The effect of pyramiding Phytophthora infestans resistance genes R Pi-mcd1 and R Pi-ber in potato

Despite efforts to control late blight in potatoes by introducing R _pi -genes from wild species into cultivated potato, there are still concerns regarding the durability and level of resistance. Pyramiding R _pi -genes can be a solution to increase both durability and level of resistance. In this study, two resistance genes, R _Pi-mcd1 and R _Pi-ber , introgressed from the wild tuber-bearing potato species Solanum microdontum and S. berthaultii were combined in a diploid S. tuberosum population. Individual genotypes from this population were classified after four groups, carrying no R _pi -gene, with only R _Pi-mcd1 , with only R _Pi-ber , and a group with the pyramided R _Pi-mcd1 and R _Pi-ber by means of tightly linked molecular markers. The levels of resistance between the groups were compared in a field experiment in 2007. The group with R _Pi-mcd1 showed a significant delay to reach 50% infection of the leaf area of 3 days. The group with R _Pi-ber showed a delay of 3 weeks. The resistance level in the pyramid group suggested an additive effect of R _Pi-mcd1 with R _Pi-ber . This suggests that potato breeding can benefit from combining individual R _pi -genes, irrespective of the weak effect of R _Pi-mcd1 or the strong effect of R _Pi-ber .     

Functional analysis of potato genes involved in quantitative resistance to Phytophthora infestans

The most significant threat to potato production worldwide is the late blight disease, which is caused by the oomycete pathogen Phytophthora infestans. Based on previous cDNA microarrays and cDNA-amplified fragment length polymorphism analysis, 63 candidate genes that are expected to contribute to developing a durable resistance to late blight were selected for further functional analysis. We performed virus-induced gene silencing (VIGS) to these candidate genes on both Nicotiana benthamiana and potato, subsequently inoculated detached leaves and assessed the resistance level. Ten genes decreased the resistance to P. infestans after VIGS treatment. Among those, a lipoxygenase (LOX; EC 1.13.11.12) and a suberization-associated anionic peroxidase affected the resistance in both N. benthamiana and potato. Our results identify genes that may play a role in quantitative resistance mechanisms to late blight.     

The R1 gene conferring race-specific resistance to Phytophthora infestans in potato is located on potato chromosome V.

Summary Late blight in potato is caused by the fungusPhytophthora infestans and can inflict severe damage on the potato crop. Resistance toP. infestans is either based on major dominantR genes conferring vertical, race-specific resistance or on minor genes inducing horizontal, unspecific resistance. A dihaploid potato line was identified which carried theR1 gene, conferring vertical resistance to allP. infestans races, with the exception of those homozygous for the recessive virulence allele of the locusV1. The F₁ progeny of a cross between this resistant parent P(R1) and P(r), a line susceptible to all races, was analysed for segregation ofR1 and of restriction fragment length polymorphism (RFLP) markers distributed on the potato RFLP map comprising more than 300 loci. TheR1 locus was mapped to chromosome V in the interval between RFLP markers GP21 and GP179. The map position ofR1 was found to be very similar to the one ofRx2, a dominant locus inducing extreme resistance to potato virus X.     

The late blight resistance locus Rpi-bib3 from Solanum bulbocastanum belongs to a major late blight R gene cluster on chromosome 4 of potato

Late blight, caused by Phytophthora infestans, is one of the most devastating diseases in cultivated potato. Breeding of new potato cultivars with high levels of resistance to P. infestans is considered the most durable strategy for future potato cultivation. In this study, we report the identification of a new late-blight resistance (R) locus from the wild potato species Solanum bulbocastanum. Using several different approaches, a high-resolution genetic map of the new locus was generated, delimiting Rpi-blb3 to a 0.93 cM interval on chromosome 4. One amplification fragment length polymorphism marker was identified that cosegregated in 1,396 progeny plants of an intraspecific mapping population with Rpi-blb3. For comparative genomics purposes, markers linked to Rpi-blb3 were tested in mapping populations used to map the three other late-blight R loci Rpi-abpt, R2, and R2-like also to chromosome 4. Marker order and allelic conservation suggest that Rpi-blb3, Rpi-abpt, R2, and R2-like reside in the same R gene cluster on chromosome 4 and likely belong to the same gene family. Our findings provide novel insights in the evolution of R gene clusters conferring late-blight resistance in Solanum spp.     

