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A pipeline for cloning genes effective against african stem rust races
- 1. The Sainsbury Laboratory 2 Blades Group Brande Wulff, TSL, Norwich UK Brande Stephen John Costas Eric Matt G Matt M Jamie 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. Cost of DNA sequencing $0.09/Mb 2
- 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. Generic Strategy Clone 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
- 6. The three rusts of wheat Brown rust Black rust Yellow rust Leaf rust Stem rust Stripe rust Puccinia tri-cina Puccinia graminis f. sp. tri-ci Puccinia striiformis var. striiformis
- 7. 30 October 2011 “The new wheat has been created by inserting genes that confer resistance to […] Ug99.” It’s also “highly drought resistant” and “fixes nitrogen!”
- 8. Germplasm Sorghum 11 Ae. sharonensis Maize Ae. longissima 2-6 Wheat Ae. bicornis 53 12 Sitopsis MYA Ae. searsii 36 Barley Ae. speltoides 47 Dvorak et al. 1997 Brachypodium Rice
- 9. Aegilops sharonensis Ae. tauschii Triticum urartu Sitopsis DD AA BB ≈ SS 400,000 years AABB Wild Emmer 10,000 years AABBDD Domestication Bread Wheat Pasta Wheat (Triticum aestivum) (Triticum turgidum ssp durum)
- 10. Ae. sharonensis distribution and habitat Shefayyim Deror Junction Haifa Port Ashdod Nahsholim P. Olivera
- 11. 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 seed N. Champouret P. Green I. Hernandez-Pinzon S. Jiang J. Song S. Banfield
- 12. Biosafety Level 3 Containment Facility University of Minnesota Jamie Kaufman
- 13. 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
- 14. Ae. sharonensis screening matrix 43 key resistant accessions 6 susceptible accessions African N. American European Accession 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
- 15. 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
- 16. Segregation for reaction to three stem rust races in F2 and F2:3 populations Reaction Observed segregation ratioa No. of Susceptible 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
- 17. 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 abd ABD AABBDD AABBDd AABbDD AABbDd AaBBDD AaBBDd AaBbDD AaBbDd ABd AABBDd AABBdd AABbDd AABbdd AaBBDd AaBBdd AaBbDd AaBbdd AbD AABbDD AABbDd AAbbDD AAbbDd AaBbDD AaBbDd AabbDD AabbDd Abd AABbDd AABbdd AAbbDd AAbbdd AaBbDd AaBbdd AabbDd Aabbdd aBD AaBBDD AaBBDd AaBbDD AaBbDd aaBBDD aaBBDd aaBbDD aaBbDd aBd AaBBDd AaBBdd AaBbDd AaBbdd aaBBDd aaBBdd aaBbDd aaBbdd abD AaBbDD AaBbDd AabbDD AabbDd aaBbDD aaBbDd aabbDD aabbDd abd AaBbDd AaBbdd AabbDd Aabbdd aaBbDd aaBbdd aabbDd aabbdd
- 18. Sequencing Aegilops sharonensis DNA from 1 accession Genome 30x illumina HiSeq ~7.5 Gb 1.5 Gb assembled seq. 2.5 million contigs N50 = 1 Kb Genes RNA from 2 accessions ~50 Mb 454 sequencing ~33,000 transcripts C. Bouyioukos B. Steuernagel ~15,000 SNPs N. Champouret I. Hernandez-Pinzon
- 19. Integrated DArT and SNP gene-based genetic map 1 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
- 20. 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
- 21. 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 L Ef1α * * Beta * * Gamma * * Bax * Mock Vector Bax-expressing A. Dawson
- 22. Acknowledgements 2Blades Group Sainsbury Laboratory Aberystwyth University Nicolas Champouret Jonathan Jones Luis Mur Andrew Dawson Sophien Kamoun John Ferguson Dan MacLean CSIRO Sixtine Flipo Michael Ayliffe Matthew Gardiner John Innes Centre Tony Pryor Phon Green Peter Dodds Lesley Boyd Inma Hernández-Pinzón John Doonan Shuzhen Jiang Graham Moore Tel Aviv University Cintia Kawashima Lucy McCann Magdalena Opanowicz Eitan Millet Matthew Moscou Cristobal Uauy Jing Song Crop Research Institute Burkhard Steuernagel NIAB Pavel Bartoš Eric Ward Rosemary Bayles Marie Westergaard Amelia Hubbard University of Minnesota Brande Wulff Alumni: Brian Steffenson Philippa Borrill Jamie Kaufman Costas Bouyioukos Stephen Cranwell Pablo Olivera Marie Dengremont Stuart Harder Gopal Jha Peter Matthews Richard Payne Zhe Wang