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

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 5.50

MOLECULAR GENETIC APPROACHES TO STUDY APOMIXIS IN HYPERICUM PERFORATUM L.

F. ARZENTON*, H. BÄUMLEIN**, F.MATZK**, F. BLATTNER**, G. BARCACCIA*, M. LUCCHIN*

*) Dipartimento di Agronomia Ambientale e Produzioni Vegetali, University of Padua, Agripolis, viale dell´Universitá 16, 35020 Legnaro, Padova, Italy

margherita.lucchin@unipd.it

**) Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany

Apomixis, FCSS, AFLP, cDNA-AFLP, H. perforatum L.

Asexual seed formation (apomixis) bypasses meiosis and fertilization of egg cell. The isolation of the apomixis genes is of great interest because this could enable the introduction of apomixis into sexual crop and thus the fixation of heterosis or of any elite genotype. St. John´s wort (Hypericum perforatum L.) reproduces mainly by facultative aposporous parthenogenesis. We consider H. perforatum a good model plant for apomixis research mainly because of its small genome size (1C=0,650pg), the existence of apomictic and sexual plants that are cross-compatible, the great variability in the mode of reproduction and its use as a medicinal plant. In order to understand the genetic control of apomixis in H. perforatum we carried out molecular analysis at the genomic (1) and at the transcriptional (2) level:

1. Several apomictic and sexual genotypes clearly characterized for the mode of reproduction by means of flow cytometric seed screen (FCSS) (Matzk et al. 2001) were analysed using the AFLP technique. The genotypes originated from different collection sites or botanical garden in Europe. Therefore they represent a very differentiated genetic background and showed a very high amount of polymorphic genomic DNA. Seven markers were selected as apomicts-specific (present in all the apomictic lines and absent in the sexuals). The large genetic variability between the genotypes and the clear discrimination of the contrasting mode of reproduction by the apomict-specific fragments support the hypothesis that this genomic level approach could lead to the isolation of markers linked to the apomixis trait. We detected the segregation patterns of the seven fragments in a triploid F₁ population obtained by crossing a completely sexual diploid with an obligate apomictic tetraploid. The mode of reproduction of the entire F₁ population is going to be determined by means of FCSS. This analysis will enable us to verify whether the seven markers are correlated with one of the two components of apomixis since the genetic control of the components of apomixis is likely to be independent in H. perforatum (Matzk et al. 2001). The subsequent conversion of the apomixis related AFLP markers into simple single locus PCR markers might provide a useful tool to greatly facilitate apomixis research in H. perforatum.

2. FCSS and molecular markers have enabled the evaluation of the mode of reproduction of 15 Italian ecotypes and the genetic variability among and within these ecotypes, respectively. The most similar ecotypes (1 and 13) showed differences in the mode of reproduction (high incidence of apomixis and reduced level of parthenogenesis in aposporous embryo sacs, respectively) and were chosen for further analysis. The reproductive organs of both ecotypes have been analysed by means of cDNA AFLP technique in order to assemble a mini library of pistil specific ESTs. All cDNA samples were investigated using cDNA pools from somatic tissues such as leaves and stems as controls. This approach enabled us to select several mRNAs specifically expressed in the pistils of both ecotypes or of just one of the two. Fragments sequencing is in progress in order to select ESTs with interesting homologies (specific of the pistil or of the reproductive phase) that will be used as probes in Southern analysis on the F₁ population.