Pyramiding potato resistance genes against late blight

Jadwiga Śliwka

Instytut Hodowli i Aklimatyzacji Roślin – Państwowy Institut Badawczy

Plant Breeding and Acclimatization Institute – National Research Institute

The G2P-SOL project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 677379

Potato Pathogens Laboratory

Phytophthora infestans Solanum tuberosum

Resistance gene mapping

Wild Solanum sp. as resistance sources

Resistance QTL analysis

Resistance gene expression study

Marker-assisted selection

Testing breeding lines

Collection of isolates

Population study

Mating type, metalaxyl resistance, mitochondrial haplotype, virulence, SSR markers

Effector diversity and expression

Resistance gene cloning

Poster 1,1 Marta Brylińska

Presentation: Emil Stefańczyk

Hot spots of resistance to P. infestans on potato genetic map

Rpi-mcq1 Rpi-edn2 R9 Rpi-dlc1 R8/Rpi-Smira2

I

VIII VII

VI V IV III II

XII XI X IX

R1

R3a R3b R5 R6 R7 R10 R11 Rpi-sto2

Rpi-pcs1 Rpi-Smira1

Rpi-vnt1.1 Rpi-vnt1.2 Rpi-vnt1.3 Rpi-nrs1 Rpi-phu1

R2, R2-like R2-likeBzura Rpi-blb3 Rpi-abpt Rpi-bst1 Rpi-mcd1 Rpi-amr3i

Rpi-blb2

Rber, Rpi-ber1 Rpi-ber2

Rpi-blb1/RB Rpi-sto1 Rpi-pta1 Rpi-plt1 Rpi-ver1-8

Rpi1

Rpi-rzc1

Rpi-mch1

Cloned Used in cisgenics Identified by IHAR-PIB Młochów

ver, mcd

ver, mcd

ver, mcd

Rpi-avl1

Rpi-cap1 Rpi-qum1 Rpi-edn3

Rpi-pur1 Rpi-vnt2

Solanum phureja

Solanum stenotomum

http://www.cgn.wageningen-ur.nl/pgr/collections/crops/potato/species.htm

Rpi-phu1

• 1970 S. stenotomum x S. phureja comes from CIP, Lima, Peru Crosses with S. tuberosum 2x • 1992 first resistant tetraploid (unreduced gametes) • 2006 mapping the gene (Śliwka et al. 2006, TAG) • 2009 sequencing, virulent isolate P. infestans EC1; MP1162? Syn. Rpi-vnt1.1 (Foster et al. 2009, MPMI) • 2010 MAS (Śliwka et al. 2010, JAG) • 2012 expression of the Rpi-phu1? • 2012 pyramiding with Sárpo Mira • 2014 GMO field tests in The Sainsbury Laboratory [Jones et al.] • ???? Cultivar???

Rpi-phu1

11.05.2016

• Originates from: Bolivia, Peru

habitat: inter-Andean valleys, barren, eroded or cultivated soil

2400-3000 m above sea level

• Syn. S. sparsipilum, re-identified as • S. brevicaule

• Selected clones:

- resistant to P. infestans – low tendency to enzymatic blackening

and after-cooking darkening

Solanum ruiz-ceballosii Cárd. 2x (2EBN) VIR8664 (VIR7370)

Ochoa 1990

rzc flower colour dH Balbina x 99-10/36 rzc

Śliwka et al. 2012 BMC Genet. 13 (1): 11

Rpi-rzc1

R2: 87.1% mean leaflet resistance 2007, 2008, 2010

Śliwka et al. 2012 BMC Genet. 13 (1): 11

New linkage map: 12-4, N=236

Brylińska et al. 2015

Are there many virulent P. infestans isolates in the field?

r R1

R5 R7 R10 R9 R8 R6

R4 R3 R2

Rpi-rzc1 ktz mch

R11 Biogold Rpi-phu1 S. Mira Bzura

ver, mcd

Virulence of 1154 P. infestans isolates collected in Poland

1 3 0 0

3 2 8

1 0 0 0

2 0 0

52 50 59 59

139 140 115 122

19 19

168 166

13 13 0

20

40

60

80

100

120

140

160

180

Rpi-phu1 Rpi-rzc1 Rpi-phu1 Rpi-rzc1 Rpi-phu1 Rpi-rzc1 Rpi-phu1 Rpi-rzc1 Rpi-phu1 Rpi-rzc1 Rpi-phu1 Rpi-rzc1 Rpi-phu1 Rpi-rzc1

2009 2010 2011 2012 2013 2014 2015

avirulent

virulent

Rpi-phu1: 12 isolates virulent (1%) Rpi-rzc1: 8 isolates virulent (0.7%) Virulent on both plants: none

