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

Eric J StockingerAMBA Barley Improvement ConferenceSan Diego, CA January 14, 2013

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.”

Winter hardiness

Bell GDH, 1944. The breeding of two-row winter-hardy barley. Journal of Agricultural Science 34, 223-38.

Partial winter kill(2006/2007)

Complete winter kill(2002/2003)

Winter hardiness

Winter typesPlants in vegetative phaseVernalization induces reproductive phase

Spring typesInherent reproductive competence

Winter vs. spring types

Barley types cultivated in different regions in 1952

Poehlman JM (1952) Breeding winter barley for hardiness and disease resistance. Econ. Bot. 6, 176-84.

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 ofVariety source 0% 1–20% 21–40% 41–60% 61–80% 81–100%

Tennessee Winter - - - 2 25 26Korea 2 10 11 19 48 1

China-Manchuria 9 14 35 37 12 2Caucasus 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 - - - -

Wiebe GA and Reid DA (1958) Comparative winter hardiness of barley varieties. Technical Bulletin. United States Department of Agriculture 1176, 20 pp.

The top 10 winter-hardy barleys(of 204 assayed 1937-1956)

1. Kearney2. Dicktoo3. OAC 4GH14. Dayton5. Nebraska 524346. Nebraska 534177. Admire8. Kansas9. OAC 30GH1010. PU B466A7772

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.

Pedigree of the most successful winter malting barley

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%

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.

* 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.

Cross or accession Percentage of F6 lines in percent survival class1–19% 20–39% 40–59% 60–79% 80–99%

MO B475 × Carstens 6 18 45 31 -Ludwig × Carstens 3 6 33 52 6MO B1300 × Carstens 49 28 23 - -MO B1400 × Carstens 37 46 17 - -MO B475 × Tschermak 11 44 33 12 -Ludwig × Tschermak 7 15 9 37 32MO B1337 × Tschermak 3 12 7 46 32MO B1400 × Tschermak 9 13 37 41 1

MO B475 - - - 33 67Carstens 42 53 5 2

Winter survival of lines selected(Columbia Missouri 1969)

Selection Pedigree Survival (%)

MO B2126 MO B475 × Tschermak 77MO B2185 MO B1400 × Tschermak 76MO B2186 MO B1400 × Tschermak 77MO B2190 MO B1400 × Tschermak 69MO B2191 MO B1400 × Tschermak 66MO B2192 MO B1400 × Tschermak 72

MO B475 Admire × Missouri Early Beardless 81Carstens 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.

Winter survival(Columbia Missouri, 1970–72)

Winter hardiness: Score field survival in Oregon and MontanaFreezing tolerance: Score regrowth after controlled freezes at 0, -4, -8, -12°CAssociated 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

Genetic analyses of winter hardiness(the ‘Dicktoo’ × ‘Morex’ population)

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.

Generate 100 doubled haploid lines from F1creating an immortalized F2 population

‘Dicktoo’LT50 = -8oC

(Six-row winter)

‘Morex’LT50 = -4oC

(Six-row spring)

F1

×

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.

Genetic analyses of winter hardiness(the ‘Nure’ × ‘Tremois’ population)

Winter hardiness: Score field survival in Northern ItalyFreezing tolerance: Score regrowth after controlled freezes at -11 and -13oC Associated biochemical traits: Score field material COR14B protein accumulation

Quantitative trait loci analysis (QTL): Test for association between traits and regions of the genome

Generate 136 doubled haploid lines from F1creating an immortalized F2 population

‘Nure’LT50 = -11oC

(Two-row winter)

‘Tremois’LT50 = -6oC

(Two-row spring)

F1

×

CBF

VRN-H1

Two loci have major effects on winter hardiness

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

FR-H2 has >13 CBFs

FROST RESISTANCE-H2

In Dicktoo and Nure, CBF2A and CBF4B are present in multiple copies in tandem array

In Morex and Tremois, CBF2 and CBF4 are present in single copy

Tremois

Nure

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

ADKnox AK, Dhillon T, Cheng H, Tondelli A, Pecchioni N, Stockinger EJ, 2010. CBF gene copy number variation at Frost Resistance-2is 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 dataAccession On 6/64”

(%)

Barley Protein

(%)

DP

(oASBC)

Alpha-amylase(20oDU)

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 standardGreen bold text = values above ideal maximum AMBA quantity standard

Accession Winter survival(Elora Research Station)

Fv/FmControl -6oC -8oC -10oC -12oC

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

Winter survival and freezing tolerance

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 winter allele:Plants in vegetative phaseVRN-1 transcript accumulation restrictedVernalization induces VRN-1 expression

Vrn-1 spring allele:Plants in reproductive phaseVRN-1 transcripts accumulateVernalization not required

CBFs and VRN-1 are inversely expressed and are differentially affected by vernalization

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.

Nonvernalized:

CBFs onVRN-1 off

Vernalized:

CBFs offVRN-1 on

CBFs and VRN-1 are inversely expressed and are differentially affected by vernalization

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.

Nonvernalized:

CBFs onVRN-1 off

Vernalized:

CBFs offVRN-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 onVRN-1 off

CBF2CBF4CBF9PHYCADA2

VRN-H1

Long days:

CBFs offVRN-1 on

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

Activity (expression) of CBF genes at FR-2 shows inverse relationship with VRN-H1

VRN-H1 may be a negative regulator of the CBFs

Key findings and prospects

Regional Barley Gene Mapping Project