Inheritable Variation In DNA Methylation And Tranposon Activation Induced By Laser-irradiation In Rice And The Possible Underlying Causes

Laser is a different natural light radiation. And Laser irradiation is a traditional method for crop improvement.Laser mutation can remarkably raise the sprouting potential and sprouting ratio of seed, accelerate the speed of growing and improve the disease resistance.DNA methylation as an important epigenetic modification plays an important role in controlling gene expression and maintaining genomic stability in animals and plants. Intrinsic DNA methylamine pattern is an integral component of the epigenetic network in many eukaryotes. Study on variation of DNA methylation pattern will help us a comprehensive understanding of function of DNA methylation on the apparent biological control. Quantitative evidences suggest that DNA methylation status is changeable when some biotic and abiotic conditions change. Therefore, the question rises whether laser irradiation could induce DNA methylation variation, through which phenotypic variation happen. In this work, we used Nd3+YAG laser-irradiating Jinong 18 rice seeds to analysis the DNA methylation alterations .The major results are as follow:We found in this study Nd3+YAG laser-irradiating rice seeds produced plants with apparent mutations in several phenotypic traits including later-heading date and dwarfism.Genome-wide fingerprinting on methylation patterns by the MSAP method revealed that the methylation alterations were actually widespred, and also mainly represent demethylation. Progeny analysis indicated that the altered MSAP patterns also were largely meiotically heritable, at least to the M2 generation. Notebly,the transgenerational continuous occurrence of multiple phenotypic and epigenetic variations from a single individual(M0) is characteristic of a transgenerational genomic instability .To further explore the possible reasons for the epigenetic variations described above,assay by q-RT-PCR of the steady-state transcript abundance of a set of genes encoding for chromatin regulation in order to explore the possible causes for the epigenetic variations,including methyltransferase MET1 and CMT3, the DNA glycosylase/ lyase DME, as wll as the important enzymes involving the genesis of small RNA, which was related to the change of methylation.The results showed that laser irradiation could cause change of DNA methylation patterns, the variation of methylation could be inherited to the progenies at a total or high frequency via meiosis although just a few sites or individuals showed recovery or further change; in the meanwhile, laser irradiation also caused the expression change of genes encoding for chromatin regulation. Perturbed homeostatic expression state of genes which involved in maintenance of chromatin structure is likely an underling cause for the laser-irradiation induced epigenetic instability.Nd3+YAG laser irradiation caused mobilization of several previously characterized transposable elements(TEs),including a MITE(mPing) and its putative transposase- encoding partner Pong .Bisulfite sequencing analysis further proved the methylation inheritance and alterations, and also indicated that the expression of Tos17 was associated with its cytosine methylation.Based upon the above observations, we conclude that Nd3+YAG laser irradiation represents a potent mutagen for rice, not only from the classic genetic point of view, but also from an epigenetic perspective by mobilizing otherwise quiescent mobile elements and by inducing heritable alterations in DNA methylation patterns.