Workpackage 06 Analysis for protein and fat and minerals

Workpackage 06Analysis for protein and fat and minerals

AGRI GENRES 061: Avena genetic resources for quality in human consumption (AVEQ). 02-04 March. 2009. Quedlinburg

Main topics

Protein content (P5 – P9 – P11)- NIRS Calibration: Kjeldahl method (P9 – P11)

sampling 10% of the whole harvest- NIRS Analyses (P5 – P9 – P11)

on the whole harvestOil content (P5 – P9 – P11)

- NIRS Calibration: Soxhlet method (P9 – P11)- NIRS Analyses (P5 – P9 – P11)

Avenins (P5)600 accessions

Micronutriments (P5 - External assistance by ENITA Clermont-Fd)

200 accessions


WP 6

FIRST AVENIN RESULTS

Audrey Didier, Jean Koenig, Gerard Branlard, Roland Chavany, François Balfourier


35 countries represented

5 to 10

10 to 20

20 to 30

30 to 40

1 to 5

312 accessions

The oat collection


Modern oat Old cultivar Oat

Landraces

Varieties struck off catalogue

Avena sativa

35 % 65 %

AACCDD

Avena sativa 88 %

AACCDDAvena strigosa 7 %

Avena byzantina 4 %

Avena abyssinica 1 %

AsAs

AACCDD

AABB

The collection


Evaluation of Agro-morphologic descriptors

WP3

• Put in place of evaluation plots

• Descriptors observation

Agro-morphologic diversity of the collection

Data analysis

Method

Avenin identification

WP6

• Avenin’s extraction protocol

• Allele identification at three loci Ave1, Ave2 et

Ave3

• Acid page gliadin pattern analysis

Avenin allelic diversity

Data analysis

Method


• Proteins block from 17 to 23 kDa • 25 genes in average by haplöid genome

• Extraction in alcohol solution

Avenins

• 3 loci: Ave1, Ave2 et Ave3

• Prolamins


A? c Ave1

B? c Ave 2

C? c Ave 3

Main locus involved in synthesis of avenin endosperm storage proteins

Avenin storage proteins are encoded by clusters of genesAvenin storage proteins are encoded by clusters of genes


Gliadins protocol - INRA Avenins protocol - Portyanko

Condition A: Incubation at 39°C without shaking in a 70% alcohol solution

Condition B: Incubation at 25°C with shaking in a 70% alcohol solution

Condition A Condition B

Elaboration of avenins extraction protocol


Three concentrations of gels:

T = 8.5% C= 4.76% T = 12% C= 4.76% T = 14% C= 4.76%

Acid page gliadin pattern analysis

T=(A+B)/V C=B/(A+B)


16 indicators of allelic pattern

Name Origin Species Name Origin Species

Astor NLD A. sat. var. aristata Manod GBR A. sat. var.

mutica

Avoine de versailles FRA A. sat. var. brunnea Margot BEL A. sat. var.

mutica

Bardsey NLD A. sat. var. mutica Maris tabard GBR A. sat. var.

mutica

Black Mesdag FRA A. sativa var montana Pelso FIN A. sat. var.

brunnea

Blanche de Wattines FRA A. sat. var. aristata Rhea FRA A. sat. var.

inermis

Boog BYS A. sativa Sano SWE A. sat. var. aristata

Hede DNK A. sativa Starblonde FRA A. sat. var. mutica

Kyto FIN A. sat. var. aurea Svalof Hellgeber SWE A. sat. var.

mutica


Identification of allelic pattern at the locus Ave1

• 5 alleles were found against 7 in Portyanko’s analysis

• 4 alleles were indentical to Portyanko’s analysis (a, b, d, g)

• 1 new allele (o)




• 3 alleles were indentical (a, d, g)

• 3 new alleles (m, o, n)




• 6 alleles were indentical (a, b, c, e, g)

• 4 new alleles (k, o, p, q)


Accessions: correspond to the 323 accessions used in WP3

Sample seeds origin:

• multiplication 2007 in Clermont-Ferrand,

• other multiplication site Quedlinburg and Radzikow

• breeders

Two analysis had been realised:

• migration of 5 seeds manually grind together

• migration of 1 manually grind seed

Migration indicators : equal mixed of Margot and Mulga lay down three times on each gel

