ABSTRACT
Previous work has shown the fixation of context-specific random amplified polymorphic DNA (RAPD) patterns in tomato cell cultures grown for 2 years in different hormonal contexts. In this work, RAPD sequences were characterised and RAPD-derived molecular markers used for a further study of variation between and within auto- and auxo-trophic tomato cultures grown in different hormonal equilibria. Results were then compared with those obtained using microsatellite markers located in noncoding regions of differentiation- and hormone-related genes and with those obtained with the external transcribed spacer (ETS) from tomato rDNA. Hybridisation of RAPDs on a tomato genomic DNA bank, or on total DNA after enzymatic digestion, suggested that the markers were repetitive in nature. Sequence analysis. however, showed that the homology between different fragments was due mainly to the presence of homo-AT nucleotide stretches. Moreover, a series of computational methods, such as an information-theory algorithm coupled with AG estimates, suggested that the RAPD fragments isolated in our experiments are noncoding. The amplification of SSR-containing RAPD-derived markers, and of other SSRs located in noncoding regions of tomato functional genes, consistently showed polymorphism between auxo- and auto-trophic somaclones (the latter being either habituated or transgenic for Agrobacterium tumefaciens oncogenes) but not within these same clones. Differences were also found between auxotrophic clones and the differentiated tissue. These findings were confirmed by restriction fragment length polymorphism (RFLP) analysis with the REII repetitive element of the ETS from tomato rDNA, which was isolated during this study. The results obtained suggest a possible role for physiological context in the selection of RAPD patterns during the evolution of tomato cells with different endogenous hormonal equilibria. The results are discussed in terms of a possible role for variation in noncoding regions of hormone-related genes in the adaptation to different physiological contexts.
