CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew.

BACKGROUND: The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene PowderyMildewResistance 4 (PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogen Oidium neolycopersici (On). RESULTS: To study whether full knock-out of the tomato PMR4 gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomato PMR4 CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomato PMR4 gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility to On based on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in the pmr4 mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants. CONCLUSION: CRISPR/Cas-9 targeted mutagenesis of the tomato PMR4 gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogen On. Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterize S-genes by generating different types of mutations.

Genome-Wide Mapping of Structural Variations Reveals a Copy Number Variant That Determines Reproductive Morphology in Cucumber.

Structural variations (SVs) represent a major source of genetic diversity. However, the functional impact and formation mechanisms of SVs in plant genomes remain largely unexplored. Here, we report a nucleotide-resolution SV map of cucumber (Cucumis sativas) that comprises 26,788 SVs based on deep resequencing of 115 diverse accessions. The largest proportion of cucumber SVs was formed through nonhomologous end-joining rearrangements, and the occurrence of SVs is closely associated with regions of high nucleotide diversity. These SVs affect the coding regions of 1676 genes, some of which are associated with cucumber domestication. Based on the map, we discovered a copy number variation (CNV) involving four genes that defines the Female (F) locus and gives rise to gynoecious cucumber plants, which bear only female flowers and set fruit at almost every node. The CNV arose from a recent 30.2-kb duplication at a meiotically unstable region, likely via microhomology-mediated break-induced replication. The SV set provides a snapshot of structural variations in plants and will serve as an important resource for exploring genes underlying key traits and for facilitating practical breeding in cucumber.

Silencing of DND1 in potato and tomato impedes conidial germination, attachment and hyphal growth of Botrytis cinerea.

BACKGROUND: Botrytis cinerea, a necrotrophic pathogenic fungus, attacks many crops including potato and tomato. Major genes for complete resistance to B. cinerea are not known in plants, but a few quantitative trait loci have been described in tomato. Loss of function of particular susceptibility (S) genes appears to provide a new source of resistance to B. cinerea in Arabidopsis. RESULTS: In this study, orthologs of Arabidopsis S genes (DND1, DMR6, DMR1 and PMR4) were silenced by RNAi in potato and tomato (only for DND1). DND1 well-silenced potato and tomato plants showed significantly reduced diameters of B. cinerea lesions as compared to control plants, at all-time points analysed. Reduced lesion diameter was also observed on leaves of DMR6 silenced potato plants but only at 3 days post inoculation (dpi). The DMR1 and PMR4 silenced potato transformants were as susceptible as the control cv Desiree. Microscopic analysis was performed to observe B. cinerea infection progress in DND1 well-silenced potato and tomato leaves. A significantly lower number of B. cinerea conidia remained attached to the leaf surface of DND1 well-silenced potato and tomato plants and the hyphal growth of germlings was hampered. CONCLUSIONS: This is the first report of a cytological investigation of Botrytis development on DND1-silenced crop plants. Silencing of DND1 led to reduced susceptibility to Botrytis, which was associated with impediment of conidial germination and attachment as well as hyphal growth. Our results provide new insights regarding the use of S genes in resistance breeding.

The Solanum demissum R8 late blight resistance gene is an Sw-5 homologue that has been deployed worldwide in late blight resistant varieties.

KEY MESSAGE: The potato late blight resistance gene R8 has been cloned. R8 is found in five late blight resistant varieties deployed in three different continents. R8 recognises Avr8 and is homologous to the NB-LRR protein Sw-5 from tomato. The broad spectrum late blight resistance gene R8 from Solanum demissum was cloned based on a previously published coarse map position on the lower arm of chromosome IX. Fine mapping in a recombinant population and bacterial artificial chromosome (BAC) library screening resulted in a BAC contig spanning 170 kb of the R8 haplotype. Sequencing revealed a cluster of at least ten R gene analogues (RGAs). The seven RGAs in the genetic window were subcloned for complementation analysis. Only one RGA provided late blight resistance and caused recognition of Avr8. From these results, it was concluded that the newly cloned resistance gene was indeed R8. R8 encodes a typical intracellular immune receptor with an N-terminal coiled coil, a central nucleotide binding site and 13 C-terminal leucine rich repeats. Phylogenetic analysis of a set of representative Solanaceae R proteins shows that R8 resides in a clearly distinct clade together with the Sw-5 tospovirus R protein from tomato. It was found that the R8 gene is present in late blight resistant potato varieties from Europe (Sarpo Mira), USA (Jacqueline Lee, Missaukee) and China (PB-06, S-60). Indeed, when tested under field conditions, R8 transgenic potato plants showed broad spectrum resistance to the current late blight population in the Netherlands, similar to Sarpo Mira.

