多样性高低进行排序,其次序为:S1>S3>S2。银白杨的Shannon信息指数（I）平均为0.3249, Nei多样性指数（h）平均为0.2122,以检测到的期望杂合度（He）为标准,将4个居群按照遗传多样性高低进行排序,其次序为:Y4>Y3>Y1>Y2。银白杨的遗传多样性水平要明显低于欧洲山杨。2.用SSR分析青杨派树种苦杨、黑杨派树种欧洲黑杨的遗传多样性发现:（1）利用筛选的12对SSR引物对苦杨共检测出89个等位基因变异,平均位点等位基因数（Na）为7.4167个;有效等位基因数（Ne）为3.8872;观察杂合度（Ho）平均为0.4249;期望杂合度（He）平均为0.3940。对欧洲黑杨共检测出24个等位基因变异,平均位点等位基因数（Na）为2.0000个;有效等位基因数（Ne）平均为1.3967;观察杂合度（Ho）平均为0.3452;期望杂合度（He）平均为0.2200。苦杨和欧洲黑杨的期望杂合度小于观察杂合度,表明其群体的杂合体过量。（2）苦杨的Shannon信息指数（I）平均为0.7659, Nei多样性指数（h）平均为0.3657,以检测到的期望杂合度（He）为标准,将6个群体按照遗传多样性高低进行排序,其次序为:K3>K1>K5>K6>K2>K4。欧洲黑杨的Shannon信息指数（I）平均为0.3199, Nei多样性指数（h）平均为0.2122,以检测到的期望杂合度（He）为标准,将4个群体按照遗传多样性高低进行排序,其次序为:H2>H3>H4>H1。欧洲黑杨的遗传多样性水平要明显低于苦杨。（3）苦杨群体间遗传分化系数（Fst）为0.0714,即有7.14%的遗传变异存在群体之间,92.86%的遗传变异存在于群体内,群体内变异是其遗传变异的主要来源;苦杨群体间的基因流（Nm）平均为3.2533。欧洲黑杨群体间遗传分化系数（Fst）为0.0243,97.57%的遗传变异存在于群体内;欧洲黑杨群体间的基因流（Nm）平均为10.0585。表明苦杨和欧洲黑杨群体间遗传分化程度小,这与群体间有较高的遗传一致度（I苦=0.9156～0.9817;I黑=0.9825～0.9972）的结果相符合。（4）苦杨的Fit和Fis均为负值,分别为-0.0860和-0.1695;欧洲黑杨的Fit和Fis也均为负值,分别为-0.5754和-0.6146。这表明苦杨和欧洲黑杨种群总体表现为杂合子过量的现象。（5）对苦杨、额河杨、欧洲黑杨3个树种共228个样本聚类分析,228个样本聚为了2个大类,第一大类是苦杨个体,第二大类是额河杨和欧洲黑杨个体,杂交种额河杨与欧洲黑杨位于同一个分支,证明额河杨应归属于黑杨组,同时也表明杂交种额河杨在基因组DNA水平上呈现黑杨的特征。苦杨种内个体间具有相当的遗传变异,而欧洲黑杨种内有许多个样本的遗传相似系数为1.00,完全聚在一起。（6）额河杨是苦杨和欧洲黑杨的天然杂种,其混生于苦杨林分中。额河杨的表型形态特征变异较大,有的外貌酷似黑杨,有的似苦杨,有的呈中间型,直接从形态上鉴别难以做到100%准确。通过表型认定为苦杨的样本K3-16与额河杨聚为一大类,通过表型认定为杂种额河杨的样本Ke2-2、Ke2-3和Ke3-18则与苦杨聚为一类,这说明额河杨与苦杨又发生了回交,导致形态多变,林分呈多代杂种的复合体,运用分子标记技术在DNA水平上鉴定物种比表型形态特征更为准确。3.采用Primer Walking法对杨属植物叶绿体DNA trnL-trnF间隔区片段进行PCR产物直接测序,对所获得的序列分析结果表明:发现有883个不变位点,108个变异位点（所占比例为11.96%）;多态性位点20个（所占比例为2.21%）,存在着插入或缺失（主要是插入或缺失A/T）以及碱基的替换（碱基替换形式有A-G/A-C/A-T/C-T/G-T）;序列G+C平均含量为32.6%。所测的杨属植物cpDNA trnL-trnF间隔区序列具有较高的单倍型多样性（Hd =0.764）和核苷酸多样性（Pi=0.00396）,表明该DNA序列片段区具有较丰富的遗传变异。中性检验所得Tajima’s D值为0.39108,该统计值在P>0.10的水平上差异不显著,接受零假设（the null hypothesis）,表明cpDNA trnL-trnF序列在杨属中呈中性进化。杨属5个树种（欧洲山杨、银白杨、苦杨、额河杨和欧洲黑杨）的cpDNA trnL-trnF间隔区序列同源性为89%,序列高度同源、为单系起源;所构建的系统发生树显示,欧洲山杨、银白杨以及苦杨划分为3个大类,欧洲黑杨位于系统树的基部;杂种额河杨与苦杨聚为一类,表明额河杨的cpDNA来自于其母本苦杨。杨属植物叶绿体DNA trnL-trnF间隔区序列鉴定出17种单倍型。白杨组中的欧洲山杨有2种单倍型（Hap₁3、Hap₁4）,单倍型Hap₁3在其群体分布中占优势地位;银白杨有4种单倍型（Hap₁4、Hap₁5、Hap₁6、Hap₁7）,单倍型Hap₁5在其群体分布中占优势地位,其中单倍型Hap₁4为两个树种所共有,但分布频率很低。黑杨组中的欧洲黑杨有2种单倍型（Hap₁、Hap₃）,单倍型Hap₁在其群体分布中占优势地位。青杨组中的苦杨鉴定出最多的单倍型,共有9种（Hap₂、Hap₅、Hap₆、Hap₇、Hap₈、Hap₉、Hap₁0、Hap₁1、Hap₁2）,单倍型Hap₂在其群体分布中占优势地位;杂种额河杨具有2种单倍型（Hap₂、Hap₄）,与苦杨居群混生的杂种额河杨主要是具有单倍型Hap₂,而与欧洲黑杨H1居群混生的杂种额河杨单株He1-8、He1-12也是具有这一单倍型,与欧洲黑杨H4居群混生的杂种额河杨单株He4-2具有单倍型Hap₄。苦杨、额河杨2个树种都具有单倍型Hap₂,而欧洲山杨和银白杨2个树种都具有单倍型Hap₁4。用2种分析方法-邻接法（neighbor-joining,NJ）和最大似然法（maximum parsimony,MP）分别对杨属5个树种的cpDNA trnL-trnF间隔区序列的17种单倍型进行系统发育分析并构建了系统树,不同方法所获得的系统树在拓扑结构上基本一致,但在自展支持率（Bootstrap value,BS值）上存在着差异。从单倍型构建的系统发生树可得知,所有杨属植物的单倍型组成了一个多系群,它们具有不同的假定祖先型;苦杨具有最多的单倍型变异;杨属各个组内的单倍型更倾向于聚为一支,但黑杨组的单倍型倾向于与青杨组的单倍型在系统进化上有更近的关系。更多还原

