摘要
毛白杨(Populus tomentosa Carr.)是我国重要的白杨派乡土树种,具有速生、材质优良、抗病性强等特点,在黄河流域发挥着重要的生态和环境保护作用,具有较高的经济、生态与社会价值,因此,对其进行遗传改良备受关注。先前的研究多集中于该树种的传统遗传学研究,如遗传多样性评价、遗传结构分析、数量性状的QTLs解析等,但未涉及表观遗传变异领域。然而目前的研究表明,表观遗传,特别是]DNA甲基化修饰在植物基因表达调控过程中扮演着重要的角色,且可以在不同世代间遗传。因此,对毛白杨群体的表观遗传变异及遗传效应进行研究具有重要的理论与应用价值。本论文首次以毛白杨432个基因型构成的自然群体和130个种内杂交子代无性系为试材,利用MSAP标记技术、多元统计和单标记分析等手段对其基因组DNA甲基化遗传变异、遗传效应进行了分析。本论文取得的主要结果如下：
1.毛白杨木质部、叶片基因组甲基化相对水平不同。自然群体木质部基因组总甲基化相对水平为26.57±5.86%,其中全甲基化相对水平为13.10±2.28%,半甲基化相对水平为13.47±4.64%；总甲基化相对水平低于非甲基化相对水平(42.71±6.73%,P<0.001),全甲基化相对水平低于半甲基化相对水平(P<0.001)。130个毛白杨种内杂交子代无性系功能叶片基因组非甲基化相对水平为71.86±1.01%,高于总甲基化相对水平(17.87±1.47%,P<0.01),全甲基化相对水平(9.26±0.96%)高于半甲基化相对水平(8.61±1.10%,P<0.01)。
2.毛白杨自然群体木质部基因组DNA甲基化存在丰富的表观遗传变异。9个种源间毛白杨基因组DNA甲基化(或非甲基化)相对水平差异显著,其中山东毛白杨总甲基化相对水平和半甲基化相对水平最大,北京毛白杨全甲基化相对水平最大,河南毛白杨非甲基化相对水平最大；同时,北京、河北、山东及河南种源内毛白杨全甲基化相对水平和半甲基化相对水平具有显著性差异,河北、山东、河南、山西和陕西种源内毛白杨DNA总甲基化相对水平和非甲基化相对水平差异显著。在表观遗传多样性方面,陕西种源毛白杨多样性最丰富(1.211),而甘肃种源多样性最小(0.811)。9个种源毛白杨表观遗传分化系数分布于0.054和0.366之间,自然群体GsT值为0.159。表观遗传结构分析显示,甲基化敏感多态性数据矩阵(MSP)组间变异占7.700%,组内变异占92.300%,第一、第二主成分共解释总惯量的64.50%。山西毛白杨除与河南毛白杨有部分交集外,与其它各种源毛白杨无交叉。而河南毛白杨则与河北、陕西等种源毛白杨有交集。甲基化类型数据矩阵(CG-CNG)组间变异占9.000%,组内占91.000%,第一、第二主成分共解释总惯量的73.11%,河南毛白杨与其它多数种源毛白杨在一定程度上有交集,而河北、山西种源毛白杨相互独立。MSP和CG-CNG矩阵具有极显著正相关性,典型对称协惯量分析认为MSP和CG-CNG矩阵分布具有相似性,第一、第二主成分分别解释了总的协惯量的68.51%、4.26%,并且CG-CNG的贡献率较大。由于植物材料定植于相同地点和取材时间的一致,消除了环境和发育阶段特异表达带来的误差,实验推测毛白杨木质部甲基化可经有丝分裂过程被继承,其表观遗传变异与遗传变异并不完全一致,并推测河南可能是毛白杨自然分布中心。
3.毛白杨自然群体木材品质等9个表型性状呈正态分布,它们之间存在显著相关性,并且树高与非甲基化相对水平呈显著负相关、与全甲基化相对水平呈显著正相关；单标记分析共检测到1101个多态性MSAP标记位点与木质素含量等9个表型性状相关联,每一标记可解释表型遗传变异1.057%(P<0.043)-7.784%(P<0.001),其中470个标记位点与两个或两个以上上表型相关联。
4.创制毛白杨种内杂交子代新种质295份,其中130个无性系功能叶片净光合速率(Pn,14.83士3.76μmol m-2S-1)、气孔导度(Gs,0.29±0.09mol m-2s-1)、胞间二氧化碳浓度(Ci,264.50±30.94μmol mol-1),幼苗高生长(H,133.59±50.44cm)和地径(D,16.29±5.20mm)等表型数据呈正态分布,且Pn分别与H、D间呈正相关(P<0.05)；表型性状Pn、Gs、H、D均与总甲基化相对水平、半甲基化相对水平显著正相关。MSAP单标记分析共检测到81个多态性MSAP标记位点与光合特征因子显著连锁,每一标记可解释表型遗传变异的3.395%(P=0.036)-18.982%(P<0.001),其中13个标记同时与生长性状显著连锁。对以上81个候选标记位点进行克隆与测序,成功获得59条基因序列,它们与编码原叶绿索酸脂还原酶的基因、或与编码细胞色素P450CYP4/CYP19/CYP26亚家族的基因、或与编码参与光合系统Ⅱ(photosystem Ⅱ)有关功能蛋白的基因等具有同源性。因此,毛白杨基因组光合基因响应基因组甲基化,并且可以影响生长性状,具有表观遗传效应。
Chinese white poplar{Populus tomentosa Carr), belonging to the section under Populus Duby, with fast-growing, high quality timber characteristics, and disease resistance, is one important native tree species in China. It plays an important role in ecological and environmental protection along the Yellow River and has great economic, ecologica and social values. Therefore, researchers focus much attention to the improvement of this tree species. Previous researches focus on genetic variation in P. tomentosa, such as evaluation of genetic diversity, genetic structure and QTLs linkage analysis, etc. However, little knowledge is known about on the epigenetic variant which plays an important role in the process of plant gene expression and regulation and can be inherited that revealed by emerging evidence. This dissertation firstly studied the genomic DNA methylation in432individuals from natural population in P. tomentosa, and one intraspecific hybridization population including130genotypes.The methylation sensitive amplification polymorphism (MSAP) techniques, multivariate statistical analysis and single marker analysis were used for exploring the epigenetic variant in DNA methylation and its effects on phenotype variation in population. The major results of this dissertation were as follows:
1. The genomic DNA methylation relative levels in xylem and leaves were not the same. Within the432individuals of natural population from P. tomentosa, the relative total methylation and non-methylation levels were26.57±5.86%and42.71±6.73%, respectively. The relative non-methylation level was significantly higher than the relative total methylation level (P<0.001). Also, compared to the relative CG methylation level (13.10±2.28%), the relative CNG methylation level (13.47±4.64%) was higher (P<0.001). In the population of130hybrids, the genomic non-methylation relative level was71.86±1.01%, which was larger than the relative total methylation level (17.87±1.47%)(P<0.001).The relative full methylation level was higher than the relative hemi-methylation level(8.61±1.10%)(F<0.001).
