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 GroupThursday 21st – Friday 22nd October 2010

Bucharest, Romania

P8 Agricultural Research Council CRA-GPG Fiorenzuola, Italy

WP 9- FROST TOLERANCE IN OAT

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

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?

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

WINTERHARDINESSEXISTS IN OAT

Range between 3 and 9 (mean

5.8)

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

AVEQ PROJECTWorkpackage 9:

PARTECIPANTSP8 Agricultural Research Council CRA-GPG Fiorenzuola, Italy (Valeria Terzi)P9 Sucaeva Genebank, Sucaeva, Romania (Danela Muraru)P10 IPGR “K.Malkow”, Sadovo, Bulgaria (Nadia Antonova)

OBJECTIVESField screening for cold tolerance for up to 500 accessionsGrowth chamber tests for cold tolerance for 100 accessions

hardening treatment (3/1°C) for 4 weeks

Plants at first leaf stage cold acclimated during 4 weeks at 3/1° C (day/night), 200E 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

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.

Fv Fm-F0

--- = ---- Fm Fm

The ratio of variable to maximal fluorescence (Fv/Fm) 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

= 0.8in dark-adapted healthy plants

Rizza et al. (Plant Breed.120: 389-396, 2001).

one further reason of interest....

Compare the reliability of direct evaluations of FROST TOLERANCE and WINTER HARDINESSIntermediate-high tolerance

Optimize a simple and reliable protocol for PHENOTYPING LARGE POPULATIONS

Breeding

Biodiversity

Association mapping

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 MAPThird leaf

First leaf

Fr-H1 is closely linked to Vrn-H1 vernalization response locus

THE SELECTED GENOTYPESgrowth chamber experiments 2008

104 oat genotypes (included 9 standards) out of 317 of the set 1

5

4

1

812

4

8

2

6

2 18

4

14

6

9

16 Holder countries

Holder

n.of genotyp

es moderncultivat

edstandar

d spring winterinterme

diate

origin from the

Holder country

origin from other

countries

germinability

n.of selecte

d genotyp

es*

AUT 11 11 - - 11 - - 11 - high 5

BGR 7 1 5 1 6 - 1(w) 4+?   variable 4

BOR 1 1 - -       1(FIN) - high 1

CZE 25 13 11 1 13   1 19 6 variable 8

DEU 65 18 46 1 56     31 1? 33 variable 12

EST 12 2 9 1 9     12 - variable 4

FRA 37 15 21 1 25 1 1+3(w) 32 4 variable 8

GBR 3 3 - - 2 1 - 3 - high 2

ITA 8 6 - 2       8 - high 6

LTU 6 2 4 - 4     3 3 variable 2

LVA 2 - 2 - 1     2 - high 1

POL 38 16 22 - 23     24 14 variable 8

ROM 9 - 8 1 8     9 - variable 4

RUS 47 - 47 - 46 - 1(s) 6 41 variable 14

SVK 16 2 14 - 14     7 9 variable 6

SWE 29 15 13 1 13     20 9 high 9

USA 2 - 2 - 1 - 1 - 2 variable 1

THE SELECTED GENOTYPESgrowth chamber experiments 2008

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, LSD0.05):

6 genotypes were identified as the most susceptible

11 “ “ “ tolerant (4 with Fv/Fm close to 0.78)

The best genotypesFv/Fm 24h recovery

Mean 104 genotypes** 0.45

Range (min-max) 0.11 ÷ 0.77

Accession number

NAME FAO code - 12°C

A7BM0005 83/200-CR BGR001 0.77

200113379 Local 7 RUS001 0.75

200110905 Kinelskjj RUS001 0.73

605 Donata ITA037 0.73

1005 Vendelin SVK001 0.70

AVE 4659 Lueneburgher Kley Neue Zucht DEU146 0.68

19903 Gagybatory K Tajfajta SVK001 0.68

200107910 Local 3 RUS001 0.67

708 Novella Antonia ITA037 0.66

200111655 Local 5 RUS001 0.66

Millennium Millennium GBR016 0.65

breeding line

The same group (104) was tested in the field (P9)

-30.0

-20.0

-10.0

0.0

10.0

20.0

30.0

0 50 100 150 200

°C

maximum

minimum

9

87

6

Suceava meteorological Center, Romania 2009-2010

visual score

A7BM0005 83/200-CR BGR001 6

200113379 Local 7 RUS001 6

Millennium Millennium GBR016 7

10 Ava ITA037 7

605 Donata ITA037 7

set 1: correlation between Fv/Fm (24h recovery) with the field data of Bulgaria 2008-09 and Romania 2009-2010

ROMANIA field 2009-10 visual

score (0-9, Rizza et al.)

