AFLP-DERIVED METHODS AS A TOOL FOR STUDY OF GENOMIC, EPIGENOMIC AND TRANSCRIPTOMIC CHANGES IN STRESSED GRAPEVINE PLANTS

Abstract

Many studies describing phenotypic and genetic variation among standard and stressed plants were already published. A possibility to estimate stress-induced changes in genome, epigenome and transcriptome arose by establishing three AFLP-based methods within our laboratory (standard AFLP, methylation-sensitive AFLP and cDNA-AFLP). Three variants of grapevine plants after different stress actions (stress by in vitro cultivation, in vitro thermotherapy and virus infection) were prepared for this study. In case of standard AFLP application, comparison of obtained spectra via 0/1 binary matrix is usually performed. Within our group of variants the results show insignificant changes in its genomes. MS-AFLP spectra evaluation needs more detailed analysis of results to extract more precise information about DNA methylation characters and impact of individual stress factors on epigenetic changes. From the point of view of functional methodology, the cDNA-AFLP appeared as the most difficult to establish. During first experiments only short PCR amplicons (at most 150 bp) were generated. Further, the calculated similarity coefficients showed surprisingly low values. These facts signalised certain unspecific RNA degradation before its conversion onto ds cDNA. The significant improvement of obtained results was recorded by using RNA later reagent, SpectrumTM Plant Total RNA Kit, RNase Out enzyme and MINT cDNA synthesis kit in cDNA-AFLP protocol. In the case of cDNA-AFLP spectra evaluation, it is possible either to prepare 0/1 binary matrix or differences can be estimated on the base of ratio between intensities of individual peaks. This fact should be taken into account if cDNA-AFLP results are compared with other fingerprinting methods as RAPD, SSR or standard AFLP.