【Abstract】 Large areas of the populus natural forests were distributed along the Erqis River in Xinjiang, which including the four Populus sections of Leuce、Aigeiros、Tacamahaca、Turanga, and the Populus tremula、Populus alba、Populus canescens、Populus nigra、Populus laurifolia、Populus×jrtyschensis just distribute naturally in this region of China. The Erqis River has abundant gene resource and idiographic value of the ecological and genetic research. The distributing pattern、breeding methods、genetic diversity and phylogenetic relationship of Populus natural forests were studied based on field surveys and experiments. Major results are described as follows:1. We analyzed the clonal structure and diversity of Populus sections of Leuce, then the results following:（1） Through the field survey, we found that the Leuce natural forests present a independently distributing pattern of male or female patch, many independent patches along the valley formed a large patch, and a number of major patches then formed a population. The study results at a microgeographical scale of single patch showed that all swatches in a single distributing patch of male or female were a identical clone, the single patch had only one genet, that was to say the structure of patch is monoclonal, and the Leuce natural forests propagated offspring by asexual clonal reproduction means （rooting） to maintain the population continuation.（2） The populations of Populus tremula and Populus alba had an abundant clonal diversity with the mean Simpson’s index was 0.987 and 0.983 based on a macrogeographical scale of the livelong valley. The high clonal diversity might be maintained if the populations were initially founded by multiple genets that differed genetically and the existent frond was the progeny of clonal genets by root clone.（3） Because the independently distributing patch of male or female was monoclonal structure and there was genetic variation among patches along the valley, we described the distributing pattern of the Leuce natural forests along the Erqis River based on the studying at microgeographical scale （single patch） and macrogeographical scale （the livelong valley）. Because of human activities and environmental degradation, then habitat fragmentation made a large patch form many adjacent patches which would have been belonged to the same clone. In addition, the disjunct patches within the same populations was a same clone system, showed that the distance of root tillering of Populus tremula and Populus alba was long, and the clonal spatial distribution was large.（4） The Shannon index（I） and Nei index（h） of Populus tremula was 0.7659 and 0.3657 and the 3 populations were ranked as S1>S3>S2 according to genetic diversity based on He; The Shannon index（I） and Nei index（h） of Populus alba was 0.3199 and 0.2122 and the 4 populations were ranked asY4>Y3>Y1>Y2 according to genetic diversity based on He. The genetic diversity of Populus alba was significantly lower than the Populus tremula.2. We analyzed the population genetic diversity of Populus nigra and Populus laurifolia by SSR molecular marker, then the results following:（1） For Populus nigra, 89 alleles were detected based on 12 SSR primers and the average number of alleles per locus Na= 7.4167, the effective