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

• Published in: Theoretical and applied genetics 1994-08, Vol.88 (6-7), p.845-851
• Main Authors: PHILLIPS, W. J, CHAPMAN, C. G. D, JACK, P. L
• Format: Article
• Language: English
• Subjects: adn; Agronomy. Soil science and plant productions; Biological and medical sciences; clonacion molecular; clonage moleculaire; dna; dna hybridization; enzima de restriccion; enzyme de restriction; Fundamental and applied biological sciences. Psychology; Genes. Genome; genetic polymorphism; hibridacion de adn; hybridation d' adn; lycopersicon esculentum; Molecular and cellular biology; molecular cloning; Molecular genetics; nucleotide sequence; polimorfismo genetico; polymorphisme genetique; restriction enzymes; secuencia nucleotidica; sequence nucleotidique
• ISSN: 0040-5752; 1432-2242
• DOI: 10.1007/BF01253995

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 kb Hinf1 fragment within the original 15 kb lambda insert. This 1349 bp subclone (pTGACA-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 ten major high molecular weight (over 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.