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

Name der Einrichtung/Institut Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019

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

Seite 2 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 2

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 3 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 3

Pathotypes and Plant Resistance

Federal Plant Variety Office, 2018

Seite 4 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 4

Localisation of 18(T1) Resistance Gene Locus

4n Karolin (1,2,6,18) [R1r2r3r4]

X

2n Solanum phureja IVP101 or IVP35 Seedlings Identification of diploid genotypes 2n Karolin Identification of recombinant genotypes between c1_4319 and c2_33712 Evaluation of potato wart resistance (6 and 18) Narrow down the resistance locus

Seite 5 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 5

Localisation of 18(T1) Resistance Gene Locus

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

Chromosome XI

Seite 6 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 6

Localisation of 18(T1) Resistance Gene Locus

Modified after Bartkiewicz et al., 2018 Modified after Bartkiewicz et al., 2018

Seite 7 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 7

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 8 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 8

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 9 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 9

Pathotype Identification - 18(T1) Transcriptome

7008 Chytrid related contigs RNASeq of wart tissue infected by 18(T1) 136.409 No Hit contigs 13.037 Transcriptome contigs 130.380 No Hit contigs

Seite 10 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 10

Pathotype Identification – SCAR Markers

SCAR marker 14425P1 (Busse et al., 2017) 14425P1 14425

Seite 11 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 11

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 12 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 12

Pathotype Identification – SCAR Markers

• SCAR marker for pathotype 18(T1): 122283P18

Seite 13 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 13

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 14 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 14

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 15 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 15

Pathotype Identification – Microsatellites

1(D1) 2(G1) 6(O1) 18(T1) SSR 7 I II I III SSR 77 I I II I

SSR 7

1(D1) 2(G1) 6(O1) 18(T1) I II I III 1(D1) 2(G1) 6(O1) 18(T1)

SSR 77

I I II I

Seite 16 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 16

Pathotype Identification – Microsatellites

SSR 27

1(D1) 2(G1) 6(O1) 8(F1) 18(T1) 3(M1) 39(P1) 2(Ch1) I II II I I II II I

Seite 17 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 17

18(T1) Transcriptome – Effector Genes

7008 Chytrid related contigs Signal Peptide Prediction: SignalP; Phobius Secretome: 475 proteins 112 proteins < 50 aa Effector Prediction: EffectorP 178 proteins 66 putative effectors + 2 genes from previous analyses

Seite 18 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 18

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 19 Name der Einrichtung/Institut … Dennis Reckwell, Development of Diagnostic Tools, Wageningen 27.06.2019 Slide 19

Thank you for your Attention!

Department of Molecular Plant Breeding