number of alleles Ne= 3.8872, the mean observed heterozygosity Ho= 0.4249, the mean expected heterozygosity He= 0.3940; Populus laurifolia, 24alleles were detected based on 12 SSR primers and the average number of alleles per locus Na=2.0000, the effective number of alleles Ne=1.3967, the mean observed heterozygosity Ho=0.3452, the mean expected heterozygosity He=0.2200. the mean expected heterozygosity （He） of Populus laurifolia and Populus nigra was less than the mean observed heterozygosity（Ho）, indicating that their heterozygote was excess .（2） The Shannon index（I） and Nei index（h） of Populus laurifolia was 0.7659 and 0.3657 and the 6 populations were ranked as K3>K1>K5>K6>K2>K4 according to genetic diversity based on He; The Shannon index（I） and Nei index（h） of Populus nigra was 0.3199 and 0.2122 and the 4 populations were ranked as H2>H3>H4>H1 according to genetic diversity based on He. The genetic diversity of Populus nigra was significantly lower than the Populus laurifolia.（3） A low level of genetic differentiation among Populus laurifolia populations was detected（Fst=0.0714） ,which indicated that the variation within groups is a major source of genetic variation, and a higher estimate of gene flow（Nm=3.2533） coincided with a high level of genetic identity（I） among the populations（from 0.9156 to 0.9817）; analogously, a low level of genetic differentiation among Populus nigra populations was detected（Fst=0.0243）, which indicated that the variation within groups was a major source of genetic variation, and a higher estimate of gene flow（Nm=10.0585） coincided with a high level of genetic identity（I） among the populations（from 0.9825 to 0.9972）.（4） The index of Fit and Fis of Populus laurifolia was -0.0860 and -0.1695 and the index of Fit and Fis of Populus nigra was -0.5754 and -0.6146, which indicated that their heterozygote was excess.（5） The clustering analysis suggested that 228 swatches were classified two filiations: the first filiation was Populus laurifolia and the second filiation was Populus nigra and Populus×jrtyschensis, which proved that Populus×jrtyschensis should be ranged to the Populus nigra group and it presented the characteristics of Populus nigra at the genomic DNA level. There were considerable genetic variation among the species of Populus laurifolia and the genetic similarity coefficient of many Populus nigra individuals was 1.00.（6） Populus×jrtyschensis, a natural hybrid of Populus laurifolia and Populus nigra, had considerable phenotypic variation. Some individuals looked like Populus nigra, some looked like Populus laurifolia and some were intermediate type, so it was difficult to identify from the modality. The sample of K3-16, originally identified be Populus laurifolia through the phenotype, was clustered to Populus×jrtyschensis, and the samples of Ke2-2 and Ke2-3 and Ke3-18, originally identified be Populus×jrtyschensis through the phenotype, were clustered to Populus laurifolia. This showed that there had backcross