Potato genome editing using the CRISPR/Cas9 system to knock out the StDMR6-1 and StCHL1 genes involved in late blight resistance

Improving potato late blight (LB) resistance is essential for ensuring food security, particularly using wild relatives endemic to Phytophthora infestans‐prone regions. However, reproductive barriers can impede the transfer from potato wild relatives (PWR) to the cultivated genepool. This study sought to incorporate potentially novel LB resistance from wild diploid accessions of Series Piurana (Solanum chiquidenum, Solanum paucissectum, and Solanum piurae), Tuberosa (Solanum cajamarquense), and Megistracroloba (Solanum sogarandinum) into tetraploid potato. Trough rescue pollination, 699 diploid interspecific hybrids were obtained, of which 385 displayed LB resistance in two endemic environments. Based on a comprehensive evaluation, including assessing 2n pollen production, 14 diploid interspecific hybrids were selected to continue the introgression process. These pre‐bred stocks were then used in interploidy (4x‐2x) crosses to incorporate their LB resistance in the cultivated tetraploid background. Assessment of 1978 genotypes resulting from interploidy crosses under endemic disease pressure yielded 717 hybrids with moderate to high levels of LB resistance. Evaluation of pollen viability and ploidy revealed moderate fertility and predominantly tetraploid genotypes. Reassessment of LB resistance on this new subset of hybrids further identified 214 genotypes with sustained resistance. Among them, 12 tetraploid hybrids with low glycoalkaloid content, favorable agronomic, and post‐harvest attributes were identified as crossing‐friendly stocks. Notably, 11 of these hybrids were derived from S. cajamarquense and one from S. sogarandinum. These promising 4x hybrids are now primed to be incorporated into potato breeding programs.

Background. The harmful organisms affecting potato cause great economic damage in all areas where the crop is grown. One of the most economically important pathogens is the potato late blight (agent Phytophthora infestans Mont. (de Bary)). Also, Potato Virus Y (PVY) and potato cyst nematode (PCN) of the Ro1 pathotype, the only one that occurs in the Russian Federation, cause significant yield losses. Materials and methods. Phytopathological and molecular screening was carried out on original interspecific potato hybrids obtained in 11 crossing combinations, seven of which were studied for the first time. Hybrids resulted from crosses that involved Mexican and South American potato species as sources of late blight and PVY resistance. Field observations of late blight resistance were conducted during three seasons under high infection pressure. A part of plant material was screened in laboratory tests. PCR tests employed 9 DNA markers of genes associated with resistance to Ph. infestans, PVY and PCN of Ro1 pathotype used in our previous study. Results. In field evaluation, hybrid clones mostly expressed high, and a part of them moderate late blight resistance. Of the 6 markers of the used Rpi genes, at least two were detected in most clones, while some had 3 or 4 markers. Some clones were found to have markers of PCN resistance genes and of several genes for resistance to PVY. The cytoplasm type was determined for all plant material. Conclutions. Within a small but pedigree-diverse set of interspeсific hybrids, the markers of resistance genes to all three pests were found. A part of clones obtained by using the sources of PVY and late blight resistance which we had identified, were found to contain two or more resistance genes to these pathogens. According to the results of molecular tests, the clones were found to carry several markers of late blight and PVY resistance genes. Original interspecific potato hybrids with determined late blight resistance levels, cytoplasm type and availability of markers of genes for resistance to three pests can be used in further hybridization.

