KALANCHOE × HOUGHTONII SSH AND MICROARRAY ANALYSIS TO SCREEN GENES INVOLVED IN VIVIPARY

Vivipary, referred here as the formation of novel complete plantlets on mature organs, has been reported in many families as an asexual propagation strategy. In K. × houghtonii (Crassulaceae), viviparous plantlets are formed on leaf margin notches in response to a long day photoperiod and their appearance follow a basipetal fashion. To identify genes involved in this process, suppression subtractive hybridisation libraries (SSH) were prepared. Two hundred c-DNA clones were classified and grouped into 14 functional categories according to Goldberg database (http://estdb.biology.ucla.edu/PcEST). Six hundred thirty sequences (200 SSH library, 48 database Kalanchoe genus, 382 other species database genes) were used as probes for microarray analysis according to the CombiMatrix technology and a 4x2K Custom Array ™ was synthesized. RNA was extracted from margin of leaves at 7 stages of development before buds emission (5 to 50 mm) during long-day photoperiod (permissive conditions). Three replications for each sample were prepared. From double strand cDNAs antisense RNAs (a RNA) were synthesized and amino-allil-UTP incorporated and coupled with Alexa Fluor ® 647. Microarray was hybridized according to CombiMatrix protocol. Data were extracted with CombiMatrix Microarray Imager software and exported into Microsoft Excel for computing of mean, median and standard deviation. Person's correlation was computed and data normalized. After background removal, probes were reduced to 484. "Fold change" method (FC=2) was used to compare different levels of gene expression of samples. Significance Analysis of Microarrays Statistic (SAM method) generated 263 significant modulated genes with a False Discovery Rate (FDR) of 5%.