Brande Wulff, The Sainsbury Lab

1. The Sainsbury Laboratory 2 Blades Group
Brande Wulff, TSL, Norwich UK
Brande Stephen John Costas Eric Matt G Matt M
Jamie Stuart Andrew Richard Cintia Nico Shuzhen Burkhard
Jing Inma Phon
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.”

2. Cost of DNA sequencing
$0.09/Mb
2

3. Principles of plant-microbe interactions
PAMPs
PAMP
receptor
Fungus/ Bacterium
Oomycete
Effector
Immunity
Effector Immunity
Haustorium
Resistance protein
Plant Cell
Dodds & Rathjen 2010

4. Generic Strategy
Clone and pyramid R genes
Find Map genes Identify and
germplasm; with next- test
Pyramid
make mapping generation candidate
populations genetics genes
★ No linkage drag ★ R genes cannot be separated ★ Durable?
Identify cognate effectors
• 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
4

6. The three rusts of wheat
Brown
rust
Black
rust
Yellow
rust
Leaf
rust
Stem
rust
Stripe
rust
Puccinia
tri-cina
Puccinia
graminis
f.
sp.
tri-ci
Puccinia
striiformis
var.
striiformis

7. 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!”

8. Germplasm
Sorghum
11
Ae. sharonensis
Maize
Ae. longissima
2-6 Wheat Ae. bicornis
53 12 Sitopsis
MYA Ae. searsii
36
Barley Ae. speltoides
47 Dvorak et al. 1997
Brachypodium
Rice

9. Aegilops sharonensis
Ae. tauschii Triticum urartu Sitopsis
DD AA BB ≈ SS
400,000
years
AABB Wild Emmer
10,000
years
AABBDD
Domestication
Bread Wheat Pasta Wheat
(Triticum aestivum) (Triticum turgidum ssp durum)

10. Ae. sharonensis distribution and habitat
Shefayyim Deror Junction
Haifa Port
Ashdod Nahsholim
P. Olivera

11. Ae. sharonensis plant care
 Seed drying to break dormancy
 Seed peeling: Peeled => 95% germination
Non-peeled => 60% germination
 Vernalisation
Ÿ Bagging to prevent cross-pollination
Ÿ 6-8 months from seed to seed
N. Champouret
P. Green
I. Hernandez-Pinzon
S. Jiang
J. Song
S. Banfield

12. Biosafety Level 3 Containment Facility
University of Minnesota
Jamie Kaufman

13. Aegilops sharonensis reaction to Ug99
0; ; 1; 1+ 2- 2+3 3 3+4
Resistant Susceptible
Reaction of 107 accessions to Ug99
Resistant 69%
P. Olivera,
Susceptible 13% J. Kaufman,
B. Steffenson
Intermediate/Heterogenous 18% Uni. Minnesota
13

14. Ae. sharonensis screening matrix
 43 key resistant accessions
 6 susceptible accessions
African N. American European
Accession TTKSK PTKST TTTTF TPKMC TRTTF JRCQC GO4CD GO41 G5/1826 G425
1644 R R R R R R R R Weak S
2189 S S S S S S n.d. n.d. n.d. n.d.
N. Champouret
B. Steffenson
J. Kaufman
P. Olivera
Z. Pretorius
P. Bartos

15. Ae. sharonensis population development
43 key resistant accessions
97 crosses
6 different susceptible
F1 F2 F2:3 … F4:5
Ae. sharonenesis 97 42 (+26) 4 (+2) 2
Ae. longissima 9
Ae. bicornis 11
Ae. searsii 3
N. Champouret
E. Millet
P. Olivera
15

16. Segregation for reaction to three stem rust
races in F2 and F2:3 populations
Reaction Observed segregation ratioa
No. of
Susceptible Resistant progeny 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)
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 N. Champouret
eTwo R genes; both semi-dominant, but when either is homozygous it confers full immunity P. Olivera
J. Kaufman
16

17. Segregation of resistance to Ug99 in 1644 × 2189 F2:3
1644 x 2189 F2.3 Obs Exp* χ2
Non-segregating (resistant) 22 21 0.04
Non-segregating (intermediate), Segregating 110 112 0.03
Non-segregating (susceptible) 3 2 0.38
* 10:53:1
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

18. Sequencing Aegilops sharonensis
DNA from 1 accession
Genome 30x illumina HiSeq
~7.5 Gb 1.5 Gb assembled seq.
2.5 million contigs
N50 = 1 Kb
Genes RNA from 2 accessions
~50 Mb 454 sequencing
~33,000 transcripts
C. Bouyioukos
B. Steuernagel ~15,000 SNPs
N. Champouret
I. Hernandez-Pinzon

19. Integrated DArT and SNP gene-based genetic map
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
1. 1644 x 1193
2. 1644 x 2189
3. 1644 x 2232
Chromosome
group
1
2
3
4
5
6
7
1Ssh 2Ssh 3Ssh 4Ssh 5Ssh 6Ssh 7Ssh
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
Moscou, Champouret, Hernández-Pinzón, Bouyioukos, Olivera, Steffenson

20. QTL mapping of Ug99 resistance in 1644 × 2189
F2 genotyping F3:4 genotyping
F2:3 phenotyping F3:4 phenotyping
A 3:1 5.5 cM in barley
1Ssh ?
2Ssh
3Ssh
4Ssh
B 3:1 37 cM in barley
5Ssh
6Ssh
7Ssh
M. Moscou
N. Champouret
Relative LOD S. Flipo

21. Ae. sharonensis infected with BSMV
WT MI EV Bax
BMV
RNA
RT1
RT2
H 2O
L 1 2 3 1 2 3 1 2 3 1 2 3 L
Ef1α * *
Beta * *
Gamma * *
Bax *
Mock Vector Bax-expressing
A. Dawson

22. Acknowledgements
2Blades Group Sainsbury Laboratory Aberystwyth University
Nicolas Champouret Jonathan Jones Luis Mur
Andrew Dawson Sophien Kamoun
John Ferguson Dan MacLean
CSIRO
Sixtine Flipo Michael Ayliffe
Matthew Gardiner John Innes Centre Tony Pryor
Phon Green Peter Dodds
Lesley Boyd
Inma Hernández-Pinzón
John Doonan
Shuzhen Jiang
Graham Moore Tel Aviv University
Cintia Kawashima
Lucy McCann Magdalena Opanowicz Eitan Millet
Matthew Moscou Cristobal Uauy
Jing Song Crop Research Institute
Burkhard Steuernagel NIAB Pavel Bartoš
Eric Ward
Rosemary Bayles
Marie Westergaard
Amelia Hubbard University of Minnesota
Brande Wulff
Alumni: Brian Steffenson
Philippa Borrill Jamie Kaufman
Costas Bouyioukos
Stephen Cranwell Pablo Olivera
Marie Dengremont
Stuart Harder
Gopal Jha
Peter Matthews
Richard Payne
Zhe Wang