2. The genome methylation in the xylem of natural P. tomentosa population showed great epigenetic variation. Firstly, significant differences among different natural populations were detected. Among the nine populations, the relative methylation/non-methylation levels were significantly different. The relative total methylation level and relative hemi-methylation level in the Shandong population were the greatest among the9populations. The relative full methylation level in the population of Beijing displayed the greatest value and the largest relative non-methylation level was found in the population of Henan. It was also found significant differences between relative CG methylation level and relative CNG methylation level within the populations of Beijing (P=0.026), Hebei (P=0.018), Shandong (P=0.002) and Henan (P=0.001), as well as significant differences between relative total methylation level and relative non-methylation level within the populations of Hebei (P=0.001), Shandong (P=0.012), Henan (P<0.001), Shanxi (P<0.001), Shaanxi (P<0.001), respectively. Secondly, the Shaanxi population had the maximum value in epigenetic diversity and thus it showed higher variation than other populations. However, the Gansu population displayed the least Shannon's diversity. Coefficients of epigenetic differentiation (GST) within each population were distributed from0.054to0.366and a GST=0.159was obtained within the432genotypes. According to the methylation sensitive matrix (MSP), the first two principal components explained64.50%of the variant which could be partitioned into between-(7.700%) and within-(92.300%) populations. The Shanxi population was non-overlapping with other populations except for a partial intersection with the Henan population which also intersected with Hebei, Shaanxi, and other populations. While the first two principal components, which could also be partitioned into between-(9.000%) and within-(91.000%) populations, of methylation pattern matrix (CG-CNG) explained73.11%of the variation in total inertia. The Henan population intersected with other populations to some degree, but the populations of Hebei and Shanxi were independent of each other. The relationship between MSP and CG-CNG profiles was significantly positively correlated. And the distributions of the two profiles were similarly that was detected by a typical symmetric co-inertia analysis. The first two principal components explained68.51%,4.26%of the total inertia, respectively, and CG-CNG token great contribution. Eliminating environmental-and stage-specificity factors, it was suggested that DNA methylation could be fixed and inherited though mitosis. Compared to genetic structure, data also suggested that epigenetic and genetic variation do not completely match.
3. The phenotype values in the content of lignose and other eight traits were displayed normal distribution, and they correlated in linear. The hight of tree were correlated negatively with relative non-methylation level, but gave positive correlation with relative CG methylation level, respectively. Using the single MSAP molecular marker analysis, we obtained that1101markers were associated with the nine timber traits.125markers were associated with content of Iignose,127markers were associated with content of holocellulose,72markers were associated with content of a-cellulose,190markers were associated with fiber length,134markers were associated with fiber width,140markers were associated with microfiber angle,312markers were associated with diameter at breast height,414markers were associated with height of tree, and345markers were associated with volume of timber, respectively. The contribution of each associated marker to explain the variant in phenotype was ranged in a percentage from1.057%(P<0.043) to7.784%(P<0.001). And470markers were associated with more than one phenotype trait.
4. New germplasm in intra-hybridization were created and295offspring were obtained. In total,1532clones were reproduced from200genotypes. Research on the130offspring including390clones, it showed that the photosynthetic characteristics data and growth traits were normally distributed. The net photosynthetic rate (Pn,14.83±3.76μmol m-2s-1),stomatal conductance (Gs,0.29±0.09mol m-2s-1), and intercellular CO2concentration (Ci,264.50±30.94μmol mol-1) showed the similar trends. Positive correlations were found between Pn and height of the seedling(H,133.59±50.44cm), and basal diameter (D,16.29±5.20mm), respectively. Positive correlations between phenotype (such as Pn, Gs, H and D) and total relative methylation level and relative CNG methylation level were investigated. Eighty one candidate markers were linked with Pn, Gs, and/or Ci,13of which were also linked with growth traits detected by using the single MSAP molecular marker analysis. Each marker could explain3.395%{P=0.036)-18.982%{P<0.001) of the variant in phenotype. Sequencing and BLAST analysis showed that candidate markers were linked to genes encoding protochlorophyllide reductase and proteins of cytochrome P450CYP4/CYP19/C YP26subfamilies, and linked to genes taking part in, e.g., photosystem Ⅱ. Therefore, the regions defined by the MSAP candidate markers are linked to genes that are essential for photosynthetic characteristics that respond to DNA methylation and subsequently affect growth traits.
引文
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