A relative quantitative Methylation-Sensitive Amplified Polymorphism (MSAP) method for the analysis of abiotic stress

Abstract

BackgroundWe present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics such as demethylation, de novo methylation, and preservation of methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied the technique to analyze aluminum (Al)-tolerant and non-tolerant control and Al-stressed inbred triticale lines. The approach is based on detailed analysis of events affecting HpaII and MspI restriction sites in control and stressed samples, and takes advantage of molecular marker profiles generated by EcoRI/HpaII and EcoRI/MspI MSAP platforms.MethodsFive Al-tolerant and five non-tolerant triticale lines were exposed to aluminum stress using the physiologicaltest. Total genomic DNA was isolated from root tips of all tolerant and non-tolerant lines before and after Al stress following metAFLP and MSAP approaches. Based on codes reflecting events affecting cytosines within a given restriction site recognized by HpaII and MspI in control and stressed samples demethylation (DM), de novo methylation (DNM), preservation of methylated sites (MSP), and preservation of nonmethylatedsites (NMSP) were evaluated. MSAP profiles were used for Agglomerative hierarchicalclustering (AHC) based on Squared Euclidean distance and Ward’s Agglomeration method whereas MSAP characteristics for ANOVA.ResultsRelative quantitative MSAP analysis revealed that both Al-tolerant and non-tolerant triticale lines subjected to Al stress underwent demethylation, with demethylation of CG predominating over CHG. The rate of de novo methylation in the CG context was ~3-fold lower than demethylation, whereas de novo methylation of CHG was observed only in Al-tolerant lines.ConclusionsOur relative quantitative MSAP approach, based on methylation events affecting cytosines within HpaII–MspI recognition sequences, was capable of quantifying de novo methylation, demethylation, methylation, and non-methylated status in control and stressed Al-tolerant and non-tolerant triticale inbred lines. The method could also be used to analyze methylation events affecting CG and CHG contexts, which were differentially methylated under Al stress. We cannot exclude that the methylation changes revealed among lines as well as between Al-tolerant and non-tolerant groups of lines were due to some experimental errors or that the number of lines was too small for ANOVA to prove the influence of Al stress. Nevertheless, we suspect that Al tolerance in triticale could be partly regulated by epigenetic factors acting at the level of DNA methylation. This method provides a valuable tool for studies of abiotic stresses in plants.

Precise evaluation of tissue culture-induced variation during optimisation of in vitro regeneration regime in barley

“…Methylation-sensitive amplified polymorphism (MSAP) (Baranek et al 2016) uses HpaII and MspI endonuclease to study methylation changes within DNA sequences at the restriction sites. This approach was recently extended, enabling quantification of only the site DNA methylation alterations (Bednarek et al 2017). Methylation amplified fragment length polymorphism (metAFLP) enables quantitative analysis of both sequence and DNA methylation changes affecting Acc65I-KpnI restriction sites (Bednarek et al 2007;Chwedorzewska and Bednarek 2012;Machczyńska et al 2014b;Wódkiewicz et al 2018).…”

The contrasting response to drought and waterlogging is underpinned by divergent DNA methylation programs associated with gene expression in sesame

“…Under drought treatment, a decrease of DNA methylation was observed in leaf tissues of two faba bean genotypes (Abid et al, 2017). Moreover, Bednarek et al (2017) observed an elevated demethylation in both non-tolerant and tolerant plants, with de novo methylation occurring less frequently than demethylation under Aluminum stress in triticale lines.…”

“…The MSAP profiles showing reproducible results between replicates were transformed into a binary matrix, with 1 as a presence of band and 0 the absence of band from either EcoRI/MspI or EcoRI/HpaII digestion and analyzed according to the protocol developed by Bednarek et al (2017). In general, these fragments could be divided into four types representing four types of DNA methylation status of the restriction sites (5′-CCGG-3′):…”

“…The methylation-sensitive amplified polymorphism (MSAP) technique developed by Reyna-López et al (1997) is an adaptation of amplified fragment length polymorphism analysis and has proven to be a powerful tool for analyzing DNA methylation. The MSAP technique has been applied to study CpG methylation in the genome of plants, somaclonal epigenetic variation, cytosine methylation during various developmental stages and resistance to biotic and abiotic stresses (Ashikawa, 2001;Matthes et al, 2001;Portis et al, 2004;Sha et al, 2005, Ruiz-Garcia et al, 2005Tang et al, 2014;Zhang et al, 2015;Bednarek et al, 2017;Abid et al, 2017). Different approaches have been proposed to interpret MSAP outputs (Schulz et al, 2013;Fulneček and Kovařik, 2014), but none takes into account the multiple events reflecting various methylation patterns that must take place simultaneously to explain the differences in individual digestion patterns between control and stressed materials.…”

“…The arrangement of all possible events explaining the sixteen four-bit binary MSAP codes (adapted fromBednarek et al, 2017) …”