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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

‘REVERSE GENETICS’ IN PETUNIA HYBRIDA

TORNIELLI G.B., TURCI M., DE BATTISTI C., ZENONI S., AVESANI L., PEZZOTTI M.

Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada le Grazie 15, 37134 Verona

pezzotti@sci.univr.it

Petunia hybrida, reverse genetic, dTph1, transposon insertion mutants

A direct way for obtaining information on the function of a gene for which the sequence is known, is by creating a loss-of-function mutation and studying the phenotype of the resulting mutant. A highly efficient procedure for obtaining mutants in a specific gene with known sequence is screening large populations of plants for insertion of transposable elements.

With the molecular isolation of the dTph1 transposable element, insertional mutagenesis has become feasible for Petunia hybrida. Adopting a particular sample pooling system each individual plant is sampled three times and identified by three coordinates, which facilitates the screening of large populations enormously. The sensitivity of the PCR technique using a transposon- and a gene-specific primer allows the subsequent detection of a single gene hit within a pool of hundreds of individuals. Selfing all plants is required to preserve population that, in this way, can be screened for insertions in many genes over a prolonged period.

We constructed and screened two petunia populations of 512 and 4096 plants to seek for insertion mutants for some target genes. Several independent insertions of dTph1 were found in each target analysed gene. Four different petunia gene, involved in different biological phenomena, were analysed: a cyclin (Pethy-CycB1), involved in cell cycle regulation; a meiotic gene (Pethy-mei2); an expansin (Pethy-Exp1), involved in cell wall expansion; a vetispiradiene synthase (Pethy-VS), involved in terpene metabolism. Insertion alleles, obtained in the two populations, were analysed and molecularly characterised. Homozygous insertions in coding region of dTph1 were obtained by selfing heterozygous plants. Genetic and molecular analyses in the segregating family were performed. A visible altered phenotype (whole plant size reduction, flower size reduction, slower growing) was observed in plants carrying homozygous insertion of dTph1 in Pethy-Exp1. Molecular characterization of the expansin mutant phenotype is reported.