【作者】 杜庆章；

【导师】 张德强；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2014， 博士

【摘要】 森林作为陆地生态系统最重要的组成部分,蕴含了约82%的生物量和50%的生物多样性,是人类可持续发展与可再生利用的主要资源。但我国森林覆盖率低,国内所需木材原料大多依赖进口,木材资源的战略安全及国家林木育种工程面临严峻挑战；而“林以种为本,种以质为先”,常规的杂交育种策略虽为国家的速生丰产林建设提供了大批良种,但林木遗传杂合度高、育种周期长且重要经济性状多为数量性状等因素严重阻碍了林木遗传改良进程,当前林业生产所用品种的生产力难以满足工业发展对木材产量与质量的需求。因此,深入研究木材形成的生物学基础,解析木材形成的遗传调控机制,是加速林木良种化进程的根本。为此,本论文以我国北方重要的用材及生态型树种毛白杨(Populus tomentosa Carr.)的自然群体与杂交群体为材料,在毛白杨木材形成相关候选基因内微卫星标记资源大规模开发的基础上,首次开展了以连锁与连锁不平衡联合作图为策略的研究工作,剖析了控制林木生长及木材品质性状的等位遗传变异,并建立了林木基因标记辅助育种技术。主要研究内容及结论如下：1.利用直接测序法从138个毛白杨木材形成相关候选基因内开发出544个SSR标记位点,其中75%的位点位于基因的启动子或内含子等调控区域。利用这些标记在毛白杨自然群体中筛选出包含861个等位位点的188对多态性SSR引物,每一标记平均等位位点数目为2-7个。随机选择30对基因内SSR引物对杨树属内五个杨派26个种间个体(馒头柳作为类外群)进行种间转移检测发现,72%的基因内SSR标记可以在胡杨派中有效扩增,而在白杨派种间扩增率为100%；Neighbor-Joining(NJ)聚类分析确定了符合杨属传统分类的6个谱系分组。另外,通过比较220对包含基因内SSR位点的毛白杨与毛果杨同源序列,确定了种间SSR变异的主要原因是SSR位点侧翼序列的突变及重复基元类型的改变。进一步的杂交群体遗传分离检测及单标记关联分析表明开发的基因内SSR标记资源在关联作图、遗传连锁作图及近缘种比较基因组学等方面均具有较大的应用潜力。2.群体遗传多样性及群体结构评价是开展连锁不平衡作图及构建核心种质资源的前提。本文利用20对毛白杨特异性SSR标记对选择于毛白杨种质资源基因库的460株个体进行了遗传多样性评价,共确定了99个等位位点,其平均观测等位位点数目为4.05,显示了该群体具有中等遗传多样性水平。群体遗传结构分析表明,毛白杨种质资源可以划分为11个亚群体(K=11)。对STRUCTURE模型确定的K=3情况下的亚群体组成进行分析,表明了毛白杨三个气候区可以分别作为相对独立的遗传群体进行相关研究。对不同气候区的遗传多样性检测表明南部气候区具有最丰富的遗传多样性水平(N=93,Np=11,HE=0.445,F=-0.102),东北及西北气候区次之,表明南部气候区是当前毛白杨生长分布的中心区域。利用建立的“UPGMA-聚类分析逐次随机取样”与“群体结构重检测”联合分析法对毛白杨种质资源进行核心种质群体构建,确定15%(69)的比例选择出来的毛白杨核心种质群体是最理想的。3.进一步的连锁不平衡研究显示,在毛白杨候选基因内1,200bp距离内连锁不平衡水平已衰退至不明显,显示了基于候选基因的连锁不平衡作图策略在林木中具有更高的效力。利用混合线性模型进行单标记关联分析,确定了来自于39个候选基因的48个SSR标记位点与10个生长与木材品质性状显著连锁,构成80个关联(Q≥0.10),每一标记位点可解释表型变异的1.20%-14.5%；基于单倍型的关联分析确定了来自于32个高连锁区域(代表20个基因)的64个单倍型与10个性状的80个关联(Q≤0.10)。比较单标记及单倍型的关联结果显示单倍型效应主要来自于其包含的显著单标记位点,基于单倍型的关联分析并没有显现出更高的作图分辨率。4.利用1200株银腺杨×毛白杨杂交F1连锁作图群体构建了首张基于候选基因内共显性标记的毛白杨高密度、高解析度遗传连锁图谱。其中,在LOD≥5.0,r≤0.25条件下,确定了包含1270个标记位点(309个SSR、929个AFLP和32个InDel)的19个较大的连锁群,连锁位点覆盖毛白杨基因组总长约2758.6cM,标记间平均遗传距离为2.3cM。341个基因内共显性标记在连锁图谱的标定代表了候选基因的定位,为后期开展重要性状的QTL作图提供了科学的遗传平台。5.基于遗传图谱信息,利用复合区间作图法对光合相关性状、生物量、叶片性状和木材品质等28个数量性状进行了QTL分析。当LOD值≥2.5时,共检测到与25个表型性状相关的179个QTLs,每一标记可解释表型变异的1.27%-30.02%,显示不同复杂数量性状具有不同的遗传控制机制。所有179个QTL按照光合相关性状、叶片性状、生物量及木材品质分组后确定的QTL数目是10-84个,不均匀地分布到18个连锁群上,平均9.4个。LG VI和LG XII是控制这些性状的重要区域,LG_XIII上没有发现与所有28个数量性状相关的QTL。进一步研究发现80%的QTLs区间内包含基因内共显性标记,其隶属的基因主要包括与细胞生长分化相关的重要调控基因或转录因子,如类核酸剪切调控蛋白基因、蛋白酶体调控亚基S3蛋白家族基因和锌指蛋白家族基因等,另外一部分是控制木材形成及生长的功能基因,如纤维素合酶基因家族成员(CesA3、CesA7和CesA9),葡萄糖-6-磷酸脱氢酶基因与EXP1基因等。6.利用连锁与连锁不平衡联合作图策略,确定了来自于27个候选基因的29个SSR与8个生长与木材品质性状组成的39个显著关联(Q≤0.10)。进一步比较了每个显著标记在两个作图群体内对应的基因型效应,最终选择基因型效应一致的31个关联组合。随后,筛选出控制每个性状极高及极低表型值的优势等位位点联合体,并根据育种需要组合利用多个优势位点联合体建立基因标记辅助选择育种技术。利用该技术分别在毛白杨种质资源库(关联群体)及杂交作图群体内选择了10个具有潜在应用价值的优良单株。更多还原

【Abstract】 Forest trees as foundation species in ecosystems that cover vast areas of the earth’s surface, constituting roughly82%of the continental biomass and comprising more than50%of the terrestrial biodiversity They are a source for many of the essential needs and sustainable development of humans. However, the shortage of forests resource and imports of most wood raw material of our essential needs make a severe challenge for the strategic security of wood material and the national forest breeding program. The seed quality is the key for trees breeding. Conventional breeding programs may provide many fine varieties for national fast-growing and high-quality forest program, but these resources can not be sufficient to meet the industrial needs for wood produces. Especially, the