Mapping the R10 and R11 genes for resistance to late blight (Phytophthora infestans) present in the potato (Solanum tuberosum) R-gene differentials of black

The R10 and R11 late blight differentials of Black (tetraploid clones 3681ad1 and 5008ab6) were crossed with the susceptible potato (Solanum tuberosum) cultivar Maris Piper and the progeny were assessed for blight resistance in a whole plant glasshouse test using race 1,2,3,4,6,7 of Phytophthora infestans. The disease scores for the R10 population displayed a continuous distribution whereas the progeny in the R11 population could be categorised as resistant or susceptible. A bulk segregant analysis using amplified fragment length polymorphism assays was done on the ten most resistant and ten most susceptible progeny in each population and two closely linked markers were found to be associated with resistance. R11 mapped to 8.5 cM from marker PAG/MAAG_172.3 and R10 mapped as a quantitative trait locus in which marker PAC/MATC_264.1 explained 56.9% of the variation in disease scores. The results were consistent with R10 and R11 being allelic versions of genes at the R3 locus on chromosome 11. The implications are discussed for mapping R-genes which fail to give complete immunity to a pathogen.     

An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated potato and tomato

Late 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 specificity has not yet been demonstrated, was mapped in an intraspecific S. bulbocastanum population on chromosome 8, 0.3 cM from marker CT88. Molecular analysis of a bacterial artificial chromosome (BAC) clone spanning the Rpi-blb1 locus identified 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 Rpi-blb1 gene, was able to complement the susceptible phenotype in a S. tuberosum and tomato background, demonstrating the potential of interspecific 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 R3 resistance to Phytophthora infestans in potato is conferred by two closely linked R genes with distinct specificities

The R3 locus of potato (Solanum tuberosum L.) confers full resistance to avirulent isolates of Phytophthora infestans, the causal agent of late blight. R3 resides in the distal part of chromosome 11 and segregates in a potato mapping population, from which a well-saturated amplified fragment length polymorphism map is available. Using a population of 1,748 plants, we constructed a high-resolution genetic map at the R3 locus. Using the combination of fine mapping and accurate disease testing with specific P. infestans isolates, we detected that the R3 locus is composed of two genes with distinct specificities. The two genes R3a and R3b are 0.4 cM apart and have both been introgressed from S. demissum, the 'donor' species of most characterized race-specific R genes to P. infestans. A natural recombinant between R3a and R3b was discovered in one accession of S. demissum. The synteny between the R3 locus and the tomato I2 locus is discussed.     

Detection of a quantitative trait locus for both foliage and tuber resistance to late blight [Phytophthora infestans (Mont.) de Bary] on chromosome 4 of a dihaploid potato clone (Solanum tuberosum subsp. tuberosum)

Linkage analysis, Kruskal–Wallis analysis, interval mapping and graphical genotyping were performed on a potato diploid backcross family comprising 120 clones segregating for resistance to late blight. A hybrid between the Solanum tuberosum dihaploid clone PDH247 and the long-day-adapted S. phureja clone DB226(70) had been crossed to DB226(70) to produce the backcross family. Eighteen AFLP primer combinations provided 186 and 123 informative maternal and paternal markers respectively, with 63 markers in common to both parents. Eleven microsatellite (SSR) markers proved useful for identifying chromosomes. Linkage maps of both backcross parents were constructed. The results of a Kruskal–Wallis analysis, interval mapping and graphical genotyping were all consistent with a QTL or QTLs for blight resistance between two AFLP markers 30 cM apart on chromosome 4, which was identified by a microsatellite marker. The simplest explanation of the results is a single QTL with an allele from the dihaploid parent conferring resistance to race 1, 4 of P. infestans in the foliage in the glasshouse and to race 1, 2, 3, 4, 6, 7 in the foliage in the field and in tubers from glasshouse raised plants. The QTL was of large effect, and explained 78 and 51% of the variation in phenotypic scores for foliage blight in the glasshouse and field respectively, as well as 27% of the variation in tuber blight. Graphical genotyping and the differences in blight scores between the parental clones showed that all of the foliage blight resistance is accounted for by chromosome 4, whereas undetected QTLs for tuber resistance probably exist on other chromosomes. Graphical genotyping also explained the lack of precision in mapping the QTL(s) in terms of lack of appropriate recombinant chromosomes.     

