The Sainsbury Laboratory 2 Blades Group        Brande Wulff, TSL, Norwich UK      Brande Stephen   John    Costas Eric    ...
Cost of DNA sequencing                  $0.09/Mb                             2
Principles of plant-microbe interactions                                PAMPs                                             ...
Generic StrategyClone and pyramid R genes     Find             Map genes        Identify and  germplasm;          with nex...
The three rusts of wheatBrown	  rust	            Black	  rust	                                   Yellow	  rust	  Leaf	  ru...
30	  October	  2011	  “The new wheat has been created by inserting genesthat confer resistance to […] Ug99.”It’s also “hig...
Germplasm                           Sorghum                11                                          Ae. sharonensis    ...
Aegilops sharonensisAe. tauschii                Triticum urartu                     SitopsisDD                           A...
Ae. sharonensis distribution and habitatShefayyim                 Deror Junction             Haifa PortAshdod             ...
Ae. sharonensis plant care         Seed drying to break dormancy         Seed peeling:     Peeled => 95% germination    ...
Biosafety Level 3 Containment Facility       University of Minnesota                      Jamie Kaufman
Aegilops sharonensis reaction to Ug99      0;      ;        1;   1+   2-   2+3    3    3+4           Resistant            ...
Ae. sharonensis screening matrix                             43 key resistant accessions                             6 s...
Ae. sharonensis population development            43 key resistant accessions          97 crosses             6 different ...
Segregation for reaction to three stem rust             races in F2 and F2:3 populations        Reaction                  ...
Segregation of resistance to Ug99 in 1644 × 2189 F2:3       1644 x 2189 F2.3                              Obs      Exp*   ...
Sequencing Aegilops sharonensis                                     DNA from 1 accessionGenome                            ...
Integrated DArT and SNP gene-based genetic map1    2     3   1    2     3   1    2     3   1      2     3    1    2       ...
QTL mapping of Ug99 resistance in 1644 × 2189           F2 genotyping          F3:4 genotyping           F2:3 phenotyping ...
Ae. sharonensis infected with BSMV           WT     MI    EV   Bax                                                        ...
Acknowledgements2Blades Group           Sainsbury Laboratory   Aberystwyth UniversityNicolas Champouret      Jonathan Jone...
A pipeline for cloning genes effective against african stem rust races
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A pipeline for cloning genes effective against african stem rust races

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A pipeline for cloning genes effective against african stem rust races

