Distribution of P1(D1) wart disease resistance in potato germplasm and GWAS identification of haplotype-specific SNP markers

Key message: A Genome-Wide Association Study using 330 commercial potato varieties identified haplotype specific SNPmarkers associated with pathotype 1(D1) wart disease resistance. Abstract: Synchytrium endobioticum is a soilborne obligate biotrophic fungus responsible for wart disease. Growing resistant varieties is the most effective way to manage the disease. This paper addresses the challenge to apply molecular markers in potato breeding. Although markers linked to Sen1 were published before, the identification of haplotype-specific single-nucleotide polymorphisms may result in marker assays with high diagnostic value. To identify hs-SNP markers, we performed a genome-wide association study (GWAS) in a panel of 330 potato varieties representative of the commercial potato gene pool. SNP markers significantly associated with pathotype 1 resistance were identified on chromosome 11, at the position of the previously identified Sen1 locus. Haplotype specificity of the SNP markers was examined through the analysis of false positives and false negatives and validated in two independent full-sib populations. This paper illustrates why it is not always feasible to design markers without false positives and false negatives for marker-assisted selection. In the case of Sen1, founders could not be traced because of a lack of identity by descent and because of the decay of linkage disequilibrium between Sen1 and flanking SNP markers. Sen1 appeared to be the main source of pathotype 1 resistance in potato varieties, but it does not explain all the resistance observed. Recombination and introgression breeding may have introduced new, albeit rare haplotypes involved in pathotype 1 resistance. The GWAS approach, in such case, is instrumental to identify SNPs with the best possible diagnostic value for marker-assisted breeding.</p

Supplementary files from the PhD thesis "Warts Wars: The resistant potatoes strike back"

Supplementary files from the experimental chapters of the PhD thesis of Charlotte Prodhomme untitled "Warts Wars; The resistant potatoes strike back

Warts wars : The resistant potatoes strike back

Potato wart disease, caused by the obligate biotrophic Chytrid fungus Synchytrium endobioticum, is one of the most important quarantine diseases of potato. This disease was named after the symptoms caused by the pathogen, which are the proliferation of meristematic tissues leading to the formation of warts, mainly on the below-ground sprouts of potato plants. The quarantine status of S. endobioticum is due to the production of spores that can remain viable in the soil for more than 40 years, the lack of chemical control and the severe yield losses. In Europe, more than 40 different pathotypes of S. endobioticum have been recorded and only resistance to pathotype 1 is commonly deployed in the breeding germplasm. The breeding and cultivation of potato varieties resistant to a wider spectrum of pathotypes is crucial for quarantine practice to reduce the propagation of the pathogen. Therefore, the identification of genes bringing resistance to the most frequent pathotypes of the pathogen and the development of diagnostic markers for marker assisted selection (MAS) is urgently needed. In this thesis, genes involved in resistance to pathotypes 1, 2, 6 and 18 of S. endobioticum were identified to make an inventory of the different resistance sources at hand for potato breeders. In Chapter 2, we investigated the distribution of the pathotype 1 resistance in a variety panel representative of the potato breeding material. Breeding programs of the 20th century were very successful in producing varieties resistant to pathotype 1 as 77% of the panel varieties were found to be resistant. To identify markers linked with pathotype 1 resistance, we used previously produced genotypic and phenotypic data to perform a Genome-Wide Association Study (GWAS). The GWAS resulted in the identification of markers associated with pathotype 1 resistance on the north arm of chromosome 11. In this region, the major effect gene Sen1 was previously identified. Sen1 is the main source of pathotype 1 resistance in the variety panel and no common ancestral donor could be identified due to the inability to define identity-by-descent (IBD). As we faced limitations to design markers fully diagnostic for pathotype 1 resistance using the GWAS approach, we aimed to develop new tools to identify haplotype specific SNPs. In Chapter 3, we developed a new set of workflows, called Comparative Subsequence Sets Analysis (CoSSA), for the genetic analysis of traits of interest and the identification of haplotype specific SNPs. CoSSA can be used for any crop as it is suitable for polyploids and can be used with or without a reference genome. We applied CoSSA to identify Sen3, a dominant gene conferring resistance to all tested pathotypes. Sen3 was fine-mapped to the resistance gene cluster C76 on the north arm of chromosome 11. Furthermore, we used CoSSA for the fine-mapping of Sen1 in Chapter 4. Sen1 was mapped to the same R gene cluster as Sen3. We performed a candidate gene analysis and showed that Sen1 encodes a nucleotide-binding domain, leucine rich containing (NLR) protein from the TNL group. The two identified candidate genes were cloned and tested in complementation assays with AvrSen1, the S. endobioticum effector protein which triggers Hypersensitive Responses (HR) in Sen1 plants. These findings will serve as novel tools to study the interactions between potato and S. endobioticum. In Chapter 5, we made an, as complete as possible at this moment, inventory of the dominant potato wart disease resistance (Sen) genes and QTLs present in the potato breeding germplasm. We combined the GWAS and CoSSA strategies to identify two new major genes, Sen4 and Sen5, which are involved in resistance to pathotypes 2, 6 and 18. We also identified several wart disease resistance QTLs which, in combination with the dominant genes, can contribute to improve resistance to the higher pathotypes. To avoid any confusion between the previously and newly identified QTLs, we introduced a new naming system which allows to differentiate each resistant haplotype identified. Finally, we screened a broad panel of potato varieties and wild Solanum species for the genes Sen1, Sen2, Sen3, Sen4 and Sen5. To put it in a nutshell, a complete picture of the major potato wart disease resistance sources present in the breeding germplasm is given in this thesis. Haplotype specific markers have been designed for all the major genes and QTLs mapped, which will facilitate the breeding of resistant varieties. Finally, the development of CoSSA will facilitate the mapping of traits of interest and the design of haplotype specific markers for any crop