R6 and R7 alleles of potato conferring race-specific resistance to Phytophthora infestans (Mont.) de Bary identified genetic loci clustering with the R3 locus on chromosome XI

In potato, 11 resistance alleles (R1–R11) are known which confer race-specific resistance to the fungus Phytophthora infestans. R1 has been mapped previously to potato chromosome V and R3 to chromosome XI. Here we report on the localization of the R6 and R7 alleles on the genetic map of potato. Differential resistant strains of tetraploid Solanum tuberosum, clones MaR6 and MaR7, were used as parental plants for the parthenogenetic induction and selection of diploid genotypes containing the R6 or the R7 resistance allele to P. infestans. One resistant dihaploid from MaR7 could be used directly as a parent to produce diploid F₁ progeny suitable for phenotypic and RFLP analysis. MaR6 did not produce useful dihaploids directly. After crossing MaR6 with a tetraploid susceptible genotype, resistant F₁ clones were selected. The resistant genotypes were then used as parents for the induction of dihaploids. Six dihaploids bearing R6 were identified that could be crossed with a diploid susceptible genotype. Two diploid F₁ populations, segregating for R6 and R7, respectively, were analysed with RFLP markers known to be linked with previously identified R genes. Markers linked with R3 were found also to be linked with R6 and R7. The resistance alleles R6 and R7 mapped to a similar distal position on chromosome XI as the R3 allele.     

A reexamination of infraspecific taxa of a wild potato,Solanum microdontum (Solanum sect.Petota:Solanaceae)

Current taxonomic interpretations ofSolanum microdontum Bitter partition the species into two or three infraspecific taxa, variously recognized as subspecies or varieties. The present study reexamines these taxa using morphological data from four individuals each of 69 accessions from most of the range of the species, planted in a common field plot. Our results show that the character states used to recognize infraspecific taxa inS. microdontum often vary within accessions and have no correlation with geography. We conclude that past hypotheses have used typological concepts and that infraspecific taxa are not warranted. This study questions other hypotheses of infraspecific taxa in sect.Petota.     

Biochemical reactions of different tissues of potato (Solanum tuberosum) to zoospores or elicitors from Phytophthora infestans

The time courses of sesquiterpenoid phytoalexin accumulation were examined in compatible and incompatible interactions of leaves and tubers from five different R genotypes of potato (Solanum tuberosum) with corresponding pathotypes of Phytophthora infestans, as well as in non-host interactions of all five potato cultivars with Phytophthora megasperma f. sp. glycinea and in elicitor-treated tubers from five, and cell suspension cultures from two, of the cultivars. In tubers, rishitin and several structurally related sesquiterpene derivatives accumulated rapidly in non-host incompatible interactions, less rapidly in host incompatible interactions, and more slowly in compatible interactions. Treatment of tubers or cell cultures with fungal culture filtrate or arachidonic acid elicited in most cases a transient accumulation of the sesquiterpenoid phytoalexins. None of these compounds was detectable under any of the applied conditions either in infected or in elicitortreated leaves. Sesquiterpenoid phytoalexins might therefore be helpful, but appear not to be essential, in disease resistance of potato.Abbreviations CF concentrated culture filtrate of Pi - cv. cultivar - Pi Phytophthora infestans (numbering indicates pathotypes corresponding to R genes in potato) - Pmg Phytophthora megasperma f. sp. glycinea     

The R1 gene for potato resistance to late blight (Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant resistance genes

Late blight caused by the oomycete Phytophthora infestans is the most destructive disease in potato cultivation worldwide. New, more virulent P. infestans strains have evolved which overcome the genetic resistance that has been introgressed by conventional breeding from wild potato species into commercial varieties. R genes (for single-gene resistance) and genes for quantitative resistance to late blight are present in the germplasm of wild and cultivated potato. The molecular basis of single-gene and quantitative resistance to late blight is unknown. We have cloned R1, the first gene for resistance to late blight, by combining positional cloning with a candidate gene approach. The R1 gene is member of a gene family. It encodes a protein of 1293 amino acids with a molecular mass of 149.4 kDa. The R1 gene belongs to the class of plant genes for pathogen resistance that have a leucine zipper motif, a putative nucleotide binding domain and a leucine-rich repeat domain. The most closely related plant resistance gene (36% identity) is the Prf gene for resistance to Pseudomonas syringae of tomato. R1 is located within a hot spot for pathogen resistance on potato chromosome V. In comparison to the susceptibility allele, the resistance allele at the R1 locus represents a large insertion of a functional R gene.     

Autotetraploids and genetic mapping using common AFLP markers: the R2 allele conferring resistance to Phytophthora infestans mapped on potato chromosome 4

 Due to the complexity of tetrasomic inheritance, mapping studies in potato (Solanum tuberosum L.) are generally conducted at the diploid level. In the present study we tested the feasibility of Bulked Segregant Analysis (BSA) using a tetraploid offspring for the identification of AFLP markers linked to the R2 allele, which confers race-specific resistance to Phytophthora infestans. Eleven bulk-specific AFLP markers, detected in fingerprints of 205 AFLP primer combinations, could be mapped in a linkage group encompassing the R2 locus. The efficiency of BSA at the tetraploid level, determined by the frequency of single-dose restriction fragments (SDRF), was much higher than expected on the basis of overall genetic dissimilarity between the parental clones. The fortuitous detection of AFLPs with linkage to the R2 allele is explained on the basis of specific genetic dissimilarity between cultivated potato and the chromosomal segment introgressed from S. demissum carrying the resistant R2 allele. AFLP markers common to those with linkage to R2 were visually recognized by their electrophoretic mobility in the AFLP fingerprint in a parental clone of a reference mapping population. Using these common AFLP markers we anchored the linkage group comprising the R2 allele to potato chromosome 4. Received: 30 October 1997 / Accepted: 6 November 1997     

