CLONING OF CONSTITUTIVELY DIFFERENTIALLY EXPRESSED GENES IN STEM TISSUE OF TWO LEMON CULTIVARS EXHIBITING DIFFERENCE IN COLD AND PHOMA TRACHEIPHILA RESISTANCE

Lemon (Citrus limon Burm. f.) cultivation in the Mediterranean and Black Sea areas suffers by two major problems: the destructive mal secco disease, caused by Phoma tracheiphila (Petri) Kant. & Gik. and the susceptibility to low temperatures. It has been established that the Greek lemon cultivar ‘Adamopoulou’ (derived from the Portuguese cultivar ‘Lisbon’) is more tolerant to mal secco disease and cold than ‘Lisbon’. A molecular analysis leading to the isolation of differentially expressed genes in the stems of a resistant and a susceptible lemon cultivar could aid in further understanding of the resistance mechanisms against mal secco and cold. For this purpose, the suppression subtractive hybridization (SSH) technique was employed. Two subtractions were performed, namely a forward subtraction (‘Adamopoulou’ - ‘Lisbon’) and a reverse subtraction (‘Lisbon’ - ‘Adamopoulou’) and the PCR products were cloned producing two subtractive cDNA libraries. A total of 587 clones (both cDNA libraries) were sequenced and the obtained sequences were subjected to VecScreen (NCBI) resulting in 81 clones containing solely lemon cDNA sequences. Sequence comparisons were performed using BLAST (Blastn, Blastn-EST, Blast-x) and homology to previously identified genes involved in defence mechanisms against biotic and abiotic stresses was revealed. More specifically, significant homology was found with: pathogenesis-related proteins, resistance gene analogs, genes homologue to Pto, genes implicated in phytoalexin biosynthesis, polygalacturonase inhibiting protein, polyphenol oxidases, fatty acid hydroperoxide lyase, cell wall proteins, aldehyde dehydrogenase; and lower homology to: ACC oxidase, a dehydrin, lipoxygenases, a ribosome-inactivating protein, late embryogenesis abundant proteins and COR proteins. Further characterization of the 81 clones is underway.