Josef Strahwald

Tagging Quantitative Trait Loci for Maturity-Corrected Late Blight Resistance in Tetraploid Potato with PCR-Based Candidate Gene Markers

Late blight caused by the oomycete Phytophthora infestans is the economically most important and destructive disease in potato cultivation. Quantitative resistance to late blight available in tetraploid cultivars is correlated with late maturity in temperate climates, which is an undesirable characteristic. A total of 30 DNA-based markers known to be linked to loci for pathogen resistance in diploid potato were selected and tested as polymerase chain reaction-based markers for linkage with quantitative trait loci (QTL) for late blight resistance and plant maturity in two half-sib families of tetraploid potatoes. Most markers originated from within or were physically closely linked to candidate genes for quantitative resistance factors. The families were repeatedly evaluated in the field for quantitative resistance to late blight and maturity. Resistance was corrected for the maturity effect. Nine of eleven different map segments tagged by the markers harbored QTL affecting maturity-corrected resistance. Interactions were found between unlinked resistance QTL, providing testable strategies for marker-assisted selection in tetraploid potato. Based on the linkage observed between QTL for resistance and plant maturity and based on the genetic interactions observed between candidate genes tagging resistance QTL, we discuss models for the molecular basis of quantitative resistance and maturity.

Single Nucleotide Polymorphisms in the Allene Oxide Synthase 2 Gene Are Associated With Field Resistance to Late Blight in Populations of Tetraploid Potato Cultivars

The oomycete Phytophthora infestans causes late blight, the most relevant disease of potato (Solanum tuberosum) worldwide. Field resistance to late blight is a complex trait. When potatoes are cultivated under long day conditions in temperate climates, this resistance is correlated with late plant maturity, an undesirable characteristic. Identification of natural gene variation underlying late blight resistance not compromised by late maturity will facilitate the selection of resistant cultivars and give new insight in the mechanisms controlling quantitative pathogen resistance. We tested 24 candidate loci for association with field resistance to late blight and plant maturity in a population of 184 tetraploid potato individuals. The individuals were genotyped for 230 single nucleotide polymorphisms (SNPs) and 166 microsatellite alleles. For association analysis we used a mixed model, taking into account population structure, kinship, allele substitution and interaction effects of the marker alleles at a locus with four allele doses. Nine SNPs were associated with maturity corrected resistance (P < 0.001), which collectively explained 50% of the genetic variance of this trait. A major association was found at the StAOS2 locus encoding allene oxide synthase 2, a key enzyme in the biosynthesis of jasmonates, plant hormones that function in defense signaling. This finding supports StAOS2 as being one of the factors controlling natural variation of pathogen resistance.

Single nucleotide polymorphism (SNP) genotyping as basis for developing a PCR-based marker highly diagnostic for potato varieties with high resistance to Globodera pallida pathotype Pa2/3

Globodera pallida is a parasitic root cyst nematode of potato, which causes reduction of crop yield and quality in infested fields. Field populations of G. pallida containing mixtures of pathotypes Pa2 and Pa3 (Pa2/3) are currently most relevant for potato cultivation in middle Europe. Genes for resistance to G. pallida have been introgressed into the cultivated potato gene pool from the wild, tuber bearing Solanum species S. spegazzinii and S. vernei. Selection of resistant genotypes in breeding programs is hampered by the fact that the phenotypic evaluation of resistance to G. pallida is time consuming, costly and often ambiguous. DNA-based markers diagnostic for resistance to G. pallida would facilitate the development of resistant varieties. A tetraploid F1 hybrid family SR-Gpa segregating for quantitative resistance to G.␣pallida was developed and evaluated for resistance to G. pallida population ‘Chavornay’. Two subpopulations of 30 highly resistant and 30 susceptible individuals were selected and genotyped for 96 single nucleotide polymorphism (SNP) markers tagging 12 genomic regions on 10 potato chromosomes. Seven SNPs were found significantly linked to the nematode resistance, which were all located within a resistance ‘hotspot’ on potato chromosome V. A haplotype model for these seven SNPs was deduced from the SNP patterns observed in the SR-Gpa family. A PCR assay ‘HC’ was developed, which specifically detected the SNP haplotype c that was linked with high levels of nematode resistance. The HC marker was only found in accessions of S.␣vernei. Screening with the HC marker 34 potato varieties resistant to G. pallida pathotypes Pa2 and/or Pa3 and 22 susceptible varieties demonstrated that the HC marker was highly diagnostic for presence of high levels of resistance to G. pallida pathotype Pa2/Pa3.

