Characterization of resistance genes to Cladosporium fulvum on the short arm of chromosome 1 of tomato

Abstract

Plant breeders generally use qualitative resistance that is associated with a hypersensitive reaction (HR) to obtain cultivars that are resistant to pathogens and pests. The genetics of this resistance is based on the gene-for-gene relationship, which involves the product of a plant resistance gene and the product of an avirulence gene of the pathogen occurs. The interaction between leaf mold ( Cladosporium fulvum ) and its solely host, tomato ( Lycopersicon esculentum ), complies with this model. In the last few years, the isolation of several avirulence ( Avr ) and resistance ( R ) genes have contributed to an increase in our knowledge on this interaction. Several resistance genes to C. fulvum ( Cf genes) have been isolated from tomato. These Cf genes are located on two different clusters on the tomato genome, which contain not only functional Cf genes, but also several homologs with yet unknown function.. The short arm of Chromosome 1 harbors one of these clusters, designated "Milky Way", comprising functional Cf genes ( Cf-4 , Cf-4A , Cf-9 ). Moreover, two other clusters are located on the short arm of Chromosome 1, designated "Northern Lights" and "Southern Cross", which only harbor homologs ( Hcr9 s), but no functional Cf genes. Also, there are several reports about the presence of other Cf genes on the short arm of Chromosome 1.To increase our knowledge on the genetic and molecular organization of Cf genes on the short arm of Chromosome 1, an experimental approach was chosen to identify Cf genes with novel specificities that map on the short arm of Chromosome 1. To saturate the tomato genome with molecular markers, an integrated high-density AFLP-RFLP map was constructed using two different L. esculentum x L. pennellii F 2 mapping populations. Although 1175 AFLP markers were mapped on the tomato genome, covering 1482 cM, only nine AFLP markers were detected between the RFLP markers CT233 and TG51, which mark a 23.6 cM interval, comprising several Hcr9 clusters, on the short arm of Chromosome 1. This relatively low number of markers is due to the clustering of most Eco RI/ Mse I AFLP markers around the centromeres (Chapter 2).Testcross populations of 66 C. fulvum resistant Lycopersicon accessions were obtained by crossing these accessions with the near isogenic line Moneymaker-Cf4 and subsequent crossing of the F 1 with the susceptible tomato cultivar Moneymaker (Chapter 3). Using disease resistance tests with C. fulvum race 0 on only 24 plants of these testcross populations, susceptible plants were identified. An under-representation of susceptible plants identified Cf resistance linked to Cf-4 , and hence location of the unknown resistance on the short arm of Chromosome 1. Out of the 21 resistant accessions that have been tested in this way, ten showed a Cf-4 linked Cf gene. Among these ten accessions, one accession specifically recognized the extracellular protein ECP5 of C. fulvum and the corresponding gene was designated Cf-ECP5 . This gene was more accurately mapped using a testcross population of 338 plants and an F 2 population of a cross between Moneymaker and CfECP5, consisting of 233 individuals. Cf-ECP5 mapped 4 cM proximal to the Hcr9 locus Milky Way and the corresponding Cf locus was designated Aurora. An amplification product that cosegregated completely with the Cf-ECP5 gene, was cloned and nine clones were sequenced (Chapter 6). These nine clones could be classified into four groups, indicating that the Aurora locus comprises several Hcr9 s.Of the 66 resistant Lycopersicon accessions mentioned above, 64 have been screened for the presence of Cf-4 and/or Cf-9 , using PVX:: Avr4 and PVX:: Avr9 , respectively. A relatively large proportion of these accessions all harbored the functional genes Cf-4 and Cf-4A (Chapter 4). Sequence analysis of the 3' end of Cf-4 and part of the 3' untranslated region of Cf-4 showed no differences from the previously published Cf-4 sequences, hence these lines contain an introgression fragment with identical Cf-4 and Cf-4A genes. Since several of these lines were previously designated with different Cf digits, a change in nomenclature is proposed.Five out of the 66 accessions studied, showed an HR upon specific recognition of ECP2 and therefore harbor the corresponding resistance gene Cf-ECP2 (Chapter 5). Using two different testcross populations and one F 2 population from a cross between Moneymaker and CfECP2, representing in total 282 individuals, Cf-ECP2 was accurately mapped. Cf-ECP2 cosegregates with the molecular marker CT116, which is located proximal to the Milky Way and Aurora clusters, but distal to the Southern Cross locus. Southern hybridization, using Cf-9 as a probe, showed a hybridizing band of7.5 kb cosegregating with Cf-ECP2 , indicating that Cf-ECP2 is a member of a previously unidentified Hcr9 locus, that has been designated Orion.Studies in Chapters 3, 5 and 6 show that functional Cf genes can be located on several different Hcr9 loci on the short arm of Chromosome 1 and that these Hcr9 loci are highly polymorphic.</p