between Populus×jrtyschensis and Populus laurifolia, then resulting in complicated morphological variation and the stand was the compound types of many hybrid generations. Identification of species by using the DNA molecular markers was more accurate than phenotypic characteristics.3. The Populus plant chloroplast DNA trnL-trnF intergenic space sequence was amplified and sequenced by the primer walking technique based on PCR product fragments. The results showed that: there were 883 invariable loci and 108 variable loci （11.96%）. The 20 polymorphic loci （2.21%） had insertion or deletion （A / T） and substitution （AG / AC / AT / CT / GT） and the content of sequence G + C was 32.6%. Relatively high level of haplotype diversity （Hd = 0.764） and nucleotide diversity （Pi = 0.00396） were detected in Populus cpDNA trnL-trnF sequence,which indicated that this DNA sequence had rich genetic variation. The Tajima’s D index of neutral test was 0.39108 and accepted the null hypothesis, its difference was not significant at the level of P> 0.10, which showed that the evolution of cpDNA trnL-trnF sequence in Populus was neutral.The cpDNA trnL-trnF intergenic space sequence of 5 Populus species （Populus tremula、Populus alba、Populus laurifolia、Populus×jrtyschensis、Populus nigra） was highly homologous and monophyletic origin . The phylogenetic tree was clustered into three filiations of Populus tremula, Populus alba and Populus laurifolia, and the Populus nigra was situated at the base of the tree. Populus jrtyschensis, this hybrid was clustered together with Populus laurifolia, which showed that the cpDNA of Populus×jrtyschensis had an origin from its female parent Populus laurifolia. 17 haplotypes were identified based on nucleotide variation. Populus tremula had 2 haplotypes （Hap₁3, Hap₁4）, and Hap₁3 was dominant; Populus alba had 4 haplotypes （Hap₁4, Hap₁5, Hap₁6, Hap₁7）, and Hap₁5 was dominant, and the Hap₁4 was shared by the two species but its frequency was low; Populus nigra had 2 haplotypes （Hap₁, Hap₃）, and Hap₁ was dominant; Populus laurifolia had the largest number of identified haplotype （Hap₂, Hap₅, Hap₆, Hap₇, Hap₈, Hap₉, Hap₁0, Hap₁1, Hap₁2）, and Hap₂ was dominant; Populus×jrtyschensis had 2 haplotypes （Hap₂, Hap₄）. The 2 species of Populus laurifolia and Populus jrtyschensis all had the Hap₂ haplotype, and Populus tremula and Populus alba all had the Hap₁4 haplotype.We analyzed and constructed phylogenetic tree of 17 haplotypes based on the neighbor-joining method （NJ） and the maximum likelihood method （MP）. The phylogenetic tree obtained by different methods was basically coincident but had differences for Bootstrap value （BS）. The results showed that all haplotypes formed a multiphyly cluster and had different presumptive ancestors; Populus laurifolia had the largest number of haplotype variation; the haplotypes in the same Populus Sect. were tend to cluster together and the haplotypes of Aigeiros and Tacamahaca had more closer phylogenetic relationship.更多还原