Research On Molecular Mechanism Of Triploid Vigor In Populus Euramericana

Poplars are important economic tree species with great ecological effect, and are also main planting tree species of China’s protection forest. Natural triploid poplars were found in actual breeding work, and some of them triploid showed better growth advantage and resistance to stress, compared with diploid. However, little is known about molecular mechanism of fast-growing traits formation in triploid trees. Here, Populus euramericana diploid and triploid, generated from crosses between the maternal parent P. deltoides and the pollen parent P. nigra (Triploid shows obvious growth vigor), were employed to analysis molecular mechanism of fast-growing advantage fromation in triploid on gene expression level, DNA methylation level and miRNA expression level, using MSAP technology, gene chip, small RNA sequenceing and trmsgenic Ambidopsis. Main results are as follows:1. MSAP analysis detected significantly different dynamic methylation changes between triploid and diploid black poplar in shoot apical buds, young leaves and mature leaves. CHG methylation of increased from3.23%to5.83%, and CG methylation fell from21.31%to14.41%, then rised to19.28%during leaves development of triplid. CHG methylation of triploid was significantly higher than diploid, and MET1involved in control of CG methylation, CMT involved in control of CHG methylation and DRM involved in control of de nove methylation were also significantly differentially expressed between diploid and triploid, and positively correlated to their corresponding methylation patterns. Twenty-one fragments representing different DNA methylation variations between diploid and triploid trees were sequenced and eighteen fragments were proved to occur in gene-coding regions. DNA methylation levels of shoot apical buds and young leaves were significantly correlated to the down-regulated genes in triploid black poplars. The results suggested that DNA methylation was involved in gene expression regulation of black poplar triploid, and CG and CHG methylation may participate in the different regulatory mechanisms. CG methylation was involved in maintaining genomic stability and CHG methylation was involved in specific gene expression regulation.2. Gene expression changes between diploid and triploid balck poplars were analysed useing poplar gene chip.1564up-regulated transcripts and2015down-regulated transcripts were detected in triploid, compared to diploid. Singular enrichment analysis (SEA) of differentially expressed gene found151up regulated GO terms and94down regulated GO terms. These up-regulated genes were mostly involved in hormone synthesis and metabolism, carbon and nitrogen cycling metabolism and cell wall synthesis. Down-regulated genes were major related to metabolism of nucleic acid. The results preliminary showed that gene expression of triploid black poplar was under the influence of ploidy changes, and its fast-growing properties were related to gene expression changes.3. NimbleGene poplar12x135K gene chip were empolyed to analysis gene expression changes between diploid and triploid balck poplars in shoot apical buds, young leaves, woody stems and woody roots. About21,000genes were detected for expression changes among four tissues, but some of genes were observed for tissue distribution changes. Different amount of differentially expressed genes were found in four tissues, and stems were detected for the most abundant differentially expressed genes, which consisted of2,307up-regulated genes and down-regulated genes. And these differentially expressed genes in stems of triploid contianed1125(up) and768(down) tissues-special variation genes.4. GO terms of these differentially expressed genes were similar to the former gene chip analysis. Up-regulated genes of shoot apical meristems were major involved in genes related to shoot development, and auxin and cell wall synthesis were enhanced; Auxin homeostasis and genes cell wall synthesis genes were also enriched in young leaves; Stems were detected for abundant transcripts involved in biomass regulation, cell wall components synthesis, modification, thickening, sedimentary processes; Genes related to root growth were up-regulated. Comparsion of differentially expressed genes among shoot apical buds, young leaves, stems and roots showed that gene expressions related auxin were active in buds and young leaves; genes related to cell wall synthesis were active in buds, young leaves and stems; PCD process and defense response genes are active in leaves, stems and roots. The comprehensive analysis of the black poplar triploid showed gene expression basis of fast-growing advantage, especially massive expression changes of transcription factors genes in stems of members which may be related to rapid growth of stems.5. BES1, a transcription factor involved in BRs signal pathway, which was proved to positively promote stem elongation, was detected for abuntant in stems of triploid black poplar, as well as BRs signal receptors and kinase genes in BRs signal pathway, which suggested that BRs signal pathway enhacement was responsible to rapid growth of stems. And activation of PCD process in several tissues was also associated with BRs pathway. Therefore, gene expression changes of all tissues in triploid black poplar were mainly related to activation of auxin and BRs signaling pathways.6. sRNA features of diploid and triploid balck poplars were scaned by Solexa sequencing technology. Amount of sRNA, miRNA clustered onto pre-miRNA of known plants miRNA and miRNA clustered onto pre-miRNA of P.trichocarpa in miRBase were detected abuntant in triploid, especially the24nt length sRNA. Compared with the known miRbase,773miRNA were detected in pre-miRNA of known plant miRNA. Results indicated that a great number of sRNAs were generated from genome changes of black poplar triploid, including siRNA and miRNAs. And activation of lots of siRNA may be associated with changes of DNA methylation level, while miRNA expression changes may be associated with gene expression regulation.7. Expression of29known miRNA were analysed among five different development stages of leaves and stem in diploid and triploid black poplars. Most of miRNA in triploid were detected in less exprssion levels in the former development stages of leaves but in higher expression levels in later development stages of leaves, compared with the diploid. However, most of miRNA in stems of triploid were higher than diploid in five detected development stages, which may be responsible to the massive genes expression changes of transcription factors in triploid stems. Eight new prediction miRNA were detected in diploid and triploid. Four new miRNA were possible after validation by pre-miRNA clone, expression analysis and stem-loop prediction. Target genes of pec-miR3x-2predicted by miRDeep, was gene encoding a subunit of RNA polymerase Ⅳ involved in transcription of sRNA. And expression levels of pec-miR3x-2target genes were negtive correlated to the expression levels of pec-miR3x-2.8. Seven high expression genes and regulation genes were cloned. Six of them were insrted into plant over-expression vector (pRI101-PtSIP3, pRI101-PtISPS, pRI101-PtAIMl, pBI121-PtGDSL, pBI121-PtClpCl, pRI101-Pt674). Except the pRI101-Pt674, the rest of genes were transfromated into Arabidopsis. So far, the PtSIP3transgenic plants were observed the remarkable phenotypic changes with inhibition of embryo and capsule development, improvement root development.Comprehensive analysis of DNA methylation analysis, genes expresion analysis of several tissues, sRNA sequencing, miRNA expression detection and diploid and triploid genome structure analysis suggested that black poplar triploid genome structure did not change in mass, and black poplar fast-growing character formation was caused by activation of auxin and BRs related signaling pathways, while the activation effect may be related to sRNA changes. siRNA may be responsible to DNA methylation changes of triploid black poplar, and expression changes of miRNA were related to regulation of growth related gene. sRNA activation was caused by genome fusion and genome duplications.