Wild potato species contain many traits of economic importance. Late blight (LB) resistance and cold chipping are traits desired in potato cultivars. These traits could be co-currently introgressed if they occurred together in wild potato species. Our research objectives were (1) to determine if variation for cold chipping exists between potato species, accessions within species, and plants within accessions all having foliar LB resistance, and (2) to identify wild potato genotypes combining LB resistance and cold chipping. Materials include 665 genotypes from 43 LB-resistant accessions of 12 potato species having Endosperm Balance Numbers (EBN) of 1, 2, and 4, and 59 LB-resistant genotypes retained from these accessions for breeding. Potato chips were made from greenhouse-grown tubers following storage at 4 C for 6 months. Chip color was scored 1–10, ≤ 4 is acceptable by industry standards. Most of the variation for chip color was due to differences between species. Species ranged in the percentage of acceptably chipping genotypes (0% – 67%) with nine of 12 species having cold-chipping genotypes. Appreciable variation was present within accessions as well. The best chipping accessions wereS. verrucosum plant introduction (PI) 161173 – 4.33 / 0.67 (mean / proportion acceptable genotypes),S. stoloniferum PI 250510 -4.36 / 0.64,S. pinnatisectum PI 347766 -4.65 / 0.35 and 275233 -4.73 / 0.44, andS. megistacrolobum PI 195210 -5.14 / 0.29. Eleven 1EBN genotypes fromS. pinnatisectum andS. trifidum and five 2EBN genotypes fromS. verrucosum, S. fendleri,S. stoloniferum, andS. microdontum were identified that combined LB resistance and cold chipping. Co-current introgression would require fewer breeding cycles than other breeding methods to identify hybrid genotypes possessing both traits.

Potato production in developing countries is expanding to warmer environments as farmers search for income opportunities and food security. Meanwhile, climate change is already affecting weather patterns in traditional potato-growing areas, where unpredictable rains and pressure from pests and disease are increasing farmers' risk. Since 2004, the International Potato Center (CIP) has sought to develop new, more heat-tolerant generations of its tropical highland-adapted late-blight resistant population. Late-blight resistant parents were crossed with early maturing and virus resistant progenitors, and selection practised under warm temperatures, water deficit and mid-latitude conditions. During the 2005-2006 summer season (January-March) 20,000 genotypes were exposed to heat in a screenhouse at CIP's experimental station in San Ramon, a warm rain forest environment at 800 m above sea level (masl) and latitude 11° 08′ S. Selected clones were assessed in the field in the same location, where average night and day temperatures during tuberization were 21°C and 27°C, respectively; the resulting heat tolerant clones were exposed to high, endemic late blight pressure in Oxapampa (mid-elevation humid tropics) in replicated trials conducted over 4 years. Selected heat tolerant, late-blight resistant clones were evaluated for yield components in the spring-summer season in La Molina (12° 05° S) and drought sensitivity in Majes (16° 28° S) along the arid coast of Peru. Sixty-one advanced clones were assessed again for yield in San Ramon, La Molina and Majes and yield stability analysed. All trials were conducted in a simple lattice design, using Désirée as a heat tolerant control. Harvesting was performed at 90 days. The 61 clones were screened for resistance to potato virus X (PVX) and potato virus Y (PVY), by mechanical inoculation and grafting under greenhouse conditions. Analysis of variance for marketable tuber yield showed significant differences among clones. In San Ramon, yields were in the range of 16.01-28.43 (t/ha), significantly exceeding the control. Of the 40 mid-maturing elite clones selected from the new 'LBHT' (late blight resistant heat tolerant) population, with resistance to late blight, tolerance to heat, 11 carry extreme resistance to PVY, 25 to PVX and seven show tolerance to drought. Eleven mid-maturing clones with heat tolerance, resistance to late blight and PVY that are suitable for mid-elevation zones and climate change, and all 40 clones, are available for variety development and further use in breeding.

In recent years, skin blemish diseases of potato (including black dot (BD) caused by Colletotricum coccodes) have begun to be important for global potato marketing, since consumers often reject tubers with an imperfect appearance, which results in financial losses caused by the disposal of unwanted potatoes. Like for many non-fatal plant diseases, BD severity may depend on the immune status of plants influenced by other infectious agents. Using a set of 98 potato cultivars differing in their late blight (LB) resistance, we examined the correlation between the intensity of their infection with LB determined by their LB resistance and the occurrence of the BD disease under field conditions with a high background level of both diseases. Using LB-susceptible (Arizona) and moderately susceptible (Sante) cultivars, we also evaluated the effect of a crop protection against LB on BD development under the same field conditions. A strong negative correlation (r = −0.81, p < 0.05) between the LB resistance and the BD occurrence has been revealed. An experiment using the two cultivars, chemically protected against LB, showed a significant reduction in BD occurrence of 30% (cv. Arizona) and 20% (cv. Sante) compared to the untreated controls; the total yield and marketability of potatoes increased by 103.6 and 62.5% for cv. Arizona and by 65.9 and 43.8% for cv. Sante. The reduction in the LB affection of potato is one of the key factors improving the immune status of potato cultivars in relation to BD infection, so methods of LB protection should be included in a complex approach to BD control.