breeding and improvement progress has been hindered by trees large size, long generation times, and the natural quantitative for economic traits of interest. Understanding of the molecular mechanisms underlying allelic variation of tree growth and wood formation is considered as one of the main prerequisites for tree genetic breeding improvement. Hence, on the basis of lager-scale development of microsatellites in candidate genes related to wood properties in the Chinese white poplar(Populus tomentosa Carr.), one of the most potential indigenous tree species used for industrial needs and renewable bioenergy in northern China, we first carried out dissection of the molecular mechanisms underlying allelic variation of growth and wood formation by using joint linkage-linkage disequilibrium (LD) mapping, and then constructed molecular marker-assisted selection (MAS) breeding approach.The main results and conclusion in this study as follows:1. We first characterized544genic SSR loci derived from138candidate genes involved in wood formation, distributed throughout the genome of P. tomentosa. Of these SSRs, three-quarters were located in the promoter or intron regions. By screening15wild P. tomentosa ecotypes, we identified188polymorphic genic SSRs with861alleles, two to seven alleles for each marker. Transferability analysis of30random genic SSRs, testing whether these SSRs work in26genotypes of five genus Populus sections (outgroup, Salix matsudana Koidz), showed that72%of the SSRs could be amplified in Turanga and100%could be amplified in Leuce. Based on genotyping of these26genotypes, a neighbor-joining (NJ) analysis showed the expected six phylogenetic groupings. In silico analysis of SSR variation in220sequences that are homologous between P. tomentosa and Populus trichocarpa suggested that genic SSR variations between relatives were predominantly affected by repeat motif variations or flanking-sequence mutations. Inheritance tests and single-marker associations demonstrated the power of genic SSRs in linkage mapping and association studies, as well as comparative genomic studies.2. An understanding of genetics diversity and population structure is important for developing association studies and constructing core collections for tree breeding. Using20species-specific microsatellite markers, we identified99alleles with a mean of4.95observed alleles per locus in the460native P. tomentosa individuals, indicating a moderate level of polymorphism across all individuals. A model-based population structure analysis divided P. tomentosa into11subpopulations (K=11). The pattern of individual assignments into the subsets (K=3) provides reasonable evidence for treating climatic zones as genetic regions for population genetics. The highest level of genetic variation was found in the southern climatic region (i.e., N=93, NP=11,HE=0.445, F=-0.102), followed by the northeastern and northwestern regions. Thus, the southern region is probably the center of the current species distribution. Finally, joint "Stepwise UPGMA clustering procedure" and "Population structure test" selection approach were firstly designed to construct the core collections and identify the core collection comprising15%(69) of the initial collection was the optimal one representing the genetic diversity of P. tomentosa genetic resources.3. Linkage disequilibrium (LD) test shows a clear and rapid decline of LD with distance in base pairs within candidate gene (r2≥0.1, within1200bp), indicating that LD did not extend over the entire gene region. The low LD indicated that candidate gene-based LD mapping approach may be having a higher mapping resolution. Using mixture linar models,80significant associations with all ten phenotypic traits were identified at the threshold of Q<0.10, representing48SSRs from39cadidate genes. These loci explained a small proportion of the phenotypic variance, ranging from1.20%to14.50%. Using haplotype-based association test, we identified80signifcant assocaitons at the threshold of Q≤0.10, representing60significant haplotypes from32high-LD regions (20cadidate genes) with all ten traits. Haplotype analysis did not improve the effect of candidate gene-based association mapping, and the significance is mainly derived from its single markers.4. As an further dissection for important traits in forest tree, a hybrid population consists of1,200individuals, were randomly selected and used for constructing a high-density and high-resolution genetic linkage map, which included19major linkage groups (2758.6cM) comprising1,270markers (929AFLP,309genic-SSRs and32InDels). The average marker interval was2.30cM.341genic-codominant markers order in the linkage map represent the positions of all candidate genes, which provide an important tool for QTLs mapping for traits of interest.5. Using composite interval mapping, QTLs mapping of28quantitative traits were carried out, including photosynthesis, leaf, growth biomass and wood-property triats. A total of179putative QTLs were detected for these25traits at LOD (Logarithm of Odds)≥2.5, with a small proportion of phenotypic variance explained, from1.27%to30.02%; each quantitative trait had its specific genetic model. The number of QTLs of all four groups varied from10to84, unevenly assigned to18linkage groups. LG_VI and LG_XII contained the hot putative QTL regions for multiple traits of interest, whereas no QTL were detected on LG_XIII. In addition,80%of all QTLs linked to genic-codominant markers, and the corresponding candidate genes not only included key function genes or transcription factor related to cell growth and development, such as Nuclear speckle splicing regulatory protein1-like, Proteasome regulatory subunit S3family protein gene and zinc finger (DHHC type) family protein gene, also the functaional genes involved to wood formation and tree growth were identified, such as CesA gene family (CesA3、CesA7and CesA9), Glucose-6-phosphate1-dehydrogenase and EXP1.6. A total of39common associations were detected in both populations using joint linkage-LD mapping approach, representing29SSRs from27candidate genes associations with eight growth and wood-property traits. On the basis of the genotypic effects for each marker tested in both populations,31associations with the same effect patterns were identified. Subsequently, the excellent allele-block controlling the highest and lowest phenotypic values were detected and further used for constructing the molecular marker-assisted selection (MAS) breeding approach. Using MAS breeding approach, we selected10high-quality and fast-growing germplasm from both association and linkage populations. The clonal propagation experiment for new germplasm of Populus tomentosa was carried out.更多还原