Silencing of six susceptibility genes results in potato late blight resistance.

Phytophthora infestans, the causal agent of late blight, is a major threat to commercial potato production worldwide. Significant costs are required for crop protection to secure yield. Many dominant genes for resistance (R-genes) to potato late blight have been identified, and some of these R-genes have been applied in potato breeding. However, the P. infestans population rapidly accumulates new virulent strains that render R-genes ineffective. Here we introduce a new class of resistance which is based on the loss-of-function of a susceptibility gene (S-gene) encoding a product exploited by pathogens during infection and colonization. Impaired S-genes primarily result in recessive resistance traits in contrast to recognition-based resistance that is governed by dominant R-genes. In Arabidopsis thaliana, many S-genes have been detected in screens of mutant populations. In the present study, we selected 11 A. thaliana S-genes and silenced orthologous genes in the potato cultivar Desiree, which is highly susceptible to late blight. The silencing of five genes resulted in complete resistance to the P. infestans isolate Pic99189, and the silencing of a sixth S-gene resulted in reduced susceptibility. The application of S-genes to potato breeding for resistance to late blight is further discussed.

Down-regulation of Arabidopsis DND1 orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew.

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death 1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.

Characterisation of the late blight resistance in potato differential MaR9 reveals a qualitative resistance gene, R9a, residing in a cluster of Tm-2 (2) homologs on chromosome IX.

KEY MESSAGE: The durable late blight resistance in potato plant Ma R9 is genetically characterized. A novel R -gene is mapped. The monogenic nature and map positions of R9 are negated and rectified. Late blight of potato (Solanum tuberosum), caused by Phytophthora infestans, can effectively be managed by genetic resistance. The MaR9 differential plant provides durable resistance to a broad spectrum of late blight strains. This resistance is brought about by at least seven genes derived from S. demissum including R1, Rpi-abpt1, R3a, R3b, R4, R8 and, so far uncharacterized resistance gene(s). Here we set out to genetically characterize this additional resistance in MaR9. Three BC1 populations derived from MaR9 were identified that segregated for IPO-C resistance but that lacked R8. One BC1 population showed a continuous scale of resistance phenotypes, suggesting that multiple quantitative resistance genes were segregating. In two other BC1 populations resistance and susceptibility were segregating in a 1:1 ratio, suggesting a single qualitative resistance gene (R9a). A chromosome IX PCR marker, 184-81, fully co-segregated with R9a. The map position of R9a on the distal end of the lower arm of chromosome IX was confirmed using PCR markers GP101 and Stm1021. Successively, cluster-directed profiling (CDP) was carried out, revealing six closely linked markers. CDP(Sw)58, CDP(Sw)59 and CDP(Sw5)10 flanked the R9a gene at the distal end (5.8 cM) and, as expected, were highly homologous to Sw-5. CDP(Tm2)2 flanked R9a on the proximal side (2.9 cM). CDP(Tm2)6 and CDP(Tm2)7 fully co-segregated with resistance and had high homology to Tm-2 (2) , showing that R9a resides in a cluster of NBS-LRR genes with homology to Tm-2 (2) . Besides R9a, additional resistance of quantitative nature is found in MaR9, which remains to be genetically characterized.

Development of late blight resistant potatoes by cisgene stacking.

