1. Avena Genetic Resources for Quality in Human Consumption (AVEQ) Final project meeting Tuesday 19th – Thursday 21st October 2010 6th Meeting of the ECPGR Avena Working Group Thursday 21st – Friday 22nd October 2010 Bucharest, Romania P8 Agricultural Research Council CRA-GPG Fiorenzuola, Italy WP 9- FROST TOLERANCE IN OAT

2. Among winter cereals, oat is the most frost sensitive and its insufficient level of winterhardiness is the main factor limiting winter sowing in many major oat growing regions in Europe Fiorenzuola 1996-’97 - 15°C without snow

3. Frost resistance not only improves winter survival but is also a fundamental component in high yielding winter cultivars IS THERE GENETIC VARIABILITY FOR THIS TRAIT?

4. Sixty-two genotypes with different origin (Europe, North America) were grown in Northern Italy (Fiorenzuola) in 1996-’97 Frost damage was estimated by a visual score (0: no damage 9: all plants killed) A BROAD GENETIC VARIABILITY FOR WINTERHARDINESS EXISTS IN OAT Range between 3 and 9 (mean 5.8)

5. LARGE GENETIC VARIABILITY FOR BOTH TRAITS IN CEREALS (Ex. barley, diploid model for the Triticeae ) In winter-sown cereals the acquisition of frost tolerance is associated with the occurrence of a cold HARDENING adaptive process at low non- freezing temperature FROST TOLERANCE, the main component of winter hardiness is a crucial factor for cereal cultivation, not only improves winter survival but is also a fundamental component in high yielding winter cultivars WINTER HARDINESS is assessed in the field as ability to survive throughout the winter

6. AVEQ PROJECT Workpackage 9: PARTECIPANTS P8 Agricultural Research Council CRA-GPG Fiorenzuola, Italy (Valeria Terzi) P9 Sucaeva Genebank, Sucaeva, Romania (Danela Muraru) P10 IPGR “K.Malkow”, Sadovo, Bulgaria (Nadia Antonova) OBJECTIVES Field screening for cold tolerance for up to 500 accessions Growth chamber tests for cold tolerance for 100 accessions

7. hardening treatment (3/1°C) for 4 weeks Plants at first leaf stage cold acclimated during 4 weeks at 3/1° C (day/night), 200  E m -2 s -1. Hardened plants are subjected to a freezing treatment at temperature from -10 to –13°C FROST TOLERANCE EVALUATION AT FIRST LEAF STAGE

8. Chlorophyll fluorescence responds to changes in PSII photochemistry and therefore represents a convenient and rapid tool to evaluate the capacity of the photosynthetic machinery at low temperature. F v F m -F 0 --- = ---- F m F m The ratio of variable to maximal fluorescence (F v /F m ) in dark-adapted state measures the maximum quantum yield for PSII photochemistry and represents a diagnostic probe for measuring low temperature stress- induced injury of photosynthesis PAM-2000, Walz, Effeltrich, Germany = 0.8 in dark-adapted healthy plants Rizza et al. (Plant Breed.120: 389-396, 2001).

9. one further reason of interest.... Compare the reliability of direct evaluations of FROST TOLERANCE and WINTER HARDINESS Intermediate-high tolerance Optimize a simple and reliable protocol for PHENOTYPING LARGE POPULATIONS Breeding Biodiversity Association mapping

10. Fr-H1 Fr-H2 Francia et al.,Theor Appl Genet (2004) 108:670–680 Two QTLs for frost tolerance were mapped on barley chromosome 5H. QTL for three distinct measures of low-temperature resistance are coincident ‘NURE’ x ’TREMOIS’ LINKAGE MAP Third leaf First leaf Fr-H1 is closely linked to Vrn-H1 vernalization response locus

11. THE SELECTED GENOTYPES growth chamber experiments 2008 104 oat genotypes (included 9 standards) out of 317 of the set 1 5 4 1 8 12 4 8 2 6 2 1 8 4 14 6 9 16 Holder countries

12. Holder n.of genotyp esmodern cultivat ed standar dspringwinter interme diate origin from the Holder country origin from other countri es germina bility n.of selecte d genotyp es* AUT11 -- -- -high5 BGR71516-1(w)4+? variable4 BOR11-- 1(FIN)-high1 CZE251311113 1196variable8 DEU651846156 31 1?33variable12 EST122919 -variable4 FRA37152112511+3(w)324variable8 GBR33--21-3-high2 ITA86-2 8-high6 LTU624-4 33variable2 LVA2-2-1 2-high1 POL381622-23 2414variable8 ROM9-818 9-variable4 RUS47- -46-1(s)641variable14 SVK16214- 79variable6 SWE2915131 209high9 USA2-2-1-1-2variable1 THE SELECTED GENOTYPES growth chamber experiments 2008

13. FREEZING STRESS AT TWO MINIMUM TEMPERATURES -10°C discrimination of the most frost susceptible genotypes -12°C good discrimination of genotype with intermediate or superior FT THE SELECTED GENOTYPES growth chamber experiments 2008 On the basis of the two experiments (ANOVA, LSD 0.05 ): 6 genotypes were identified as the most susceptible 11“ “ “ tolerant (4 with F v /F m close to 0.78)

14. The best genotypes Fv/Fm 24h recovery Mean 104 genotypes** 0.45 Range (min-max) 0.11 ÷ 0.77 breeding line

15. The same group (104) was tested in the field (P9) 9 8 7 6 Suceava meteorological Center, Romania 2009-2010 visual score

