1. Evolution de novo of stable, heritable resistance to leaf rust ( Puccinia triticina Eriks.) in susceptible wheat following cycles of infection with WSMV ( Wheat Streak Mosaic Virus ) Steve Haber 1, D.L. Seifers 2, P. Seto-Goh 1 & B. McCallum 1 1 AAFC-Cereal Research Centre, 195 Dafoe Rd., Winnipeg MB R3T 2M9 2 Agricultural Research Center, KSU-Hays, Hays KS 67601-9228, U.S.A.

2. …Yeah, looks good, farmers like it, farmers like it, low polyphenol oxidase, low polyphenol oxidase, makes great Korean noodles… makes great Korean noodles…

3. …BUT, terrible rust resistance, and it sure gets hammered and it sure gets hammered by WSMV… by WSMV…

4. Whadda we gonna do???

5. Wheat streak mosaic virus naturally transmitted by wheat curl mite (Aceria tosichella) experimentally transmitted by rub-inoculation

6. 6 The Tiller Effect expression Variable expression of traits WSMV (e.g. resistance to WSMV) in the same plant …expansion of range of variation??

7. Extreme variationWSMV Extreme variation in response to WSMV: Tiller Effect Generation grown from seed experiencing Tiller Effect

8. Could we harness this heritable variation in expression to evolve resistance to Leaf Rust de novo???

9. Lakins progress Lakins progress KSU-ARCH natural WSM 1 st cycle 100 healthy plants WSM (x1) 100 WSM (x1) plants of plants in field affected by ( < 1% of plants in field affected by WSM ) WSM (x1) Seed from WSM (x1) plants healthy Seed from healthy plants Field2001-02

10. Lakins progress Lakins progress Field2001-02 1 st cycle 2 nd cycle Indoors2002-03 Grow out plants rub-inoculate @ 2-leaf stage vernalize 6 weeks WSM (x2) 572 surviving WSM (x2) plants WSM (x2) 572 WSM (x2) sublines WSM(x1) Seed from WSM(x1) plants

11. Lakins progress Lakins progress 1 st cycle 2 nd cycle 3 rd cycle WSM (x2) 572 WSM (x2) sublines 572 headrowsHealthy controls ( little ) natural disease pressure * Harvest seed from 200 headrows200 rows 169g bulk seedBulk seed WSM(x1) Seed from WSM(x1) plants Field2001-02 Indoors2002-03 Field2003-04

12. 12 Lakins progress Lakins progress 3 rd cycle 4 th cycle 169g bulk seedBulk seed 2m x 60m plot heavy disease pressure * Leaf Rust * Stripe Rust * Identify individuals resisting R1R1R1R1 R2R2R2R2 R3R3R3R3 R4R4R4R4 R5R5R5R5 R1R1R1R1 ---n/a--- R1 Field2003-04 Field2004-05

13. Lakins progress Lakins progress Leaf Rust } 4 th cycle 5 th cycle 2005-06 indoors indoors R1R1R1R1 Stripe Rust} resistant contd R1 34 R1 seed vernalize 6 weeks R1 34 R1 plants R1 30 R1 seed transplant R1 21 R1 plants R1 55 R1 plants survivors grown indoors to maturity R1-derived 55 R1-derived sublines ProgressiveNecrosis 5 plants #2#2 #3#3 #1#1 #4#4 Fred Field2004-05 Field

14. Lakins progress Lakins progress 2006-07 6 th cycle Indoor seedling tests Field Test contd R1-derived 55 R1-derived sublines Leaf Rust Stripe Rust all Susc. 12 mixed 35 all Res. 8 55 all Susc. mixed all Res. Necrosis 12358 22228 3 3121 #3#3 Fred Lakin Lakin (orig.)

