Molecular cloning and analysis of one member of a polymorphic family of GACA-hybridising DNA repeats in tomato

Abstract

Simple sequence repeat oligonucleotides were used to probe the tomato genome for elements displaying variability amongst commercial cultivars. The oligonucleotide (GACA)₄ was found to be particularly informative on genotype screening blots, hybridising to a highly polymorphic family of elements, and was used to clone one such member from a lambda library. The GACA-hybridisation was localised to a 1.3-kbHinfI fragment within the original 15-kb lambda insert. This 1,349-bp subclone (pT-GACA-2:1.3) was used to probe 27 Californian processing varieties and found to be capable of distinguishing all from each other, thus demonstrating its utility as a genetic fingerprinting probe for cultivar identification. Hybridisation occurred to approximately 10 major high molecular weight (> 4-kb) bands, most of which segregated independently in F₂ populations, as well as a large number of less clearly resolvable smaller fragments. Sequence analysis of the cloned element reveals that it is almost entirely composed of GACA or GATA repeats. These tetranucleotides are organised into distinct repetitive domains, consisting either of tandem arrays of each tetranucleotide or interspersions of GACA and GATA to form dodecanucleotides that are then further repeated. The boundaries between domains contain sufficient departures from the concensus repeat to allow construction of unique polymerase chain reaction (PCR) primers. Amplification from two such contiguous regions identifies length variation in both, thus yielding a genotype screen appropriate for high-throughput applications, such as assessment of purity in F₁ hybrid seed lots.