Inma

Brande Eric Cintia Nico Stuart

Matt M Costas

Phon

Stephen Andrew

John Richard

Matt G Shuzhen

Jing Jamie Burkhard

Jonathan Swift, 1726 “Whosoever could make two ears of corn or two blades of grass to grow upon a spot of ground where only one grew before, would deserve better of mankind, and do more essential service to his country, than the whole race of politicians put together.”

The Sainsbury Laboratory 2 Blades Group Brande Wulff, TSL, Norwich UK

$0.09/Mb

2

Cost of DNA sequencing

Bacterium Fungus/ Oomycete

Haustorium

Effector

PAMP receptor

Immunity

Effector

Resistance protein

Immunity

PAMPs

Plant Cell

Dodds & Rathjen 2010

Principles of plant-microbe interactions

Find germplasm;

make mapping populations

Map genes with next-generation genetics

Identify and test

candidate genes

Generic Strategy

Pyramid

4

★ No linkage drag ★ R genes cannot be separated ★ Durable?

•  Prioritize R genes with best odds of durability •  Ensure cloning R genes with distinct specificities •  Confirm function of R transgenes •  Mine R genes in non-host species •  Molecular breeding of new R genes

Identify cognate effectors

Clone and pyramid R genes

Black  rust  Stem  rust  Puccinia  graminis  f.  sp.  tri-ci  

Yellow  rust  Stripe  rust  Puccinia  striiformis  var.  striiformis  

Brown  rust  Leaf  rust  Puccinia  tri-cina  

The three rusts of wheat

30  October  2011  

“The new wheat has been created by inserting genes that confer resistance to […] Ug99.”

It’s also “highly drought resistant” and “fixes nitrogen!”

Germplasm

11

Maize

Sorghum

Wheat

Brachypodium

Rice

36

47

53 MYA

Barley

12 Sitopsis 2-6 Ae. bicornis

Ae. speltoides Ae. searsii

Ae. sharonensis

Ae. longissima

Dvorak et al. 1997

400,000  years  

10,000  years  

Wild Emmer

Triticum urartu Ae. tauschii

Bread Wheat (Triticum aestivum)

AA BB

AABB

DD

AABBDD

Pasta Wheat (Triticum turgidum ssp durum)

Domestication

SS Sitopsis

≈

Aegilops sharonensis

Shefayyim

Ashdod

Deror Junction

Nahsholim

Ae. sharonensis distribution and habitat

Haifa Port

P. Olivera

Seed drying to break dormancy Seed peeling: Peeled => 95% germination

Non-peeled => 60% germination Vernalisation �  Bagging to prevent cross-pollination

�  6-8 months from seed to seed

Ae. sharonensis plant care

N. Champouret P. Green I. Hernandez-Pinzon S. Jiang J. Song S. Banfield

Biosafety Level 3 Containment Facility University of Minnesota

Jamie Kaufman

Aegilops sharonensis reaction to Ug99

13

Reaction of 107 accessions to Ug99 Resistant 69% Susceptible 13% Intermediate/Heterogenous 18%

P. Olivera, J. Kaufman, B. Steffenson Uni. Minnesota

0; ; 1; 1+ 2- 2+3 3 3+4

Resistant Susceptible

Ae. sharonensis screening matrix

TTKSK PTKST TTTTF TPKMC TRTTF JRCQC GO4CD GO41 G5/1826 G425 R R R R R R R R Weak S 1644

Accession

African N. American European

2189 S S S S S S n.d. n.d. n.d. n.d.

43 key resistant accessions 6 susceptible accessions

N. Champouret B. Steffenson J. Kaufman P. Olivera Z. Pretorius P. Bartos

Ae. sharonensis population development

6 different susceptible

15

F1 F2 F2:3 … F4:5 Ae. sharonenesis 97 42 (+26) 4 (+2) 2

N. Champouret E. Millet P. Olivera

43 key resistant accessions

97 crosses

Ae. longissima 9 Ae. bicornis 11 Ae. searsii 3

Segregation for reaction to three stem rust races in F2 and F2:3 populations

aR, Resistant; IR, Intermediate Resistant; I, Intermediate; IS, Intermediate Susceptible; S, Susceptible; HR, Homozygous resistant; Seg, Segregating, HS, Homozygous susceptible bTwo R genes; both semi-dominant and when combined confer full immunity cTwo R genes; one dominant and one recessive dThree R genes; all semi-dominant with any two conferring full immunity eTwo R genes; both semi-dominant, but when either is homozygous it confers full immunity

16

Reaction No. of progeny

Observed segregation ratioa Susceptible Resistant TTKSK (Ug99) TTTTF TPMKC Model

2189 575 94 F2 22R:49IR:17I:6S 4:8:3:1b (p=0.97) 94 F2 77R:17S 13:3c (p=0.87)

