Genetic and epigenetic instabilities induced by tissue culture in wild barley (Hordeum brevisubulatum (Trin.) Link)

Abstract

A simple tissue culture protocol was developed for efficient plant regeneration from young inflorescence-derived calli in wild barley, Hordeum brevisubulatum (Trin.) Link, an important pasturage grass. Genetic and epigenetic instabilities in the regenerated plants (regenerants) were assessed by three molecular markers AFLP, S-SAP and MSAP. Two pools of calli derived from young inflorescences of a single donor plant and 44 randomly chosen regenerants were subjected to AFLP analysis. Results showed that 74 out of 793 scored bands were polymorphic among the studied samples, giving rise to a genetic variation frequency of 9.3%. The number of variant bands as compared to the donor plant varied greatly among the regenerants, with a small number of regenerants accumulated a large number of variant bands (maximum 55), while the majority of regenerants showed only 2-3 variant bands. A subset of regenerants together with the two pools of calli were selected for S-SAP and MSAP analysis to detect possible retrotranspositional activity of a prominent retroelement family, BARE-1, in the genomes of Hordem species, and possible alterations in cytosine methylation. S-SAP analysis showed that of the 768 scored bands, 151 were polymorphic among the analyzed samples, giving rise to a genetic variation frequency of 19.7%, albeit no evidence for retrotranspositional event was obtained based on locus-specific PCR amplifications. MSAP analysis revealed that tissue culture has caused cytosine methylation alterations in both level and pattern compared with the donor plant. Sequencing of selected variant bands indicated that both proteincoding genes and transposon/retrotransposons were underlying the genetic and epigenetic variations. Correlation analysis of the genetic and epigenetic instabilities indicated that there existed a significant correlation between MSAP and S-SAP (r = 0.8118, 1,000 permutations, P < 0.05), whereas the correlation between MSAP and AFLP (r = 0.1048) is not statistically significant.Electronic supplementary material The online version of this article (

Assessment of genetic and epigenetic changes during cell culture ageing and relations with somaclonal variation in Coffea arabica

“…Similarly, low levels of methylation variation were found in freesia somatic seedlings derived from short-term cultures (Gao et al 2010) and pea long-term multiple shoot cultures (Smýkal et al 2007). Many studies with methylation-sensitive molecular markers that reported a high level of epigenetic variation reported no effect on phenotype or morpho-agronomic traits (Bednarek et al 2007;Li et al 2007;Schellenbaum et al 2008;Fiuk et al 2010). Methylation changes contribute poorly to global estimations when they do not occur at high frequencies (Smulders and Klerk 2011).…”

Genetic and Epigenetic Variations in Barley Calli Cultures

“…taylor et al (18) indicated that there might be epigenetic changes although there are few polymorphisms. li et al (11) searched for genetic and epigenetic similarities in tissue culture regenerants of wild barley (Hordeum brevisubulatum (trin.) link) with various markers such as AFlP, S-SAP and MSAP.…”

Analysis of genetic stability of in vitro propagated potato microtubers using DNA markers

“…Aversano et al (2009) demonstrated that under in vitro culture conditions Solanum genotype affects the integrity of the genome and absence of polymorphism at plastid level confirms the greater genetic stability of cytoplasmic DNA. Li et al (2007) demonstrated genetic and epigenetic instabilities induced by tissue culture in wild barley using AFLP, S-SAP and MSAP markers. Based on our results, it is concluded that AFLP is the best method followed by SSR, ISSR and RAPD for detection of genetic stability of in vitro conserved potato microtubers.…”

“…AFLP can be generated for any organism without initial investment in primer/probe development and sequence analysis. Like RAPD and ISSR, in the past SSR and AFLP markers have also been used to detect genetic fidelity in many plants such as Elaeis guineensis (Matthes et al 2001), Hordeum brevisubulatum (Li et al 2007), Solanum tuberosum (Zarghami et al 2008;Dann and Wilson 2011), Pinus pinaster (Marum et al 2009) and Ocotea catharinensis (Hanai et al 2010). The aim of the present study was to assess the genetic stability of in vitro conserved potato microtubers by RAPD, ISSR, SSR and AFLP markers and also confirmed in vitro conservation protocol.…”