Effect of different restriction enzymes, probe source, and probe length on detecting restriction fragment length polymorphism in tomato

Abstract

Since the construction and use of RFLP genetic maps depends on the ability of cloned sequences to detect polymorphism, we have attempted to determine conditions under which maximum levels of polymorphism can be detected. Forty cloned nuclear sequences from three different libraries (cDNA, EcoRI genomic, and Pstl genomic) were hybridized to total DNA from 149 plants representing eight species of the tomato genus Lycopersicon. Five different restriction enzymes were employed in this study. We examined the relationship between polymorphism (number of restriction patterns) and clone size, restriction enzyme, size of hybridizing restriction fragments, and clone source (library). We found no relationship between clone size (ranging from 0.4 to 5.3 kb) and polymorphism. There was a strong positive relationship (r ² = 0.79) between polymorphism and the average size of the fragments produced by each restriction enzyme. cDNA clones hybridized to larger fragments compared to genomic clones. cDNAs also detected significantly more polymorphism (approximately 25% more) than genomic clones — possibly indicating high levels of sequence variability in introns and/or areas flanking coding regions.