Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress
Salsomaggiore Terme, Italy - 26/29 September, 2001
SEARCH FOR THE GENES INVOLVED IN SEXUAL DIFFERENTIATION OF CANNABIS SATIVA L.
MOLITERNI V.M.C.*, CATTIVELLI L.**, MANDOLINO G.*, RANALLI P.*
* Istituto Sperimentale per le Colture Industriali. Via di Corticella 133, 40129 Bologna
** Istituto Sperimentale per la Cerealicoltiura, Sezione di Fiorenzuola d'Arda, Via S. Protaso 302, Fiorenzuola d'Arda, Piacenza
Cannabis sativa L., sexual differentiation, cDNA AFLP
Cannabis sativa is a naturally dioecious species with heterogametic males ( 2n = 18+XY ) and homogametic females ( 2n = 18+XX ). The sexual differentiation of C. sativa is strongly influenced by environmental factors such as temperature and photoperiod. Anomalies also occur in floral development like the presence of reproductive structures of the opposite sex, or the development of bisexual inflorescences (monoecious phenotype).
By means of optical microscopy, we have identified the earliest step of apex sexual differentiation as the leaves of the fourth node rise on. More than 50% of the samples observed at this stage have indeed developed some floral meristem buds.
In order to identify the genes involved in these earliests stages of the sexual differentiation of C. sativa, we have carried out an analysis of gene expression by means of the cDNA AFLP technique. Apices from the fourth node of male and female plants, grown in controlled conditions were collected, than the mRNA was extracted and used as template for the cDNA synthesis. Double stranded cDNA was digested with the restriction enzymes BstY1 and Mse1. Analysis of amplified fragments obtained using 60 combinations of the BstY+1 and Mse+3 primers enabled us to identify few hundreds of fragments with an apparent differential expression in male and female samples. In order to verify their actual differential expression, these fragments were eluted from the gel and re-amplified with the same primers combinations that were used to generate them. Subsequently they were blotted in double copies and than hybridised respectively with the total cDNA from males and females apices collected at the fourth node. This approach allowed the reduction of the fragments number to 12. Subcloning and sequencing will permit to track back to the genes having a differential expression at this stage of sexual development of C. sativa and that may be involved in its regulation.