Material used


Allelic pattern identification

Identify locus Number of accession Presence percentage

Ave1 29 9.1

A. byzantina subsp. byzantina (2)A. sativa (16)

A. sativa var.nigra (2)A. sativa subsp. sativa (8)

A. strigosa (1)

Ave2 15 4.7

A. abyssinica (1)A. sativa (9)

A. sativa subsp. nudisativa (2)A. sativa subsp. sativa (3)

Ave3 20 6.3

A. byzantina (1)A. sativa (8)

A. sativa var. nigra (2)A. sativa subsp. nudisativa (3)

A. sativa subsp. sativa (6)

Ave1 + Ave2 18 5.6

A. sativa (9) A. sativa subsp. nudisativa (2) A. sativa subsp. sativa (7)

Ave1 + Ave3 63 19.7

A. sativa (39)A. sativa var. nigra (1)



Allelic pattern identification

Identify locus Number of accession Presence percentage

Ave2 + Ave 3 44 13.7

A. abyssinica (1)A. byzantina subsp. byzantina (2)

A. sativa (19)A. sativa var. macrantha (1)


Ave1 + Ave2 + Ave3 91 28.4

A. byzantina subsp. byzantina (1)A. sativa (39)


A. sativa var. brachytricha (1)A. strigosa subsp. brevis (1)

None 40 12.5

A. byzantina (1)A. byzantina subsp. byzantina(1)A. sativa (13) A. sativa subsp.nudisativa (1)

A. sativa subsp.sativa (11)A. strigosa (5)A. strigosa subsp.brevis (1)A. strigosa var.nigra (1)

A. strigosa var.typica (3)A. strigosa subsp.strigosa var.gilva (3)


18% of accessions shown difference between one seed and five seeds pattern.

This 18% are composed by 80% landraces and 20% modern cultivars.

Hypothesis:

• genetic heterogeneity• mix of accessions

Results


Allele repartitions to the 3 locus (in %)

Ave 1 Ave2 Ave3

a 0 21 11

b 55 - 38

c - - 34

d 18 10 -

e - - 0

g 2 12 9

j - - 2

k - - 1

m - 12 -

n - 10 -

o 25 35 4

p - - 0

q - - 1

100 100 100

91 accessions


• Difficulty to identify the allelic pattern at the three loci

Different technical conditions

Variation of accession in function of donor origin

• Analysis of avenin, on a 324 accessions set, did not allow an individually differentiation of the accessions

•Further studies on relation between agro-morphological characters and avenins

Conclusions and Perspectives


What’s next?

NIRS analysis (P5 – P9 – P11)Dehulling in process

Who sent to who?What delay expected

Start of NIRS analysis 2009 /2010Calibration for protein and oil content 2009/2010

Avenins (P5) 2010 ?

Micronutriments 2010


Evaluation of Agro-morphologic descriptors

• Put in place of evaluation plots

• Descriptors observation

Agro-morphologic diversity of the collection

Data analysis

Method

Avenin identification

• Avenin’s extraction protocol

• Allele identification at three loci Ave1, Ave2 et

Ave3

• Acid page gliadin pattern analysis

Avenin allelic diversity

Data analysis

Method


Observations in evaluation plots (2008)

Panicles descriptors (scale 1 to 3)

Disease and lodgingdescriptors (scale 1 to 9)

Plant height(cm)

Heading date (day)

april june jully augustmay

Sowing Harvest

Yield (q/ha)

TGW (g)

Glume color (scale 1 to 6)


Principal Components Analysis

2 first axis explain 51% of diversity variation

Correlation:

• Positive between lodging and plant height

• Negative between (lodging plus plant height) and sensibility to black rust

• Negative between yield and heading date

Descriptors projection on factorial plan (1 x 2)Active and Additional Variables

Active Suppl.

Crown rust

Yield

-1,0 -0,5 0,0 0,5 1,0

Axis 1 : 35,16%

-1,0

-0,5

0,0

0,5

1,0

Axis 2 : 16,25%

Height

Lodging

Black rust

TGW

PanicleColor

Oïdium

Heading


Less diversity in modern cultivars with trend to better yield and hight TGW

Diversity of agro-morphologic descriptors in the collection

Modern cultivars

Old cultivars


Allelic pattern

Ave1: b Ave2: a Ave3: b 12 %

Ave1: o Ave2: o Ave3: c 19 %

Ave1: b Ave2: o Ave3: c13 %

Comparison between agro-morphologic and avenins diversity (91 accessions)

No relation between the both

Documents

Workpackage 06 Analysis for protein and fat and minerals