【作者】 白卉；

【导师】 姜静； 邢玉娟；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2010， 博士

【摘要】 山杨(Populus Davidiana.Dode)是我国广泛分布的乡土树种之一,也是白杨派的重要山地造林树种。其材质优良、木材色白、质轻而软、结构均匀、纹理通顺,是优质的工业原料。本研究以建立在黑龙江省林口县青山林场的山杨种源家系保存林为试验材料,从表型和DNA水平测定了山杨遗传多样性,分析了种源间以及种源内家系间遗传多样性。同时依据208个单株SSR分子标记遗传距离,结合单株材积的性状,筛选出核心库并进行了评价,建立了核心种质资源的离体保存体系,并对其进行利用进行了的初步研究。旨在为山杨基因资源评价、保护与保存、遗传改良策略制定等提供科学理论依据,并为优良山杨基因资源的利用提供技术支撑。通过以上研究,得出如下主要研究结果：1.山杨表型性状的变异非常丰富,在种源间和种源内许多性状均存在显著的遗传变异,尤其是树高、胸径和材积等生长性状,种源间差异达到极显著水平。山杨种群表型性状频率多样度为0.664,种源间为0.018,种源内为0.646；总体水平的Shnnaon信息指数(Ⅰ)为1.469,种源间为0.096,种源内为1.373。根据表型多样性综合指标的聚类结果,可将山杨6个种源划分为两大类,方正、苇河聚为一类,湖上、江山娇、曙光、铁力聚为一类。2.利用6对SSR引物对6个种源的山杨的遗传关系进行了SSR分析,在引物预期产物大小片段处共扩增出29个位点,其中所有位点均具有多态性,多态位点比率达到100%。利用Shannon指数与Nei指数估算6个种源山杨的遗传变异,山杨总体的Shannon指数为1.1001,各个种源的Shannon多态性信息指数中,方正种源最大,达到1.1111,铁力种源最小,为0.7508。根据Shannon指数的大小将各种源排序为：方正>湖上>苇河>曙光>江山娇>铁力。所研究的山杨种源总的Nei遗传多样性指数为0.5957,每个种源的Nei指数分布在0.4421-0.5944范围内。所有种源根据Nei指数排列的顺序与根据Shannon指数排列的顺序基本一致。3.6个山杨种源间的基因分化指数Gst=0.1143,基因流系数Nm为1.9366。山杨种源内的遗传多样性占总群体的88.57%,种源间遗传多样性占总群体的11.43%,这说明山杨种源内变异占较大比例。山杨种源间遗传分化指数Gst=0.1143,说明种群间遗传分化较大。山杨6个种群间的基因流Nm为1.9366,证明山杨种群间存在一定的基因流动,能够降低局部变异,防止适应性分化。4.利用SSR分子标记构建的山杨6个种源间的遗传关系聚类图,根据Nei’s遗传距离利用MEGA软件构建种群遗传关系聚类图。方正、苇河、曙光、铁力聚为一类,江山娇和湖上聚为一类。5.对208份山杨种质材料以UPGMA法进行聚类,并采取逐步聚类随机取样法、逐步聚类优先取样法和分组聚类法,分别在30%、25%、20%、15%、10%的比率下抽取15个候选子集,经过对候选子集遗传多样性参数的比较研究,初步筛选出逐步聚类优先取样法20%抽样比率下的核心库T3的42份山杨核心种质资源为最佳核心种质,经分子多样性评价核心库有39种基因型,等位基因Na*4.6667,有效等位基因Ne*2.7288,Shannon指数I*1.1173,Nei指数H*0.6067。核心库表型性状多样性评价,均值差异百分率MD%为15.42%,极差符合率CR79.09%,变异系数变化率VR为90.00%.,达到了核心库构建的标准,’因此认为核心库T3应作为山杨种源家系保存林208个个体的分子遗传多样性的核心种质资源进行保存。6.构建了山杨组培体系,筛选出山杨芽最佳消毒方法是70%-75%乙醇浸泡10-20s,0.1%氯化汞消毒3min左右；最佳繁殖途径是以腋芽作为外殖体离体快繁；山杨芽最适分化培养基为NT+6-BA0.5mg/L+KT0.3 mg/L,分化率最高可达86%,分化系数达到4.32；最佳增殖培养基为WM+6-BA0.5mg/L+NAA0.01mg/L+2%蔗糖；组织培养中高温、透气性差的塑料膜、光强不足等因素均显著促进玻璃化苗的发生,其中封口材料是玻璃化发生的主要因子；最佳生根培养基为WM+IBA1.5mg/L+NAA0.01-0.02mg/L+2%蔗糖,生根率高达93.2%,每苗生根数可高达14条。由遗传因素决定,山杨不同无性系生根能力存在显著差异；移栽应选择茎基部无愈伤组织,根生长粗壮、无污染的健壮苗,移栽基质为1/2珍珠岩+1/2壤土,移栽后5天内湿度维持在70%以上,并且每天喷雾2次,成活率最高可达90%。并确定山杨组培苗在WM+6-BA3.0mg/L+6.5g/L琼脂+2%蔗糖培养基中,可以将保存130天。7.对于山杨核心库优良基因资源采取工厂化育苗的方式直接加以利用。明确了山杨工厂化育苗生产流程,简化了山杨组培技术体系,将嫩枝扦插方法引入组织培养中,使原有的初始分化—继带增殖培养—生根培养—营养杯已在四个阶段减少到初始分化培养—继代增殖培养—试管外生根三个阶段,缩短生产周期1-2个月；针对不同移栽容器采取不同的移栽方法,并对山杨容器苗造林技术进行规范。更多还原

