Genetic mapping of resistance gene analogs in an interspecific population of tomato segregating for early blight resistance

by Nino-Liu, David Orlando.

Limited sources of genetic variation exist within the cultivated tomato, Lycopersicon esculentum Mill., for resistance to one of its most destructive foliar diseases, early blight (EB), which is caused by the imperfect fungus Alternaria solani Sorauer. Current breeding strategies are trying to identify specific chromosomal regions in wild tomato relatives carrying genes that significantly contribute to an enhanced resistance response. The introgression of quantitative trait loci (QTLs) for EB resistance into the cultivated varieties is greatly facilitated by innovative molecular approaches such as marker-assisted selection (MAS). A recent trend in crop breeding is the utilization of gene-markers with assigned functions or putative roles in the expression of the trait under evaluation. Plant resistance genes (R-genes) are some of the most attractive gene-markers because of their direct involvement in vertical resistance and their presumed contribution to quantitative and non-specific resistance. In the past few years the structural similarities between R-gene proteins have been efficiently exploited in the identification, mapping and cloning of new R-gene analog (RGA) sequences and specificities. A purpose of this study was to develop PCR-based markers by using the conserved domains of proteins encoded by R-genes as template for primers capable of detecting RGAs in tomato. Ninety-nine polymorphic marker loci were detected after the separation of the total amplification products of these primers by high-resolution polyacrylamide gel electrophoresis. These markers were added to a genetic map of tomato which was previously developed in our laboratory based on a BC1 population of a cross between a L. esculentum (EB susceptible) and a L. hirsutum (EB resistant) lines. The mapped markers were initially considered as RGAs because of their observed resemblance to R-genes’ genomic organization and their coincidence with the locations of QTLs for EB resistance in the genetic map. A second purpose of this study was to determine whether the markers mapped in the tomato BC1 population corresponded to regions of R-genes or R-gene analogs. For this purpose, all markers were isolated from dried gels, cloned, sequenced and compared to the thousands of nucleotide and protein sequences stored in the GeneBank. Blast homology searches revealed that, despite their genomic organization and coincidence with R-genes and QTLs, very few of the markers were genuine RGAs. These results indicate that, contrary to what has been continuously assumed and reported by other investigators, most markers produced by R-gene-derived primers are not in fact R-gene analogs. However, the R-gene primer approach appears as a valuable tool for the development of new molecular markers and saturation of targeted chromosomal regions such as QTLs for disease resistance. Furthermore, while the nature of clustering of these markers remains unclear, the R-gene primer approach may provide useful markers associated with QTLs for use in MAS. iv v