| Wide hybridization occurs frequently in plants,which may promote genome evolution and induce genetic and epigenetic instabilities in the resultant hybrids, allopolyploids and introgressants.It remains unclear however whether pollination by alien pollens of an incompatible species may impose a "stress" even in the absence of genome-merger or genetic introgression,whereby genetic and/or epigenetic instability of the maternal recipient genome might be provoked.In this study,we uncovered a rice mutator-phenotype(named Rb0) from a set of rice plants derived from a crossing experiment involving two remote and apparently incompatible species,Oryza sativa L. and Echinochloa crusgalli L..We observed the fertilization behavior of barnyard grass pollens on the rice stigma via microscopic observation.We found that barnyard grass pollen tube can geminate and grow on the stigma of rice and the pollen tube can enter into rice micropyle after pollination.However,gel-blotting by total genomic DNA of the pollen-donor showed no evidence for introgression in the resultant mutant plant(Rb0). Characterization of Rb0 and its selfed progenies(S1) ruled out contamination(via seed or pollen) or polyploidy as a cause for its dramatic phenotypic changes,but revealed mobilization of several previously characterized transposable elements(TEs),including a MITE(mPing) and its putative transposase-encoding partner Pong and two LTR retrotransposons(Osr42 and Tos17) and methylation variation of two LTR retrotransposons(Tos17 and Osr36).Genome-wide analysis by the AFLP and SSR fingerprinting markers revealed extensive,transgenerational alterations in genetic variation in Rb0 and/or its immediate progenies S1 by alien pollens of barnyard grass,including base mutation,deletion and insertion,as well as the number of microsatellite repeat sequence changes.Meanwhile, genome-wide analysis by MSAP revealed an enhanced DNA methylation level and changing of site-specific patterns.Notebly,the transgenerational continuous occurrence of multiple phenotypic,genetic and epigenetic variations from a single individual(Rb0) is characteristic of a "transgenerational genomic instability".To further explore the possible reasons for the genetic variations described above, assay by q-RT-PCR of the steady-state transcript abundance of a set of genes encoding for the various putative mismatch repair(MMR) genes showed inheritance of the altered expression of these genes in Rb0,S1 sefled progenies and S2 sefled progenies relative to the rice parental line.At the same time,we analyzed the expression-state of genes which involved in chromatin regulation in order to explore the possible causes for the epigenetic variations. We also found expression of these genes changed and inherited.Finally,in oder to detect whether perturbed homeostatic expression-state of genes is prevalent in this "stress" or not,we investigated these genes in another rice mutator-phenotype (Tong211-LP) that produced by pollinating the rice stigma with pollens of O.biennis L. Strikingly,the expression-state of these genes were also found heritably changed in Tong211-LP and its selfed progenies as well as in S1 that produced by pollinating the rice stigma with pollens of barnyard grass.Perturbed homeostatic expression-state of genes which involved in maintenance of chromatin structure is likely an underlying cause for the alien pollination-induced epigenetic/genetic instability,and which occurred apparently without entailing genome merger or genetic introgression.Our results suggest that pollination by unrelated alien pollens in plants might impose a stress condition and induce genetic and epigenetic instabilities in the maternal genome.Given simplicity of this cross-pollination experiment,it is not difficult to imagine that similar incidents may occur under natural conditions.At the same time,the results of this study have provided a theoretical basis for the possible utility of "incompatible wide-hybridization" in crop breeding.
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