Mapping And Characterization Of A New Tomato Resistance Gene Cf-ECP3 Against Cladosporium Fulvum

A new tomato resistance gene against Cladosporium fulvum has been identified . The resistance gene recognizes the extracellular protein ECP3, and was designated Cf-ECP3. A F-, population composed of 192 plants was used for segregation analysis of the resistance. The segregation ratio showed that this gene inherits as a monogenic dominant trait. Cf-ECP3 cosegragated with the chromosome 1 Cleaved Amplified Polymorphic Sequences(CAPS) marker CT1 16 and mapped accurately at Orion, a locus harbouring Cf-ECP2 in other genotypes. RFLP anaysis with a Hcr9(Homologous of Cladosporium resistance gene Cf-9, Hcr9) probe further demonstrated that a 1.6kb BgJlI and 1.6 kb SstI fragement cosegregated with Cf-ECP3 and indicates that this gene is supposed to be a Hcr9. Thus in addition to the Milky Way locus harbouring the Cf4 and Cf-9, Orion is another complex locus on the short arm of chromosome I that encodes multiple resistance specificities to C. fulvum. In order to isolate this gene and the Orion cluster at which Cf-ECP3 and Cf-ECP2 were mapped, a combined map-based cloning and candidate-gene approach was employed. A 5 genomic equivalent s phage genomic library of Cf-ECP3 was constructed and screened with a Hcr9 probe. 37 positive clones containing Hcr9 were identified, and after restriction mapping, DNA hybridization and DNA fingerpringting these clones containing at least one Hcr9 were aligned into three contigs, while there are still five separate clones containing part of Hcr9 or incomplete Hcr9 that didn抰 fit into any contigs mentioned above. One of these seperate clones, clone 38 ,can hybridize with both CTI 16 and Hcr9, which implied that this clone is a part of Orion cluster, and Cf-ECP3 may be present in this clone. A PCR strategy was employed to amplify Hcr9 or intergenic region of each contigs and separate 3 clones. The PCR products were partially sequenced and aligned with all the known Hcr9 locus. The results showed that four Hcr9s present in contig 1 share very high homology with the Hcr9 members at NL locus of MoneymakerCf-O, NL-OA, NL-OC, NL-OD, NL-OE respectively, but NL- OB was absent in contig 1. The DNA sequence of Hcr9 present in a separate clone (clone 6) is very similar with NL-OC. These results combined with other data from different tomato genotypes imply that gene duplication and gene translocation play a great role in diversifying the Hcr9s on the shari arm of chromosome 1. This knowledge is very helpful to understand the evolution of Hcr9s and the novel variation of Cf genes.