MUTATIONAL ANALYSIS IN CITRUS. A PROPOSAL FOR THE DEVELOPMENT OF GENOMIC TOOLS

Carretera Moncada - Náquera, Km. 4,5, Moncada (Valencia), E46113, Spain

We have generated several collections of citrus mutants at the Instituto Valenciano de Investigaciones Agrarias to identify and characterize unknown genes of agronomic interest. The collections include more than 10.000 plants and were generated from material treated with increasing doses of the mutagen agent, either chemical (ethyl methanesulfonate, EMS) or physical (gamma rays and fast neutrons), to produce both point mutations (base pair change) and chromosomal alterations (deletions). The physiological data and the agronomic parameters registered during the last years indicated that several of these genotypes maintained statistical differences with the average regarding productivity, fruit quality and ripening. Furthermore, studies on tolerance to environmental stresses such as salinity and water stress are also pursued. However, the molecular characterization of citrus mutants is extremely complex since these species are highly heterozygous and, in addition, most popular approaches to detect mutations and polymorphisms have been developed as reverse genetics tools, such as denaturing High Performance Liquid Chromatography (d-HPLC) or the high-throughput TILLING (Targeting Induced Local Lesions in Genomes) method. For the characterization of citrus mutations we will use genomic tools. We intend to accomplish functional analyses through cDNA (partial and full length) microarrays and to obtain information on the genes whose activity is directly or indirectly affected by a gene perturbation in specific processes. The deletion mutants can be utilized to detect DNA losses by array CGH (Comparative Genomic Hybridization) using cDNA and BAC clones. Other possibility is the generation of oligonucleotide arrays, that may be of particular interest for point mutations.