Association genetics in Solanum tuberosum provides new insights into potato tuber bruising and enzymatic tissue discoloration

BackgroundMost agronomic plant traits result from complex molecular networks involving multiple genes and from environmental factors. One such trait is the enzymatic discoloration of fruit and tuber tissues initiated by mechanical impact (bruising). Tuber susceptibility to bruising is a complex trait of the cultivated potato (Solanum tuberosum) that is crucial for crop quality. As phenotypic evaluation of bruising is cumbersome, the application of diagnostic molecular markers would empower the selection of low bruising potato varieties. The genetic factors and molecular networks underlying enzymatic tissue discoloration are sparsely known. Hitherto there is no association study dealing with tuber bruising and diagnostic markers for enzymatic discoloration are rare.ResultsThe natural genetic diversity for bruising susceptibility was evaluated in elite middle European potato germplasm in order to elucidate its molecular basis. Association genetics using a candidate gene approach identified allelic variants in genes that function in tuber bruising and enzymatic browning. Two hundred and five tetraploid potato varieties and breeding clones related by descent were evaluated for two years in six environments for tuber bruising susceptibility, specific gravity, yield, shape and plant maturity. Correlations were found between different traits. In total 362 polymorphic DNA fragments, derived from 33 candidate genes and 29 SSR loci, were scored in the population and tested for association with the traits using a mixed model approach, which takes into account population structure and kinship. Twenty one highly significant (p < 0.001) and robust marker-trait associations were identified.ConclusionsThe observed trait correlations and associated marker fragments provide new insight in the molecular basis of bruising susceptibility and its natural variation. The markers diagnostic for increased or decreased bruising susceptibility will facilitate the combination of superior alleles in breeding programs. In addition, this study presents novel candidates that might control enzymatic tissue discoloration and tuber bruising. Their validation and characterization will increase the knowledge about the underlying biological processes.

Validation of candidate gene markers for marker-assisted selection of potato cultivars with improved tuber quality

Tuber yield, starch content, starch yield and chip color are complex traits that are important for industrial uses and food processing of potato. Chip color depends on the quantity of reducing sugars glucose and fructose in the tubers, which are generated by starch degradation. Reducing sugars accumulate when tubers are stored at low temperatures. Early and efficient selection of cultivars with superior yield, starch yield and chip color is hampered by the fact that reliable phenotypic selection requires multiple year and location trials. Application of DNA-based markers early in the breeding cycle, which are diagnostic for superior alleles of genes that control natural variation of tuber quality, will reduce the number of clones to be evaluated in field trials. Association mapping using genes functional in carbohydrate metabolism as markers has discovered alleles of invertases and starch phosphorylases that are associated with tuber quality traits. Here, we report on new DNA variants at loci encoding ADP-glucose pyrophosphorylase and the invertase Pain-1, which are associated with positive or negative effect with chip color, tuber starch content and starch yield. Marker-assisted selection (MAS) and marker validation were performed in tetraploid breeding populations, using various combinations of 11 allele-specific markers associated with tuber quality traits. To facilitate MAS, user-friendly PCR assays were developed for specific candidate gene alleles. In a multi-parental population of advanced breeding clones, genotypes were selected for having different combinations of five positive and the corresponding negative marker alleles. Genotypes combining five positive marker alleles performed on average better than genotypes with four negative alleles and one positive allele. When tested individually, seven of eight markers showed an effect on at least one quality trait. The direction of effect was as expected. Combinations of two to three marker alleles were identified that significantly improved average chip quality after cold storage and tuber starch content. In F1 progeny of a single-cross combination, MAS with six markers did not give the expected result. Reasons and implications for MAS in potato are discussed.

