Insight into the genetic basis of winter hardiness and the potential this has to alter US malting-quality barley

Eric J Stockinger AMBA Barley Improvement Conference San Diego, CA January 14, 2013 Insight into the genetic basis of winter hardiness and the potential this has to alter US malting-quality barley

Winter hardiness The plant breeder who developed Maris Otter and Director of the Plant Breeding Institute in Cambridge England, Dr. George Douglas Hutton Bell (G.D.H. Bell), 1944: there is no absolute difference between spring and winter types, and between hardy and non-hardy types, but the differences are essentially quantitative showing a gradation from extreme early spring types to extreme winter types, and from lack of hardiness to extreme winter hardiness. Bell GDH, 1944. The breeding of two-row winter-hardy barley. Journal of Agricultural Science 34, 223-38.

Winter hardiness Complete winter kill (2002/2003) Partial winter kill (2006/2007)

Winter vs. spring types Winter types Plants in vegetative phase Vernalization induces reproductive phase Spring types Inherent reproductive competence

Barley types cultivated in different regions in 1952 Poehlman JM (1952) Breeding winter barley for hardiness and disease resistance. Econ. Bot. 6, 176-84.

Screening winter barley germplasm (Columbia MO, winter 1942-1943) Number of varieties with survival of Variety source 0% 1 20% 21 40% 41 60% 61 80% 81 100% Tennessee Winter - - - 2 25 26 Korea 2 10 11 19 48 1 China-Manchuria 9 14 35 37 12 2 Caucasus 24 16 52 80 34 - Western Europe 39 3 3 3 11 - Mediterranean 32 14 4 3 1 - Ethiopia 91 1 2 4 1 - Japan 2 6 4 1 1 - India 12 1 - - - - Poehlman JM (1952) Breeding winter barley for hardiness and disease resistance. Econ. Bot. 6, 176-84.

The top 10 winter-hardy barleys (of 204 assayed 1937-1956) 1. Kearney 2. Dicktoo 3. OAC 4GH1 4. Dayton 5. Nebraska 52434 6. Nebraska 53417 7. Admire 8. Kansas 9. OAC 30GH10 10. PU B466A7772 Wiebe GA and Reid DA (1958) Comparative winter hardiness of barley varieties. Technical Bulletin. United States Department of Agriculture 1176, 20 pp.

Pedigree of the most successful winter malting barley Fischbeck G (1992) Barley cultivar development in Europe - success in the past and possible changes in the future. In Barley Genetics VI; Proceedings of the Sixth International Barley Genetics Symposium. Munck L (ed.), (ed.), Vol. II, pp. 885-901, Munksgaard International Publishers, Ltd, Copenhagen, Denmark, Helsingborg, Sweden.

Potential for winter malting barley in the US Variety Row On 6/64" Winter survival (%) *, (%) * Tschermak 2 46% 12% Carstens 2 52% 23% MO B475 6 9.3% 57% * Based on three Missouri locations 1962 1963 Minimum acceptable: 6-row 70%; 2-row 80% Duelos LA, Poehlman JM, and Hoskins PH (1970) Breeding 2-row winter-type malting barley. In Barley genetics II. Proceedings of Second International Barley Genetics Symposium. (ed.), Vol. pp. 283-86, Washington State University, Pullman.

Winter survival of lines selected (Columbia Missouri 1969) Cross or accession Percentage of F 6 lines in percent survival class 1 19% 20 39% 40 59% 60 79% 80 99% MO B475 Carstens 6 18 45 31 - Ludwig Carstens 3 6 33 52 6 MO B1300 Carstens 49 28 23 - - MO B1400 Carstens 37 46 17 - - MO B475 Tschermak 11 44 33 12 - Ludwig Tschermak 7 15 9 37 32 MO B1337 Tschermak 3 12 7 46 32 MO B1400 Tschermak 9 13 37 41 1 MO B475 - - - 33 67 Carstens 42 53 5 2 Duelos LA, Poehlman JM, and Hoskins PH (1970) Breeding 2-row winter-type malting barley. In Barley genetics II. Proceedings of Second International Barley Genetics Symposium. (ed.), Vol. pp. 283-86, Washington State University, Pullman.

