DNA methylation in sugarcane somaclonal variants assessed through methylation-sensitive amplified polymorphism

Abstract

ABSTRACT. Micropropagation is an important tool for large-scale multiplication of plant superior genotypes. However, somaclonal variation is one of the drawbacks of this process. Changes in DNA methylation have been widely reported as one of the main causes of somaclonal variations in plants. In order to investigate the occurrence of changes in the methylation pattern of sugarcane somaclonal variants, the MSAP (methylation-sensitive amplified polymorphism) technique was applied to micro-propagated plantlets sampled at the third subculture phase. The mother plant, in vitro normal plantlets, and in vitro abnormal plantlets (somaclonal variants) of four sugarcane clones were screened against 16 MSAP selective primers for EcoRI/MspI and EcoRI/HpaII restriction enzymes. A total of 1005 and 1200 MSAPderived markers with polymorphism percentages of 28.36 and 40.67 were obtained for EcoRI/HpaII and EcoRI/MspI restriction enzyme combinations, respectively. The genetic similarity between the mother plant and the somaclonal variants ranged from 0.877 to 0.911 (EcoRI/ MspI) and from 0.928 to 0.955 (EcoRI/HpaII). Most of the MASPs among mother plant and micro-propagated plantlets were derived from EcoRI/MspI restriction enzymes suggesting alteration due to gain or loss of internal cytosine methylation. A higher rate of loss of methylation (hypomethylation) than gain of methylation (hypermethylation) was observed in the abnormal in vitro sugarcane plantlets. Although changes in the methylation pattern were also observed in the in vitro normal plantlets, they were lower than those observed for the in vitro abnormal plantlets. The MASP technique proved to be a promising tool to early assessment of genetic fidelity of micro-propagated sugarcane plants.

Abnormalities in somatic embryogenesis caused by 2,4-D: an overview

“…Genetic similarity between mother plant and abnormal plantlets was compared and the range of similarity was 87.7-91.1% for EcoRI/MspI and 92-95% for EcoRI/HpaII. These results confirm that there is a relationship between abnormalities in in vitro plantlets and DNA methylation (Francischini et al 2017).…”

“…DNA methylation is an epigenetic event that consists of the addition of a methyl group at the 5′-position of the cytosine in the contexts of CpG (5′-C-phosphate-G-3′), CHG or CHH (with H being A/T/G) repetitive sequences in the plant DNA (Finnegan et al 2000;Zhang et al 2010). In tissue culture, mainly in somatic embryogenesis there is a growing appreciation that DNA methylation is an important mechanism influencing SV in the resultant plantlets altering their physiology and phenotype and it can be inherited by generations (Chakrabarty et al 2003;Tchorbadjieva and Pantchev 2004;Francischini et al 2017). Chromatin remodeling is also related to SV when sudden variation in the gene expression can cause phenotypic and physiologic alterations in the plantlets generated by in vitro culture.…”

Whole-Genome Bisulfite Sequencing Reveals a Role for DNA Methylation in Variants from Callus Culture of Pineapple (Ananas comosus L.)

“…Changes in DNA methylation have been observed in abnormal sugarcane plantlets. The rate of hypomethylation is higher than that of hypermethylation [30].…”

Sugarcane mosaic virus mediated changes in cytosine methylation pattern and differentially transcribed fragments in resistance-contrasting sugarcane genotypes

“…MSAP markers were obtained according to Lei et al [33] and adapted for sugarcane, as described by Francischini et al [34]. In brief, each DNA pool was digested separately with 2.5 U of EcoRI-HpaII or EcoRI-MspI restriction enzyme combination at 37˚C for 4 h, and adaptor ligation was performed at 37˚C for 16 h. The reaction was diluted 6× in Mili-Q water for the pre-selective amplification reaction, and then 5 μL aliquot of this reaction was diluted 10x in Mili-Q water for selective amplification by using HpaII-MspI/EcoRI primers with three selective nucleotides at the 3 0 -terminal and fluorescently labeled with IRD 700 or IRD 800 dyes.…”