Genetic analysis of resistance of Solanum tuberosum L. to potato wart disease

The obligate biotrophic soil-borne fungus Synchytrium endobioticum is the causal agent of potato wart disease and is classified as a quarantine pest by the European and Mediterranean Plant Protection Organization. It produces sporangia with several hundred motile zoospores which infect meristematic tissue of below-ground parts of the plants, like tubers, stolons and stems of the potato, causing yield losses of up to 50-100 %. Typical symptoms are the formation of cauliflower-like irregular galls on the below-ground parts of the plant. The thick-walled resting sporangia are able to survive in the soil for several decades. More than 40 different pathotypes of S. endobioticum have been described. Pathotypes 1, 2, 6 and 18 are the most common and most aggressive forms of the fungus in Europe. Chemical control is difficult because accessibility of winter sporangia is limited and chemicals cannot penetrate the thick walls of the sporangium. Chemicals reported to be effective against S. endobioticum have also been described as phytotoxic and harmful to the environment. Strict phytosanitary measures and breeding of resistant potato cultivars are currently the methods of choice to control the disease. Another difficulty is the phenotypic resistance assessment of potato cultivars. To determine the resistance approximately 20 tubers have to be inoculated per pathotype and genotype, which are then evaluated microscopically and assigned to different resistance classes, ranging from extremely resistant to extremely susceptible. These amounts of tubers become available only after several years within the breeding process. Molecular markers that could detect resistant genotypes early in the breeding process, independently of the availability of tubers, would greatly facilitate breeding of potato wart resistant cultivars. Several resistance loci have been identified on different potato chromosomes with different resistance loci dependent on the genetic background of the used plant material. One major resistance locus has been identified in almost all genetic studies on wart resistance: the Sen1 locus on chromosome 11. In this study, we generated a dihaploid potato population derived from a resistant tetraploid cultivar by a so-called prickle pollination with a wild potato species, Solanum phureja, which is known to possess high dihaploid induction ability, to analyze resistance against potato wart disease by reducing the genetic complexity implemented when working with tetraploid potato with its tetrasomic inheritance. Using genotyping data of a 12.8 k SNP array we could show that genetic analysis in dihaploids is much easier showing simpler segregation ratios in the progeny. Simultaneously the number of simplex markers is increased when compared to conventional crosses between two tetraploid genotypes. Putative introgressions of the pollinator genome in the dihaploid progeny were present in almost all genotypes and on almost all chromosomes. However, introgressions occurred as single events and did not disturb genetic analysis of the dihaploid genotypes. SNP marker data was used to generate 45 linkage maps, representing almost all of the 48 potato chromosomes. QTL mapping was performed for different phenotypic traits such as shoot length, number of nodes, number of tubers and tuber weight. QTL mapping revealed new quantitative trait loci but also confirmed already known QTLs described in the literature. Tubers of the dihaploid genotypes were tested for resistances to S. endobioticum pathotypes 6 and 18. Qualitative resistance mapping positioned the major resistance locus for both pathotypes on chromosome 11 in the Sen1 region. The development of additional molecular markers further improved the mapping resolution, narrowing the resistance locus to less than 800 kilobase pairs. Eight molecular markers were segregating without recombination to resistance in our population. Two markers showed high diagnostic values in a small association panel, consisting of 50 German and Polish potato varieties. The markers were diagnostic in 89.5 % of the cultivars for resistance to pathotype 18 and in 86.6 % of the cultivars for resistance for pathotype 6. The markers represent the first diagnostic markers for the pathotypes 18 and 6. Sequencing of different pathotypes of S. endobioticum was performed to develop molecular markers to allow differentiation of the pathotypes. However, sequencing revealed only a very low polymorphism rate between the pathotypes. Markers developed in this study, allowed the distinction between pathotypes 1, 2 and 6 and pathotypes 8 and 18

Improved genetic resolution for linkage mapping of resistance to potato wart in monoparental dihaploids with potential diagnostic value in tetraploid potato varieties

Key message: We achieved improved mapping resolution of the major wart resistance locus Xla-TNL containing also Sen1 in a dihaploid population using SNP data and developed additional markers with diagnostic value in tetraploid varieties. Abstract: We analyzed a segregating monoparental dihaploid potato population comprising 215 genotypes derived from a tetraploid variety that is highly resistant to Synchytrium endobioticum pathotypes 18 and 6. The clear bimodal segregation for both pathotypes indicated that a major dominant resistance factor in a simplex allele configuration was present in the tetraploid donor genotype. Compared to that in previous analyses of the same tetraploid donor in conventional crosses with susceptible tetraploid genotypes, a segregation pattern with a reduced genetic complexity of resistance in dihaploids was observed here. Using the 12.8 k SolCAP SNP array, we mapped a resistance locus to the Xla-TNL region containing also Sen1 on potato chromosome 11. The improved mapping resolution provided by the monoparental dihaploids allowed for the localization of the genes responsible for the resistance to both pathotypes in an interval spanning less than 800 kbp on the reference genome. Furthermore, we identified eight molecular markers segregating without recombination to pathotype 18 and pathotype 6 resistance. Also, two developed markers display improved diagnostic properties in an independent panel of tetraploid varieties. Overall, our data provide the highest resolution mapping of wart resistance genes at the Xla-TNL locus thus far