16. set 1: correlation between Fv/Fm (24h recovery) with the field data of Bulgaria 2008-09 and Romania 2009-2010 n=104 for laboratory experiments n=318 field experiments --

17. THE SELECTED GENOTYPES growth chamber experiments 2010 106 oat genotypes (included 11 standards) out of 309 of the set 2 3 7 10 20 1 8 9 9 4 1 1 11 1 1 4 17 Holder countries 3 2

18. FREEZING STRESS AT TWO MINIMUM TEMPERATURES: -11°C,-12°C good discrimination between intermediate and tolerant genotypes THE SELECTED GENOTYPES growth chamber experiments 2010

19. The 13 best genotypes -11°C THE SELECTED GENOTYPES growth chamber experiments 2010

20. n=106F v /F m 24h -11°CF v /F m 24h -12°C mean 2 experiments F v /F m 24h -11°C F v /F m 24h -12°C0.523*** Bulgaria field 2009-‘10 scale 0-9-0.476***-0.369***-0.493*** Bulgaria field 2009-‘10 scale 0-7 (IBPGR)-0.400***-0.248*(*)-0.385*** THE SELECTED GENOTYPES growth chamber experiments 2010

21. Correlation between the laboratory and field xperiments Sodovo, Bulgaria 2009-2010 F v /F m after stress -11°C F v /F m after stress - 12°C mean 2 experime nts F v /F m after stress - 11°C F v /F m after stress - 12°C-0.601*** Bulgaria field 2009-‘10 scale 0-9-0.396***-0.305**-0.396*** Bulgaria field 2009-‘10 scale 0-7 (IBPGR)-0.352***-0.205*-0.298** Romania prova campo 2010-11

22. To which extent the evaluation of the plant response in EARLY GROWTH STAGE or EARLY ACCLIMATION state plays a critical role in determining the potential (maximum) frost hardiness? Can we further simplify our evaluation of frost tolerance? in progress............some more questions... CAN WE CONTRIBUTE IN UNDERSTANDING THE BASIS OF THE FROST TOLERANCE CAPACITY? Is there genetic variability for the plant capacity for “early acclimation?”

23. The ability to induce early hardening is of advantage under field conditions, where temperature falls progressively, because it enables resistant plants to prepare for cold before the susceptible ones. The TRESHOLD TEMPERATURE for COR14 accumulation is higher in the resistant cv Crosatti et al. Planta 196:458-463, 1995 Winter, Resistant Spring, Susceptible SUBOPTIMAL HARDENING TEMPERATURE

24. THE SELECTED GENOTYPES growth chamber experiments 2010 104 oat genotypes (included 9 standards) out of 317 of the set 1 Plants at first leaf stage wereexposed 3 weeks at suboptimal hardening temperature12/7°C. Freezing stress at -6°C and -7°C

25. Freezing -7°C after acclimation at suboptimal hardening temperature (12/7°C, day/night). SET 1 Mean 104 genotypes** 0.70 0.29 Range (min ÷ max) 0.66 ÷ 0.78 0.02 ÷ 0.75

26. Comparison between laboratory tests (“early” or “standard” hardening) and with the field data for set 1 n=104-106F v /F m 24h -6°CF v /F m 24h -7°C mean 2 experiments - 6 -7°C F v /F m 24h -7°C0.656*** F v /F m 24h -12° e-10°C means0.477***0.453***0.512*** F v /F m 24h -12°0.446***0.422***0.477*** ROMANIA field 2009-10 visual score (0-9)-0.435***-0.623***-0.588*** BULGARIA field 2009-10 scale 0-9-0.234* -0.291** BULGARIA field 2009-10 scale 0-7 (IBPGR)-0.305**-0.361***-0.369***

27. - one more stress temperature to test frost tolerance under “standard”hardening conditions (common publication) WP9. The experiments planned in the AVEQ project are concluded, with the only exception of the second field in Romania for the evalaution of winter hardiness in the second set of 106 accessions (set 2) This is due to a modified organization of the laboratory tests (200 accessions instead of 100) and a consequent oganization of the field experiments (environment subjected to severe frost) about the AVEQ project...... beyond the objectives..... - effect of hardening at suboptimal hardening conditions (12/7°) for the set 2 - interest for analysing wild species, landraces...(adaptability, breeding)

28. .........and beyond? POSSIBLE QUESTIONS frost tolerance and winter hardiness: how to dissect the components of WINTER HARDINESS (development traits. vernalization, photoperiod, earliness per se) in a large number of genotypes? GEOGRAPHYCAL ORIGIN BREEDING EFFECTS use of SELECTED MOLECULAR MARKERS for traits affecting winter hardiness (vernalization and photoperiod).The best approach? What plants need for adaptability to the PRESENT and FUTURE environmental conditions?

29. A B TREMOIS A B ladder NURE Phenotype laboratory tests Genotype. Selection ofadequate molecular markers Field validation genes for vernalizatio n/photoperio d response In barley

30. Growth at 12/7°C, associated to higher light intensity (700  E) implied a morphological adaptation in frost resistant and frost sensitive cultivars Frost resistantFrost sensitive

31. P9 Sucaeva Genebank, Sucaeva, Romania (Danela Muraru) P10 IPGR “K.Malkow”, Sadovo, Bulgaria (Nadia Antonova) P8 Agricultural Research Council CRA-GPG Fiorenzuola, Italy Valeria Terzi Michele Stanca Luigi Cattivelli Donata Pagani Flavio Astesano Thank You!