Methylation-sensitive RFLPs: characterisation of two oil palm markers showing somaclonal variation-associated polymorphism

Abstract

The occurrence of "mantled" somaclonal variants (approx. 5%), which display alterations in floral organ structure, among populations of somatic embryo-derived oil palms ( Elaeis guineensis Jacq.) currently hampers any scaling-up of clonal plant micropropagation. As a first step towards the identification of abnormality-discriminating markers, we have screened a set of 27 oil palm cDNA probes for methylation-sensitive restriction fragment length polymorphisms (RFLPs) using callus genomic DNA digested with the isoschizomeric enzymes MspI and HpaII. Only two probes (CPHO62 and -63) were found to differentiate reproducibly in two different genotypic backgrounds between nodular compact calli (NCC) and fast-growing calli (FGC), which generate 5% and 100% "mantled" plantlets, respectively. Comparative analyses were then conducted on DNA from inflorescences and leaves of normal and abnormal adult regenerants. With both probes, the observed methylation patterns were strongly clone-dependent and monomorphic with respect to the phenotype of the regenerants, except for the type-specific banding pattern obtained with the CPHO62 probe on material from the LMC3 clonal offspring. The results presented here mirror the higher difference in genomic DNA methylation observed between normal and abnormal embryogenic calli when compared to more differentiated plant material. Moreover, they reinforce the paramount interest of NCC and FGC callus lines as a material of choice in the search for early epigenetic markers of the "mantled" somaclonal variation. The potential use of methylation-sensitive RFLPs for the early detection of somaclonal variation at early stages of the micropropagation process is discussed.

Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications

“…When over-expressed in tobacco, the gene was able to confer cell division and callus formation properties, instead of root organogenesis in an auxin-rich medium, Leal et al (2006) Fluorescent in situ hybridization (FISH) Gernand et al (2007) 2 DNA sequence changes Restriction fragment length polymorphism (RFLP) Andreev et al (2005) Inter-simple sequence repeats markers (ISSR) Sreedhar et al (2007) Random amplified polymorphic DNA (RAPD) Jin et al (2008) Microsatellites or simple sequence repeat (SSR) Jin et al (2008) Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) Kour et al (2009) Amplified fragment length polymorphism (AFLP) Li et al (2007) Next generation sequencing of genomic DNA libraries Jiang et al (2011) 3 DNA sequence-specific changes Inter-retrotransposon amplified polymorphism (IRAP) Smýkal et al (2007) Sequence-specific amplification polymorphism (SSAP) Kour et al (2009) Transposon display Ngezahayo et al (2009) 4 RNA expression changes Differential display Linkiewicz et al (2004) Suppression subtractive hybridization Zeng et al (2006) EST array or Gene Chip hybridization Che et al (2006a, b) cDNA macroarray hybridization Singla et al (2007) Genome-scale microarray hybridization Bao et al (2009) Kaeppler and Phillips (1993a, b) and Jaligot et al 2002 Methylation-sensitive restriction fragment length polymorphism (metRFLP) Bednarek et al (2007) Methylation-sensitive amplification polymorphism (MSAP) Kour et al (2009) Based on treatment with the restriction enzyme McrBC, which only cleaves DNA at 5-methylcytosine Tanurdzic et al (2008) DNA degradation followed by reversed-phase high-performance liquid chromatography (RP-HPLC) Kubis et al (2003) DNA degradation followed by high performance capillary electrophoresis (HPCE) Berdasco et al (2008) Based on bisulfite treatment, which changes un-methylated cytosines into uracil, and sequencing Ngezahayo et al (2009) Transposon methylation display (TMD) Ngezahayo et al (2009) 9 Chromatin condensation Fluorescent in situ hybridization (FISH) Koukalova et...…”

Somaclonal variation in plants: causes and detection methods

“…RFLP was one of the first techniques used to study somaclonal variation and has been widely used in several species. For example, Jaligot et al (2002) described methylation-sensitive RFLP markers that differentiated between normal and abnormal embryogenic calli of oil palm. The markers were useful for the early detection of somaclonal variation.…”

Flow Cytometry, HPLC-RP, and metAFLP Analyses to Assess Genetic Variability in Somatic Embryo-Derived Plantlets of Gentiana pannonica Scop

“…Many marker systems have been applied to study somaclonal variation. RFLP techniques have been used to assess polymorphic changes (Müller et al 1990;Sabir et al 1992;Chowdhury and Vasil 1992) and evaluate alterations of the DNA methylation patterns (Jaligot et al 2002). In contrast to the RAPD technique which exclusively identifies sequence changes, its application to study somaclonal variation remains controversial (Munthali et al 1996).…”