Late blight and early blight, caused by Phytophthora infestans and Alternaria solani, respectively, are the two most widely occurring foliar diseases of potato in the U.S.A. Resistance to both diseases is necessary if growers are to reduce fungicide applications. Field resistance to late blight has previously been reported in an accession of Solanum hougasii (2n = 72). The putative aneuploid clone E53.61, derived from (S. hougasii x S. tuberosum) x S. tuberosum was obtained from C.R. Brown and crossed with three S. tuberosum clones. Thirty-five hybrid clones were evaluated for foliar late blight resistance at the Russell E. Larson Agricultural Research Center near State College, PA along with the susceptible check ‘Atlantic’ and for foliar early blight resistance in Presque Isle, ME along with the susceptible check ‘Harley Blackwell’ for 3 years (2012 to 2014). The experimental design was a randomized complete block design with two to three replications each year. The US-23 genotype of P. infestans occurred naturally and/or was used in inoculations in PA and plants were infected naturally with A. solani in ME. Relative area under the disease progress curve (RAUDPC) values were calculated based on visual assessment of foliar disease four to five times late in the season each year and subjected to statistical and stability analyses. There were significant differences among clones and the clone x environment interaction was significant for both diseases. Of the 35 hybrid clones evaluated for late blight, 16 were more resistant, 7 were more susceptible, and 12 were as susceptible as ‘Atlantic’. Of those same hybrid clones evaluated for early blight, 23 were more resistant than ‘Harley Blackwell’; the rest were as susceptible. Late blight resistance or susceptibility was independent of the stability of resistance, however, early blight resistance was associated with greater stability. Fourteen clones were more resistant than the check varieties for both late blight and early blight, suggesting that resistance genes for both late blight and early blight have been combined in this genetic material; three of these clones also had high specific gravity and acceptable chip color out of 10 °C storage.

New races of Phytophthora infestans rapidly defeat potato late blight (LB) resistance based on Solanum demissum germplasm, and breeders search for new sources of durable LB resistance. We developed and verified six sequence characterized amplified region markers recognizing the race-specific genes R1 and R3 of S. demissum and the broad-spectrum resistance gene RB of S. bulbocastanum and the germplasms of these species and used them to screen 209 accessions of 21 wild Solanum species. In addition to S. demissum, homologues of R1 and R3 were found in several species of series Demissa,Longipedicellata and diploid Tuberosa; R3 homologues were also detected in S. bulbocastanum,S. cardiophyllum and S. ehrenbergii. The RB homologues were found in a wider range of Solanum species. The markers of R1 and R3 genes reliably discerned between germplasms of S. tuberosum ssp. tuberosum and wild sources of LB resistance. Following introgression, the species-specific markers of demissum and bulbocastanum germplasm were rapidly lost, whereas the markers of R1 and R3 genes lasted through several meiotic generations and were maintained at high frequencies in modern potato cultivars. The presence of these markers in demissoid potato cultivars was significantly associated with LB resistance, presuming that both genes contribute to overall defence response.

Late blight, caused by the oomycete Phytophthora infestans (Mont) de Bary, is the most important disease of cultivated potato (Solanum tuberosum L.). An efficient long-term strategy for controlling late blight infection involves integration of host plant resistance to disease management programs. In this study, a diploid potato population segregating for late blight resistance was generated from an interspecific cross (MSX902) between S. tuberosum x S. chacoense hybrid (84SD22) and a late blight resistant Solanum berthaultii hybrid (Ber83). A total of 129 progeny and two parents were tested for foliar resistance against the US-23 P. infestans genotype using detached-leaf bioassays and inoculated field trials (2014 and 2015). Distribution of foliar late blight resistance and susceptibility was bimodal in both types of phenotype evaluation, suggesting a major R-gene may be associated with the resistance. A major QTL for late blight resistance on chromosome 10 was detected using both detached leaf bioassay and field trial data. The SNP marker solcap_snp_c1_15219 (26.5 cM) was at the peak of the QTL. Additionally, a minor QTL on chromosomes 5 was linked to SNPs solcap_snp_c1_3793. These SNP markers closely linked to the late blight resistance QTL can be used for marker-assisted breeding.

