Screening of viviparous plantlet formation-related genes in Kalanchoe daigremontiana by SSH analysis

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. daigremontiana (Crassulaceae), viviparous plantlets are formed on leaf margin notches in response to a long day photoperiod and their appearance follows a basipetal fashion. To identify genes involved in this process, suppression subtractive hybridisation libraries (SSH) were prepared. For this purpose, RNA was extracted from tissue forming buds and from tissue not forming buds. cDNAs were synthesized and selectively amplified to enrich differentially expressed sequences. The cDNAs of forward-subtracted library, created using tissue forming buds as tester were directly inserted in a plasmid vector and the ligation products were transformed into E. coli. The clones were screened for differential expression by hybridization using either forward or reverse libraries as probes. Up-regulated and down-regulated cDNA clones were identified and sequenced. On the basis of TBLASTX algorithm analysis and after correcting for redundancy, 135 cDNA clones with a putative differential expression pattern between the two kinds of tissues were classified and grouped into fourteen functional categories according to the Goldberg database (http://estdb.biology.ucla. edu/PcEST/) and functional classes that could be involved in the vivipary process were identified.