【作者】 卫尊征；

【导师】 李百炼； 张德强； 张金凤；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2010， 博士

【摘要】 小叶杨(Populus simonii)是我国北方地区的主要乡土树种,具有耐瘠薄、抗逆性强、适应性广、易繁殖、寿命长和杂交可配性好等特点,是目前我国“三北”地区的主要绿化生态建设树种以及培育优良抗逆无性系的重要亲本材料。由于干旱缺水、沙漠化和滥砍滥伐等自然灾害和人为因素的影响,我国的小叶杨遗传种质资源正在急剧减少,迫切需要对现有的小叶杨种质资源进行评价与保存。为此,在对小叶杨基因遗传资源进行踏查的基础上,对我国小叶杨主要分布区内11个省(市)自治区的16个群体进行了资源收集,共收集保存了528个基因型个体。在此基础上,在表型性状、生理生化和分子水平上系统研究了小叶杨自然群体的遗传多样性,揭示了在不同种源的遗传变异规律,最终构建了小叶杨核心种质保存的样本方案,为保护现存的小叶杨遗传资源提供了理论依据。同时,利用基于自然群体的连锁不平衡作图策略,进行了SSR分子标记位点与表型及生理生化共25个性状的联合遗传学研究,研究结果为筛选优良等位变异、发掘和创新种质资源及制定遗传改良策略提供了科学理论依据,具有重要的应用价值。主要研究结果如下：(1)在表型水平上对小叶杨自然群体进行了遗传多样性研究,结果显示,小叶杨15个表型性状在种源间和种源内均存在丰富的遗传变异。叶、茎和根三类表型性状的变异系数分别为22.91%、35.68%和40.90%,其中以叶面积变异系数(48.79%)最高,叶柄长(45.32%)次之,叶厚(12.89%)和侧脉角(7.38%)最低,叶片生长性状高于其它生长性状；种源内的表型变异系数最大的为河北张家口(32.15%),河南洛宁(31.89%)次之,山东沂水(25.13%)和青海都兰(24.80%)最小；各地区种源以分布中心区的华北(31.92%)和华中地区(29.99%)种源变异系数最高,其它边缘群体西北(28.36%)、东北(28.25%)和西南(26.96%)次之,华东地区(25.13%)最小。重复力计算表明叶、茎和根表型性状主要受中度和强度遗传与固定环境效应控制,变异范围为0.456-0.798；各种源重复力均值差异不大,多在0.5-0.6范围内波动。表型分化系数为0.4518,种源内变异(54.82%)高于种源间变异(45.18%),建议种源内无性系间的遗传差异是小叶杨遗传多样性的主要来源。相关分析显示仅有叶宽等3个性状与海拔等4个生理生态因子存在显著或极显著关系。UPGMA聚类结果可将16个小叶杨种源划分成四个主要类群,河北张家口和陕西洛川各自形成一个类群,青海的互助、祁连和都兰形成第三类群,其余种源构成第四类群。(2)在生理生化水平上,对小叶杨10个生理性状在自然群体中的遗传多样性研究表明,在种源间和种源内存在丰富的遗传变异。光合、呼吸和抗逆3种类型生理性状变异系数分别为22.94%、36.61%和40.25%,其中以丙二醛含量变异系数(56.47%)最高,苯丙氨酸解氨酶(54.48%)次之,叶绿素A(22.01%)和类胡萝卜素(20.31%)最低；各种源平均变异系数差异不大,多数在20%-30%之间,其中以陕西洛川(40.71%)和陕西富县(36.61%)最高,青海互助(24.78%)最低；同样,以小叶杨分布中心区的华北(35.63%)、华中(34.39%)及东北地区(34.59%)变异系数最大,其它边缘地区次之,西北种源变异最小(33.74%)。所有生理指标都受强度遗传与固定环境效应控制,各个生理指标的重复力大小为0.694-0.986；各种源重复力差异不明显,多在0.8-0.9范围内,以山西宁武(0.929)和四川康定(0.925)最高,内蒙通辽(0.840)和辽宁朝阳(0.826)最低。生理分化系数为23.93%,即小叶杨生理变异中种源内贡献占76.07%,说明种源内遗传资源比种源间丰富。相关分析中,过氧化氢酶等4个生理指标与除经度外的多数地理生态因子呈显著或极显著相关。UPGMA聚类结果可将16个小叶杨种源分为四个主要类群,其中山西宁武和辽宁朝阳分别组成前两个类群,青海互助、青海祁连和陕西洛川3个种源组成第三类群,青海都兰与甘肃迭部等13个种源形成第四类群。(3)在分子水平上对小叶杨自然群体进行了遗传变异研究,利用20对SSR引物共扩增出306SSR标记位点,每一SSR可扩增出8～28个多态性位点,所有位点观察等位基因数(Na)、有效等位基因数(Ne)、观察杂合度(Ho)、期望杂合度(He)、多态信息量(PIC). Nei’s基因多样性(h)、Shannon信息指数(I)、固定指数(Fit)和近交指数(Fis)分别为6.981、3.877、0.511、0.691、0.637、0.677、1.443、0.348和0.244。群体内遗传多样性十分丰富,各遗传参数分别为Na=6.981, Ne=3.877, Ho=0.511,He=0.691, PIC=0.637,h=0.677,I=1.443,基因流(Nm)=1.588。分子方差分析结果显示群体内差异是遗传变异的主要来源,群体内方差分量占85%以上,群体间不足15%。各群体Nei’s无偏遗传距离和遗传一致度的变化范围为0.442～0.818和0.115-0.542；聚类结果显示小叶杨主要分为3个类群,山东沂水群体单独形成一类,青海群体、陕西富县及山西宁武群体聚为第二类,其它地区群体形成第三类。(4)对小叶杨表型、生理和DNA分子标记3个层次的研究内容进行耦合,结果显示三者在揭示小叶杨遗传多样性水平方面有一定的差异,仅在群体遗传结构和分化、种群遗传多样性变异及群体聚类等方面有一定的吻合。表型与生理遗传多样性参数不相关,而与SSR分子标记具有相关性；群体内差异是遗传变异的主要来源,群体内遗传多样性大于群体间遗传多样性；陕西中部与河南西北伏牛山区等的遗传变异最为丰富,遗传多样性最高,山东沂蒙、青海都兰和互助等地区的遗传变异最小,遗传多样性最低；青海互助和祁连、内蒙通辽及吉林通榆等群体的表型、生理和SSR标记多样性聚类结果比较一致。(5)结合表型、生理生化和分子标记3方面的研究结果,通过综合比较拟合出小叶杨群体间及群体内个体间的样本策略,即在小叶杨全分布区内随机抽取12个以上的群体,每群体保存36个以上的单株。同时,营建了小叶杨异地保存初级核心种质资源库,对充分保护小叶杨资源遗传多样性、防止基因丢失及筛选优异种质具有重要的理论和实践意义。(6)在对小叶杨自然群体进行遗传亚结构分析的基础上,进行了SSR标记基因型与表型及生理生化性状的连锁分析。对于表型性状,共检测到17个SSR标记位点与其显著连锁。与每一性状显著连锁的SSR标记位点数为1-8个,每一位点可解释表型变异的1.84%-23.39%：而对于生理生化性状,关联分析仅检测到与叶绿素B等4个性状显著连锁的6个SSR标记位点,每性状关联位点数为1-3个,关联位点对生理生化表型变异的贡献率为11.33%～26.46%；关联分析中存在“一因多效”及“多效一因”现象。(7)以携带“无效等位基因(null allele)”材料表型均值为对照,进一步分析了与性状关联位点的等位变异,估计了等位变异的潜在表型效应增量(减量),并利用该信息估计了位点增效(减效)等位变异的平均效应。结果表明,关联位点正、负效应等位变异均值间有差异；多性状关联位点的等位变异在不同性状间具有各自表型效应的方向和大小；等位变异在相关性状效应上方向、大小的异同解释了性状间正、负相关的遗传原因；研究鉴别出了一批与表型及生理性状关联的优异位点和等位变异及携带优异等位变异的载体材料。更多还原