BACKGROUND: Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties. RESULTS: We pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), but lacking the bacterial kanamycin resistance selection marker (NPTII) was transformed to the three selected potato varieties using Agrobacterium-mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated. CONCLUSIONS: We developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.

Vector integration in triple R gene transformants and the clustered inheritance of resistance against potato late blight.

Genetic transformation with resistance (R) genes is expected to enhance resistance durability against pathogens, especially for potato, a vegetatively propagated crop with tetrasomic inheritance and a long-term breeding program. In this study, 128 potato transformants were analysed for the presence of vector T-DNA genes, borders and backbone sequences. They were harvested after transformation using a construct containing neomycin phosphotransferase II (nptII) and three R genes against potato late blight (Phytophthora infestans). Our analysis revealed that 45 % of the R gene-containing transformants possessed a low T-DNA copy number, without the integration of vector backbone and borders. The integration of vector backbone sequences was characterized using eight genes, and backbone gene tetA was selected for the early prediction of plants with backbone sequence integration. Three transformants, two plants harbouring one T-DNA copy and one plant harbouring three T-DNA copies, were crossed with susceptible cv. Katahdin. Based on our results, we conclude that all four T-DNA genes were inherited as one cluster and segregated in a Mendelian fashion. The three T-DNA inserts from the transformant harbouring three T-DNA copies were statistically proven to be un-linked and inherited into the offspring plants independently. All of the R genes were functionally expressed in the offspring plants as in their parental transformants. This functional gene stacking has important implications towards achieving more durable resistance against potato late blight.

Qualitative and quantitative late blight resistance in the potato cultivar Sarpo Mira is determined by the perception of five distinct RXLR effectors.

Potato defends against Phytophthora infestans infection by resistance (R)-gene-based qualitative resistance as well as a quantitative field resistance. R genes are renowned to be rapidly overcome by this oomycete, and potato cultivars with a decent and durable resistance to current P. infestans populations are hardly available. However, potato cultivar Sarpo Mira has retained resistance in the field over several years. We dissected the resistance of 'Sarpo Mira' in a segregating population by matching the responses to P. infestans RXLR effectors with race-specific resistance to differential strains. The resistance is based on the combination of four pyramided qualitative R genes and a quantitative R gene that was associated with field resistance. The qualitative R genes include R3a, R3b, R4, and the newly identified Rpi-Smira1. The qualitative resistances matched responses to avirulence (AVR)3a, AVR3b, AVR4, and AVRSmira1 RXLR effectors and were overcome by particular P. infestans strains. The quantitative resistance was determined to be conferred by a novel gene, Rpi-Smira2. It was only detected under field conditions and was associated with responses to the RXLR effector AvrSmira2. We foresee that effector-based resistance breeding will facilitate selecting and combining qualitative and quantitative resistances that may lead to a more durable resistance to late blight.

Genetic analysis of metabolites in apple fruits indicates an mQTL hotspot for phenolic compounds on linkage group 16.

Apple (Malus×domestica Borkh) is among the main sources of phenolic compounds in the human diet. The genetic basis of the quantitative variations of these potentially beneficial phenolic compounds was investigated. A segregating F1 population was used to map metabolite quantitative trait loci (mQTLs). Untargeted metabolic profiling of peel and flesh tissues of ripe fruits was performed using liquid chromatography-mass spectrometry (LC-MS), resulting in the detection of 418 metabolites in peel and 254 in flesh. In mQTL mapping using MetaNetwork, 669 significant mQTLs were detected: 488 in the peel and 181 in the flesh. Four linkage groups (LGs), LG1, LG8, LG13, and LG16, were found to contain mQTL hotspots, mainly regulating metabolites that belong to the phenylpropanoid pathway. The genetics of annotated metabolites was studied in more detail using MapQTL®. A number of quercetin conjugates had mQTLs on LG1 or LG13. The most important mQTL hotspot with the largest number of metabolites was detected on LG16: mQTLs for 33 peel-related and 17 flesh-related phenolic compounds. Structural genes involved in the phenylpropanoid biosynthetic pathway were located, using the apple genome sequence. The structural gene leucoanthocyanidin reductase (LAR1) was in the mQTL hotspot on LG16, as were seven transcription factor genes. The authors believe that this is the first time that a QTL analysis was performed on such a high number of metabolites in an outbreeding plant species.

