Isolation of tissue culture-induced polymorphisms in bananas by representational difference analysis

Somaclonal variation is a widespread phenomenon in tissue cultured plants. The frequency with which these variants arise depends on a number of variables including the source of the explant, propagation medium, and the number of propagation cycles. A variety of molecular approaches have been applied to develop molecular markers to monitor and/or identify this variation. Included in this repertoire are Restriction Fragment Length Polymorphisms, Random Amplified Polymorphic DNAs, Amplified Fragment Length Polymorphisms and sequenced amplified characterized regions. Each of these techniques had been applied to normal and variant individuals in order to identify polymorphisms. A drawback of each of these techniques is that only a small portion of the genome is screened with any one probe or set of primers. Therefore, if variation is rare it is difficult to identify, while if it is common, it is difficult to identify the most useful or relevant polymorphism. We will describe experiments in which representational difference analysis was applied to flax and banana variants to identify polymorphic sequences. Subtractions were done using the DNA from two individuals to obtain the comparison between single lines, and with pooled DNA from many variants to obtain polymorphisms shared by a number of variants. In each case the variant sequences were identified and sequence tagged sites were developed. These tags could then be used in a PCR-based assay to identify variants at any stage of the propagation process. In addition, it was possible to identify the molecular events associated with the generation of the polymorphism. The general applicability of this approach to other micropropagation systems will also be considered.