Development of Diagnostic Tools for Resistance Screening and - PowerPoint PPT Presentation

Name der Einrichtung/Institut Development of Diagnostic Tools for Resistance Screening and Pathotype Differentiation Dennis Reckwell, Friederike Chilla, Annette Bartkiewicz, Diro Terefe-Ayana, Marian Meyer, Kerstin Flath, Marcus Linde and Thomas Debener Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019

Name der Einrichtung/Institut … Outline - Localization of a resistance locus against S.endobioticum pathotypes 6(O1) and 18(T1) in the potato cultivar Karolin - Characterisation of putative resistance genes against pathotypes 6(O1) and 18(T1) - Development of molecular tools to distinguish pathoytpes 1(D1), 2(G1), 6(O1) and 18(T1) - Identification of putative effector proteins of pathotype 18(T1) Seite 2 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 2

Name der Einrichtung/Institut … Pathotypes and Plant Resistance Federal Plant Variety Office, 2018 Seite 3 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 3

Name der Einrichtung/Institut … Localisation of 18(T1) Resistance Gene Locus 4n Karolin (1,2,6,18) 2n Solanum phureja X [R 1 r 2 r 3 r 4 ] IVP101 or IVP35 Identification of diploid Seedlings genotypes Identification of recombinant genotypes 2n Karolin between c1_4319 and c2_33712 Narrow down the Evaluation of potato wart resistance locus resistance (6 and 18) Seite 4 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 4

Name der Einrichtung/Institut … Localisation of 18(T1) Resistance Gene Locus Chromosome XI 215 genotypes of the dihaploid population were checked for recombinants - 4 recombinants -18(T1) Resistance localised to an intervall of 777 kbp (Bartkiewicz et al., 2018) 394 additional dihaploid genotypes were checked for recombinants - 12 genotypes showed recombination between the SNP markers. Resistance is under evaluation. Modified after Bartkiewicz et al., 2018 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 5 Seite 5

Name der Einrichtung/Institut … Localisation of 18(T1) Resistance Gene Locus Modified after Bartkiewicz et al., 2018 Modified after Bartkiewicz et al., 2018 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 6 Seite 6

Name der Einrichtung/Institut … Identification of Resistance Genes -The highly resistant dihaploid genotype B35B-1 was sequenced both by Illumina and PacBio. - The Illumina read contigs were mapped to the identified resistance locus on the S. phureja DM1-3 genome. - PacBio contigs were used to span gaps between the Illumina contigs. 79 % of the 777 kbp were identified - These sequences are under inverstigation to identify putative resistance genes Seite 7 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 7

Name der Einrichtung/Institut … Evaluation of a putative Resistance Gene The identified resistance marker sequence Kc8103 is part of a putative resistance gene: TIR-NBS-LRR gene -Kc8103gene was amplified and cloned from a resistant dihaploid genotype -The gene was cloned into a binary vector for agrobacteria-mediated transformation under the control of a double CaMV35S Promoter -Stable transformation of susceptible potato cultivars will evaluate an resistance function against 18(T1). Seite 8 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 8

Name der Einrichtung/Institut … Pathotype Identification - 18(T1) Transcriptome RNASeq of wart tissue infected by 18(T1) 7008 Chytrid related contigs 136.409 No Hit contigs 130.380 No Hit contigs 13.037 Transcriptome contigs Seite 9 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 9

Name der Einrichtung/Institut … Pathotype Identification – SCAR Markers 14425 14425P1 SCAR marker 14425P1 (Busse et al., 2017) Seite 10 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 10

Name der Einrichtung/Institut … Pathotype Identification – SCAR Markers  Draft genome sequence of S. endobioticum strain MB42 1(D1) (Jack Vossen; WUR).  Transcriptome of 18(T1) and No Hit sequences mapped against MB42 genome  Contigs with sequence differences between 1(D1) and 18(T1) were choosen for evaluation, 7 were confirmed by sequencing.  Pathotypes 2(G1) and 6(O1) did not differ from pathotype 1(D1) Seite 11 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 11

Name der Einrichtung/Institut … Pathotype Identification – SCAR Markers  SCAR marker for pathotype 18(T1): 122283P18 Seite 12 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 12

Name der Einrichtung/Institut … Pathotype Identification – Microsatellites Whole Genome Shotgun Sequencing of wart tissue infected by 18(T1) 915.972 DNA contigs Mapping to the Transcriptome contigs 423 DNA contigs Screening for microsatellites: SSR Locator 389 microsatellite motifs ( ≥ 3 Repeats) Seite 13 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 13

Name der Einrichtung/Institut … Pathotype Identification – Microsatellites - Published results: - 2 Clusters: 1, 2, 6 and 8 +18 - Differentiation between pathotypes 1, 2 and 6 unsolved. - 100 additional SSR motifs were investigated Modified after Busse et al., 2017 Seite 14 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 14

Name der Einrichtung/Institut … Pathotype Identification – Microsatellites SSR 7 SSR 77 I II I III I I II I 1(D1) 2(G1) 6(O1) 18(T1) 1(D1) 2(G1) 6(O1) 18(T1) 1(D1) 2(G1) 6(O1) 18(T1) SSR 7 I II I III SSR 77 I I II I Seite 15 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 15

Name der Einrichtung/Institut … Pathotype Identification – Microsatellites SSR 27 I II II I I II II I 1(D1) 2(G1) 6(O1) 8(F1) 18(T1) 3(M1) 39(P1) 2(Ch1) Seite 16 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 16

Name der Einrichtung/Institut … 18(T1) Transcriptome – Effector Genes 7008 Chytrid related contigs Signal Peptide Prediction: SignalP; Phobius Secretome: 475 proteins Effector Prediction: EffectorP 178 proteins 112 proteins < 50 aa 66 putative effectors + 2 genes from previous analyses Seite 17 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 17

Name der Einrichtung/Institut … Conclusion - 18(T1) and 6(O1) resistance of cultivar Karolin colocalizes on chromosome XI in an intervall of 777 kbp, which is going to be narrowed down to less than 500 kbp. - The putative resistance gene Kc8103gene is under evaluation. Sequence data are available to identify further candidate resistance genes - Microsatellites to distinguish pathoytpes 1(D1), 2(G1), 6(O1) and 18(T1) are identified. Diagnostic value needs to be checked with further isolates and pathotypes - Four putative effector proteins of pathotype 18(T1) were identified Seite 18 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 18

Name der Einrichtung/Institut … Thank you for your Attention! Department of Molecular Plant Breeding Seite 19 Dennis Reckwell , Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 19