Broad spectrum late blight resistance in potato differential set plants MaR8 and MaR9 is conferred by multiple stacked R genes.

Phytophthora infestans is the causal agent of late blight in potato. The Mexican species Solanum demissum is well known as a good resistance source. Among the 11 R gene differentials, which were introgressed from S. demissum, especially R8 and R9 differentials showed broad spectrum resistance both under laboratory and under field conditions. In order to gather more information about the resistance of the R8 and R9 differentials, F1 and BC1 populations were made by crossing Mastenbroek (Ma) R8 and R9 clones to susceptible plants. Parents and offspring plants were examined for their pathogen recognition specificities using agroinfiltration with known Avr genes, detached leaf assays (DLA) with selected isolates, and gene-specific markers. An important observation was the discrepancy between DLA and field trial results for Pi isolate IPO-C in all F1 and BC1 populations, so therefore also field trial results were included in our characterization. It was shown that in MaR8 and MaR9, respectively, at least four (R3a, R3b, R4, and R8) and seven (R1, Rpi-abpt1, R3a, R3b, R4, R8, R9) R genes were present. Analysis of MaR8 and MaR9 offspring plants, that contained different combinations of multiple resistance genes, showed that R gene stacking contributed to the Pi recognition spectrum. Also, using a Pi virulence monitoring system in the field, it was shown that stacking of multiple R genes strongly delayed the onset of late blight symptoms. The contribution of R8 to this delay was remarkable since a plant that contained only the R8 resistance gene still conferred a delay similar to plants with multiple resistance genes, like, e.g., cv Sarpo Mira. Using this "de-stacking" approach, many R gene combinations can be made and tested in order to select broad spectrum R gene stacks that potentially provide enhanced durability for future application in new late blight resistant varieties.

Functional stacking of three resistance genes against Phytophthora infestans in potato.

Functional stacking of broad spectrum resistance (R) genes could potentially be an effective strategy for more durable disease resistance, for example, to potato late blight caused by Phytophthora infestans (Pi). For this reason, three broad spectrum potato R genes (Rpi), Rpi-sto1 (Solanum stoloniferum), Rpi-vnt1.1 (S. venturii) and Rpi-blb3 (S. bulbocastanum) were selected, combined into a single binary vector pBINPLUS and transformed into the susceptible cultivar Desiree. Among the 550 kanamycin resistant regenerants, 28 were further investigated by gene specific PCRs. All regenerants were positive for the nptII gene and 23 of them contained the three Rpi genes, referred to as triple Rpi gene transformants. Detached leaf assay and agro-infiltration of avirulence (Avr) genes showed that the 23 triple Rpi gene transformants were resistant to the selected isolates and showed HR with the three Avr effectors indicating functional stacking of all the three Rpi genes. It is concluded that Avr genes, corresponding to the R genes to be stacked, must be available in order to assay for functionality of each stack component. No indications were found for silencing or any other negative effects affecting the function of the inserted Rpi genes. The resistance spectrum of these 23 triple Rpi gene transformants was, as expected, a sum of the spectra from the three individual Rpi genes. This is the first example of a one-step approach for the simultaneous domestication of three natural R genes against a single disease by genetic transformation.

Silencing susceptibility genes in potato hinders primary infection of Phytophthora infestans at different stages.