Recent trends in genetics studies and molecular breeding of potato

International audienceThe development of marker-assisted selection in the 1990s offered new possibilities to fasten potato breeding and make it more efficient. A turning point in molecular potato breeding was the release of the first potato reference genome in 2011. The aim of this chapter is to give a comprehensive overview of the progress made in potato genetic studies and molecular breeding since the publication of the first potato genome. With the continuous progress made in sequencing technologies, the number of potato genomic datasets made available to the scientific community is blooming, opening new possibilities for a better comprehension and an optimized utilization of the large potato genetic diversity. The development of high-throughput genotyping methods and of software and tools especially designed for autopolyploid species also led to an important leap forward in potato genetic studies. These important developments caused changes in the plant material used in potato genetic studies, allowing switching from less complex diploid biparental populations to tetraploid panels less distant from the breeding material. Finally, an overview of the recent advances made in marker-assisted selection for resistance to potato cyst nematodes, potato virus Y, wart disease, and late blight is given. For each of these economically important traits, the lastly developed diagnostic markers at hand for potato breeders are describe

Synchytrium endobioticum , the potato wart disease pathogen

International audiencePotato wart disease is considered one of the most important quarantine pests for cultivated potato and is caused by the obligate biotrophic chytrid fungus Synchytrium endobioticum. This review integrates observations from early potato wart research and recent molecular, genetic, and genomic studies of the pathogen and its host potato. Taxonomy, epidemiology, pathology, and formation of new pathotypes are discussed, and a model for molecular S. endobioticum-potato interaction is proposed. Taxonomy Currently classified as kingdom: Fungi, phylum: Chytridiomycota, class: Chytridiomycetes, order: Chytridiales, family: Synchytriaceae, genus: Synchytrium, species: Synchytrium endobioticum, there is strong molecular support for Synchytriaceae to be transferred to the order Synchytriales. Hosts and disease symptoms Solanum tuberosum is the main host for S. endobioticum but other solanaceous species have been reported as alternative hosts. It is not known if these alternative hosts play a role in the survival of the pathogen in (borders of) infested fields. Disease symptoms on potato tubers are characterized by the warty cauliflower-like malformations that are the result of cell enlargement and cell multiplication induced by the pathogen. Meristematic tissue on tubers, stolons, eyes, sprouts, and inflorescences can be infected while the potato root system seems to be immune. Pathotypes For S. endobioticum over 40 pathotypes, which are defined as groups of isolates with a similar response to a set of differential potato varieties, are described. Pathotypes 1(D1), 2(G1), 6(O1), and 18(T1) are currently regarded to be most widespread. However, with the current differential set other pathogen diversity largely remains undetected. Pathogen-host interaction A single effector has been described for S. endobioticum (AvrSen1), which is recognized by the potato Sen1 resistance gene product. This is also the first effector that has been described in Chytridiomycota, showing that in this fungal division resistance also fits the gene-for-gene concept. Although significant progress was made in the last decade in mapping wart disease resistance loci, not all resistances present in potato breeding germplasm could be identified. The use of resistant varieties plays an essential role in disease manageme

Comparative Subsequence Sets Analysis (CoSSA) is a robust approach to identify haplotype specific SNPs; mapping and pedigree analysis of a potato wart disease resistance gene Sen3