15. Lakin R1-descendants Lakin v R1-descendants LineKSU-ARCH Stripe Rust Field testKSU-ARCH L.Rust (MKPS) Field testCRC-Wpg LR (TDBG) Seedling test Necrosis Lakin (1-7-5) SS 15/15 mesothetic (;/3+) NO 2-6-2n/a 15/15 Resistant (0;) Uniform 2-4-5 RR 10/10 Resistant (0;) NO 4-3-3 R S 8 mesothetic (;/3+) 7 Susceptible (3+) NO 4-12-3 RR 13 Resistant (0;) 6 mesothetic (;/3+) 1 Susceptible (3+) NO

16. ...OK…Evolving resistance to Leaf Rust de novo looks promising for Lakin…

17. ...BUT…Will it work for other wheats, including spring wheats???

18. ...Lets try it with Thatcher !? Thatcher !?

19. Thatchers progress Thatchers progress 4-wk vernalization quasivernalization 1 st cycle CRC-Winnipeg 10 Thatcher (spring wheat) seeds 10 Thatcher seedlings WSMV rub-inoculate WSMV @ tillering WSMV-resistance Tillers expressing WSMV-resistance A071 qvA071ws1.2m Indoors2008

20. 20 Thatchers progress Thatchers progress 1 st cycle 2 nd cycle A071 qvA071ws1.2m WSMV Altered response to WSMV 30 seeds 30 seedlings rub-inoculate WSMV rub-inoculate WSMV @ tillering WSMV-resistance Tillers expressing WSMV-resistance A267 A267ws3.1tB Indoors2008 Indoors2008-09

21. 21 Thatchers progress Thatchers progress A267 A267ws3.1tB Indoors2008 1 st cycle 2 nd cycle Indoors2008-09 3 rd cycle Field2009 Altered response to WSMV A071 qvA071ws1.2m Altered response to WSMV FHB nursery Head-row (21 seeds) FHB nursery erFH09_i011 Selectspecificheads A505

22. 22 Thatchers progress Thatchers progress 4 th cycle Indoors2009-10 contd A505 24 seeds 24 seedlings WSMV rub-inoculate WSMV @ tillering Thatcher Breeders seed ThatcherA505 WSMV-resistance 22/24 plants express WSMV-resistance 14 10 ThatcherA505ws1.07 TDBG Leaf Rust 21/24 express Adult-plant Resistance

23. Thatchers progress Thatchers progress 4 th cycle 5 th cycle contd de novo WSMV resistance de novo Leaf Rust (TDBG) resistance A505 A505rw1.07m A699 14 seedlings TPMK Stem Rust @ 2-leaf stage A699rs1.2 Thatcher 2+ ;1- TDBG Leaf Rust @ Adult-plant stage Thatcher 33- A699rs1.2 0; 11/14 (more) Resistant 11/14 Resistant Indoors2009-10 Indoors2010

24. Thatchers progress Thatchers progress 5 th cycle 6 th cycle contd de novo Leaf Rust (TDBG) resistance (Adult plant) de novo Stem Rust (TPMK) resistance (seedling) A699rs1.2 A829 10 seedlings 0 TDBG Leaf Rust @ 2-leaf stage WSMV rub-inoculate WSMV @ tillering @ Adult-plant stage Indoors2010 Indoors2010 Thatcher 3+ A829rs1.7 1-2= TDBG Leaf Rust 0 A829rs1.7 ;1-2=

25. ...OK…Evolving resistance to Leaf Rust de novo appears to work for Lakin…AND… Thatcher… Thatcher…

26. BUT…it would sure help the case if we had an idea how the de novo Leaf Rust resistance is inherited !?

27. ...Yeah… Gotta make crosses and look at progeny!

28. 28 Inheritance of de novo LR-resistance Inheritance of de novo LR-resistance LineKSU-ARCH Stripe Rust Field testKSU-ARCH L.Rust (MKPS) Field testCRC-Wpg Lr (TDBG) Seedling test Necrosis 4-3-3 R S 8 mesothetic (;/3+) 7 Susceptible (3+) NO 4-12-3 RR 13 Resistant (0;) 6 mesothetic (;/3+) 1 Susceptible (3+) NO......choosing parents for a cross

29. 4-3-3 4-12-3 99359938 0 9935rs1.69938rw1.2 (;1-/3+ mesothetic) WSM (0; Resistant no WSM symptoms) X A124 (F 1 )( 24/24 0;) Lr-TDBG Lr-TDBG Resistant Seed Seedlings Lr-TDBG Leaf Rust @ 2-leaf stage WSMV WSMV @ tillering WSM ( 3/8 no WSM symptoms ) WSMV WSMV Resistant