The potato originated in southern Peru and north-western Bolivia (South America). However, native accessions have also been cultivated in India for many years. Late blight, caused by the fungus Phytophthora infestans, is the most devastating potato disease, while potato cyst nematode (Globodera spp.) (PCN) is another economically significant quarantine-requiring pest in India. In this study, we have generated a new Indian native collection of 94 potato accessions collected from different parts India. These accessions were screened against late blight and potato cyst nematode resistance by using gene-based molecular markers and phenotypic screening methods. Marker assisted selection using R1 gene-specific marker CosA210 revealed a late blight resistance gene in 11 accessions. PCN resistance bands were found in 3 accessions with marker TG689141, 5 accessions with marker 57R452, and 1 accession having Gro1-4-1602 marker for G. rostochiensis (Ro1,4), while 64 accessions amplified marker HC276 indicating G. pallida (Pa2,3) resistance gene (GpaVvrn QTL). On the other hand, phenotypic screening against late blight resistance under natural epiphytic conditions (hot-spot) revealed three accessions with high resistance, while others were resistant (1 accession), moderately resistant (5 accessions), susceptible (29 accessions), and highly susceptible (56 accessions). For G. rostochiensis (golden cyst nematode) and G. pallida (white cyst nematode) resistance, accessions were grouped into highly resistant (3, 3), resistant (0, 2), moderately resistant (6, 29), susceptible (32, 30), and highly susceptible (53, 30), respectively, for the two PCN species. Collectively, we identified promising accessions with high resistance to late blight (JG-1, Kanpuria Safed, and Rangpuria), and also highly resistant to both Globodera species (Garlentic, Jeevan Jyoti, and JG-1). Our findings suggested that these accessions would be useful for late blight and PCN resistance breeding, as well as future molecular studies in potatoes.

Keywords: Earliness, Foliage maturity type, Late blight, Phytophthora infestans , Potato, QTL analysis, Resistance, Solanum tuberosumPotato ( Solanum tuberosum ) is grown throughout the world and is among the most important crops for global food supply. Late blight, caused by Phytophthora infestans , is the most important and destructive disease wherever potato is grown. Potato varieties with resistance to late blight can be used to contest the disease. The application of race-specific resistance has turned out not to be durable, because P. infestans is able to overcome this type of resistance. Race-non-specific resistance is expected to be more durable but, unfortunately, this type of resistance against P. infestans is consistently associated with late foliage maturity. The purpose of the research described in this thesis was to unravel the nature of the association between race-non-specific resistance to late blight and foliage maturity type in potato. The main goal was to determine whether the association between the two traits is genetic or physiological. As part of the physiological studies, single-node cuttings of potato were tested for their potential to serve as a model to predict foliage maturity type at any time during the cropping season. Single-node cuttings did not reflect the stage of tuber development or tuber induction of the plants from which they were taken, and they do not appear to reflect the physiological state of the whole plant adequately. Consequently, single-node cuttings cannot be used to predict foliage maturity type.A possible physiological association between late blight resistance and foliage maturity type was studied by relating the effects of plant age, leaf age, and leaf position on resistance. Leaf position had the largest and most significant effect: apical leaves were far more resistant to late blight than basal leaves. Plant age and leaf age had only minor effects. Thus, the resistance of a specific leaf remained about the same during its entire lifetime.Genetic studies were performed with seven different diploid potato progenies. Recombinant genotypes with early foliage maturity and resistance against P. infestans were not identified. Therefore, it cannot be concluded that the association between late blight resistance and foliage maturity type is due to closely linked genes. QTL analyses revealed loci for resistance to late blight on chromosomes 3, 5, and 10, and for foliage maturity type on chromosome 5. The locus for foliage maturity type could not be distinguished from the most important QTL for resistance: the allele of molecular marker GP21 that is associated with late blight resistance is also associated with late foliage maturity.