【Abstract】 Populus simonii, indigenous to north China, is one of key forest resources for shelterbelt with a lot of characteristics, such as higher tolerance with drought, low temperature and sterile soil and other adaptability. It is propagating easily, and has long life and higher ability to reproduce with hybridization and other characteristics. Therefore, it is the main tree species in ecological protection and the important parent materials for cultivation of excellent resistance germplasm in "Three North" area of China. The genetic resource of P. simonii is being reducing dramatically owing to the reasons of natural disasters, including drought, water shortage, desertification, deforestation, and man-made factors. Thus, it is urgent to evaluate and conservate the genetic resource of natural population in P. simonii. For this purpose, in this thesis, a total of 528 individuals were collected from 16 provenances, covering 11 provinces or autonomous regions, and then established ex situ conservation pool of P. simonii. The genetic diversity of natural populations of P. simonii were systematically studied at the phenotype, physiology and biochemistry, and DNA levels, and the law of genetic variation of different sources and diversity of the state is revealed, while a sample of the core genetic resource conservation programs is constructed. The results provide the necessary theoretical basis and reference for the rescue and protection of existing P. simonii resources. Interestingly, the association between SSR genotypes and phenotypes of 25 economic traits were conducted by the linkage disequibrium mapping based on natural population in P. simonii. The SSR markers significantly associated with commercial traits provide the solid foundation for screening good alleles, discovering and developing genetic resource and making genetic of policies and strategies in P. simonii. The main research results were as follows:1. The genetic diversity of natural population of P. simonii was studied at the phenotypic level, and the result indicated that variation of 15 phenotypic traits of P. simonii is significant different among provenances and among clones within provenances. The phenotypic coefficients of variation of leaves, stems and roots were 22.91%,35.68% and 40.90%, respectively; In which, coefficient of variation for leaf area (48.79%) was the highest, followed by petiole length (45.32%), leaf thickness (12.89%) and the lateral veins angle (7.38%) were the lowest, and leaf growth characteristics is higher than the others; As for the phenotypic coefficient of variation, the largest one is located in Zhangjiakou (32.15%), and the second one is located in Henan Luoning (31.89%), the minimum one from Shandong Yishui (25.13%) and Qinghai Dulan (24.80%) provenances; the variation of measured characteristics is as following among provenances:North China where are the distribution center (31.92%) and Central China (29.99%) were the highest and other marginalized groups in the Northwest (28.36%), Northeast (28.25%) and Southwest (26.96%) were followed, East China (25.13%) was minimum. Repeatability values showed that leaf, stem and root phonotypical trait is mainly affected by fix environment and gene in moderate and high degree. The scope of variation is 0.456-0.798; the average of all repeatability is no substantial difference which ranged from 0.5 to 0.6. Phenotypic differentiation coefficient is 0.4518; variation (54.82%) among provenances is higher than variation (45.18%) among clones with in provenances, which indicates that clones within provenances, is the major source of genetic diversity of P. simonii. Correlation analysis showed those only 3 traits of leaf width and 4 physiological and ecological factors of elevation have significant relationships. Clustering of 16 P. simonii provenances can be divided into four main groups though UPGMA testing, Zhangjiakou in Hebei and Luochuan in Shanxi each form a separate group, Huzhu, Qilian and Dulan in Qinghai form the third