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University of Minnesota
Rust Resistance Gene Cloning perfect markers and cassette development
Peter Dodds BGRI technical workshop March 2014
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
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
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
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
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
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
Fielder #19 #13
Leaf Rust Stem Rust
Sr50 transgenics express stem rust resistance
Rohit Mago
R Cloning Targets: SrR = Sr50
• 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
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
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
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
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 ??
Transformation efficiency Fielder = 41% Gladius = 32% Westonia = 45% Mace = 25%
R gene cis-stacks: wheat transformation
Mick Ayliffe
Transformed into durum wheat cultivar Stewart
Yr36 Lr21 RB (D genome)
10kb 7kb
17kb
Not in cultivated durum
T1 T2 Control 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
RB Lr21
10.2 kb S N
5’
S
3’
7kb
3’ Yr36 5’
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
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
• 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