Stacking of resistance loci to give full resistance to higher - - PowerPoint PPT Presentation

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Stacking of resistance loci to give full resistance to higher - - PowerPoint PPT Presentation

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Stacking of resistance loci to give full resistance to higher pathotypes of S. endobioticum June 27 th 2019, Charlotte Prodhomme Inventory of resistances known so far 1999: 1 st mapping of Sen1 (Hehl et al ., 1999) Adapted from Obidiegwu et al .,

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Stacking of resistance loci to give full resistance to higher pathotypes of S. endobioticum

June 27th 2019, Charlotte Prodhomme

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Inventory of resistances known so far

2 Adapted from Obidiegwu et al., 2015

1999: 1st mapping of Sen1 (Hehl et al., 1999) 2011: Mapping of several QTLs (Ballvora et al., 2011) 2013: Mapping of several QTLs (Groth et al., 2013) 2006: Mapping of Sen1-4 (Brugmans et al., 2006) 2015: Mapping of several QTLs (Obidiegwu et al., 2015) 2018: Mapping of Sen2 (Plich et al., 2018)

Sen2

2018: Mapping of Sen3 (Bartkiewicz et al., 2018 ;

Prodhomme et al., 2019) Sen3

> Qualitative and quantitative resistance loci identified > Broad / specific spectrum of resistance How to use these resistance loci in breeding? What is the pathotype R spectrum for Q management?

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Population segregating for pathotypes 2, 6 and 18 resistance

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Resistant parent Kuba P1, 2, 6, 18 Susceptible parent Ludmilla P1

n = 328

20 40 60 80 100 120 140 160 [1 - 1.5[ [1.5 - 2[ [2 - 2.5[ [2.5 - 3[ [3 - 3.5[ [3.5 - 4[ [4 - 4.5[ [4.5 - 5] P2 P6 P18

R S

Glynne-Lemmerzahl

Bimodal distribution of resistance to pathotypes 2, 6 and 18 > P18 resistance skewed toward susceptibility

Segregating ratio χ2 χ2 test significance P2 (G1) 1:1 0.30 ns P6 (O1) 1:1 1.48 ns P18 (T1) 1:1 30.49 ***

Prodhomme et al., 2019 (Plant Methods)

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Principal Component Analysis of phenotypes

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Individuals fully resistant to P2, P6 and P18 n = 67 Individuals fully susceptible to P2, P6 and P18 n = 170 Individuals fully resistant to P2 and P6, weaker resistance to P18 n = 93 50% 50%

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Hypothesis about the R loci segregating

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1/ There is one gene (or several tightly linked

genes) giving full resistance to P2 and P6

and partial resistance to P18  1:1 segregation ratio 2/ One or several other loci are required by the first gene to bring full resistance to P18 50% 50%

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Strategy to identify the R loci segregating

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Major gene + P18 R loci Major gene Major gene P18 R loci?  Whole Genome Sequencing of the three bulks + 2 parents P2P6-R- bulk P2P6P18

  • R-bulk

S-bulk

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Comparative Subread Sets Analysis = CoSSA

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Whole Genome Sequencing

ACGTGATGACGTGACGTACCGG… ACGTG CGTGA GTGAT TGATG GATGA ATGAC K-mer analysis in each sample = quick and easy comparisons Reads Subreads = k-mers

Prodhomme et al., 2019 (Plant Methods)

S-bulk R-bulk R-P2P6- bulk

Whole Genome Sequencing

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CoSSA method to identify sequence variants linked to the major gene segregating

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P2P6P18-R-bulk S-bulk

k-mers k-mers k-mers k-mers k-mers k-mers k-mers k-mers k-mers k-mers k-mers k-mers

Major gene + P18 R loci Major gene P18 R loci? Major gene (+ P18 R loci?)

