Inma
Brande
Lucy
Cintia Matt M Jan
Phon
The Sainsbury Laboratory 2 Blades Group
Andrew
Marie Sue
Matt G
Shuzhen
Guotai Burkhard
Antony
$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
3
Find
germplasm;
make mapping
populations
Map genes
with next-
generation
genetics
Identify and
test
candidate
genes
Generic Strategy
Pyrami
d
4
No linkage drag R genes cannot be separated Durable?
• Ensure cloning R genes with distinct specificities
• Confirm function of R transgenes
Identify matching effectors
Clone and pyramid R genes
5
Black rust Stem rust Puccinia graminis f. sp. tritici
Yellow rust Stripe rust Puccinia striiformis var. striiformis
Brown rust Leaf rust Puccinia triticina
The three rusts of wheat
6
Norman Borlaug
7
Spread of African stem rust
Ug99
2007 sr31 sr36 2006 sr31 sr24
1999 sr31
2005
2008
2007 sr31 sr24
8
9
“The new wheat has been created by inserting
genes that confer resistance to […] Ug99.”
It’s also “highly drought resistant” and “fixes nitrogen!”
30 October 2011
Seed drying to break dormancy
Seed peeling: Peeled => 95% germination
Non-peeled => 60% germination
Bagging to prevent cross-pollination
6-8 months from seed to seed
7.5 Gb genome
Diploid
Ae. sharonensis – a model “non-model”
N. Champouret
P. Green
I. Hernandez-Pinzon
S. Jiang
J. Song
S. Banfield
G. Cox 10
Biosafety Level 3 Containment Facility
University of Minnesota
Jamie Kaufman 11
Aegilops sharonensis reaction to Ug99
12
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 population development
6 different susceptible
13
F1 F2 F2:3 … F5:6
Ae. sharonenesis 97 68 6 2
43 key resistant accessions
97 crosses
R genes TTKSK TRTTF TTTTF
1 (2) 3 1 2 3
3 2 Unclear 10
Segregation in F2 or F2:3
N. Champouret
J. Kaufman
P. Olivera
E. Millet
B. Steffenson
G. Yu
ABD ABd AbD Abd aBD aBd abD abd
AB
D 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
14
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
15
Relative LOD
1Ssh
2Ssh
3Ssh
4Ssh
5Ssh
6Ssh
7Ssh
QTL mapping of Ug99 resistance in 1644 × 2189
M. Moscou
N. Champouret
S. Flipo
G. Yu
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
16
Wheat stripe rust severity (2000-2009)
Wellings et al. (2010) BGRI Workshop
5% yield
loss
10% yield loss
Molecular Basis of Non-host Resistance to
Wheat Stripe Rust in Barley
11
Maize
Sorghum
Wheat
Brachypodium
Rice
36
47
53
MYA
Barley
12 Sitopsis
2-6
Matt Moscou
Why are most barley
genes for resistance to
barley stripe rust recessive?
Do barley stripe rust
resistance genes function
against wheat stripe rust in
barley?
Can we exploit a molecular
understanding of this
interaction to make wheat
resistant to wheat stripe
rust?
Host to barley stripe rust Marginal host to wheat stripe rust
Stripe rust on Barley
Morex Steptoe HOR 1428 Trumpf
Full lifecycle Varying degrees of
colonisation Penetration
Little colonisation
Matt Moscou
HOR 1428 x Morex F2 population inoculated with Pst 08/501
0.0 1.0 2.0 3.0
0
20
40
30
10
Chlorosis
Fre
quency
Morex
HOR 1428
Morex
Matt Moscou
HOR 1428
HOR 1428 x Morex F2 population inoculated with Pst 08/501
SNP Genotyping - Illumina GoldenGate Oligo Pooled Assay
Matt Moscou
Composite interval mapping on the
HOR 1428 x Morex F2 population inoculated with Pst 08/501
QTL 1
QRpst-1H
QTL 2
spst1
QTL 3
QRpst-6H
Chlo
rosis
M
icro
scopy
S R R R R
Resistant Resistant parent
HOR 1428
Matt Moscou
QTL 3
Morex Het HOR
1428
13.0%
6.6%
QTL 2
Morex Het HOR
1428
49.1%
45.3%
QTL 1
Morex Het HOR
1428
0
0.5
1.0
1.5
0
1.0
2.0
3.5 12.5%
2_01451_02081_0409
1_1345
1_0490
1_0113 1_07382_05571_0223
1_1199
2_0210 1_01322_0302
2_0680 2_1418 2_0777
2_13971_0793
2_1490 2_09391_0261 1_1405 2_02691_0093 1_0942 1_0028
1_10421_1114 2_0289
1_1244 2_0610 1_08811_0262
2_0482 2_0472 2_0363 2_0412 2_1481 2_03612_14001_0509
2_0723 2_0453 2_1087 2_0062 2_1273 1_12071_0010 1_0606
1_04671_1500 1_1004 2_1353
2_06701_0724
2_0689 1_07232_01781_13981_05882_03842_08382_12431_12922_05152_04541_0510
1_0334 2_1111
1_01231_0712
1_1299 2_0701 2_1035
2_0007
2_0272
2_0013
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
CH
L
pIN
F
mIN
F
4H
4H
Mlo
0 1 2 3 4
05
10
15
Fre
quency
Infection phenotype
Bgh CC148
Segregation for resistance in a HOR
1428 x Morex F2 population
HOR 1428 F1 Morex
23
Not Mla
Not mlo
Colocalization of race-specific and species specificity
resistance loci to multiple pathogens
Mla
mlo
Mlf
Rpst1
spst
Rpst2
Mlsp
MlLa
Mlg Rmo1 QRmo
QRmo
QRmo
Barley powdery mildew
Wheat stripe rust
Rice blast
Rmo2
QRpst
QRpst-6H
QRpst-5H-2
QRpst-3H
QRpst-4H
mlo
Barley stripe rust rps2
Rps-5H
Rps-3H
Ml?