group, and the remaining species constitute the fourth source group.2. The genetic diversity of natural population of P. simonii also detected at the physiological and biochemical levels, and the results revealed that the genetic variation was significant different among population and among clones within provenances for 10 physiological traits of P. simonii. Coefficients of variation of photosynthesis, respiration, and stress resistance was 22.94%,36.61% and 40.25% respectively, of which malondialdehyde (own coefficients of variation 56.47%) is the highest, phenylalanine ammonia lyase (54.48%) followed, chlorophyll A (22.01%) and carotenoids (20.31%) is minimum; various original average coefficient of variation is insignificant, the majority is between 20% and 30%, of which Luochuan (40.71%) and Fuxian (36.61%) in Shanxi province is the highest, Huzhu in Qinghai (24.78%) is minimum; Similarly, the coefficient of variation were the highest among North China which are the distribution center (35.63%), Central China (34.39%) and the northeast (34.59%), other marginalized groups followed, northwest (33.74%) was minimum. All the physiological indicators were mainly affected by fix environment and gene in high degree. The repeatability of each physiological indicator was 0.694-0.986; each original repeatability is no big difference which ranged from 0.8 to 0.9, of which Ningwu in Shanxi (0.929) and Kangding (0.925) in Sichuan was the highest, Tongliao (0.840) in Inner Mongolia and Chaoyang (0.826) in Liaoning is the lowest. Physiological differentiation coefficient was 23.93%,that was P. simonii physiological variation accounted for 76.07% among clones within population, indicating the genetic resource among clones within population is richer than among population. Correlation analysis showed that four physiological indices including catalase and the geographical and ecological factors excepting longitude were highly significant correlation. Clustering of 16 P. simonii provenances can be divided into four main groups though UPGMA testing:Ningwu in Shanxi and Chaoyang in Liaoning formed the first two groups, Huzhu and Qilian in Qinghai and Luochuan in Shaanxi formed the third group, other 13 kinds of provenances including Dulan in Qinghai and Diebu in Gansu was the fourth group; Mantel’s tests show phenotype relationship is not significant between Euclidean distance and geographical distance3. The genetic diversity of natural population of P. simonii was studied at the molecular level. In total,306 polymorphic SSR loci were detected using 20 pairs of SSR primers, of which each SSR marker produced polymorphic loci alleles ranged from 8 to 28, and all sites observed number of alleles, effective number of alleles, observed heterozygosity, expected heterozygosity, polymorphism information content, Nei’s gene diversity, Shannon information index, fixation index and inbreeding index were 6.981,3.877,0.511,0.691,0.637,0.677,1.443,0.348 and 0.244, respectively. It was very rich in genetic diversity within populations, the genetic parameters were Na = 6.981, Ne= 3.877, Ho= 0.511, He= 0.691, PIC= 0.637, h= 0.677,I= 1.443, Nm= 1.588. Analysis of molecular variance showed that differences in populations was the main source of genetic variation, variance within groups accounted for more than 85%, less than 15% between groups. Nei’s unbiased genetic distance and genetic identity varied from 0.442-0.818 and 0.115-0.542 in each group; P. simonii was divided into three groups by clustering, Yishui in Shangdong provenance formed a group of individual, provenances of Qinghai, Fuxian and Ningwu