Multiple alleles for resistance and susceptibility modulate the defense response in the interaction of tetraploid potato (Solanum tuberosum) with Synchytrium endobioticum pathotypes 1, 2, 6 and 18

The obligate biotrophic, soil-borne fungus Synchytrium endobioticum causes wart disease of potato (Solanum tuberosum), which is a serious problem for crop production in countries with moderate climates. S. endobioticum induces hypertrophic cell divisions in plant host tissues leading to the formation of tumor-like structures. Potato wart is a quarantine disease and chemical control is not possible. From 38 S. endobioticum pathotypes occurring in Europe, pathotypes 1, 2, 6 and 18 are the most relevant. Genetic resistance to wart is available but only few current potato varieties are resistant to all four pathotypes. The phenotypic evaluation of wart resistance is laborious, time-consuming and sometimes ambiguous, which makes breeding for resistance difficult. Molecular markers diagnostic for genes for resistance to S. endobioticum pathotypes 1, 2, 6 and 18 would greatly facilitate the selection of new, resistant cultivars. Two tetraploid half-sib families (266 individuals) segregating for resistance to S. endobioticum pathotypes 1, 2, 6 and 18 were produced by crossing a resistant genotype with two different susceptible ones. The families were scored for five different wart resistance phenotypes. The distribution of mean resistance scores was quantitative in both families. Resistance to pathotypes 2, 6 and 18 was correlated and independent from resistance to pathotype 1. DNA pools were constructed from the most resistant and most susceptible individuals and screened with genome wide simple sequence repeat (SSR), inverted simple sequence region (ISSR) and randomly amplified polymorphic DNA (RAPD) markers. Bulked segregant analysis identified three SSR markers that were linked to wart resistance loci (Sen). Sen1-XI on chromosome XI conferred partial resistance to pathotype 1, Sen18-IX on chromosome IX to pathotype 18 and Sen2/6/18-I on chromosome I to pathotypes 2,6 and 18. Additional genotyping with 191 single nucleotide polymorphism (SNP) markers confirmed the localization of the Sen loci. Thirty-three SNP markers linked to the Sen loci permitted the dissection of Sen alleles that increased or decreased resistance to wart. The alleles were inherited from both the resistant and susceptible parents.

Comparative transcript profiling by SuperSAGE identifies novel candidate genes for controlling potato quantitative resistance to late blight not compromised by late maturity

Resistance to pathogens is essential for survival of wild and cultivated plants. Pathogen susceptibility causes major losses of crop yield and quality. Durable field resistance combined with high yield and other superior agronomic characters are therefore, important objectives in every crop breeding program. Precision and efficacy of resistance breeding can be enhanced by molecular diagnostic tools, which result from knowledge of the molecular basis of resistance and susceptibility. Breeding uses resistance conferred by single R genes and polygenic quantitative resistance. The latter is partial but considered more durable. Molecular mechanisms of plant pathogen interactions are elucidated mainly in experimental systems involving single R genes, whereas most genes important for quantitative resistance in crops like potato are unknown. Quantitative resistance of potato to Phytophthora infestans causing late blight is often compromised by late plant maturity, a negative agronomic character. Our objective was to identify candidate genes for quantitative resistance to late blight not compromised by late plant maturity. We used diagnostic DNA-markers to select plants with different field levels of maturity corrected resistance (MCR) to late blight and compared their leaf transcriptomes before and after infection with P. infestans using SuperSAGE (serial analysis of gene expression) technology and next generation sequencing. We identified 2034 transcripts up or down regulated upon infection, including a homolog of the kiwi fruit allergen kiwellin. 806 transcripts showed differential expression between groups of genotypes with contrasting MCR levels. The observed expression patterns suggest that MCR is in part controlled by differential transcript levels in uninfected plants. Functional annotation suggests that, besides biotic and abiotic stress responses, general cellular processes such as photosynthesis, protein biosynthesis, and degradation play a role in MCR.