30. A124 ( F 1 ) Inheritance of de novo Lr-resistance...Reassortment in daughter generations (24/24 0;) Resistant MKPS 78 Res. 29 Susc. ~ 3 : 1 TDBG 100 Res. 7 mesothetic 18 Susc. (3+) ~ 3 : 1 F2F2 A124ws3.2 t A MKPS F 2 plants @ 2-leaf stage 9 Res. 4 Susc. TDBG 11 Res. 2 mesothetic 1 Susc. (3+) Seed from F 1 plant WSMV @ tillering A368ws1.1m Lr-TDBG Lr-TDBG Resistant WSMV WSMV Resistant Lr-TDBG

31. Inheritance of combined de novo Lr WSMV Lr & WSMV Resistance...Example of selection for true-breeding line A368ws1.1m F 3 plants TDBG @ 2-leaf stage 15/15 ( 0; ) WSMV @ tillering 1/3 Res. 25 seeds 25 seeds to KSU-ARCH 2009/10 Trial Row14 Seed from F 2 plant A486 LakinA486 A486ws1.1

32. Inheritance of combined de novo Lr WSMV Lr & WSMV Resistance...Example of selection for true-breeding line WSMV Half-row rub-inoculated Fall 2009 KSU-ARCH 2009/10 Trial Row 14 F 3 plants Seed from F 2 plant April 12 (2010)

33. Inheritance of combined de novo Lr WSMV Lr & WSMV Resistance...Example of selection for true-breeding line WSMV Half-row rub-inoculated Fall 2009 KSU-ARCH 2009/10 Trial Row 14 F 3 plants Seed from F 2 plant April 26

34. Inheritance of combined de novo Lr WSMV Lr & WSMV Resistance...Example of selection for true-breeding line WSMV Half-row rub-inoculated Fall 2009 KSU-ARCH 2009/10 Trial Row 14 F 3 plants Seed from F 2 plant May 11

35. Inheritance of combined de novo Lr WSMV Lr & WSMV Resistance...Example of selection for true-breeding line WSMV Half-row rub-inoculated Fall 2009 KSU-ARCH 2009/10 Trial Row 14 F 3 plants Seed from F 2 plant May 27 Lakin Row 14 8/8 Resistant

36. Inheritance of combined de novo Lr WSMV Lr & WSMV Resistance...Example of selection for true-breeding line WSMV Half-row rub-inoculated Fall 2009 8/8 Resistant KSU-ARCH 2009/10 Trial Row 14 F 3 plants Naturalinoculum Stripe Rust Row 14Lakin Seed from F 2 plant May 27

37. Inheritance of combined de novo Lr WSMV Lr & WSMV Resistance...Example of selection for true-breeding line WSMV Half-row rub-inoculated Fall 2009 8/8 Resistant KSU-ARCH 2009/10 Trial Row 14 F 3 plants 0 Naturalinoculum Stripe Rust Row 14Lakin Seed from F 2 plant May 27 Lakin Row 14 Leaf Rust June 3

38. Summary Lakin Row 14 Row 14 Leaf Rust Field test (MKPS) (TDBG) Stripe Rust Field test Seedling WSMV Indoor test Field test MS (2) R (0) S (3+) R (0) R (0;) S R S S R R Thatcher A829 A829 Leaf Rust (TDBG) Seedling Adult R (0;1=) WSMV Indoor test R S Stem Rust Seedling (TPMK) MR (;1=) MS (2+) S (3+) MR (;12=)

39. Conclusions Epigenetics discovers & identifies …Genetics fixes & exploits …Genetics fixes & exploits Modifying expression Modifying expression of existing gene alleles affords a new tool gene introgression a new tool to complement gene introgression Susceptible progenitor Resistant descendants Cycles of selfing Selection and advance under pressure from systemic stress(es)

40. 40 Acknowledgements Brian Gillis Jeff Ackerman Pat Seto-Goh Taye Zegeye KANSAS WHEAT COMMISSION for funding support …and