Late blight (LB) caused by the oomycete Phytophthora infestans, is a major disease of potato and tomato worldwide and can cause up to 100% yield losses. The devastating economic impact of this disease intensified the related pathology and genetics research since the occurrence of Irish famine in 1840s, with a side gain of major scientific discoveries. For example, many of the crucial steps involved in LB defense response in host plants have been elucidated through the use of modern cytological and molecular biology techniques. Also, genetic and biochemical studies have revealed differences between oomycetes and pathogenic fungi, which has led to more selective use of chemicals for LB control. Furthermore, the discovery of P. infestans two mating types and the resultant generation of more aggressive lineages by sexual recombination stresses the need for an integrated and sustainable approach to LB control. These measures would include the use of cultural practices, selective fungicide applications, and genetic resistance. In potato at least a dozen major resistance genes and several quantitative trait loci (QTLs) for LB resistance have been identified, and most modern cultivars have been bred with one or more resistance genes. In tomato, though most commercial cultivars are susceptible to LB, a few major resistance genes and several QTLs have been identified and several breeding programs around the world are developing breeding lines and commercial cultivars with LB resistance. Most recently, a few fresh-market tomato hybrid cultivars with LB resistance were released by the North Carolina State University Tomato Breeding Program in the United States. There is, however, an insufficient number of potato and tomato cultivars with LB resistance, resulting in continued expensive as well as the hazardous and increasingly ineffective use of chemicals for disease control. In an era when both host plants and P. infestans genomes are sequenced and considerable genomic information is available, it is not unexpected that a more sustainable solution to controlling LB is on the horizon. In this review, we summarize the recent achievements in better understanding of the P. infestans pathogenesis, host-pathogen interactions, and the progress made in developing genetic resistance in potato and tomato.

Late blight (LB), caused by the oomycete pathogen Phytophthora infestans, is a devastating disease of potato that is necessary to control by regularly treatment with fungicides. Silicon (Si) has been used to enhance plant resistance against a broad range of bacterial and fungal pathogens; however, the enhanced LB resistance and the molecular mechanisms involving the plant hormone pathways remain unclear. In this study, Si treatment of potato plants was found to enhance LB resistance in both detached leaves and living plants accompanied by induction of reactive oxygen species (ROS) production and pathogenesis-related genes expression. Regarding the hormone pathways involved in Si-mediated LB resistance, we found a rapidly increased content of ethylene (ET) 15 min after spraying with Si. Increased jasmonic acid (JA) and JA-Ile and decreased salicylic acid (SA) were identified in plants at 1 day after spraying with Si and an additional 1 day after P. infestans EC1 infection. Furthermore, pretreatment with Me-JA enhanced resistance to EC1, while pretreatment with DIECA, an inhibitor of JA synthesis, enhanced the susceptibility and attenuated the Si-mediated resistance to LB. Consistent with these hormonal alterations, Si-mediated LB resistance was significantly attenuated in StETR1-, StEIN2-, StAOS-, StOPR3-, StNPR1-, and StHSP90-repressed plants but not in StCOI1- and StSID2-repressed plants using virus-induced gene silencing (VIGS). The Si-mediated accumulation of JA/JA-Ile was significantly attenuated in StETR1-, StEIN2-, StOPR3- and StHSP90-VIGS plants but not in StCOI1-, StSID2- and StNPR1-VIGS plants. Overall, we reveal that Si can be used as a putative alternative to fungicides to control LB, and conclude that Si-mediated LB resistance is dependent on the ET/JA-signaling pathways in a StHSP90- and StNPR1-dependent manner.

