Plant Molecular Biology 52: 527–536, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 527 Labile DNA sequences in flax identified by combined sample representational difference analysis (csRDA) Thomas J. Oh 1 and Christopher A. Cullis 2,∗ 1 NovoMark Technologies, 2915 Southington Road, Shaker Heights, OH 44120, USA; 2 Department of Biology, Case Western Reserve University 10900 Euclid Ave., Cleveland, OH 44106 USA ( ∗ Author for correspondence; E-mail: cac5@po.cwru.edo) Received 9 May 2002; accepted in revised form 2 January 2003 Key words: aberrant segregation, environmental stress, flax, plasticity, RAPD, representational difference analysis Abstract Flax (Linum usitatissimum) has a genome in which changes have been associated with environmental factors. The inbred flax variety, Stormont Cirrus (Pl), served as the parent, and several lines (termed genotrophs) were derived from this parent. The phenotypes of the genotrophs were stable in a number of different growth environments, unlike the original Pl line in which changes associated with environmental factors continued to occur. These genotrophs differed from the original line in a number of characteristics, but the only known phenotypic character- istic that is shared by all the genotrophs and different from the parental, Pl, line is the lack of changes associated with the original environmental factors. However, some of these genotrophs have changed in both phenotype and nuclear DNA subsequent to their original growth and differentiation from Pl. Representational difference analysis (RDA) has been used to identify differences between Pl and all the genotrophs in an attempt to identify the loci controlling these aspects of plasticity. Subtractions between Pl DNA as a tester (target) and one of the genotrophs (individual RDA) or a mixture of different types of genotroph (L6, S6, C2, and LH) DNAs as a driver were done (combined sample RDA; csRDA). In addition, contrary RDA, where of the genotroph DNA was used as a tester and Pl DNA as a driver, was also executed. Three difference clones (163-4-2, 123-5-2, and 163-13), from 74 primary clones obtained after three rounds of subtractions with Pl DNA as tester were further characterized. In addition, 2 difference products (213-r1 and 213-r9) were characterized from contrary RDA. The clones 163-4-2 and 163-13 from the csRDA showed polymorphisms between Pl and all the genotrophs when PCR was done with primers derived from sequences of the clones, but only the clone 163-13 polymorphism was confirmed by Southern blot analysis. Four of 5 clones (163-4-2, 123-5-2, 163-13 and 213-r9) that have been characterized appear to be associated with structural changes in the DNA. From the contrary csRDA, it was observed that no clones could be recovered from subtractions between a mixture of genotrophs as a tester and Pl as a driver, and several possible explanations have been proposed. Introduction Flax (Linum usitatissimum) is agronomically impor- tant for fiber (linen) and oil (linseed oil). The diploid chromosome number is 30, comprised of 15 pairs of small similarly sized chromosomes. A variety of environmental conditions have been shown to be as- sociated with phenotypic, biochemical, and molecular changes in the first generation progeny of treated plants, so making it a useful model system to inves- tigate factors involved in genome stability (Durrant, 1962, 1971; Cullis, 1977, 1985). The original plant material was derived from the flax variety, Stormont Cirrus, and it has been denoted as plastic (Pl) due to its behavior after environmental treatments of ei- ther imbalances of nutrients or specific temperature regimes (Durrant, 1962). The resulting stable lines were termed genotrophs. The genotrophs have been referred to as stable since, unlike the original Pl line, they breed true when grown in a number of different