2189 1644 139 F2.3 26HR:110Seg:3HS 10:53:1d (p=0.46) 2232 1644 90 F2 63R:27S 3:1 (p=0.71)

114 F2:3 29HR:82Seg:3HS 10:53:1d (p=0.01) 548 1995 88 F2 65R:23S 3:1 (p=0.81) 548 2205 76 F2 54R:22S 3:1 (p=0.43) 548 2233 74 F2 32R:15IR:21IS:6S 7:4:4:1e (p=0.77) 548 6793 63 F2 57R:6S 15:1 (p=0.28)

N. Champouret P. Olivera J. Kaufman

ABD ABd AbD Abd aBD aBd abD abd

ABD AABBDD AABBDd AABbDD AABbDd AaBBDD AaBBDd AaBbDD AaBbDd

ABd AABBDd AABBdd AABbDd AABbdd AaBBDd AaBBdd AaBbDd AaBbdd

AbD AABbDD AABbDd AAbbDD AAbbDd AaBbDD AaBbDd AabbDD AabbDd

Abd AABbDd AABbdd AAbbDd AAbbdd AaBbDd AaBbdd AabbDd Aabbdd

aBD AaBBDD AaBBDd AaBbDD AaBbDd aaBBDD aaBBDd aaBbDD aaBbDd

aBd AaBBDd AaBBdd AaBbDd AaBbdd aaBBDd aaBBdd aaBbDd aaBbdd

abD AaBbDD AaBbDd AabbDD AabbDd aaBbDD aaBbDd aabbDD aabbDd

abd AaBbDd AaBbdd AabbDd Aabbdd aaBbDd aaBbdd aabbDd aabbdd

Non-segregating (intermediate), Segregating Non-segregating (resistant)

Non-segregating (susceptible)

* 10:53:1

1644 x 2189 F2.3 Obs Exp* χ2

22 21 0.04 110 112 0.03 3 2 0.38

Segregation of resistance to Ug99 in 1644 × 2189 F2:3

Genome ~7.5 Gb

Sequencing Aegilops sharonensis

Genes ~50 Mb

RNA from 2 accessions

454 sequencing ~33,000 transcripts

~15,000 SNPs

DNA from 1 accession 30x illumina HiSeq

1.5 Gb assembled seq. 2.5 million contigs

N50 = 1 Kb

C. Bouyioukos B. Steuernagel N. Champouret I. Hernandez-Pinzon

1Ssh 2Ssh 3Ssh 4Ssh 5Ssh 6Ssh 7Ssh

Chromosome group

1 2 3 4 5 6 7

1. 1644 x 1193

3. 1644 x 2232 2. 1644 x 2189

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

Integrated DArT and SNP gene-based genetic map

Moscou, Champouret, Hernández-Pinzón, Bouyioukos, Olivera, Steffenson

1644 x 1193 1644 x 2232 1644 x 2189 Consensus F2 population 182 177 175 534 Total cM 885 972 888 1115 DArT markers 495 791 750 1598 Gene SNP markers 0 49 40 52 SSR markers 15 0 0 15

Relative LOD

1Ssh

2Ssh

3Ssh

4Ssh

5Ssh

6Ssh

7Ssh

QTL mapping of Ug99 resistance in 1644 × 2189

M. Moscou N. Champouret S. Flipo

A

B

?

F2 genotyping F2:3 phenotyping

F3:4 genotyping F3:4 phenotyping

3:1

3:1 5.5 cM in barley

37 cM in barley

Ef1α

Beta

Gamma

1 RT1

3 2 1 3 2 1 3 2 1 3 2 WT MI EV Bax

RT2

RN

A

H2O

BM

V

*

*

*

**

*

L L

*Bax

Mock Vector Bax-expressing

A. Dawson

Ae. sharonensis infected with BSMV

2Blades Group Nicolas Champouret Andrew Dawson John Ferguson Sixtine Flipo Matthew Gardiner Phon Green Inma Hernández-Pinzón Shuzhen Jiang Cintia Kawashima Lucy McCann Matthew Moscou Jing Song Burkhard Steuernagel Eric Ward Marie Westergaard Brande Wulff Alumni: Philippa Borrill Costas Bouyioukos Stephen Cranwell Marie Dengremont Stuart Harder Gopal Jha Peter Matthews Richard Payne Zhe Wang

Acknowledgements Sainsbury Laboratory Jonathan Jones Sophien Kamoun Dan MacLean

University of Minnesota Brian Steffenson Jamie Kaufman Pablo Olivera

Crop Research Institute Pavel Bartoš

CSIRO Michael Ayliffe Tony Pryor Peter Dodds

NIAB Rosemary Bayles Amelia Hubbard

Tel Aviv University Eitan Millet

Aberystwyth University Luis Mur

John Innes Centre Lesley Boyd John Doonan Graham Moore Magdalena Opanowicz Cristobal Uauy