Standard strategies to identify genomic regions involved in a specific trait variation are often limited by time and resource consuming genotyping methods. Other limiting pre-requisites are the phenotyping of large segregating populations or of diversity panels and the availability and quality of a closely related reference genome. To overcome these limitations, we designed efficient Comparative Subsequence Sets Analysis (CoSSA) workflows to identify haplotype specific SNPs linked to a trait of interest from Whole Genome Sequencing data. As a model, we used the resistance to Synchytrium endobioticum pathotypes 2, 6 and 18 that co-segregated in a tetraploid full sib population. Genomic DNA from both parents, pedigree genotypes, unrelated potato varieties lacking the wart resistance traits and pools of resistant and susceptible siblings were sequenced. Set algebra and depth filtering of subsequences (k-mers) were used to delete unlinked and common SNPs and to enrich for SNPs from the haplotype(s) harboring the resistance gene(s). Using CoSSA, we identified a major and a minor effect locus. Upon comparison to the reference genome, it was inferred that the major resistance locus, referred to as Sen3, was located on the north arm of chromosome 11 between 1,259,552 and 1,519,485 bp. Furthermore, we could anchor the unanchored superscaffold DMB734 from the potato reference genome to a synthenous interval. CoSSA was also successful in identifying Sen3 in a reference genome independent way thanks to the de novo assembly of paired end reads matching haplotype specific k-mers. The de novo assembly provided more R haplotype specific polymorphisms than the reference genome corresponding region. CoSSA also offers possibilities for pedigree analysis. The origin of Sen3 was traced back until Ora. Finally, the diagnostic power of the haplotype specific markers was shown using a panel of 56 tetraploid varieties. CoSSA is an efficient, robust and versatile set of workflows for the genetic analysis of a trait of interest using WGS data. Because the WGS data are used without intermediate reads mapping, CoSSA does not require the use of a reference genome. This approach allowed the identification of Sen3 and the design of haplotype specific, diagnostic markers

Comparative Subsequence Sets Analysis (CoSSA) is a robust approach to identify haplotype specific SNPs; mapping and pedigree analysis of a potato wart disease resistance gene Sen3

Standard strategies to identify genomic regions involved in a specific trait variation are often limited by time and resource consuming genotyping methods. Other limiting pre-requisites are the phenotyping of large segregating populations or of diversity panels and the availability and quality of a closely related reference genome. To overcome these limitations, we designed efficient Comparative Subsequence Sets Analysis (CoSSA) workflows to identify haplotype specific SNPs linked to a trait of interest from Whole Genome Sequencing data. As a model, we used the resistance to Synchytrium endobioticum pathotypes 2, 6 and 18 that co-segregated in a tetraploid full sib population. Genomic DNA from both parents, pedigree genotypes, unrelated potato varieties lacking the wart resistance traits and pools of resistant and susceptible siblings were sequenced. Set algebra and depth filtering of subsequences (k-mers) were used to delete unlinked and common SNPs and to enrich for SNPs from the haplotype(s) harboring the resistance gene(s). Using CoSSA, we identified a major and a minor effect locus. Upon comparison to the reference genome, it was inferred that the major resistance locus, referred to as Sen3, was located on the north arm of chromosome 11 between 1,259,552 and 1,519,485 bp. Furthermore, we could anchor the unanchored superscaffold DMB734 from the potato reference genome to a synthenous interval. CoSSA was also successful in identifying Sen3 in a reference genome independent way thanks to the de novo assembly of paired end reads matching haplotype specific k-mers. The de novo assembly provided more R haplotype specific polymorphisms than the reference genome corresponding region. CoSSA also offers possibilities for pedigree analysis. The origin of Sen3 was traced back until Ora. Finally, the diagnostic power of the haplotype specific markers was shown using a panel of 56 tetraploid varieties. CoSSA is an efficient, robust and versatile set of workflows for the genetic analysis of a trait of interest using WGS data. Because the WGS data are used without intermediate reads mapping, CoSSA does not require the use of a reference genome. This approach allowed the identification of Sen3 and the design of haplotype specific, diagnostic markers

