Genetic and epigenetic instability of amplification-prone sequences of a novel B chromosome induced by tissue culture in Plantago lagopus L.

Abstract

Gene amplification is prevalent in many eukaryotes and has been found linked to various phenomena such as ontogenesis, carcinogenesis, in vitro culturing, neoplasia and drug resistance. Earlier, we reported a novel B chromosome in Plantago lagopus L., which was found to have arisen as a result of massive amplification of 5S rDNA. In addition, the chromosome is also composed of 45S rDNA and transposable elements. While the importance of gene amplification cannot be underestimated, its mechanism of origin is still unclear. Therefore, the aim of the present study was to determine whether amplification can be reactivated in the novel B chromosome. For this purpose, in vitro culture was used as stress. Three modes of tissue culture, i.e., direct, indirect and somatic embryogenesis were used for raising in vitro cultures. The variations due to genetic and epigenetic mechanisms were assessed in regenerants using molecular techniques, namely, PCR-RFLP, SSAP and MSAP. The retrotransposon-based molecular markers were applied to detect the polymorphism within transposable elements of in vitro regenerated and mother plants. We detected the variations that may be due to genetic changes either because of element recombination or activation of transposable elements which can lead to increase in the copy number. MSAP analysis revealed the differences in the DNA methylation pattern of the regenerants derived from novel chromosome bearing mother plants. Some regenerated plants were associated with increase and decrease in DNA methylation of both internal and external cytosine of the CCGG sequence.

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

“…The magnitude of DNA sequence and methylation variation present in in vitro regenerated plants has been analyzed in diverse crops such as barley (Bednarek et al 2007;Li et al 2007), cocoa (López et al 2010), grapevine (Schellenbaum et al 2008b), hop (Peredo et al 2006), pea (Smýkal et al 2007), Doritaenopsis ), plantago (Kour et al 2009), Arabidopsis (Yu et al 2010), maize ) and medicinal plants such as Codonopsis ). The frequency of these variations increased with culture age, although some studies also show a subsequent decline in very advanced culture regenerants, possibly due to the loss of totipotency and regeneration potential of extremely mutated cells as observed in cocoa cultures (López et al 2010).…”

“…An extensive increase in copy number of mainly 5S ribosomal DNA sequences and transposable elements in the medicinal plant Plantago lagopus resulted in the formation of an independent, supernumerary 'apparent B chromosome'. Genetic instability as visualized by recombination and transposition could be re-induced in these B chromosomes by means of in vitro stress (Kour et al 2009). …”

“…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...…”

Morphological and RAPD-marker characterization of Melia volkensii (Grke) in vitro plants regenerated via direct and indirect somatic embryogenesis

“…Epigenetic variations are due to changes in gene expression arising from DNA methylation, histone modifications and small RNA-mediated regulation (Miguel and Marum, 2011;Neelakanda and Wang, 2012). DNA sequence changes can be detected by a variety of molecular markers such as random amplified polymorphic DNA (RAPD) and microsatellites or simple sequence repeats (SSR) (Jin et al, 2008), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (Kour et al, 2009) and amplified fragment length polymorphism (AFLP) (Li et al, 2007).…”

B chromosome in Plantago lagopus Linnaeus, 1753 shows preferential transmission and accumulation through unusual processes

“…The primers selected for the analysis were MKD-4, MKD-5, MKD-9, MDK-11, ASW-8, ASW-9 and ASW-10 ( Kour et al 2009). A total of twelve different combinations were made using these primers (Suppl.…”