QRpst-5H-1
Black Sigatoka of banana Banana
4th most important crop after wheat, rice, maize
Staple for 400m people in the tropics
90% of bananas are grown on small farms and eaten locally
Export worth $5 billion a year
Cladosporium fulvum
Tomato leaf mold ECP2 Avr4
ECP6
v
Can C. fulvum R gene host range be extended?
Bolton et al. 2008
Stergiopoulos et al. 2010
Marshall et al. 2011
Mycosphaerella fijiensis
Black Sigatoka of banana
Mycosphaerella graminicola
Septoria leaf blotch
Stagonospora nodorum
Glume blotch
Pyrenophora tritici-repentis
Tan spot
Alternaria brassicicola
Brassica dark leaf spot
v v v
Cf-Ecp6
Cf-Ecp2
Cf-4
?
?
?
27
~40 Kb in Heinz
11G (110 Kb)
7B (117 Kb)
4B (140 Kb)
M1 M2 M4 M5
Cf-Ecp2
Mutant 1
Mutant 2
Cf-Ecp2 map interval in tomato
M3
✓
✗
✓ ✓ ✓
✓ ✓ ✓
✓ ✓ ✓ ✗
✓
✓
✓
✓
✓
✓
28
Collaborations
Ayele Badebo, Debre Zeit Agricultural Research Center, Ethiopia
Brian Steffenson, University of Minnesota1, USA
Colwyn Thomas, UEA, UK
Cristobal Uauy, JIC, UK
Diane Saunders, Sophien Kamoun, TSL, UK
Eitan Millet, Tel Aviv University, Israel
Graham Moore, JIC, UK
Jesse Poland, USDA-ARS, Manhattan, KS, USA
John Doonan, University of Aberystwyth, UK
John Rathjen, Australian National University, Australia
Jorge Dubcovsky, UC Davis, USA
Klaus Mayer, MIPS, Munich, Germany
Manash Chatterjee, BenchBio, Gujarat, India
Mario Caccamo, TGAC, UK
Mick Ayliffe1, CSIRO, Australia
Mogens Hovmøller, Aarhus University, Denmark
Nick Talbot, University of Exeter, UK
Parveen Chhuneja, Punjab Agricultural University, India
Paul Nicholson, JIC, UK
Pavel Bartoš, Prague
Peter Dodds1, CSIRO, Australia
Rients Niks, Wageningen, Netherlands
Robbie Waugh, JHI, UK
Robbie Waugh, JHI, UK
Robert Park, University of Sydney, Australia
Rosemary Bayles, NIAB, Cambridge
Ruth Waynera, Kenyan Agricultural Research Institute, Kenya
Wendy Harwood, JIC, UK
Wolfgang Spielmeyer, CSIRO, Australia
Xianming Chen, USDA-ARS, Pullman, WA, USA
Zakkie Pretorius, University of Freestate, South Africa
2Blades Group
Sue Banfield
Jan Bettgenhaeuser
Andrew Dawson
Matthew Gardiner
Phon Green
Inma Hernández-Pinzón
Georgina Cox
Shuzhen Jiang
Cintia Kawashima
Lucy McCann
Matthew Moscou
Jing Song
Burkhard Steuernagel
Marie Westergaard
Brande Wulff
Guotai Yu
Alumni: Costas Bouyioukos
Nicolas Champouret
Stephen Cranwell
John Ferguson
Gopal Jha
Eric Ward
Acknowledgements