Application of Molecular Markers in Breeding Zucchini Yellow Mosaic Virus (ZYMV) Tolerant Pumpkin
(Cucurbita pepo L.) Varieties in Austria

T. Lelley and S.M. Henglmüller

Institute for Agrobiotechnology. Department of Biotechnology in Plant Production,
Konrad-Lorenz Str. 20, A-3430 Tulln, Austria

M. Riedle-Bauer

Federal Office and Research Centre of Agriculture, Spargelfeldstrase 191, A-1226 Vienna, Austria

Additional index words. zucchini yellow mosaic virus, RAPD, backcross

Abstract. Growing pumpkin for seed oil production has a long standing tradition in Austria. The hull less seeded pumpkin Cucurbita pepo var. styriaca has been cultivated for >100 years, consumption of seed oil is part of the culture. In 1997, for the first time, a strong infection by ZYMV caused severe yield losses. The extent of damage prompted a breeding program to introduce virus tolerance into Austrian varieties. The project is financially supported by the Ministry of Agriculture and the mostly affected Federal States. Tolerant material was collected including the varieties Tigress, Puma, and Jaguar of Harris Moran Seed Co., Dividend and Revenue of Novartis USA, Nicklow's Delight of Nicklow's Vegetables, and the variety Whitaker provided by Professor Robinson, Cornell University. Initially, an infection test was made in the greenhouse using a virus inoculum isolated from infected fields in Austria. In this experiment, the varieties Tigress, Puma, Jaguar, Dividend, and Revenue along with five Austrian breeding lines were included. The two coleoptiles and later the first leaf were inoculated. While the Austrian lines immediately showed strong symptoms and did not survive the experiment, the American ones expressed good tolerance until the second scoring, 6 weeks after inoculation. A crossing program was set up, where these varieties were crossed as pollinators with selected Austrian breeding lines. DNA was isolated from all crossing partners and a RAPD analysis was carried out to detect polymorphism. A comparison was made for the separation of the amplification products using either 1.5% agarose or 10 % polyacrylamide gel. In agarose gel, bands were visualized by ethidiumbromide, and in polyacrylamide, they were stained with silver nitrate. Only polymorphic bands occurring in the potential donor varieties were considered. Compared to 113 polymorphic loci identified in agarose gel, 266 were detected in polyacrylamide gel. Clearly, band separation in polyacrylamide gel is superior to agarose gel. Moreover, in agarose gel only fragments >1000 bp produce clear bands, whereas in polyacrylamide gel bands <1000 bp are well visible. RAPD primers amplify more fragments in the latter size category. F1 plants were harvested and seeds are being grown at present to produce an F2 generation. F2-derived F3 populations will be used for selecting F2 plants homozygous for tolerance or susceptibility. From such plants DNA will be pooled for bulked segregant analysis. Parallel to selfing, F1s will also be backcrossed to the respective Austrian strains. The project aims at the introduction of alleles for tolerance against ZYMV into Austrian breeding material. Later, backcrossing will be done by breeders, while our institute will assist in selection by using molecular markers.

Cucurbitaceae '98