Most potato cultivars are susceptible to late blight disease caused by the oomycete pathogen Phytophthora infestans. A new source of resistance to prevent or diminish pathogen infection is found in the genetic loss of host susceptibility. Previously, we showed that RNAi-mediated silencing of the potato susceptibility (S) genes StDND1, StDMR1 and StDMR6 leads to increased late blight resistance. The mechanisms underlying this S-gene mediated resistance have thus far not been identified. In this study, we examined the infection process of P. infestans on StDND1-, StDMR1- and StDMR6-silenced potato lines. Microscopic analysis showed that penetration of P. infestans spores was hampered on StDND1-silenced plants. On StDMR1- and StDMR6-silenced plants, P. infestans infection was arrested at a primary infection stage by enhanced cell death responses. Histochemical staining revealed that StDMR1- and StDMR6-silenced plants display elevated ROS levels in cells at the infection sites. Resistance in StDND1-silenced plants, however, seems not to rely on a cell death response as ROS accumulation was found to be absent at most inoculated sites. Quantitative analysis of marker gene expression suggests that the increased resistance observed in StDND1- and StDMR6-silenced plants relies on an early onset of SA- and ET-mediated signalling pathways. Resistance mediated by silencing StDMR1 was found to be correlated with the early induction of SA-mediated signalling. These data provide evidence that different defense mechanisms are involved in late blight resistance mediated by functional impairment of different potato S-genes.

Recognition of Pep-13/25 MAMPs of Phytophthora localizes to an RLK locus in Solanum microdontum.

Pattern-triggered immunity (PTI) in plants is mediated by cell surface-localized pattern recognition receptors (PRRs) upon perception of microbe-associated molecular pattern (MAMPs). MAMPs are conserved molecules across microbe species, or even kingdoms, and PRRs can confer broad-spectrum disease resistance. Pep-13/25 are well-characterized MAMPs in Phytophthora species, which are renowned devastating oomycete pathogens of potato and other plants, and for which genetic resistance is highly wanted. Pep-13/25 are derived from a 42 kDa transglutaminase GP42, but their cognate PRR has remained unknown. Here, we genetically mapped a novel surface immune receptor that recognizes Pep-25. By using effectoromics screening, we characterized the recognition spectrum of Pep-13/25 in diverse Solanaceae species. Response to Pep-13/25 was predominantly found in potato and related wild tuber-bearing Solanum species. Bulk-segregant RNA sequencing (BSR-Seq) and genetic mapping the response to Pep-25 led to a 0.081 cM region on the top of chromosome 3 in the wild potato species Solanum microdontum subsp. gigantophyllum. Some BAC clones in this region were isolated and sequenced, and we found the Pep-25 receptor locates in a complex receptor-like kinase (RLK) locus. This study is an important step toward the identification of the Pep-13/25 receptor, which can potentially lead to broad application in potato and various other hosts of Phytophthora species.

Genome architecture and tetrasomic inheritance of autotetraploid potato.

Potato (Solanum tuberosum) is the most consumed non-cereal food crop. Most commercial potato cultivars are autotetraploids with highly heterozygous genomes, severely hampering genetic analyses and improvement. By leveraging the state-of-the-art sequencing technologies and polyploid graph binning, we achieved a chromosome-scale, haplotype-resolved genome assembly of a cultivated potato, Cooperation-88 (C88). Intra-haplotype comparative analyses revealed extensive sequence and expression differences in this tetraploid genome. We identified haplotype-specific pericentromeres on chromosomes, suggesting a distinct evolutionary trajectory of potato homologous centromeres. Furthermore, we detected double reduction events that are unevenly distributed on haplotypes in 1021 of 1034 selfing progeny, a feature of autopolyploid inheritance. By distinguishing maternal and paternal haplotype sets in C88, we simulated the origin of heterosis in cultivated tetraploid with a survey of 3110 tetra-allelic loci with deleterious mutations, which were masked in the heterozygous condition by two parents. This study provides insights into the genomic architecture of autopolyploids and will guide their breeding.

Cloning and characterization of r3b; members of the r3 superfamily of late blight resistance genes show sequence and functional divergence.