Targeted and Untargeted Approaches Unravel Novel Candidate Genes and Diagnostic SNPs for Quantitative Resistance of the Potato (Solanum tuberosum L.) to Phytophthora infestans Causing the Late Blight Disease

The oomycete Phytophthora infestans causes late blight of potato, which can completely destroy the crop. Therefore, for the past 160 years, late blight has been the most important potato disease worldwide. The identification of cultivars with high and durable field resistance to P. infestans is an objective of most potato breeding programs. This type of resistance is polygenic and therefore quantitative. Its evaluation requires multi-year and location trials. Furthermore, quantitative resistance to late blight correlates with late plant maturity, a negative agricultural trait. Knowledge of the molecular genetic basis of quantitative resistance to late blight not compromised by late maturity is very limited. It is however essential for developing diagnostic DNA markers that facilitate the efficient combination of superior resistance alleles in improved cultivars. We used association genetics in a population of 184 tetraploid potato cultivars in order to identify single nucleotide polymorphisms (SNPs) that are associated with maturity corrected resistance (MCR) to late blight. The population was genotyped for almost 9000 SNPs from three different sources. The first source was candidate genes specifically selected for their function in the jasmonate pathway. The second source was novel candidate genes selected based on comparative transcript profiling (RNA-Seq) of groups of genotypes with contrasting levels of quantitative resistance to P. infestans. The third source was the first generation 8.3k SolCAP SNP genotyping array available in potato for genome wide association studies (GWAS). Twenty seven SNPs from all three sources showed robust association with MCR. Some of those were located in genes that are strong candidates for directly controlling quantitative resistance, based on functional annotation. Most important were: a lipoxygenase (jasmonate pathway), a 3-hydroxy-3-methylglutaryl coenzyme A reductase (mevalonate pathway), a P450 protein (terpene biosynthesis), a transcription factor and a homolog of a major gene for resistance to P. infestans from the wild potato species Solanum venturii. The candidate gene approach and GWAS complemented each other as they identified different genes. The results of this study provide new insight in the molecular genetic basis of quantitative resistance in potato and a toolbox of diagnostic SNP markers for breeding applications.

Selection and validation of potato candidate genes for maturity corrected resistance to Phytophthora infestans based on differential expression combined with SNP association and linkage mapping

Late blight of potato (Solanum tuberosum L.) caused by the oomycete Phytophthora infestans (Mont.) de Bary, is one of the most important bottlenecks of potato production worldwide. Cultivars with high levels of durable, race unspecific, quantitative resistance are part of a solution to this problem. However, breeding for quantitative resistance is hampered by the correlation between resistance and late plant maturity, which is an undesirable agricultural attribute. The objectives of our research are (i) the identification of genes that condition quantitative resistance to P. infestans not compromised by late plant maturity and (ii) the discovery of diagnostic single nucleotide polymorphism (SNP) markers to be used as molecular tools to increase efficiency and precision of resistance breeding. Twenty two novel candidate genes were selected based on comparative transcript profiling by SuperSAGE (serial analysis of gene expression) in groups of plants with contrasting levels of maturity corrected resistance (MCR). Reproducibility of differential expression was tested by quantitative real time PCR and allele specific pyrosequencing in four new sets of genotype pools with contrasting late blight resistance levels, at three infection time points and in three independent infection experiments. Reproducibility of expression patterns ranged from 28 to 97%. Association mapping in a panel of 184 tetraploid cultivars identified SNPs in five candidate genes that were associated with MCR. These SNPs can be used in marker-assisted resistance breeding. Linkage mapping in two half-sib families (n = 111) identified SNPs in three candidate genes that were linked with MCR. The differentially expressed genes that showed association and/or linkage with MCR putatively function in phytosterol synthesis, fatty acid synthesis, asparagine synthesis, chlorophyll synthesis, cell wall modification, and in the response to pathogen elicitors.

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. [Formula: see text] Copyright