1. FR-H3: A New QTL to Assist in the Development of Fall-Sown Barley with Superior Low Temperature Tolerance

2. Outline Introduction Germplasm Linkage Maps Phenotyping & QTL Analysis Conclusions and Implications

3. Introduction

4. Fall-Sown Malting Barley New to the U.S. Two varieties approved by AMBA ◦Both two-row, lack LTT 49% malting barley planted has been six- row over the past 5 years Most selection has been done within a spring growth habit framework Advantages ◦Higher Yielding ◦Conserves Water

5. Adaptation to Target Regions Winter Hardiness Disease Resistance

6. Winter Hardiness Complex Trait Vernalization Sensitivity Photoperiod Sensitivity Low Temperature Tolerance

7. Vernalization Sensitivity The requirement of a period of low temperature in order to transition from the vegetative to reproductive state

8. Photoperiod Sensitivity Flowering is dependent on sufficient day length ◦Short Day ◦Long Day

9. Low Temperature Tolerance The capacity to survive low temperatures Induced response Reduced after reproductive transition

10. Defined by LTT, VS, & PS Winter ◦Varying LTT, VS, PS varies Facultative ◦Varying LTT, No VS, PS varies Spring ◦Lack LTT and VS, Short PS irrelevant Growth Habits

11. Winter Hardiness: VS Winter Genotype: Vrn-H2_/vrn-H1vrn-H1/vrn-H3vrn-H3

12. Winter Hardiness: PS PPD-H1 ◦Recessive Allele Insensitivity to long photoperiods PPD-H2 ◦Deletion Short PS

13. Winter Hardiness: LTT FR-H1 ◦VRN-H1? FR-H2 ◦≥11 C-repeat binding factor genes ◦Copy number variation

14. Low Temperature Tolerance Studies FR-H1 first reported Dicktoo x Morex (Hayes et al. 1993) FR-H2 discovered Nure x Tremois (Francia et al. 2004) FR-H1 and FR-H2 validated through GW- AM (von Zitzewitz et al. 2011) Galiba et al. 1995 Skinner et al. 2005 Francia et al. 2007 Stockinger et al. 2007 Galiba et al. 2009

15. Is LTT due only to FR-H1 and FR- H2 or are there other, undiscovered determinants of this critical trait?

16. Resistance to Biotic Stresses Barley Stripe Rust ◦Incited by Puccinia striiformis f. sp. hordei ◦Prominent in Pacific NW  First reported in Oregon in 1995 ◦Spread by wind QTL and Genes Identifies Chen et al. 1994 Hayes et al. 1996 Toojinda et al. 2000 Castro et al. 2003 Vales et al. 2005 Rossi et al. 2006

17. Scald ◦Incited by Rhynchosporium secalis ◦Spread by splashing rain ◦Reduce functional green leaf tissue on plant Many Genes and QTL identified Fungicides effective but more effective to deploy resistance genes Pyramiding resistance genes ◦Effective and durable Resistance to Biotic Stresses Abbott et al. 1992 Garvin et al. 1997 Jensen et al. 2002 Cheong et al. 2006 Li and Zhou 2011

18. Overall Scheme Two doubled haploid populations Genotyping ◦Custom 384-OPA Linkage Maps (JoinMap 4) Phenotyping ◦LTT ◦VS (FT & FLN) ◦BSR ◦Scald QTL analysis (Windows QTL Cartographer) Pooled data when appropriate

19. Germplasm Two doubled haploid populations (half-sibs) ◦NB3437f/OR71 (N=111) and NB713/OR71 (N=124) OR71 ◦Facultative, Moderate LTT, BSR resistant, good malting quality NB3437f ◦Facultative, High LTT, BSR susceptible, poor malting quality NB713 ◦Winter, High LTT, BSR susceptible, poor malting quality

20. NB3437f NB3437 bulk seed lot grown <1% flowered without vernalization VRN-H2 deletion confirmed

21. Genotyping Parents previously genotyped with Barley OPA 1 & 2 (Barley CAP) ◦3,072 SNPs total Custom 384-OPA ◦Barley consensus map (Close et al. 2009) ◦Maximize polymorphism, genome coverage, focus on specific regions/genes of interest  Concurrent MAS study

22. Linkage Mapping NB3437f/OR71 (220 SNPs) NB713/OR71 (217 SNPs) Combined population (157 SNPs)

23. NB3437f/OR71 3_1324 0.0 1_1345 2.8 1_0319 5.6 1_0738 23.8 3_08391_0881 53.8 2_0482 54.7 2_0453 56.5 1_1224 62.2 2_0451 64.1 2_0815 66.9 2_0670 77.9 1_1213 83.6 2_0384 89.4 3_0142 94.1 3_0554 113.7 2_0119 121.5 1_0510 124.2 2_0974 152.6 3_0385 154.4 1_1233 155.3 1_0697 160.0 2_1210 162.8 1_0387 176.2 3_1422 177.1 2_1221 0.0 2_0206 0.9 2_0010 12.0 3_0167 19.7 3_0531 24.5 3_0654 0.0 1_1260 2.8 2_1536 6.5 3_0538 8.4 2_1275 11.1 3_0080 16.9 2_1133 22.6 2_0096 25.4 2_0645 31.1 3_0850 68.1 2_0795 70.8 3_1278 0.0 2_0653 13.7 1_1490 0.0 3_0162 47.4 2_0686 57.4 3_0577 0.0 1_1364 0.9 1_0401 2.8 1H2H 3H4H5Ha6H7H 5Hb 5Hc 5Hd 5He