ROMANIA field 2009-10 number

survived plant (total row 1,2)

BULGARIA field 2008-09 visual

score (0-9, Rizza et al.) Mean

replications I,II

BULGARIA field 2008-09 visual

score (0-9 IBPGR)

Fv/Fm means two laboratory experiments

0.447*** 0.432*** -0.224* -0.121ns

ROMANIA field 2009-10 visual score (0-9, Rizza et al.) -0.951*** 0.267** 0.287**

ROMANIA field 2009-10 number survived plant (total

row 1,2) -0.271*** -0.257** BULGARIA field 2008-09

visual score (0-9, Rizza et al.) Mean replications I,II 0.340***

n=104 for laboratory experimentsn=318 field experiments

- -

THE SELECTED GENOTYPESgrowth chamber experiments 2010

106 oat genotypes (included 11 standards) out of 309 of the set 2

Holdern.of

genotypes*

origin from the Holder

countryn.of selected genotypes*

AUT 6 3 3BGR 17 5 7CZE 40 5 10DEU 79 12 20ESP 10 3 3EST 1 1 1FRA 28 8? 8GBR 37 6 9HUN 36 8 9IRL 5 4 4ITA 1 1 1NDL 1 1 1POL 67 6 11PRT 1 1 1ROM 1 1 1RUS 2 2 2SWE 4 4 4

335 95

*standard excluded 11 STANDARDS

3

7

1020

1

8

9

9

4

1

1 11

1

1

4

17 Holder countries3

2

FREEZING STRESS AT TWO MINIMUM TEMPERATURES:

-11°C ,-12°Cgood discrimination between intermediate and

tolerant genotypes

THE SELECTED GENOTYPESgrowth chamber experiments 2010

Accession number status FAO CODE

after stress

24h recovery

Fv/Fm Fv/FmA7BM0002 BREEDING LINE cultivated BGR001 0.79 0.81A7BM0006 BREEDING LINE cultivated BGR002 0.78 0.8019632 Cimarron cultivated FRA040 0.78 0.7837 Luilbreg (YUG) cultivated GBR011 0.78 0.7836 Belyski (YUG) cultivated GBR011 0.77 0.77CPVO19981528 EVORA standard FRA261 0.77 0.75A7BM0003 BREEDING LINE cultivated BGR001 0.79 0.75BGR 24983 K06-08 local BGR002 0.78 0.758 ARGENTINA standard ITA037 0.76 0.751126 AJ 145/114 cultivated GBR011 0.77 0.74RCAT012421 Kulsovati B cultivated HUN003 0.78 0.7419620 La Gaillarde cultivated FRA040 0.76 0.74AVE 1994 grisea DEU146 0.76 0.72

Means 106 genotypes 0.75 0.52Range (min-max) 0.66 - 0.79 0.16 - 0.81

The 13 best genotypes -11°C

THE SELECTED GENOTYPESgrowth chamber experiments

2010

 n=106 Fv/Fm 24h -11°C Fv/Fm 24h -12°Cmean 2

experiments

Fv/Fm 24h -11°C    

Fv/Fm 24h -12°C 0.523***    

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 GENOTYPESgrowth chamber experiments

2010

Correlation between the laboratory and field xperimentsSodovo, Bulgaria 2009-2010

 

Fv/Fm after stress

-11°C

Fv/Fm after

stress -12°C

mean 2 experime

nts

Fv/Fm after stress -

11°C      

Fv/Fm 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

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?”

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

THE SELECTED GENOTYPESgrowth 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

Accession namber variety name FAO code after stress 24h recovery

Millennium Millennium GBR016 0.783 0.749A7BM0005 83/200-CR BGR001 0.775 0.747200113379 Local 7 RUS001 0.782 0.746605 Donata ITA037 0.755 0.742St. 4 Evora EVORA FRA040 0.782 0.72710 Ava ITA037 0.759 0.716200105652 Local 1 RUS001 0.745 0.673200110905 Kinelskjj RUS001 0.706 0.61419303 Neu Grosse FRA040 0.745 0.594Breton Breton POL040 0.725 0.590200111655 Local 5 RUS001 0.723 0.5908-ARGENTINA ARGENTINA ITA037 0.736 0.589AINTREE Aintree FRA261 0.743 0.583200107022 Local 2 RUS001 0.74 0.558200107910 Local 3 RUS001 0.735 0.531

Fv/Fm

Freezing -7°C after acclimation at suboptimal hardening temperature (12/7°C, day/night). SET 1

Mean 104 genotypes** 0.70 0.29Range (min ÷ max) 0.66 ÷ 0.78 0.02 ÷ 0.75

Comparison between laboratory tests (“early” or “standard” hardening) and with the field data for set 1

n=104-106 Fv/Fm 24h -6°C Fv/Fm 24h -7°C

mean 2 experiments -

6 -7°C

Fv/Fm 24h -7°C 0.656***    

Fv/Fm 24h -12° e-10°C

means 0.477*** 0.453*** 0.512***

Fv/Fm 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** -0.291**

BULGARIA field 2009-10 scale 0-7 (IBPGR) -0.305** -0.361*** -0.369***

- 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)

.........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 ORIGINBREEDING 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?

AB

TR

EM

OIS

A B

lad

de

r

NU

RE

NU

RE

Phenotypelaboratory tests

Genotype. Selection ofadequate molecular markers

Field validation

genes for vernalization/photoperiod response

In barley

Growth at 12/7°C, associated to higher light intensity (700E) implied a morphological adaptation in frost

resistant and frost sensitive cultivars

Frost resistant

Frost sensitive

P9 Sucaeva Genebank, Sucaeva, Romania (Danela Muraru)P10 IPGR “K.Malkow”, Sadovo, Bulgaria (Nadia Antonova)

P8 Agricultural Research Council CRA-GPG Fiorenzuola, Italy

Valeria TerziMichele StancaLuigi CattivelliDonata PaganiFlavio Astesano

Thank You!