| Epigenetic modification refers to changes in the chromatin leading to stable and heritable phenotypic variations,not involving changes in the DNA sequence.These changes include various types of modification such as DNA covalent modification,modification of histones,chromatin remodeling,and non-coding RNA regulation.Among these,the archetypical and most-shared modification in eukaryotic genomes is DNA cytosine methylation,which plays an important role in regulating gene expression during growth and different developmental stages of plants.In this study,the genomic DNA methylation in 432 individuals from nine natural populations of P.tomentosa,ten distinct types of organ or tissue,and 552 individuals from the progeny population of?Populus aba × Populus glandulosa?"YX01" and P.tomentosa "LM50" were detected using the methylation sensitive amplified polymorphism?MSAP?technique.Also,multivariate statistical analysis and single marker association analysis were used for exploring the epigenetic variation in DNA methylation and its effects on phenotypic variation in populations.The major results of this dissertation were as follows:1.Within the 432 individuals from nine natural populations of P.tomentosa,the relative total methylation level?6.22%-22.40%,average 12.60 ± 2.51%?of mature xylem was significantly higher than the relative non-methylation level?6.19%-15.77%,average 11.21± 1.52%?,the relative full methylation level?3.10%-15.10,average 6.31 ± 1.79%?was higher than the relative hemi-methylation level?2.86%-12.87%,average 6.29±1.75%?.For the ten distinct types of organ or tissue,compared to the average value of the relative total methylation level?10.71%?,the average value of the relative non-methylation level?36.17%?was higher,also the average relative full methylation level?11.90%?was higher than the average relative hemi-methylation level?9.52%?.In the population of 552 hybrids from?Populus alba × Populus glandulosa?"YX01" and P.tomentosa "LM50",the relative total methylation level?16.40%-45.98%,average 26.58±4.40%?of mature xylem was significantly higher than the relative non-methylation level?6.59%-23.52%,average 15.35±2.63%?,the relative full methylation level?7.80%-33.19%,average 14.26±3.77%?was higher than the relative hemi-methylation level?8.01%-23.76%,average 12.32±2.08%?.The difference was significant for the genomic DNA methylation of mature xylem between natural population and progeny population,the relative total methylation level of the natural population was lower than the progeny population.The genomic DNA methylation in mature xylem of natural population of P.tomentosa showed great epigenetic variation,significant differences of the relative methylation/non-methylation levels among nine different natural populations were detected.The relative non-methylation level in the Henan population was the greatest among the nine populations.The relative hemi-methylation level and relative total methylation level in the population of Beijing displayed the greatest value and the largest relative full methylation level was found in the population of Hebei.The difference between relative hemi-methylation level and relative full methylation level was significant within the populations of Beijing?P<0.001?,Hebei?P<0.001?,Shandong?P<0.001?,Henan?P=0.031?,and Anhui?P=0.016?.Meanwhile,the difference between relative total methylation level and relative non-methylation level was significant within the populations of Beijing?P<0.001?,Hebei?P<0.001?,Shandong?P<0.001?,Shanxi?P<0.001?,Anhui?P=0.002?,and Jiangsu?P=0.047?.2.The epigenetic diversity?0.51 ± 0.17?of natural population of P.tomentosa was higher than the genetic diversity?0.12±0.10?.The genetic diversity index of the nine natural populations was measured between 0.14 ± 0.09?Hebei?and 0.59 ± 0.09?Jiangsu?,the epigenetic diversity index was measured between 0.49±0.17?Hebei?and 0.63±0.07?Jiangsu?.Genetic and epigenetic diversity were both significantly different among the nine natural populations?P<0.001?,the estimate of genetic and epigenetic differentiation was ST=0.073 and ST=0.112.The optimal number of subpopulation for naturally occurring population of P.tomentosa was K=3.3.The ranges of variation for the nine phenotypic traits were considerable and the distribution of frequency for each trait in the measured population of P.tomentosa followed an approximately normal distribution,and the nine phenotypic traits correlated in linear to some extent.Single marker analysis showed 1627 MSAP markers were detected to be responsible for variance of phenotypic traits with the contribution ranging from 1.00%to 22.53%.And 368 markers were associated with more than one phenotypic trait,133 markers were associated with both growth and wood property traits.Within the progeny population,the ranges of variation for the nine phenotypic traits were also considerable and the distribution of frequency for each trait followed an approximately normal distribution,and the nine phenotypic traits correlated in linear to some extent.Single marker analysis detected 2616 MSAP markers were responsible for variance of phenotypic traits with the contribution ranging from 1.79%to 15.37%.And 460 markers were associated with more than one phenotypic trait,20 markers were associated with both growth and wood property traits.4.The full-sib family population of 552 F1 individuals of popular was genotyped using MSAP markers and tested through the regression mapping algorithm and Kosambi's mapping function in JoinMap 4.1 to estimate the epigenetic linkage map?E-map?and to calculate the map distance between mapping markers.The E-map consisting of 19 major linkage groups?LG?was constructed with 2463 polymorphic MSAP markers and the number of polymorphic MSAP markers in each LG ranged from 64 to 203,with an average of 130 loci.The LGs covered a total map distance of 2426.7 cM,and LGs ranged from 72.9 to 161.7 cM with an average marker interval of 1.1 cM.LGV had the lowest density?0.45 per cM?of epiloci,while LGXIX had the highest density?2.09 per cM?.Furthermore,seven obvious gaps?>10 cM?were detected to distribute on six LGs,and LG IV had two gaps,LGV,LGVI,LGIX,LGXIV,and LGXVI each had one gap.The distribution of epiloci on individual linkage groups appeared to be extremely uneven across the E-map.5.QTLs controlling nine different traits were identified and assigned as QTLepi,a total of 163 QTLepi were detected from the E-map by the MQM analysis.These QTLepi unevenly distributed on linkage groups with explanation to phenotypic variance ranging from 0.1%to 44.5%.For tree height,25 QTLepi distributed on eight linkage groups were detected to explain the variation with the range of 0.1%to 15.7%.Nine QTLepi from five linkage groups controlled basal diameter and contributed at the level of 0.5%to 20.5%to the variation.Stem volume were controlled by 26 QTLepi on ten linkage groups with the contribution ranged from 3.18%to 10.9%.20 QTLepi from nine linkage groups were detected to control the variation of fiber length with the range of 1.7%to 15.7%.16 QTLepi from three linkage groups were detected to control the variation of fiber width with the range of 1.3%to 12.5%.Five QTLepi from three linkage groups were detected to control the variation of microfiber angle with the range of 2.7%to 44.5%.12 QTLepi from six linkage groups were detected to control the variation of lignin content with the range of 3.5%to 9.7%.3 1 QTLepi from ten linkage groups were detected to control the variation of a-cellulose content with the range of 1.1%to 14.5%.19 QTLepi from eight linkage groups were detected to control the variation of holocellulose content with the range of 0.5%to 18.6%.6.The result of gene expression analysis showed that patterns and locations of DNA cytosine methylation could be related with transcription,transcription level of hemi-methylated gene was significantly lower than full methylated and non-methylated one,and transcription level of gene with promoter methylation was also significantly lower than that with gene body methylation.By sequencing of the fragment with trait-associated and tissue-polymorphic MSAP markers,homolog protein coding genes involving in various biological processes in the genome of P.trichocarpa were detected to be regulated by methylation.Transcription levels of wood-property related genes significantly varied with different methylation patterns in distinct tissues of stem vascular and this revealed the correlation between gene expression and methylation.Furthermore,wood-property related genes with different methylation patterns displayed different genetic effect on variation of phenotypic traits. |
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