24. NB713/OR71 2_0502 0.0 3_1144 2.5 2_1174 15.3 3_0951 22.2 2_0749 38.0 1_0764 64.3 2_1134 65.9 3_1467 67.5 1_0938 72.6 3_0350 75.1 1_0293 76.8 2_1431 85.5 2_0956 88.5 3_0298 95.8 1_0006 102.7 2_0990 105.2 2_0434 113.2 2_0187 138.1 2_0021 151.6 3_0257 162.5 2_0908 168.5 1_0693 177.3 1_0782 185.2 2_0915 200.3 3_1324 0.0 1_1345 2.5 1_0319 7.6 1_1300 13.6 3_0150 16.1 3_0864 47.6 3_0993 85.6 3_0866 93.5 1_0881 95.1 2_0361 98.2 2_0453 99.2 1_1224 103.4 2_0451 105.9 1_0090 109.3 2_0197 122.1 1_1213 132.8 2_0384 139.8 2_0515 158.2 159.0 166.8 167.7 168.6 180.0 183.5 186.9 1_0510 2_0974 3_0385 1_1233 2_1210 3_0889 3_0006 3_1422 187.7 3_0543 0.0 2_0226 2.5 3_0714 10.3 3_0167 16.3 3_0654 63.8 2_1536 68.9 3_0538 73.9 1_1281 77.3 2_1275 80.6 2_0350 86.6 1_0953 87.4 2_1133 94.3 2_0096 99.4 3_0314 102.8 3_1361 116.6 3_0834 128.5 2_0850 132.7 1_0414 139.6 2_0403 142.9 2_1314 145.5 3_0456 153.3 3_0619 157.6 1_0094 194.1 2_0653 199.2 1_1490 0.0 1_1497 0.8 1_0820 5.9 2_0536 37.1 1_0736 41.4 3_0577 44.8 1_1364 49.9 1_0401 52.4 1_0894 0.0 3_1350 0.8 1H2H 3H4H5Ha6H7Ha 5Hb 7Hb

25. Combined Population 2_0502 0.0 3_1144 1.3 3_0951 16.2 1_0764 63.2 2_1134 66.3 3_1467 68.1 1_0938 73.0 3_0350 74.3 1_0293 1_0324 75.2 2_1431 83.6 2_0956 85.4 3_0298 91.2 1_0006 99.1 2_0990 100.9 2_0434 108.8 2_0187 132.9 2_0021 145.7 3_0257 158.9 2_0908 162.9 1_0782 183.3 3_1284 0.0 1_0180 6.9 2_1015 19.0 3_0432 63.9 3_0363 91.5 1_1046 101.3 2_1166 111.8 2_1144 114.4 3_1383 118.0 2_0667 120.2 2_0699 123.7 2_0340 132.1 1_0475 133.4 1_0969 133.8 1_0876 142.8 3_0049 153.9 1_0916 181.0 3_0152 186.5 1_1227 199.6 1_0315 218.7 1_1380 222.3 3_1527 231.7 2_1453 236.2 3_0378 242.1 1_1345 0.0 1_0319 4.0 1_0881 0.0 2_0361 2.0 2_0453 3.5 1_1224 8.5 2_0451 10.7 1_1213 37.2 2_0384 43.7 1_0510 74.8 2_0974 91.4 3_0385 92.7 1_1233 93.7 2_1210 103.0 3_1422 113.0 3_0654 0.0 1_1260 1.3 2_1536 5.8 3_0538 9.4 2_1275 14.3 2_1133 28.1 2_0096 32.1 3_1278 0.0 2_0653 41.4 3_0162 0.0 2_0686 39.9 3_0577 88.0 1_1364 91.1 1_0401 93.3 5Hb 2_1032 0.0 3_1485 36.2 2_0052 46.1 1_0817 58.2 1_0461 59.1 1_0003 59.5 3_1187 60.4 1_1067 66.3 2_0058 68.9 2_1298 76.3 3_0637 77.6 2_0969 88.1 3_1253 90.7 1_1246 92.9 1_1147 98.3 2_0783 101.0 1_1294 107.9 3_1353 115.3 2_0448 136.2 3_1495 140.8 2_1477 145.7 3_1392 150.2 1_0828 159.2 1H2H 3H4Ha5Ha6H7H 5Hd 4Hb 3_0850 0.0 2_0795 2.2 5Hc

26. Phenotyping LTT ◦Controlled Environment  MRI ◦Field Conditions  SPMN, LNE, FCCO, FMT Final Leaf Number ◦GH: V+/V- Flowering Time ◦GH: V+/V- ◦Field: COR Disease Resistance ◦BSR and Scald  COR

