Methylation patterns revealed by MSAP profiling in genetically stable somatic embryogenic cultures of Ocotea catharinensis (Lauraceae)

Abstract

Ocotea catharinensis is a rare tree species indigenous to the Atlantic rainforest of South America. In spite of its value as a hardwood species, it is in danger of extinction. The species erratically produces seeds showing irregular flowering and slow growth. Therefore, plants are not easily replaced. Tissue culture-based techniques are commonly used for obtaining living material for tree propagation and in vitro preservation. Therefore, a high-frequency somatic embryogenic system was developed for the species. In the present work, the genetic fidelity of cell aggregates and somatic embryos at various stages of in vitro development of O. catharinensis was investigated using RAPD and AFLP markers. Both analyses confirmed the absence of genetic variation in all developmental stages of O. catharinensis embryogenic cultures, verifying that the in vitro system is genetically stable. The cultures were also analyzed for their methylation profiles at 5′-CCGG-3′ sites by identifying methylation-sensitive amplification polymorphisms. Some of these markers differentiated cell aggregates from embryo bodies. The sequencing of ten MSAP markers revealed that four sequences showed significant similarity to genes encoding plant proteins. Particularly, the predicted amino acid sequence of the fragment designated as OcEaggHMttc155 was similar to the enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO), which is involved in the biosynthesis of ethylene, and its expression was reported to occur from the beginning to the intermediate stages of plant embryo development. Here, we suggest that this enzyme is possibly involved in the control of the earliest stages of somatic embryogenesis of O. catharinensis, and an approach to study ACO expression during somatic embryogenesis is proposed.

Study of the genetic diversity and structure of a natural population of Nectandra megapotamica (Spreng.) Mez. using RAPD markers

“…These results indicated that these 11 RAPD markers can be reliably used for differentiating N. megapotamica individuals and populations. Hanai et al (2010) identified 94 bands when they used the same RAPDs for screening in vitro cultures of Ocotea catharinensis.…”

“…The primers were selected according to the results obtained by Hanai et al (2010) using Ocotea catharinensis (Lauraceae). A total of 11 primers were selected, based on their high polymorphic profile and good reproducibility of the generated fragments ( Table 2).…”

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

“…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.…”

Comparative analysis of five DNA isolation protocols and three drying methods for leaves samples of Nectandra megapotamica (Spreng.) Mez

“…The primers (Table 2) were selected according to the results obtained by Hanai et al (2010) using Ocotea catharinensis (Lauraceae). The amplification reactions were performed in an AmpliTherm TX96 Plus TM thermocycler.…”