This document summarizes research on cloning rust resistance genes from wheat and developing gene pyramids via genetic engineering. Key points include: - Researchers at the University of Minnesota and other institutions are working to clone multiple rust resistance genes from wheat including Sr2, Sr22, Sr33, Sr35, Sr46, Sr50 and Lr67. - Cloned genes like Lr34/Yr18, Yr36, and others can be stacked together in transgenic cassettes to provide pyramided resistance in a single locus. - Preliminary work has successfully stacked two or three resistance genes in transgenic wheat. - Further work will continue cloning additional genes, validating gene function through transformation, and

1. University of Minnesota
Rust Resistance Gene Cloning
perfect markers and cassette development
Peter Dodds
BGRI technical workshop March 2014

2. Outlook: R gene pyramids via GM gene cassettes
• Stacking of multiple R genes in a single location
eg.
Segregates as one locus
Sr33 Sr35 Sr50
Need: Multiple cloned R genes (=> perfect markers)
Ability to stack into a transgene cassette

3. Outlook: R gene pyramids via GM gene cassettes
Available genes:
APR Lr34/Yr18 Krattinger et al 2009 Science 323:1360
= ABC transporter
Yr36 Fu et al 2009 Science 323:1357
= START Kinase
R Sr33 Periyannan et al 2013 Science 341:786
Sr35 Saintenac et al 2103 Science 341:783
Lr1,Lr10,Lr21
= CC-NB-LRR immune receptors
LRR
NB-ARC
CC

4. APR Cloning Targets: Lr67
Lr67 [=Yr46=Sr55=Pm46=Ltn3]
(leaf rust, stripe rust, stem rust, powdery mildew, leaf tip necrosis )
chromosome 4DL
§ cosegregation of all five phenotypes at Lr67
locus
§ mutation experiments indicates a single gene
affects all five phenotypes
R S
Lagudah

5. APR Cloning Targets: Sr2 and Lr46
Sr2 physical contig across locus fully sequenced, 1.2Mbp
3 candidates testing in transgenic wheat
new mutants and recombinants in locus
Mago, Tabe, Spielmeyer
Lr46/Yr29 multi-pathogen resistance with Ltn
mapped on 1BL
sequencing a physical contig across locus
several mutants
Lagudah

6. Gli-1, Glu-3
SrR
Sec-1
NOR
1DL.1RS (Imperial)
(Shepherd 1973)
3+ 2=
Gli-1, Glu-3
Lr26, Sr31, Yr9
Sec-1
NOR
1BL.1RS (Petkus)
Pm8
R Cloning Targets: SrR = Sr50
Rohit Mago

7. D E
F G
C B
A
Physical contig at Sr50 locus : 1RS chromosome sorted library (Dolezel)
~100kb
Extent of smallest deletion
-1 -23
A
M7 M13
CC NB LRR
Smallest deletion mutant includes 6 NB-LRR genes
2 EMS mutants carry small deletions in one NB-LRR gene
R Cloning Targets: SrR = Sr50
Rohit Mago

8. Fielder #19 #13
Leaf Rust
Stem Rust
Sr50 transgenics express stem rust resistance
Rohit Mago
R Cloning Targets: SrR = Sr50

9. • Derived from Aegilops tauschii AUS18913
• Confers all stage partial resistance to stem rust
(Australian, Ug99 race group, Ethiopian and Yemeni isolates)
• Located on chromosome 2DS
R Cloning Targets: Sr46
Sr46 susc
Lagudah & Bariana

10. Sr46
2DS 2DL
Genetic map, chr. 2DS
psr249
barc297
1 3
Sr46
Ae. tauschii
BAC contig
Brachypodium 5g region
Ae. tauschii sequence contigs
Sr46 candidate gene
altered in three mutants (CC-NB-LRR)
R Cloning Targets: Sr46

11. Effective against worldwide stem rust races
(Australia, Canada, India, US and Ug99 etc.)
Two sources:
2+ 4
R Cloning Targets: Sr22
T. boeoticum T. monococcum
Ø Middle East part of Israel
Ø Gerechter- Amitai et al. , 1971 Ø Kerber and Dyck, 1973
Ø Canada
W3534
(Standard differential for Sr22 -Australia)
Schomburgk
(Australian wheat cultivar)
Sam Periyannan

12. Schomburgk x Westonia
(1200 F2)
cfa2123
cfa2019
Sr22, cssu22 (IH81)
BE498985
BG262287, BF201318,
BG604641, AT7D7092
BG274853
BE443521
1
2
7AL
AT7D7094
2
6
4044 (Sr22) x 4069
(1200 F2)
BE498985
AT7D7094
53
Sr22
4445409
8
9
4554964
4556232
1
With Matt Rouse
U. Minnesota
T.monococcum
Sam Periyannan
R Cloning Targets: Sr22
Ø 1300 M2 heads – screened
at PBI, Cobbitty
Ø 6 Mutants
EMS mutants

13. R gene cis-stacks
Genes available
Lr34/Yr18, Lr21, Lr67/Yr46
Sr22, Sr33, Sr35, Sr46, Sr50
Yr36
R1 R2 R3 R4 R5
Resistance gene cis-stacks
Durability ??

14. Transformation efficiency
Fielder = 41%
Gladius = 32%
Westonia = 45%
Mace = 25%
R gene cis-stacks: wheat transformation
Mick Ayliffe

15. Transformed into durum wheat cultivar Stewart
Yr36 Lr21 RB
(D genome)
10kb 7kb
17kb
Not in cultivated
durum
T1 T2Control
2 confirmed transgenics with both genes
- show leaf rust resistance
- to test for stripe rust
R gene cis-stacks: a 2-gene stack
Mick Ayliffe

16. RB
Lr21
10.2 kb
S N
5’
S
3’
7kb
3’
Yr365’
N
3’
Lr34/Yr18 hyg
LB RB
16kb
Co-transformation
150 Fielder transgenics produced - screening now for co-transformation
Mick Ayliffe
R gene cis-stacks: a 3-gene stack

17. Current activities
• Complete cloning of Sr2, Lr46
• Confirm Sr22 and Sr46 by transformation
• Develop gene stacking approaches
insertion site targetting
• Understanding resistance gene function
can we tweak them to improve function?
• R genes from wider sources: Barley, Brachypodium
can’t be deployed conventionally

18. • PLANT PRODUCTION AND PROTECTION
CSIRO Plant Industry
Mick Ayliffe
Maud Bernoux
Stella Cesari
Sutha Chandramohan
Jeff Ellis
Evans Lagudah
Greg Lawrence
Rohit Mago
John Moore
Adnane Nemri
Kim Newell
Sam Periyannan
Terese Richardson
Joanna Risk*
Wendelin Schnippenkoetter
Wolfgang Spielmeyer
Linda Tabe
Narayana Upadhyaya
Libby Viccars*
Xioadi Xia
CIMMYT, Mexico
Ravi Singh
Sybil Herrera-Foessel
Julio Huerta-Espino
Caixia Lan
Plant Breeding Institute
University of Sydney
Harbans Bariana
Urmil Bansal
Colin Wellings/William Cuddy
Robert Park