Winter survival (Columbia Missouri, 1970 72) Selection Pedigree Survival (%) MO B2126 MO B475 Tschermak 77 MO B2185 MO B1400 Tschermak 76 MO B2186 MO B1400 Tschermak 77 MO B2190 MO B1400 Tschermak 69 MO B2191 MO B1400 Tschermak 66 MO B2192 MO B1400 Tschermak 72 MO B475 Admire Missouri Early Beardless 81 Carstens 58 Poehlman JM, Duclos L, Kruse C. (1973) Progress in development of two-row winter malting barley. Proceedings of the Barley Improvement Conference, Minneapolis, Minnesota, 15-21.

Genetic analyses of winter hardiness (the Dicktoo Morex population) Dicktoo LT 50 = -8 o C (Six-row winter) Morex LT 50 = -4 o C (Six-row spring) F 1 Generate 100 doubled haploid lines from F 1 creating an immortalized F 2 population Winter hardiness: Score field survival in Oregon and Montana Freezing tolerance: Score regrowth after controlled freezes at 0, -4, -8, -12 C Associated biochemical traits: Score field material for crown fructan content Quantitative trait loci analysis (QTL): Test for association between traits and regions of the barley genome Hayes PM, Blake T, Chen THH, et al., 1993. Quantitative trait loci on barley (Hordeum vulgare L.) chromosome 7 associated with components of winterhardiness. Genome 36, 66-71.

Genetic analyses of winter hardiness (the Nure Tremois population) Nure LT 50 = -11 o C (Two-row winter) Tremois LT 50 = -6 o C (Two-row spring) F 1 Generate 136 doubled haploid lines from F 1 creating an immortalized F 2 population Winter hardiness: Score field survival in Northern Italy Freezing tolerance: Score regrowth after controlled freezes at -11 and -13 o C Associated biochemical traits: Score field material COR14B protein accumulation Quantitative trait loci analysis (QTL): Test for association between traits and regions of the genome Francia E, Rizza F, Cattivelli L, et al., 2004. Two loci on chromosome 5H determine low-temperature tolerance in a 'Nure' (winter) 'Tremois' (spring) barley map. Theor. Appl. Genet. 108, 670-80.

Two loci have major effects on winter hardiness CBF VRN-H1 Francia E, Rizza F, Cattivelli L, et al., 2004. Two loci on chromosome 5H determine low-temperature tolerance in a 'Nure' (winter) 'Tremois' (spring) barley map. Theor. Appl. Genet. 108, 670-80. Skinner JS, Szucs P, Von Zitzewitz J, et al., 2006. Mapping of barley homologs to genes that regulate low temperature tolerance in Arabidopsis. Theor. Appl. Genet. 112, 832-42.

CBFs binds CRT/DREs and activate genetic pathways increasing freezing tolerance Stockinger et. al., 1997; Gilmour et. al., 1998; Jaglo-Ottosen et. al., 1998

FROST RESISTANCE-H2 FR-H2 has >13 CBFs In Dicktoo and Nure, CBF2A and CBF4B are present in multiple copies in tandem array Nure Tremois In Morex and Tremois, CBF2 and CBF4 are present in single copy Knox AK, Dhillon T, Cheng H, Tondelli A, Pecchioni N, Stockinger EJ, 2010. CBF gene copy number variation at Frost Resistance-2 is associated with levels of freezing tolerance in temperate-climate cereals. Theor. Appl. Genet. 121, 21-35. Francia E, Barabaschi D, Tondelli A, et al., 2007. Fine mapping of a HvCBF gene cluster at the frost resistance locus Fr-H2 in barley. Theor. Appl. Genet. 115, 1083-91.

CBF14 copy numbers vary across wheats (and between classes) Hard Red Wheats (Crimean landrace origins) Soft Red Wheats (W. N. European landrace origins) B A D Knox AK, Dhillon T, Cheng H, Tondelli A, Pecchioni N, Stockinger EJ, 2010. CBF gene copy number variation at Frost Resistance-2 is associated with levels of freezing tolerance in temperate-climate cereals. Theor. Appl. Genet. 121, 21-35.

Rhg1 copy number variation in soybean Cook DE, Lee TG, Guo X, et al., 2012. Copy number variation of multiple genes at Rhg1 mediates nematode resistance in soybean. Science 338, 1206-9.