  1. 1. The Sainsbury Laboratory 2 Blades Group Brande Wulff, TSL, Norwich UK Brande Stephen John Costas Eric Matt G Matt MJamie Stuart Andrew Richard Cintia Nico Shuzhen Burkhard Jing Inma Phon 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.”
  2. 2. Cost of DNA sequencing $0.09/Mb 2
  3. 3. Principles of plant-microbe interactions PAMPs PAMP receptor Fungus/ Bacterium Oomycete Effector Immunity Effector Immunity Haustorium Resistance protein Plant Cell Dodds & Rathjen 2010
  4. 4. Generic StrategyClone and pyramid R genes Find Map genes Identify and germplasm; with next- test Pyramid make mapping generation candidate populations genetics genes★ No linkage drag ★ R genes cannot be separated ★ Durable?Identify cognate effectors•  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 4
  5. 5. The three rusts of wheatBrown  rust   Black  rust   Yellow  rust  Leaf  rust   Stem  rust   Stripe  rust  Puccinia  tri-cina   Puccinia  graminis  f.  sp.  tri-ci   Puccinia  striiformis  var.  striiformis  
  6. 6. 30  October  2011  “The new wheat has been created by inserting genesthat confer resistance to […] Ug99.”It’s also “highly drought resistant” and “fixes nitrogen!”
  7. 7. Germplasm Sorghum 11 Ae. sharonensis Maize Ae. longissima 2-6 Wheat Ae. bicornis53 12 SitopsisMYA Ae. searsii 36 Barley Ae. speltoides 47 Dvorak et al. 1997 Brachypodium Rice
  8. 8. Aegilops sharonensisAe. tauschii Triticum urartu SitopsisDD AA BB ≈ SS 400,000   years   AABB Wild Emmer 10,000   years   AABBDD Domestication Bread Wheat Pasta Wheat (Triticum aestivum) (Triticum turgidum ssp durum)
  9. 9. Ae. sharonensis distribution and habitatShefayyim Deror Junction Haifa PortAshdod Nahsholim P. Olivera
  10. 10. Ae. sharonensis plant care  Seed drying to break dormancy  Seed peeling: Peeled => 95% germination Non-peeled => 60% germination  Vernalisation Ÿ  Bagging to prevent cross-pollination Ÿ  6-8 months from seed to seedN. ChampouretP. GreenI. Hernandez-PinzonS. JiangJ. SongS. Banfield
  11. 11. Biosafety Level 3 Containment Facility University of Minnesota Jamie Kaufman
  12. 12. Aegilops sharonensis reaction to Ug99 0; ; 1; 1+ 2- 2+3 3 3+4 Resistant Susceptible Reaction of 107 accessions to Ug99 Resistant 69% P. Olivera, Susceptible 13% J. Kaufman, B. Steffenson Intermediate/Heterogenous 18% Uni. Minnesota 13
  13. 13. Ae. sharonensis screening matrix  43 key resistant accessions  6 susceptible accessions African N. American EuropeanAccession TTKSK PTKST TTTTF TPKMC TRTTF JRCQC GO4CD GO41 G5/1826 G425 1644 R R R R R R R R Weak S 2189 S S S S S S n.d. n.d. n.d. n.d. N. Champouret B. Steffenson J. Kaufman P. Olivera Z. Pretorius P. Bartos
  14. 14. Ae. sharonensis population development 43 key resistant accessions 97 crosses 6 different susceptible F1 F2 F2:3 … F4:5 Ae. sharonenesis 97 42 (+26) 4 (+2) 2 Ae. longissima 9 Ae. bicornis 11 Ae. searsii 3 N. Champouret E. Millet P. Olivera 15
  15. 15. Segregation for reaction to three stem rust races in F2 and F2:3 populations Reaction Observed segregation ratioa No. ofSusceptible Resistant progeny 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) 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 N. Champouret eTwo R genes; both semi-dominant, but when either is homozygous it confers full immunity P. Olivera J. Kaufman 16
  16. 16. Segregation of resistance to Ug99 in 1644 × 2189 F2:3 1644 x 2189 F2.3 Obs Exp* χ2 Non-segregating (resistant) 22 21 0.04 Non-segregating (intermediate), Segregating 110 112 0.03 Non-segregating (susceptible) 3 2 0.38 * 10:53:1 ABD ABd AbD Abd aBD aBd abD abdABD AABBDD AABBDd AABbDD AABbDd AaBBDD AaBBDd AaBbDD AaBbDdABd AABBDd AABBdd AABbDd AABbdd AaBBDd AaBBdd AaBbDd AaBbddAbD AABbDD AABbDd AAbbDD AAbbDd AaBbDD AaBbDd AabbDD AabbDdAbd AABbDd AABbdd AAbbDd AAbbdd AaBbDd AaBbdd AabbDd AabbddaBD AaBBDD AaBBDd AaBbDD AaBbDd aaBBDD aaBBDd aaBbDD aaBbDdaBd AaBBDd AaBBdd AaBbDd AaBbdd aaBBDd aaBBdd aaBbDd aaBbddabD AaBbDD AaBbDd AabbDD AabbDd aaBbDD aaBbDd aabbDD aabbDdabd AaBbDd AaBbdd AabbDd Aabbdd aaBbDd aaBbdd aabbDd aabbdd
  17. 17. Sequencing Aegilops sharonensis DNA from 1 accessionGenome 30x illumina HiSeq~7.5 Gb 1.5 Gb assembled seq. 2.5 million contigs N50 = 1 KbGenes RNA from 2 accessions~50 Mb 454 sequencing ~33,000 transcriptsC. BouyioukosB. Steuernagel ~15,000 SNPsN. ChampouretI. Hernandez-Pinzon
  18. 18. Integrated DArT and SNP gene-based genetic map1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1. 1644 x 1193 2. 1644 x 2189 3. 1644 x 2232 Chromosome group 1 2 3 4 5 6 7 1Ssh 2Ssh 3Ssh 4Ssh 5Ssh 6Ssh 7Ssh 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 Moscou, Champouret, Hernández-Pinzón, Bouyioukos, Olivera, Steffenson
  19. 19. QTL mapping of Ug99 resistance in 1644 × 2189 F2 genotyping F3:4 genotyping F2:3 phenotyping F3:4 phenotyping A 3:1 5.5 cM in barley 1Ssh ? 2Ssh 3Ssh 4Ssh B 3:1 37 cM in barley 5Ssh 6Ssh 7Ssh M. Moscou N. Champouret Relative LOD S. Flipo
  20. 20. Ae. sharonensis infected with BSMV WT MI EV Bax BMV RNA RT1 RT2 H 2O L 1 2 3 1 2 3 1 2 3 1 2 3 LEf1α * *Beta * *Gamma * *Bax *Mock Vector Bax-expressing A. Dawson
  21. 21. Acknowledgements2Blades Group Sainsbury Laboratory Aberystwyth UniversityNicolas Champouret Jonathan Jones Luis MurAndrew Dawson Sophien KamounJohn Ferguson Dan MacLean CSIROSixtine Flipo Michael AyliffeMatthew Gardiner John Innes Centre Tony PryorPhon Green Peter Dodds Lesley BoydInma Hernández-Pinzón John DoonanShuzhen Jiang Graham Moore Tel Aviv UniversityCintia KawashimaLucy McCann Magdalena Opanowicz Eitan MilletMatthew Moscou Cristobal UauyJing Song Crop Research InstituteBurkhard Steuernagel NIAB Pavel BartošEric Ward Rosemary BaylesMarie Westergaard Amelia Hubbard University of MinnesotaBrande WulffAlumni: Brian SteffensonPhilippa Borrill Jamie KaufmanCostas BouyioukosStephen Cranwell Pablo OliveraMarie DengremontStuart HarderGopal JhaPeter MatthewsRichard PayneZhe Wang

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