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Inheritance of resistance specific variants

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Ludmilla Kuba

Mapping of the subreads to the potato reference genome

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Subread mapping reveals a single locus for P2, P6, P18 resistance

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a) R-genes in the reference genome (Jupe et al., 2013)

b) R-bulk specific k-mers from Kuba c) R-bulk specific k-mers from Ludmilla d) R-bulk specific k-mers from both parents

Prodhomme et al., 2019 (Plant Methods)

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Design of KASP markers to validate Sen3

Prodhomme et al., 2019 (Plant Methods)

Jupe et al., 2013

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Effect of chr11_1519485 on P18 resistance

R S

P2 P6 P18

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CoSSA method to identify sequence variants linked to the P18 R loci segregating

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P2P6P18-R-bulk R-P2P6-Bulk R-P2P6-Bulk

R-P2P6-bulk Major gene + P18 R loci Major gene Major gene + P18 R loci

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Inheritance of resistance specific variants

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Ludmilla Kuba

Mapping of the subreads to the potato reference genome

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Subread mapping reveals several loci for P18 resistance

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a b c d

a) R-genes in the reference genome (Jupe et al., 2013)

b) R-bulk specific k-mers from Kuba c) R-bulk specific k-mers from Ludmilla d) R-bulk specific k-mers from both parents

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Validation experiment with KASP markers

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Interaction Sen3 * chr8  p-value = 0.00974**

Pathotype 2 Pathotype 6 Pathotype 18

Chr02_30 ns ns ns Chr02_32 ns ns ns Chr05_8 ns ns ns Chr06_25 ns ns ns Chr07_10 ns ns ns Chr07_41 ns ns ns Chr08_44 ns ns 0.03401 Chr08_45 ns ns 0.04718 Chr11_1259552 < 2.2e-16 < 2.2e-16 < 2.2e-16 Chr11_1519485 < 2.2e-16 < 2.2e-16 < 2.2e-16 Chr11_1666090 < 2.2e-16 < 2.2e-16 < 2.2e-16

Sen3

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Effect of the chr8 locus on P18 resistance

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Ludmilla Kuba

R S

Pathotype 18 resistance

3 CNL clusters:

  • Clusters 59, 60 and 61
  • Rpi-blb1, Rpi-pta1,

Rpi-sto1

Jupe et al., 2013

chr8 No QTL Sen3 + chr8 Sen3

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One isolate = several genotypes

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van de Vossenberg et al., 2018

› Mitochondrial genome sequencing revealed an intra- isolate variation. › Higher diversity observed for isolates from P8, P18, P38. > Intra-isolate diversity can explain the strong or weak effect of the R loci mapped

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Use of only one R gene (Sen3)

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Sen3 SenChr8 AvrSen3 AvrSen Chr8

Incomplete resistance Pathotype 18 population New pathotype population

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Use of only one R gene (Sen3)

Sen3 SenChr8 AvrSen3 AvrSen Chr8

New pathotype population Susceptibility

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Stack of R genes (Sen3 + chr8)

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Sen3 SenChr8 AvrSen3 AvrSen Chr8

Complete resistance Pathotype 18 population

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  • Diversity of S. endobiodicum populations causal to partial

resistance:

  • Complex populations can only be contained by broad

spectrum R genes or R gene stacks

  • More research should be performed on identifying R loci to less

frequent S. endo genotypes:

  • How to identify such loci? How to design diagnostic

markers for a use in MAS?

Take home messages

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Phenotype the segregating population with the “trained” pathotype

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Use the pathotype 18 population that was multiplied on Sen3 genotypes  enrichment

  • f the S. endobioticum genotypes

recognised by the chromosome 8 QTL Re-phenotype the K x L population with the “trained” P18 Stronger effect of chr8 QTL / weaker effect

  • f Sen3 on P18 resistance  unambiguous

phenotypes  mapping and fine-mapping

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Acknowledgements

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Wageningen University and Research Plant Breeding Jack Vossen Herman van Eck Richard Visser Danny Esselink Gert van Arkel Linda Kodde Theo Borm Dirk-Jan Huigen Marjan Bergervoet Biointeractions Theo vd Lee Bart vd Vossenberg Consortium Partners TKI T&U

This project is financially supported by the Dutch Topsector Horticulture & Starting Materials. Within the Topsector, private industry, knowledge institutes and the government are working together on innovations for sustainable production of safe and healthy food and the development of a healthy green environment.

Thank you for your attention