2012 Malting quality data Accession On 6/64 (%) Barley Protein (%) DP ( o ASBC) Alphaamylase (20 o DU) Beta-glucan (ppm) Quality Score Overall Rank 'Maja' 21.8 13.2 199 70.6 52 50 1 'Endeavor' 40.0 11.4 165 *102.2 69 45 2 'Charles 51.7 11.4 137 *98.3 57 41 3 'Wintmalt' 83.4 12.2 124 52.6 165 28 11 'Maris Otter' 33.4 9.6 86 37.5 122 15 28 'Halcyon' 42.6 11.8 107 39.2 309 19 20 'Pipkin' 40.0 10.8 94 49.1 199 9 52 'Puffin' 80.6 11.8 99 42.3 311 31 6 'Signal' 61.7 13.6 155 58.0 117 31 6 'Trigger' 96.6 13.3 109 55.2 420 28 11 MO B2126 69.7 12.5 74 28.4 692 10 45 MO B2169 78.7 13.1 102 43.0 489 17 24 MO B2171 78.7 12.4 106 46.8 403 25 14 MO B2186 75.8 13.0 91 42.4 522 10 45 MO B2247 75.8 12.5 92 40.7 490 17 24 MO B2549 77.1 *14.9 163 52.3 422 28 11 Blue bold text = values below ideal minimum AMBA quality standard Green bold text = values above ideal maximum AMBA quantity standard

Winter survival and freezing tolerance Accession Winter survival (Elora Research Station) Fv/Fm Control -6 o C -8 o C -10 o C -12 o C Cassata 1.2 0.88 0.86 0.75 0.46 0.34 Flagon 0.8 0.88 0.89 0.78 0.44 0.36 Maris Otter 0.0 0.88 0.84 0.71 0.42 0.30 Pearl 6.5 0.89 0.87 0.84 0.41 0.40 Macgregor 17.5 0.88 0.87 0.76 0.33 0.45 McKellar 15.0 0.88 0.87 0.77 0.43 0.55 H260-008 13.8 0.88 0.86 0.86 0.24 0.63 H260-017 10.2 0.88 0.85 0.80 0.45 0.52 H262-021 8.0 0.88 0.86 0.84 0.48 0.46 H262-003 7.8 0.89 0.86 0.83 0.52 0.51 H262-033 9.0 0.89 0.87 0.84 0.61 0.43 H271-003 17.5 0.88 0.87 0.84 0.58 0.55 Kawartha (SB) n.a. 0.88 0.76 0.64 0.30 0.22 Maxine (WW) n.a. 0.90 0.88 0.88 0.75 0.64 Larson, R. J. A. (2012) Winter hardy spring wheat breeding: analysis of winter spring wheat germplasm and the development of selection tools. Ph.D. Thesis, University of Guelph.

What is the underlying molecular basis of FR-H1?

Insight into the relationship between VRN-1 and freezing tolerance (FR-1) Vrn-1 spring allele: Plants in reproductive phase VRN-1 transcripts accumulate Vernalization not required vrn-1 winter allele: Plants in vegetative phase VRN-1 transcript accumulation restricted Vernalization induces VRN-1 expression

CBFs and VRN-1 are inversely expressed and are differentially affected by vernalization Nonvernalized: CBFs on VRN-1 off Vernalized: CBFs off VRN-1 on Stockinger EJ, Skinner JS, Gardner KG, Francia E, Pecchioni N, 2007. Expression levels of barley Cbf genes at the Frost resistance- H2 locus are dependent upon alleles at Fr-H1 and Fr-H2. Plant J. 51, 308-21.

CBFs and VRN-1 are inversely expressed and are differentially affected by vernalization Nonvernalized: CBFs on VRN-1 off Vernalized: CBFs off VRN-1 on Stockinger EJ, Skinner JS, Gardner KG, Francia E, Pecchioni N, 2007. Expression levels of barley Cbf genes at the Frost resistance- H2 locus are dependent upon alleles at Fr-H1 and Fr-H2. Plant J. 51, 308-21.

'Dicktoo' Short days: CBFs on VRN-1 off Long days: CBFs off VRN-1 on CBF2 CBF4 CBF9 PHYC ADA2 VRN-H1 Stockinger EJ, Skinner JS, Gardner KG, Francia E, Pecchioni N, 2007. Expression levels of barley Cbf genes at the Frost resistance- H2 locus are dependent upon alleles at Fr-H1 and Fr-H2. Plant J. 51, 308-21.

Key findings and prospects Regulatory genes increasing freezing tolerance, the CBFs, are higher in copy number in winter barley lines than spring barley lines. FR-H2 in part may be a numbers game in which more is better High copy number Low copy number

Key findings and prospects Activity (expression) of CBF genes at FR-2 shows inverse relationship with VRN-H1 VRN-H1 may be a negative regulator of the CBFs

Regional Barley Gene Mapping Project