The origin and widespread occurrence of Sli based self-compatibility in potato

Self-compatible (SC) diploid potatoes allow innovative potato breeding. Therefore, the Sli gene, originally described in S. chacoense, has received much attention. In elite S. tuberosum diploids, spontaneous berry set is occasionally observed. We aimed to map SC from S. tuberosum origin. Two full-sib mapping populations from non-inbred diploids were used. Bulks were composed based on both pollen tube growth and berry set upon selfing. After DNA sequencing of the parents and bulks we generated k-mer tables. Set algebra and depth filtering was used to identify bulk-specific k-mers. Coupling and repulsion phase k-mers, transmitted from the SC parent, mapped in both populations to the distal end of chromosome 12. Intersection between the k-mers from both populations, in coupling phase with SC, exposed a shared haplotype of approximately 1.5 Mb. Subsequently we screened read archives of potatoes and wild relatives for k-mers specific to this haplotype. The well-known SC clones US-W4 and RH89-039-16, but surprisingly, also S. chacoense clone M6 were positives. Hence, the S. tuberosum source of SC seems identical to Sli. Furthermore, the candidate region drastically reduced to 333 kb. Haplotype specific KASP markers were designed and validated on a panel of diploid clones including another renown SC dihaploid G254. Interestingly, k-mers specific to the SC haplotype were common in tetraploid varieties. Pedigree information suggests that the SC haplotype was introduced into tetraploid varieties via the founder ‘Rough Purple Chili’. We show that Sli is surprisingly widespread and indigenous to the cultivated gene pool of potato

The origin and widespread occurrence of Sli based self-compatibility in potato

Self-compatible (SC) diploid potatoes allow innovative potato breeding. Therefore, the Sli gene, originally described in S. chacoense, has received much attention. In elite S. tuberosum diploids, spontaneous berry set is occasionally observed. We aimed to map SC from S. tuberosum origin. Two full-sib mapping populations from non-inbred diploids were used. Bulks were composed based on both pollen tube growth and berry set upon selfing. After DNA sequencing of the parents and bulks we generated k-mer tables. Set algebra and depth filtering was used to identify bulk-specific k-mers. Coupling and repulsion phase k-mers, transmitted from the SC parent, mapped in both populations to the distal end of chromosome 12. Intersection between the k-mers from both populations, in coupling phase with SC, exposed a shared haplotype of approximately 1.5 Mb. Subsequently we screened read archives of potatoes and wild relatives for k-mers specific to this haplotype. The well-known SC clones US-W4 and RH89-039-16, but surprisingly, also S. chacoense clone M6 were positives. Hence, the S. tuberosum source of SC seems identical to Sli. Furthermore, the candidate region drastically reduced to 333 kb. Haplotype specific KASP markers were designed and validated on a panel of diploid clones including another renown SC dihaploid G254. Interestingly, k-mers specific to the SC haplotype were common in tetraploid varieties. Pedigree information suggests that the SC haplotype was introduced into tetraploid varieties via the founder ‘Rough Purple Chili’. We show that Sli is surprisingly widespread and indigenous to the cultivated gene pool of potato

Comparative Subsequence Sets Analysis (CoSSA) is a robust approach to identify haplotype specific SNPs; Mapping and pedigree analysis of a potato wart disease resistance gene Sen3

Background: Standard strategies to identify genomic regions involved in a specific trait variation are often limited by time and resource consuming genotyping methods. Other limiting pre-requisites are the phenotyping of large segregating populations or of diversity panels and the availability and quality of a closely related reference genome. To overcome these limitations, we designed efficient Comparative Subsequence Sets Analysis (CoSSA) workflows to identify haplotype specific SNPs linked to a trait of interest from Whole Genome Sequencing data. Results: As a model, we used the resistance to Synchytrium endobioticum pathotypes 2, 6 and 18 that co-segregated in a tetraploid full sib population. Genomic DNA from both parents, pedigree genotypes, unrelated potato varieties lacking the wart resistance traits and pools of resistant and susceptible siblings were sequenced. Set algebra and depth filtering of subsequences (k-mers) were used to delete unlinked and common SNPs and to enrich for SNPs from the haplotype(s) harboring the resistance gene(s). Using CoSSA, we identified a major and a minor effect locus. Upon comparison to the reference genome, it was inferred that the major resistance locus, referred to as Sen3, was located on the north arm of chromosome 11 between 1,259,552 and 1,519,485 bp. Furthermore, we could anchor the unanchored superscaffold DMB734 from the potato reference genome to a synthenous interval. CoSSA was also successful in identifying Sen3 in a reference genome independent way thanks to the de novo assembly of paired end reads matching haplotype specific k-mers. The de novo assembly provided more R haplotype specific polymorphisms than the reference genome corresponding region. CoSSA also offers possibilities for pedigree analysis. The origin of Sen3 was traced back until Ora. Finally, the diagnostic power of the haplotype specific markers was shown using a panel of 56 tetraploid varieties. Conclusions: CoSSA is an efficient, robust and versatile set of workflows for the genetic analysis of a trait of interest using WGS data. Because the WGS data are used without intermediate reads mapping, CoSSA does not require the use of a reference genome. This approach allowed the identification of Sen3 and the design of haplotype specific, diagnostic markers.</p