Identification of a resistance gene Rpi-dlc1 to Phytophthora infestans in European accessions of Solanum dulcamara

Initial screening of 14 Solanum dulcamara accessions enabled the identification of individuals resistant and susceptible to Phytophthora infestans. Crosses between contrasting genotypes resulted in three F₂–BC₁ populations segregating for resistance to late blight in a laboratory assay and under field conditions. Genetic profiling of one of these populations using 128 AFLP primers generated three markers linked to the resistant phenotype. Blast analysis of the sequenced markers resulted in a plausible gene position on the distal end of the long arm of chromosome 9 that could be confirmed by CAPS markers. Thus, we describe a first resistant gene, named Rpi-dlc1, from S. dulcamara, a Solanum species native to Europe. In addition, one population was tested for broadness of resistance responses using a set of seven additional P. infestans isolates, varying in virulence. This indicated the possible presence of additional Rpi genes.     

A nonspecific lipid transfer protein,StLTP10, mediates resistance to Phytophthora infestans in potato

Nonspecific lipidtransfer proteins (nsLTPs), which are small, cysteine-rich proteins, belong to the pathogenesis-related protein family, and several of them act as positive regulators during plant disease resistance. However, the underlying molecular mechanisms of these proteins in plant immune responses are unclear. In this study, a typical nsLTP gene, StLTP10, was identified and functionally analysed in potato. StLTP10 expression was significantly induced by Phytophthora infestans, which causes late blight in potato, and defence-related phytohormones, including abscisic acid (ABA), salicylic acid, and jasmonic acid. Characterization of StLTP10-overexpressing and knockdown lines indicated that StLTP10 positively regulates plant resistance to P. infestans. This resistance was coupled with enhanced expression of reactive oxygen species scavenging- and defence-related genes. Furthermore, we identified that StLTP10 physically interacts with ABA receptor PYL4 and affects its subcellular localization. These two proteins work together to regulate stomatal closure during pathogen infection. Interestingly, we also found that wound-induced protein kinase interacts with StLTP10 and positively regulates its protein abundance. Taken together, our results provide insight into the role of StLTP10 in resistance to P. infestans and suggest candidates to enhance broad-spectrum resistance to pathogens in potato.     

Genetic mapping from field tests of qualitative and quantitative resistance to Phytophthora infestans in a population derived from Solanum tuberosum and Solanum berthaultii

Under controlled field conditions, a Solanum backcross population segregated for resistance to Phytophthora infestans. The population (`BCT') had been derived previously by crossing the Solanum tuberosum dihaploid USW2230 × Solanum berthaultii PI473331 to obtain the hybrid M200-30, and then backcrossing the hybrid to the S. tuberosum dihaploid HH1-9. Resistance was assessed from analyses of epidemics in small plots of each individual genotype, and data were recorded as area under the disease progress curve (AUDPC). The parents of the original cross (USW2230 and a selection from PI473331) were not included in the test, but the hybrid was incompatible and HH1-9 was compatible with the tester strain of P. infestans (US-8 lineage). Somewhat more than half of the progeny also were incompatible with the tester strain, indicating the presence of an R gene. This gene segregated from the S. berthaultii parent and mapped 4.8 cm from the RFLP marker TG63 on chromosome 10. We deduce that the R gene is not R-1, R-2, R-3, R-6, or R-7 and is probably not R-4, R-5, or R-10. Among the remaining, compatible progeny, there was a wide range of quantitative resistance. All were more resistant than the susceptible cultivar Superior, and most individuals were much more resistant than the moderately resistant cultivar Kennebec. AUDPC values among the sub-population of compatible genotypes ranged from about 400 to 1500 units the first year and from 400 to 1760 units the second year. At least five quantitative trait loci (QTLs) were detected in this sub-population in both 1997 and 1998, including one detected through segregation of alleles from both the hybrid parent and the recurrent S. tuberosum parent. A model of main and epistatic effects explained 56% and 66% of the variation observed for quantitative resistance to late blight in 1997 and 1998, respectively. Several of the QTLs for late blight resistance were located in regions of the genome to which QTLs for late maturity have previously been mapped.