Potato late blight, caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary, is the most disastrous disease on potato worldwide and also the greatest threat to potato production in China. Loss of yield and quality, and the costs of chemical control of potato late blight account for multi-billion US$ annually. Using host resistance is thought to be an economical and efficient approach to control potato late blight. To combat the disease breeders have introduced late blight resistance (R) genes from various Solanum species into the cultivated potato. The proteins encoded by these R genes can recognise specific races of the pathogen. This then triggers a cascade of defence responses ultimately leading to a localized programmed cell death called the hypersensitive response (HR) that arrests growth of the pathogen. However, once the resistant potato cultivars are released into the field, the resistance based on these genes is quickly overcome due to rapid evolution and adaption of P. infestans. In the potato-P. infestans interaction race-specific recognition by R genes is based on the 'gene-for-gene' model which predicts that resistance is governed by the (direct or indirect) interaction of an R protein with its corresponding effector, the product of an avirulence (Avr) gene. If either the R gene or the Avr gene is absent or non-functional the interaction is compatible and the host susceptible for disease. Key to a better understanding of the molecular basis of resistance in the potato-P. infestans pathosystem is the unravelling of R protein-effector interactions and, hence, cloning of more R and Avr genes is a prerequisite to study these interactions. This thesis describes the mapping and cloning of Avr genes in P. infestans, and the phenotypic and genotypic diversity in P. infestans field isolates in Northern China. To isolate P. infestans Avr genes a positional cloning strategy was adopted. Chapter 2 presents a molecular-genetic linkage map of P. infestans that was constructed based on Single Nucleotide Polymorphism (SNP) markers and Amplified Fragment Length Polymorphism (AFLP) markers. The map was generated using a mapping population of 83 F1 progeny derived from two Dutch field isolates, NL80029 and NL88133. Of 631 markers (398 SNP and 233 AFLP markers) that segregated in this population, 534 markers were positioned on 19 linkage groups spanning a total of 1144 cM and an average distance of 2.14 cM between adjacent markers. Fourteen of the linkage groups are major linkage groups that contain markers from both parents. The others are minor linkage groups with markers of only one of the two parents. In parallel, a transcriptional profiling strategy was adopted to identify avirulence-associated transcripts (Chapter 3). cDNA-AFLP was used for comparing transcripts in P. infestans isolates with different virulence phenotypes. A large number of avirulence-associated TDFs (Transcript Derived Fragments) was cloned and sequenced, and EST and genome databases were mined to generate more sequence data. To identify promising candidates, bioinformatic predictions such as the presence of signal peptides, number of cysteine residues and putative virulence functions were used as important selection criteria. Four TDFs associated with Avr loci were identified, two for Avr4 and two for the Avr3b-Avr10-Avr11 locus. Chapter 4 describes how a combined approach of genetic mapping, transcriptional profiling and BAC marker landing resulted in isolation of the P. infestans avirulence gene Avr4. PiAvr4 encodes a 287 amino acid protein that belongs to a superfamily of effectors sharing the putative host cell targeting motif RXLR-dEER. For the functional characterization P. infestans race 4 strains were transformed with PiAvr4. This resulted in transformants that were avirulent on R4 potato plants, demonstrating that PiAvr4 is responsible for eliciting R4-mediated resistance. Expression of PiAvr4 in R4 plants using PVX-agroinfection and agroinfiltration showed that PiAvr4 itself is the effector that elicits HR on R4 plants. On potato plants lacking R4, like Bintje, there was no response. The presence of the RXLR-dEER motif suggested intracellular recognition of PiAvr4 but nevertheless a hypersensitive response was observed when PiAvr4 was targeted to the outside of the cell. Deletion of the RXLR-dEER domain neither stimulated nor prevented elicitor activity of PiAvr4. Race 4 strains have frame shift mutations in the PiAvr4 gene that result in short truncated peptides, indicating that PiAvr4 is not crucial for virulence. Chapter 5 describes Avr1-associated markers that resulted from genetic mapping, transcriptional profiling and BAC-end sequences. In silico landing of these markers on the P. infestans genome sequence narrows down a 800 kb genomic interval that carries seven genes that have the hallmarks of an oomycete Avr gene. They all encode a secreted protein with a conserved RXLR-dEER domain at the N-terminus and a divergent C-terminal region. Each of these seven could be a candidate for Avr1. The seven RXLR effector genes were further characterized by bioinformatic analyses such as HMM score of the RXLR motif, and prediction of the presence of W, Y, and L motifs in the C-terminal region. Cloning and functional analyses using transient expression assays in plants carrying the resistance gene R1 should reveal whether any of the seven candidates is Avr1. Chapter 6 describes the phenotypic and genotypic diversity of P. infestans isolates collected in Northern China between 1997 and 2003, especially in Inner Mongolia. Characterization included mating type, virulence, mitochondrial DNA (mtDNA) haplotype and DNA fingerprinting patterns based on simple sequence repeats (SSR) and amplified fragment length polymorphism (AFLP). All isolates had the A1 mating type, mtDNA haplotype IIa and an identical SSR genotype (designated as SG-01-01) that differed from the SSR genotypes found in the reference isolates, including the ones representing the 'old' US-1 lineage that dominated the worldwide P. infestans population prior to 1980. In contrast, the virulence spectra differed significantly and virulence to all R genes present in the standard differential set (R1 to R11) was found. AFLP analysis revealed some diversity; eight different AFLP genotypes were found that could be grouped into two major clusters. This study shows that there is very little genotypic diversity in the P. infestans population in Northern China. The occurrence of many different races within this uniform population is discussed in the framework of recently gained insights in the molecular determinants of avirulence in P. infestans and their role in the 'gene-for-gene' interaction with potato. Finally, in Chapter 7, the implications of the findings described in this thesis are discussed with specific emphasis on Avr gene cloning, RXLR-dEER effectors, virulence diversity and durable late blight resistance. By combining various cloning strategies it becomes feasible to speed up the cloning of putative P. infestans Avr genes. Moreover, the use of high throughput effector genomics screenings will allow the identification of the corresponding R genes. The high virulence diversity that is found in P. infestans field isolates, even within one clonal lineage, might be correlated to the observation that RXLR-dEER effector genes are the most rapidly evolving genes in the genome of P. infestans. Therefore, generating potato cultivars with durable resistance to late blight seems more challenging than anticipated.

