Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 5.11

GALA APPLE TRANSFORMED FOR SCAB (VENTURIA INAEQUALIS) RESISTANCE WITH CLONED VF GENE REGION CONSTRUCT

BELFANTI E.*, BARBIERI M.*, TARTARINI S.*, VINATZER B.*, SANSAVINI S.*,

DILWORTH E.**, GIANFRANCESCHI L.**, HERMANN D.**, PATOCCHI A.**, GESSLER C.**

*) Dipartimento di Colture Arboree (DCA), University of Bologna, Italy

**) Plant Pathology, Institute of Plant Science, ETH Zurich, Switzerland

Venturia inaequalis, Malus x domestica, Vf-resistance gene cloning, genetic transformation, GM-apples

Scab is the most serious disease of apple in many districts throughout the world and can require more than 15-20 treatments per year. The monogenic resistance conferred by the Vf gene against Venturia inaequalis has been employed in traditional breeding programs for over fifty years. Despite the many cultivars that have been released from these efforts, the problem has not yet been resolved because the resulting fruit quality is less than satisfactory and because this kind of vertical resistance has been overcome by V. inaequalis races 6 and 7 in several European countries. Eight years ago, Bologna’s DCA and Zurich’s ETH undertook the cloning of the Vf gene starting from a saturated map; the markers AL07 SCAR and M18 CAPS proved to be closely linked to the Vf gene.

The BAC library derived from cv. Florina extended more than five times the size of the apple haploid genome itself. Chromosome walking was than used to screen this library with Vf marker and yielded a contig of the Vf locus of 550 kb. Recombinant analysis of the seedling population enable the restriction of the contig to 350 kb made up of 5 BAC clones, including the Vf region. The next step was the construction of a cDNA library from Florina leaves elicited with V. inaequalis conidia; this library’s subsequent screening yielded more than one hundred hybridizing cDNAs. A cluster of receptor-like genes homologous to Cf Cladosporium fulvum resistance gene family of tomato was identified, sequenced, isolated from the Vf region and named HcrVf sequences.

A plasmid construct of the HcrVf2 sequence (about 2,000 base pairs), selected among several of the same cluster, was introgressed mediated by Agrobacterium tumefaciens into Gala leaf and internode explants using the binary vector pCambia2301 (containing two nptII genes and a GUS reporter gene with its promotor CaMV35s). PCR assays on about 50 regenerated plantlets after 15 months on selection medium showed that almost 90% had HcrVf2 and the control genes. The acquired resistance was corroborated by repeated in vitro infection with V. inaequalis conidia: on the transformed plants V. inaequalis conidia germinated and formed appressoria, however on most leaflets of particular transformant lines no stroma was observed, contrary on untransformed controls extended stroma was observed in most leaflets. A particular transformant line did not express resistance and V. inaequalis was able to form stroma similar to the control plants. The definitive demonstration of acquired resistance is to be conducted in vivo with artificially and naturally inoculated plants.