Isolation and characterization of a highly variable GACA/GATA locus from L. esculentum with applications in genetic fingerprinting and hybrid screening.

Phillips W., Chapman, C., Jack, P. PBI Cambridge, Maris Lane, Trumpington, Cambridge, CB2 2LQ, ENGLAND

Molecular markers can be of great utility as tools for variety/hybrid verification and in marker assisted breeding; for these reasons we are working on developing marker systems that will be informative within L. esculentum. Early work focussed on identifying restriction fragment length polymorphisms (RFLPs), however the level of variability revealed within commercial cultivars using conventional single-copy probes was found to be very low (typically only 6% of clones showed variation), and generally only two size variants were identified, thus limiting the discriminatory power of these markers.

Work in other systems has shifted towards repetitive sequences as sources of variation (e.g. humans, mice) where both large repetitive arrays (e.g. Jeffrey's sequence) and smaller di, tri, or tetranucleotide repeat arrays (i.e. microsatellites) have been found to be highly variable yet relatively stable in their inheritance. We have investigated a number of repeat arrays, for example (GACA)4 or (GATA)4, both of which have yielded promising results in other plant species (Beyerman et al., 1992), and recently demonstrated in tomato (Vosman et al., 1992). Probings with (GACA)4 showed significant polymorphism, and this oligonucleotide was therefore used to clone GACA elements from a representative tomato genomic library. Initial probings yielded approximately 50 putative GACA clones, but 90% of these provided unstable and recalcitrant to purification.

One clone was studied in depth; the GACA element was subcloned, used to probe a selection of tomato cultivars, and found to reveal significant variation. Many loci appear to hybridize to this element, approximately 10 of which show clearly resolvable polymorphism and segregate independently. In contrast to the original (GACA)4 probings, this cloned element is robust, yielding clean reproducible signals. Sequence analysis of this 1.3 kb clone reveals that is consists almost completely of GACA/GATA repeats, flanked by short unique domains. Within this array there are minor sequence variants which have allowed the construction of unique primers which permit PCR amplification of internal regions of the element. These smaller subsections of the element amplify to yield discrete products which show polymorphism between commercial cultivars; three main size variants have thus far been noted. This PCR analysis has been used to assess hybrid purity, and further work is underway to analyze this element in segregation populations.

Literature cited:

Beyerman, B., Nurnberg, P., Weihe, A., Meixner, M., Epplen, J.T., and Borner, T. 1992. Theor. Appl. Genet. 83:691-694.
Vosman, B., Arens, P., Rus-Kortekaas, W. and Smulders, M.J.M. 1992. Theor. Appl. Genet. 85:239-244.