| 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. |
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