Late blight (LB) caused by the oomycete Phytophthora infestans (Mont.) de Bary is the greatest threat to potato production worldwide. Current potato breeding for LB resistance heavily depends on the introduction of new genes for resistance to P. infestans (Rpi genes). Such genes have been discovered in highly diverse wild, primitive, and cultivated species of tuber-bearing potatoes (Solanum L. section Petota Dumort.) and introgressed into the elite potato cultivars by hybridization and transgenic complementation. Unfortunately, even the most resistant potato varieties have been overcome by LB due to the arrival of new pathogen strains and their rapid evolution. Therefore, novel sources for germplasm enhancement comprising the broad-spectrum Rpi genes are in high demand with breeders who aim to provide durable LB resistance. The Genbank of the N.I. Vavilov Institute of Plant Genetic Resources (VIR) in St. Petersburg harbors one of the world’s largest collections of potato and potato relatives. In this study, LB resistance was evaluated in a core selection representing 20 species of seven Petota series according to the Hawkes (1990) classification: Bulbocastana (Rydb.) Hawkes, Demissa Buk., Longipedicellata Buk., Maglia Bitt., Pinnatisecta (Rydb.) Hawkes, Tuberosa (Rydb.) Hawkes (wild and cultivated species), and Yungasensa Corr. LB resistance was assessed in 96 accessions representing 18 species in the laboratory test with detached leaves using a highly virulent and aggressive isolate of P. infestans. The Petota species notably differed in their LB resistance: S. bulbocastanum Dun., S. demissum Lindl., S. cardiophyllum Lindl., and S. berthaultii Hawkes stood out at a high frequency of resistant accessions (7–9 points on a 9-point scale). Well-established specific SCAR markers of ten Rpi genes—Rpi-R1, Rpi-R2/Rpi-blb3, Rpi-R3a, Rpi-R3b, Rpi-R8, Rpi-blb1/Rpi-sto1, Rpi-blb2, and Rpi-vnt1—were used to mine 117 accessions representing 20 species from seven Petota series. In particular, our evidence confirmed the diverse Rpi gene location in two American continents. The structural homologs of the Rpi-R2, Rpi-R3a, Rpi-R3b, and Rpi-R8 genes were found in the North American species other than S. demissum, the species that was the original source of these genes for early potato breeding, and in some cases, in the South American Tuberosa species. The Rpi-blb1/Rpi-sto1 orthologs from S. bulbocastanum and S. stoloniferum Schlechtd et Bché were restricted to genome B in the Mesoamerican series Bulbocastana, Pinnatisecta, and Longipedicellata. The structural homologs of the Rpi-vnt1 gene that were initially identified in the South American species S. venturii Hawkes and Hjert. were reported, for the first time, in the North American series of Petota species.

BackgroundPhytophthora 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.ResultsWe 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.ConclusionsWe 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.