27. LTT: MRI and SPMN

28. LTT: LNE & FCCO

29. LTT QTL Results FR-H3 FR-H2 VRN-H2

30. 5H and MRI QTL Scans

31. Final Leaf Number

32. FLN QTL Results VRN-H2 VRN-H3 VRN-H2

34. Flowering Time

35. FT QTL Results VRN-H3 VRN-H2 PPD-H2

36. Disease: BSR and Scald

37. BSR & Scald QTL Results Rps4 Rrs15 Rrs2

38. Combined Population QTL Analysis VRN -H3 Rps4 FR-H3 FR-H2

39. Conclusions and Implications

40. LTT: Challenges and Opportunities Assessing LTT is challenging Lowest Temperatures ◦SPMN: -27°C; LNE: -26°C; FCCO: -25°C; FMT: -32°C; MRI: -13.5°C  No Survival at FMT  Nearly complete survival FCCO Other factors Controlled Freeze Tests $$ SPMN has been informative

41. FR Loci Expectations FR-H1 ◦~24cM region between flanking markers  All parents monomorphic within 5 cM (n = 27 markers) FR-H2 ◦Expected QTL  Marker polymorpisms  Differences in LTT ◦Complicated by 5H fragmentation

42. 5H Fragmentation Chromosome rearrangements ◦e.g. Inversion Copy Number ◦CBF genes related to degree of LTT (Stockinger et al. 2007) Heterozygosity in Parents ◦Genotyping of parents revealed no heterozygosity ◦Multiple plants used for crossing not all were genotyped ◦Retention of heterozygosity? High density mapping, cytogenetics

43. FR-H3 Discovery Genetic basis of superior LTT in NE unknown: necessary for molecular breeding Previous Reports on 1H (Hayes et al. 2003) ◦Dicktoo x Morex  Pleiotropic effect of PPD-H2  PPD-H2 distal to FR-H3 SPMN ◦Additive effect of 29% in combined population

44. Vernalization Sensitivity: validation and discovery Role of VRN-H2 Confirmed ◦NB3437f x OR71  All progeny flowered under V- conditions  VRN-H2 deletions ◦NB713 x OR71  Segregated for VS  VRN-H2 presence/absence

45. Unexpected role of VRN-H3 Variation in exotic germplasm Wong ◦Released: China 1941 ◦Pedigrees of NB3437f & NB713 NE contributed the early flowering allele VRN Model ◦Recessive vrn-H3 required for VS ◦Dominant Vrn-H3 flowers very early ◦NB713 has VS  alternative recessive allele ◦Recessive allele triggered only under long days Vernalization Sensitivity: validation and discovery

46. LTT and VS: uncoupled NB3437f x OR71 and NB713 x OR71 similar LTT values NB713 x OR71 ◦High survival with and without VS ◦VRN-H2 QTL was detected in MRI  VS may have delayed vegetative to reproductive transition through critical times

47. NB713 x OR71: Facultative Vs. Winter

48. BSR Resistance First report in fall-sown barley background Resistance donors ◦Kold or Strider  Winter six-rows released by OSU  Effective resistance since BSR first reported in the area in 1995 Large-effect QTL on 1H ◦58-88% variation Favorable 1H LTT and BSR alleles in repulsion ◦Progeny with high survival and BSR resistance ◦~20 cM apart in combined map

49. Combined Population QTL Scan

50. Scald Resistance All parents have some resistance ◦Selected for “field resistance” NB3437f x OR71 ◦Favorable alleles on 4H and 7H contributed by NB3437f NB713 x OR71 ◦Favorable alleles on 2H and 7HL contributed by OR71 Results confounded by year effect ≥4 loci segregating

51. Prospects for Disease Resistant, LT Tolerant Fall-Sown Barley Fall-sown barley advantages ◦e.g. Yield and water conservation Pre-requisites ◦Sufficient LTT, disease resistance, grain with a premium Assessment of malting quality not possible ◦BSR (COR) and LT injury (SPMN)

52. OrNe Germplasm Continuation Used in crossing ◦LTT and scald resistance Limited number incorporated in breeding program ◦OBELT Included in association mapping panel in the TCAP (http://triticeaecap.org/) ◦Assessed for multiple phenotypes including malt quality

53. Final Thoughts Multitude of favorable alleles discovered for future MAS and GS schemes Newly discovered FR-H3 and superior Nebraska FR-H2 ◦Targets for LTT introgression Finding undiscovered QTL shows that the search for more LTT QTL should continue as well as identifying candidate genes and causal polymorphisms

54. Acknowledgements OSU Barley Project ◦Patrick Hayes ◦Alfonso Cuesta-Marcos ◦Ann Corey ◦Tanya Filichkin ◦Yada Chutimanitsakun ◦Natalie Graham Committee Members ◦Glenn Howe, Tony Chen, James Hermes Collaborators ◦CSIS: Luis Cistue ◦UNL: Stephen Baenziger ◦UM: Kevin Smith ◦Anheuser-Busch: G. Hanning, J. Heward ◦MRI: Ildikó Karsai, Zoltan Bedo ◦JHI: J. Russell, Robbie Waugh USDA/AFRI

55. Questions

56. Are You Feeling OrNe Baby?