The goal: Rpi-phu1 + Rpi-rzc1

• Virulence is easily lost in storage of the isolate • Virulence test: laborious and variation in results • Marker-Assisted Selection • Tetraploid breeding lines

12

Resistance assessment

13

leaflet tests performed on individuals from the G2P populations • P. infestans isolates

• MP324 on G2P-1, G2P-2 and G2P-3 • MP324x (virulent on plants with Rpi-phu1) on G2P-2

susceptible-to-resistant ratio consistent with the expected ratio • 1:1 (G2P-1 and G2P-3; gene Rpi-rzc1)

• 3:1 (G2P-2; Rpi-rzc1 and Rpi-phu1 genes) or 1:1

We cannot distinguish plants with Rpi-phu1 + Rpi-rzc1 from plants with Rpi-rzc1

Crosses

14

resistance plants

population origin gene 1 gene 2 grown secured

G2P-1 DG 06-508 x DG 08-238 Rpi-rzc1 239 124

G2P-2 DG 97-943 x DG 99-10/36 Rpi-phu1 Rpi-rzc1 290 194

G2P-3 DG 97-2174 x DG 99-10/36 Rpi-rzc1 260 181

789 499

additional interploid (4x – 2x) crosses performed: • DG 08-238 × susceptible potato cultivars (26 seedlings obtained)

15

GP94

99-10/36 dh Balbina 05-18/3 05-18/25 DG

08-238 DG

92-227 97-30 /588

97-30 /616

Rpi-rzc1 Rpi-rzc1 Rpi-rzc1 Rpi-phu1 Rpi-phu1

99-10/36 × dh Balbina 99-10/36 × 97-943 DG 92-227 × 93-352

Marker GP94 outside the Rpi-phu1 gene, specific? recombinants

Marker phu6 inside the Rpi-phu1 gene, not specific

16

phu6

99-10/36 dh Balbina 05-18/3 05-18/25 DG

08-238 DG

92-227 97-30 /588

97-30 /616

Rpi-rzc1 Rpi-rzc1 Rpi-rzc1 Rpi-phu1 Rpi-phu1

99-10/36 × dh Balbina 99-10/36 × 97-943 DG 92-227 × 93-352

755 NBS-LRR genes in the reference potato genome Jupe et al. 2013

17

Rpi-phu1 + Rpi-rzc1: Rpi-phu1 homologs are present in Rpi-rzc1 plants

mch mch rzc mch pnt rzc rzc ktz ktz ktz + + - rzc rzc spl vrn + -

• Sequencing of the homologs • Development of more specific markers for Rpi-phu1 • Validation on diploid and tetraploid level (five F1, 500-750 plants)

18

Rpi-phu1 homologs in the Rpi-rzc1 plants

primers based on the Rpi-phu1 sequence (Rpi-vnt1.1; FJ423044.1) a) covering the whole open reading frame b) spanning shorter fragments of the gene

Rpi-phu1 2676 bp

PCR

reamplification

matrix: DNA

template: purified PCR product

Sequencing, sequence comparison and design of better marker in progress

16.05.2017 19 RenSeq1910

[HinfI]

X

99-10/36 dh Balbina 05-18/3 05-18/25 DG

08-238 DG

92-227 97-30 /588

97-30 /616

Rpi-rzc1 Rpi-rzc1 Rpi-rzc1 Rpi-phu1 Rpi-phu1

99-10/36 × dh Balbina 99-10/36 × 97-943 DG 92-227 × 93-352

Marker RenSeq1910 f Rpi-rzc1

20 RZC1

X

99-10/36 dh Balbina 05-18/3 05-18/25 DG

08-238 DG

92-227 97-30 /588

97-30 /616

Rpi-rzc1 Rpi-rzc1 Rpi-rzc1 Rpi-phu1 Rpi-phu1

99-10/36 × dh Balbina 99-10/36 × 97-943 DG 92-227 × 93-352

Marker RZC1 of Rpi-rzc1

Summary

• Introgression of Rpi-rzc1 to new breeding lines and populations

• Pyramiding Rpi-rzc1+Rpi-phu1 on diploid level • MAS is not trivial, marker optimization • Transfer of Rpi-rzc1 to tetraploid level • Pyramiding Rpi-rzc1+Rpi-phu1 on tetraploid level

21

11.05.2016 22

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Potato Pathogens Laboratory

Jadwiga Śliwka Marta Brylińska Małgorzata Frączak Anna Jarzyńska Sylwester Sobkowiak Emil Stefańczyk Paulina Smyda-Dajmund Jarosław Plich

Thank you