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