Massive resistance (R) gene stacking is considered to be one of the most promising approaches to provide durable resistance to potato late blight for both conventional and genetically modified breeding strategies. The R3 complex locus on chromosome XI in potato is an example of natural R gene stacking, because it contains two closely linked R genes (R3a and R3b) with distinct resistance specificities to Phytophthora infestans. Here, we report about the positional cloning of R3b. Both transient and stable transformations of susceptible tobacco and potato plants showed that R3b conferred full resistance to incompatible P. infestans isolates. R3b encodes a coiled-coil nucleotide-binding site leucine-rich repeat protein and exhibits 82% nucleotide identity with R3a located in the same R3 cluster. The R3b gene specifically recognizes Avr3b, a newly identified avirulence factor from P. infestans. R3b does not recognize Avr3a, the corresponding avirulence gene for R3a, showing that, despite their high sequence similarity, R3b and R3a have clearly distinct recognition specificities. In addition to the Rpi-mcd1/Rpi-blb3 locus on chromosome IV, the R3 locus on chromosome XI is the second example of an R-gene cluster with multiple genes recognizing different races of P. infestans.

Functional analysis and expression profiling of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples.

Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (P(MdRbc)) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar 'Gala' was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. 'Santana'.

SolRgene: an online database to explore disease resistance genes in tuber-bearing Solanum species.

BACKGROUND: The cultivated potato (Solanum tuberosum L.) is an important food crop, but highly susceptible to many pathogens. The major threat to potato production is the Irish famine pathogen Phytophthora infestans, which causes the devastating late blight disease. Potato breeding makes use of germplasm from wild relatives (wild germplasm) to introduce resistances into cultivated potato. The Solanum section Petota comprises tuber-bearing species that are potential donors of new disease resistance genes. The aim of this study was to explore Solanum section Petota for resistance genes and generate a widely accessible resource that is useful for studying and implementing disease resistance in potato. DESCRIPTION: The SolRgene database contains data on resistance to P. infestans and presence of R genes and R gene homologues in Solanum section Petota. We have explored Solanum section Petota for resistance to late blight in high throughput disease tests under various laboratory conditions and in field trials. From resistant wild germplasm, segregating populations were generated and assessed for the presence of resistance genes. All these data have been entered into the SolRgene database. To facilitate genetic and resistance gene evolution studies, phylogenetic data of the entire SolRgene collection are included, as well as a tool for generating phylogenetic trees of selected groups of germplasm. Data from resistance gene allele-mining studies are incorporated, which enables detection of R gene homologs in related germplasm. Using these resources, various resistance genes have been detected and some of these have been cloned, whereas others are in the cloning pipeline. All this information is stored in the online SolRgene database, which allows users to query resistance data, sequences, passport data of the accessions, and phylogenic classifications. CONCLUSION: Solanum section Petota forms the basis of the SolRgene database, which contains a collection of resistance data of an unprecedented size and precision. Complemented with R gene sequence data and phylogenetic tools, SolRgene can be considered the primary resource for information on R genes from potato and wild tuber-bearing relatives.

Mapping of the S. demissum late blight resistance gene R8 to a new locus on chromosome IX.

The use of resistant varieties is an important tool in the management of late blight, which threatens potato production worldwide. Clone MaR8 from the Mastenbroek differential set has strong resistance to Phytophthora infestans, the causal agent of late blight. The F1 progeny of a cross between the susceptible cultivar Concurrent and MaR8 were assessed for late blight resistance in field trials inoculated with an incompatible P. infestans isolate. A 1:1 segregation of resistance and susceptibility was observed, indicating that the resistance gene referred to as R8, is present in simplex in the tetraploid MaR8 clone. NBS profiling and successive marker sequence comparison to the potato and tomato genome draft sequences, suggested that the R8 gene is located on the long arm of chromosome IX and not on the short arm of chromosome XI as was suggested previously. Analysis of SSR, CAPS and SCAR markers confirmed that R8 was on the distal end of the long arm of chromosome IX. R gene cluster directed profiling markers CDP(Sw5)4 and CDP(Sw5)5 flanked the R8 gene at the distal end (1 cM). CDP(Tm2)1-1, CDP(Tm2)1-2 and CDP(Tm2)2 flanked the R8 gene on the proximal side (2 cM). An additional co-segregating marker (CDP(Hero)3) was found, which will be useful for marker assisted breeding and map based cloning of R8.