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  • 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