in Shaanxi, were the second group, others formed the third; Mantel test showed that P. simonii genetic distance and geographic distance was no correlation.4. Though three levels of the research content of coupling for P. simonii in phenotype, physiology and DNA molecular markers, it was found that the threes had certain differences in the levels of genetic diversity, it only had a certain agreement that the population genetic structure and differentiation, genetic variation and population diversity cluster, etc. Phenotype of genetic diversity and physiological parameters are not correlated but the SSR markers; the difference among population was the major source of genetic variation, populations greater the genetic diversity within populations was more than among populations; there were abundant genetic variations and the highest genetic diversity in the middle of Shaanxi and Funiu mountains of the Northwest of Henan and so on. However, it opposites in Yimeng of Shandong, Dulan and Huzhu of Qinghai and so on. Huzhu of Qinghai, Qilian, Inner Mongolia and Tongyu of Jilin and so on were consistent on phenotype, physiological and clustering results of SSR marker diversity. The genetic diversity of three markers of the reflection had focused on the situation but complement each other.。5. Combining with the data of genetic diversity at the phenotypic, physiology and molecular levels, the sampling strategy for establishing core germplasm of P. simonii was plotted within populations and between the populations, i.e., samples of more than 12 groups were chose randomly in the whole distribution area in P. simonii, each group kept more than 36 plants. Meanwhile, the genetic resource was constructed for protecting the primary core offsite collection, which has important theoretical and practical significance in the full protecting P. simonii genetic diversity, preventing gene loss and screening of elite germplasm.6. Considering sub-structure of natural population of P. simonii, the association studies between SSR genotypes and phenotypic data of phenotype, physiological and biochemical traits were performed. The results showed that 17 SSR markers associated with 8 traits, including leaf length/width, etc. For each trait, the numbers of associated loci is 1 to 8, related loci on the phenotype of rate of explanation is 1.84%-23.39%; physiological trait association mapping only detected the 6 related loci of 4 trait including chlorophyll B, each trait loci is 1 to 3, explain rate of related loci on the physiological traits’change is large ranging from 11.33% to 26.46%; there was "pleiotropic effects" and "a result of multi-effect" phenomenon in association mapping.7. The alleles of SSR markers associated with phenotype were further analysis through comparing with the phenotype average date of null allele. Alleles potential phenotypic effects of increment (decrement) was estimated, and using the information estimated the average effect of alleles of the site efficiency (less effective). The results showed that there were different among loci positive and negative effects, the average of alleles; the alleles variation of multiple trait loci has their own phenotypic direction and size among different traits. Alleles variation on the direction of effects in the relevant traits, the size of the similarities and differences between characteristics explained the positive and negative correlation of genetic causes; study identified a number of phenotypic and physiological characteristics with excellent sites and the associated alleles and carrying outstanding alleles of the carrier material.更多还原