Maximization of Markers Linked in Coupling for Tetraploid Potatoes via Monoparental Haploids

Haploid potato populations derived from a single tetraploid donor constitute an efficient strategy to analyze markers segregating from a single donor genotype. Analysis of marker segregation in populations derived from crosses between polysomic tetraploids is complicated by a maximum of eight segregating alleles, multiple dosages of the markers and problems related to linkage analysis of marker segregation in repulsion. Here, we present data on two monoparental haploid populations generated by prickle pollination of two tetraploid cultivars with Solanum phureja and genotyped with the 12.8 k SolCAP single nucleotide polymorphism (SNP) array. We show that in a population of monoparental haploids, the number of biallelic SNP markers segregating in linkage to loci from the tetraploid donor genotype is much larger than in putative crosses of this genotype to a diverse selection of 125 tetraploid cultivars. Although this strategy is more laborious than conventional breeding, the generation of haploid progeny for efficient marker analysis is straightforward if morphological markers and flow cytometry are utilized to select true haploid progeny. The level of introgressed fragments from S. phureja, the haploid inducer, is very low, supporting its suitability for genetic analysis. Mapping with single-dose markers allowed the analysis of quantitative trait loci (QTL) for four phenotypic traits

Genomic and transcriptomic resources for marker development in synchytrium endobioticum, an elusive but severe potato pathogen

Synchytrium endobioticum is an obligate biotrophic fungus that causes wart diseases in potato. Like other species of the class Chytridiomycetes, it does not form mycelia and its zoospores are small, approximately 3 μm in diameter, which complicates the detection of early stages of infection. Furthermore, potato wart disease is difficult to control because belowground organs are infected and resting spores of the fungus are extremely durable. Thus, S. endobioticum is classified as a quarantine organism. More than 40 S. endobioticum pathotypes have been reported, of which pathotypes 1(D1), 2(G1), 6(O1), 8(F1), and 18(T1) are the most important in Germany. No molecular methods for the differentiation of pathotypes are available to date. In this work, we sequenced both genomic DNA and cDNA of the German pathotype 18(T1) from infected potato tissue and generated 5,422 expressed sequence tags (EST) and 423 genomic contigs. Comparative sequencing of 33 genes, single-stranded confirmation polymorphism (SSCP) analysis with polymerase chain reaction fragments of 27 additional genes, as well as the analysis of 41 simple sequence repeat (SSR) loci revealed extremely low levels of variation among five German pathotypes. From these markers, one sequence-characterized amplified region marker and five SSR markers revealed polymorphisms among the German pathotypes and an extended set of 11 additional European isolates. Pathotypes 8(F1) and 18(T1) displayed discrete polymorphisms which allow their differentiation from other pathotypes. Overall, using the information of the six markers, the 16 isolates could be differentiated into three distinct genotype groups. In addition to the presented markers, the new collection of EST from genus Synchytrium might serve in the future for molecular taxonomic studies as well as for analyses of the host-pathogen interactions in this difficult pathosystem. © 2017 The American Phytopathological Society.Federal Ministry of Food and Agricultur

Data_Sheet_1_Maximization of Markers Linked in Coupling for Tetraploid Potatoes via Monoparental Haploids.DOCX

<p>Haploid potato populations derived from a single tetraploid donor constitute an efficient strategy to analyze markers segregating from a single donor genotype. Analysis of marker segregation in populations derived from crosses between polysomic tetraploids is complicated by a maximum of eight segregating alleles, multiple dosages of the markers and problems related to linkage analysis of marker segregation in repulsion. Here, we present data on two monoparental haploid populations generated by prickle pollination of two tetraploid cultivars with Solanum phureja and genotyped with the 12.8 k SolCAP single nucleotide polymorphism (SNP) array. We show that in a population of monoparental haploids, the number of biallelic SNP markers segregating in linkage to loci from the tetraploid donor genotype is much larger than in putative crosses of this genotype to a diverse selection of 125 tetraploid cultivars. Although this strategy is more laborious than conventional breeding, the generation of haploid progeny for efficient marker analysis is straightforward if morphological markers and flow cytometry are utilized to select true haploid progeny. The level of introgressed fragments from S. phureja, the haploid inducer, is very low, supporting its suitability for genetic analysis. Mapping with single-dose markers allowed the analysis of quantitative trait loci (QTL) for four phenotypic traits.</p