SummaryLate blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease for potato cultivation. Here, we describe the positional cloning of the Rpi-blb1 gene from the wild potato species Solanum bulbocastanum known for its high levels of resistance to late blight. The Rpi-blb1 locus, which confers full resistance to complex isolates of P. infestans and for which race speci®city has not yet been demonstrated, was mapped in an intraspeci®c S. bulbocastanum population on chromosome 8, 0.3 cM from marker CT88. Molecular analysis of a bacterial arti®cial chromosome (BAC) clone spanning the Rpi-blb1 locus identi®ed a cluster of four candidate resistance gene analogues of the coiled coil, nucleotide-binding site, leucine-rich repeat (CC-NBS-LRR) class of plant resistance (R ) genes. One of these candidate genes, designated the Rpiblb1 gene, was able to complement the susceptible phenotype in a S. tuberosum and tomato background, demonstrating the potential of interspeci®c transfer of broad-spectrum late blight resistance to cultivated Solanaceae from sexually incompatible host species. Paired comparisons of synonymous and non-synonymous nucleotide substitutions between different regions of Rpi-blb1 paralogues revealed high levels of synonymous divergence, also in the LRR region. Although amino acid diversity between Rpi-blb1 homologues is centred on the putative solvent exposed residues of the LRRs, the majority of nucleotide differences in this region have not resulted in an amino acid change, suggesting conservation of function. These data suggest that Rpi-blb1 is relatively old and may be subject to balancing selection.
The conserved sequences in the nucleotide-binding sites of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of disease resistance (R) genes have been used for PCR-based R-gene isolation and subsequent development of molecular markers. Here we present a PCR-based approach (NBS profiling) that efficiently targets R genes and R-gene analogs (RGAs) and, at the same time, produces polymorphic markers in these genes. In NBS profiling, genomic DNA is digested with a restriction enzyme, and an NBS-specific (degenerate) primer is used in a PCR reaction towards an adapter linked to the resulting DNA fragments. The NBS profiling protocol generates a reproducible polymorphic multilocus marker profile on a sequencing gel that is highly enriched for R genes and RGAs. NBS profiling was successfully used in potato with several restriction enzymes, and several primers targeted to different conserved motifs in the NBS. Across primers and enzymes, the NBS profiles contained 50-90% fragments that were significantly similar to known R-gene and RGA sequences. The protocol was similarly successful in other crops (including tomato, barley, and lettuce) without modifications. NBS profiling can thus be used to produce markers tightly linked to R genes and R-gene clusters for genomic mapping and positional cloning and to mine for new alleles and new sources of disease resistance in available germplasm.
SummaryThe necessity to develop potato and tomato crops that possess durable resistance against the oomycete pathogen Phytophthora infestans is increasing as more virulent, crop-specialized and pesticide resistant strains of the pathogen are rapidly emerging. Here, we describe the positional cloning of the Solanum bulbocastanum-derived Rpi-blb2 gene, which even when present in a potato background confers broadspectrum late blight resistance. The Rpi-blb2 locus was initially mapped in several tetraploid backcross populations, derived from highly resistant complex interspecific hybrids designated ABPT (an acronym of the four Solanum species involved: S. acaule, S. bulbocastanum, S. phureja and S. tuberosum), to the same region on chromosome 6 as the Mi-1 gene from tomato, which confers resistance to nematodes, aphids and white flies. Due to suppression of recombination in the tetraploid material, fine mapping was carried out in a diploid intraspecific S. bulbocastanum F1 population. Bacterial artificial chromosome (BAC) libraries, generated from a diploid ABPT-derived clone and from the resistant S. bulbocastanum parent clone, were screened with markers linked to resistance in order to generate a physical map of the Rpi-blb2 locus. Molecular analyses of both ABPTand S. bulbocastanum-derived BAC clones spanning the Rpi-blb2 locus showed it to harbor at least 15 Mi-1 gene homologs (MiGHs). Of these, five were genetically determined to be candidates for Rpi-blb2. Complementation analyses showed that one ABPT-and one S. bulbocastanum-derived MiGH were able to complement the susceptible phenotype in both S. tuberosum and tomato. Sequence analyses of both genes showed them to be identical. The Rpi-blb2 protein shares 82% sequence identity to the Mi-1 protein.Significant expansion of the Rpi-blb2 locus compared to the Mi-1 locus indicates that intrachromosomal recombination or unequal crossing over has played an important role in the evolution of the Rpi-blb2 locus. The contrasting evolutionary dynamics of the Rpi-blb2/Mi-1 loci in the two related genomes may reflect the opposite evolutionary potentials of the interacting pathogens.
Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease in potato. For sustainable management of this economically important disease, resistance breeding relies on the availability of resistance (R) genes. Such R genes against P. infestans have evolved in wild tuber-bearing Solanum species from North, Central and South America, upon co-evolution with cognate avirulence (Avr) genes. Here, we report how effectoromics screens with Avr2 of P. infestans revealed defense responses in diverse Solanum species that are native to Mexico and Peru. We found that the response to AVR2 in the Mexican Solanum species is mediated by R genes of the R2 family that resides on a major late blight locus on chromosome IV. In contrast, the response to AVR2 in Peruvian Solanum species is mediated by Rpi-mcq1, which resides on chromosome IX and does not belong to the R2 family. The data indicate that AVR2 recognition has evolved independently on two genetic loci in Mexican and Peruvian Solanum species, respectively. Detached leaf tests on potato cultivar ‘Désirée’ transformed with R genes from either the R2 or the Rpi-mcq1 locus revealed an overlapping, but distinct resistance profile to a panel of 18 diverse P. infestans isolates. The achieved insights in the molecular R – Avr gene interaction can lead to more educated exploitation of R genes and maximize the potential of generating more broad-spectrum, and potentially more durable control of the late blight disease in potato.
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