| Populus simonii Carr.belonging to the section under Populus Tacamahaca is wildly distributed in northern China,with drought resistance,barren tolerance and strong comprehensive resistance.It is one of the main tree species to construct the "Three North Shelter Forest Program" in China.P.simonii plays an important role in ecological and environmental protection of the Yellow River Basin and has high environmental,ecological and economic values.However,the research on P.simonii mainly focused on limited aspects,like the evaluation and the use of its genetic advantages in traditional cross breeding.Poplar owns a long span life and high heterozygosity,which hinders its own genetic improvement process.In recent years,more and more evidences have shown that epigenetics is closely related to plant growth and resistance.With the rapid development of epigenetic molecular markers and bisulfate sequencing,the role of epigenetic research in forest assisted breeding has been greatly enhanced.Here,epigenetics was introduced into the study of genetic improvement of P.simonii based on current research trends.For the first time,a natural population consisting of 505 individuals of different genotypes from 9 provenances of Populus simonii was used to study the population epigenetic characteristics of P.simonii by MSAP.The excellent resistant clone QL9 was selected for bisulfate sequencing in order to have a better understanding of its resistance mechanism and complete the epigenotyping with call SNPs.Meanwhile,Back Propaganda(BP)neural network was used to quantify the effects of different epigenotypes on gene expression.The major results of this dissertation are as follows:(1)A wide variation of genome methylation was found by performing MSAP,and 20,413 MSAP fragments were obtained.Of which 8,187(40.11%)fragments were polymorphic.The Shannon index was used to evaluate the population diversity of P.simonii from 17 provenances in 9 provinces in China.In the control group,the population epigenetic diversity ranged from 0.654±0.551 to 1.248±0.397,of which Shaanxi was the highest,while Gansu was the lowest.There were significant differences in epigenetic diversity among 9 subpopulations(?=42.179,P<0.001).Epigenetic population structure analysis showed that the natural population of P.simonii was divided into three sub-populations.The Yellow River separated the natural population into two parts:the north and the south,and the Taihang Mountains separated the north part into two sub-groups:the east and the west.It indicates that the propagation process of Populus is closely related to the geographical environment.P.simonii populations from different provenances retain the epigenetic information of their respective provenances,which we call"environmental imprinting".(2)They are all epigenetic information accumulated by P.simonii in long-term adaptation survival of habitat.After drought stress,the diversity of nine sub-populations decreased significantly,ranging from 0.1 15±0.287 in Gansu Province to 0.307 ± 0.242 in Shaanxi Province.There was no significant difference between natural populations(?2=7.603,P=0.473).The epigenetic population structure of P.simonii changed from three subgroups to one under drought stress.The analysis of meteorological data of poplar plantation in recent 60 years shows that the natural population of P.simonii has been subjected to drought stress once or several times every summer since it was transplanted to Guan County,Shandong Province in 2007,which indicates that the epigenetic information of natural population of P.simonii periodically changes with the periodic stimulation of the external environment.(3)DNA methylation mainly affects the growth and development of organisms by affecting gene expression in the modified region.The epigenotype was proposed for the first time to be used to describe the modification of DNA methylation on alleles,and introduced linear regression to quantify the effect of different epigenotypes on expression,and obtained E=14.93X-17.19Y-19.57Z+22.12.The corresponding relationship between different epigenotypes and gene expression was revealed.However,the application range of linear regression is narrow(R2=0.06),indicating that gene expression is affected by multiple factors.Then we introduce a multidimensional back propagation(BP)neural network to construct a potential model.The input data were screened by principal component analysis with SPSS 20.The ratio of homozygous methylation to heterozygous methylation explained 99.428%of the variance of the ratio and selected as the input data.Through the analysis of the least mean square error,the hidden layer is determined to be five,and the fitting success rate is 99.059%,which is much higher than 6%of the multiple linear regression.In addition,the BP neural network trained by MATLAB can be used to predict the new data,which is universal and can be applied to other treatments,even other species.Furthermore,the fitting success rates of the apparent genotype effects of heat,cold,salt,osmotic stress and control group was 43.32%,37.11%,39.41%,45.63%and 50.78%,respectively,using the BP neural network model trained under drought treatment.The result proved that BP neural network has certain universality and can be applied to other data besides training data.(4)DNA methylation plays an important role in response to environmental stimuli.However,the response pattern of poplar to different types of external stresses is still unclear.Here,four stresses(heat,cold,salinity and osmosis)were performed to P.simonii,and sequential monitoring was carried out,including short-term monitoring,0 hours,3 hours,6 hours,12 hours and 24 hours after treatment;and long-term monitoring,1 month,3 months and 6 months after treatment.The first detection of P.simonii under different abiotic stresses showed a significant increase in methylation levels,suggesting that external stimuli can promptly trigger the change of DNA methylation.Under heat treatment,the methylation level increased most significantly,which was significantly higher than that of other treatments at 3 and 6 hours.At 24 hours,DNA methylation levels under osmotic and cold stress were significantly higher than those under heat and salinity stress.The results showed that there were different response patterns for cytosine methylation under different treatments.(5)Under the four treatments,1,376 stress-specific differential methylation regions(SDMRs)were isolated and sequenced by MSAP.After removing the failed sequencing and redundant fragments,1,123 SDMRs mapped genes encoding proteins,16 mapped miRNA genes and 17 mapped long non-coding(Inc)RNA genes.The SDMRs 162 was mapped to Psi-MIR396e and PSILNCRNA00268512 simultaneously.The lncRNA could be used as a target mimic of PsiMIR396e to regulate the level of miRNAs.The methylation level of response to stress gradually declined over time,and about 35.1%of the response sites remained methylated one month after treatment,while only about 15.3%remained methylated six months later.For six months after treatment,only one miRNA,MIRNA6445a,showed long-term stability of methylation and remained methylated.The above results reveal that P.simonii as tall perennial woody plants are constantly exposed to environmental changes,requiring rapid,transient,and/or seasonal responses.So long-term stable methylation might play very important roles in the responses to environmental stimuli.For example,MIRNA6445a can improve the tolerance of P.simonii to stress. |
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