【Abstract】 Populus davidiana Dode is a wildely distributed native tree species in China, is one of the most important mountain broadleaf trees. The material quality is quite well, the wood-color is white, its wood is homogeneous, light and soft, which is the high-quality industrial material. This study used the provenance and family conservation of Populus davidiana Dode in Qingshan Forest Farm, Linkou County, Heilongjiang Province, as the test material. The phenotype and DNA levels were researched to analyze the poplar genetic diversity. At the same time each plant genetic distance based on 208 SSR markers, with the plant material product of the characters, the core collection is eatablished and evaluated, and we establish a core vitro conservation of it, and were used to conduct a preliminary study. Designed to poplar genetic resource evaluation, protection and preservation of, genetic improvement strategies that will provide^ the scientific theoretical basis, and good use of poplar genetic resources to provide technical support. The studies come to the following key findings:1. The phenotypic variation of P. davidiana Dode is very rich in provenances and provenances in many traits, there were significant genetic variation, especially in height, diameter and volume growth traits, among provenances difference was very significant. Phneotpyic diversity index was0.664 among 0.018, among Population 0.646, Shannon infmoration index was1.469 maong Provence 0.096, among Population 1.373. Comprehensive phenotypic diversity index based clustering results may be six kinds of sources Populus divided into two categories, Fangzheng, Weihe clustered together, Hushang, Jiangshanjiao and Tieli, Shuguang force together as a classs.2.6 pairs of SSR primers were used to analyze the genetic relationship of 6 provenances of-P. davidiana, there were 29 locis amplified in the expected size fragment of the product, the percent of polymaorphic loci was 100%. Estimation of the genetic variation of 6 provenances of P. davidiana according to the Shannon index and Nei’s index, the Shannon index were 1.1001 in P. davidiana, the Shannon index of FZ provenance was the highest, the number was 1.1111, the TL provenance was the lowest and the the number was 0.7508. The order or 6 provenances according to the Shannon index was:FZ>HS>WH>SG>JSJ>TL. The Nei’s index of P. davidiana was 0.5957, every Nei’s index was distributed from 0.4421 to 0.5944. The order according to the Shannon index and Nei’s index was basically the same.3. The genetic diversity within provenance was 88.57% and the genetic diversity between provenances was 11.43%. This was indicated that the variation within provenance account for a large proportion of the sourse. The coefficient of genetic differentiation was 0.1143 which showed that the higher genetic differentiation occurred between provenances. The mean of gene flow was 1.9366, it was showed that there have gene flow between provenances, it can reduce local variation and prevent adaptive differentiation.4. Constructed the dendrogram of genetic relationship between the 6 provenances using SSR molecular markers. The genetic distance between HS provenance and JSJ provenance was close, the two provenances were clustered into one group, and SG provenance and WH provenance were clustered into one group. FZ provenance was far from other 5 provenances.5. Make use of the UPGMA clustering method, the 208 Populus make up a germplasm to and cluster random sampling method to gradually step by step cluster sampling method and packet priority clustering method, respectively,30%,25%,20%,15%,10% collected under the rate of a subset of 15 candidates, after the candidate subset of parameters of genetic diversity of a comparative study of the initial screening phase cluster sampling of priority under 20% of the core collection sampling rate of 42 Populus T3 core germplasm resources the best core collection, evaluation by the core collection of molecular diversity of 39 genotypes, alleles Na * 4.6667, effective allele Ne* 2.7288, Shannon index I* 1.1173, Nei’s index H* 0.6067. Core collection phenotypic diversity assessment, the mean percentage difference was 15.42% MD%, very poor compliance rate CR79.09%, coefficient of variation was 90.00% rate of change of VR., To the core library building standards, so that the core library T30000000000000000000 Populus species should be preserved forest resources and families of 208 individuals of molecular genetic diversity of the germplasm resources preservation.6. Establish a tissue culture systerm. The optimal sterilizing method of buds was dipping into 70% to 75% alchohol for ten to twenty seconds, and then treating with 0.1% HgCl2 for about 3min; the optimal material for propogation by multiplication is of axillary buds; The optimum culture medium of differentiation was NT+6-BA1.0mg/L+KT0.3mg/L+2%sucrose, the differentiation rate was 86%, a single explant can differentiated 4.32 buds at best. The optimum proliferation culture medium was WM+6-BA1.0mg/L+NAA0.01mg/L+2%sucrose. High temperature, airtight plastic membrane and weak light can promote the vitrification, of all, the airtight plastic membrane is the most important factors. WM+IBA1.5mg/L+IAA0.01mg/L +2% surose was the optimum culture medium of rooting. The rooting rate was up to 93.2%, a single seeding has 14 roots at the most. Study on the influence of factors to rooting emphatically, There were prominent difference in the seedling age to influence on rooting rate and rooting numbers, the rooting ability which in the 30dto 35d seedling age is the most strongly.7.Populus well use for the core colledtion to factory breeding of genetic resources be used directly. Clear aspen seedlings factory production processes, simplified technology of tissue culture Populus will softwood cutting method into the tissue culture, so that the initial differentiation of the original-with the proliferation of cultured following-rooting-Nutrition Cup in four reduced to the initial phase of differentiation culture-subculture-in vitro rooting of three stages, shortening the production cycle 1 to 2 months; for different transplanting transplanting container to take a different approach, and afforestation techniques of poplar container to regulate.更多还原