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