06 wp5 progresses&results-20130221
-
Upload
fruitbreedomics -
Category
Technology
-
view
122 -
download
5
Transcript of 06 wp5 progresses&results-20130221
WP5 _ Enhancing the knowledge of genetics
underlying novel traits and providing phenotyping methods
Results achieved since the beginning of the project and plans for 2013
Main objectives of the WP5
• Develop tools for novel trait determination
• Investigate novel and complex traits
• Make novel trait analysis high throughput and applicable for the research community and the breeding industry
3 tasks
• Task 5.1 Improve Monilinia resistance in peach
• Task 5.2 Assess fruit quality Jurriaan • Apple
• Peach Remo
• Task 5.3 Select traits important for climate change adaptation Evelyne
Results achieved since the beginning of the project
Task 1. improve resistance to Monilinia in peach
• Protocols for infection tests in lab • Test of artificial infections in orchard • Microscopy analysis of infection • Biochemical analyses of fruit surface • Tests of fungicide activity of some compounds • Spore survey in the orchard
Results achieved since the beginning of the project
Task 1. improve resistance to Monilinia in peach
Spray Monilinia laxa suspension
Infection enhancement by humidity increasing: fruit covering
One week incubation Susceptibility score:
% infected fruits
Setup of high-throughput orchard brown-rot phenotyping protocol
CB2 CB3
inoculation
cove
rin
g
no yes
no C I
paper CB2 IB2
plastic CB3 IB3
Results achieved since the beginning of the project
Task 1. improve resistance to Monilinia in peach
Spray Monilinia laxa suspension
Infection enhancement by humidity increasing: fruit covering
One week incubation Susceptibility score:
% infected fruits
Setup of high-throughput orchard brown-rot phenotyping protocol
CB2 CB3
IB2 treatment allowed to distinguish between tolerant and susceptible accessions
cell collapse ( ) fungal colonization ( )
Monilinia disease progress : 8 vs. 48 hour after inoculation
Zephir 8hpi Zephir 48hpi
E. Lady 8hpi E. Lady 48hpi
Bolinha 8hpi Bolinha 48hpi
Bolinha: no fungal impact on the analysed samples
Results achieved since the beginning of the project
Task 1. improve resistance to Monilinia in peach
Biochemical analyses of fruit surface
40 60 80 100 120 140 160
050
100
150
Jours après f loraison
Masse d
'un fru
it (g
)
A
I
II
III
40 60 80 100 120 140 160
050
100
150
Jours après f loraison
Dem
i-circonfé
rence (
mm
)
B
40 60 80 100 120 140 160
0.0
0.5
1.0
1.5
2.0
Jours après f loraison
Indic
e d
e D
iffére
nce d
'Absorb
ance
C SG
ZE
Fruit growth for 2 cultivars
Days after bloom
Fru
it m
ass
(g)
02
04
06
08
01
00
25-apr 16-may 30-may 20-jun 4-jul 18-jul maturity
Infe
ctio
n p
rob
ab
ility (
%)
* ** *
SG
ZE
Infection probability for 2 cultivars
I II III
Results achieved since the beginning of the project
Task 1. improve resistance to Monilinia in peach
Cuticular conductance Biochemical analyses • surface compounds • waxes • cutins • epiderm and flesh phenolics
40 60 80 100 120 140 160
05
10
15
20
total cuticular wax quantities (mg/dm²)
DAB
wa
x a
ccu
mu
latio
n m
g/d
m²
SGZE
Total cuticular waxes accumulation
Wax
(m
g/d
m²)
Days after bloom
0 50 100 150 200 250
20
04
00
60
08
00
10
00
masse du fruit (g)
co
nd
ucta
nce
de
l'é
pid
erm
e (
cm
/h)
SG
ZE
Fruit mass (g)
Cu
ticu
lar
con
du
ctan
ce (
cm/h
)
Cuticular conductance
Results achieved since the beginning of the project
Task 1. improve resistance to Monilinia in peach
Correlations between infection probability and
surface compounds
-1-0
.75
-0.5
-0.2
50
0.2
50.5
0.7
51
Proportion d'infection
Acide oléanolique
Acide ursolique
pcoumaroyl di-hexoside penta acétylé
Pic52.5_312
Pic54.6_308
Pic64.6_307
Ole
anolic
acid
Urso
lic a
cid
pcoum
aro
yl
Pic
52.5
_312
Pic
54.6
_308
Pic
64.6
_307
Pic
65.7
_312
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
Pro
port
ion d
'infe
ction
Acid
e o
léanoliq
ue
Acid
e u
rsoliq
ue
pcoum
aro
yl di-
hexosid
e p
enta
acéty
lé
Pic
52.5
_312
Pic
54.6
_308
Pic
64.6
_307
Oleanolic acid
Ursolic acid
pcoumaroyl
Pic52.5_312
Pic54.6_308
Pic64.6_307
Pic65.7_312
inhibitory candidates
02
04
06
08
01
00
25-apr 16-may 30-may 20-jun 4-jul 18-jul maturity
Infe
ctio
n p
rob
ab
ility (
%)
* ** *
SG
ZE
Infection probability I II I
II
mg/d
m²
01
23
4
Oleanolic acid
mg/d
m²
02
46
810
Ursolic acid
Aire d
e p
ic/d
m²/
10^5
05
10
15
20
25
30
Pic52.5_312
Jours après f loraison
Aire d
e p
ic/d
m²/
10^5
05
10
15
Pic64.6_307
40 60 80 100 120 140 160
05
10
15
20
25
30
Jours après f loraison
Aire d
e p
ic/d
m²/
10^5
Pic65.7_312
40 60 80 100 120 140 160
05
10
15
20
25
Aire d
e p
ic/d
m²/
10^5
p-coumaroyl derivative
SG
ZE
days after bloom
Evolution of fruit surface compounds along fruit growth
Days after bloom
Task5.2 Assess fruit quality
State of progress
Research topic in task 5.2
• Texture/maturation/quality tools evaluation – Compare, use and evaluate common used methods (colour cards, pm, ss, tta,
AWETA, DA) – Acoustic method for crispiness (FC, NZ) – Juice/juiciness analysis by punching and study tissue structure (NZ) – New methods for Fibre content (ML)
• Gene expression based methods for analysing fruit texture trait – Ethylene pathway dedicated approach to unravel ripening and texture trait
biomarkers (DLO) – Differential cultivars for harvest time, storage quality, shelf-life and meatiness
(JP)
• Storage stress test to select and early predict storage performance of new
lines – Standard measurements and inspection (DLO) – Gene expression analysis (JP, DLO)
Texture/maturation/quality tools evaluation
Quality measurements (n=20) • At harvest • After 1 week regime + 1 week shelf life (18°C, 75% RH) • After 1 month regime + 1 week shelf life (18°C, 75% RH) • After 2 months regime + 1 week shelf life (18°C, 75% RH)
Monitored quality parameters: • Fruit weight • Ethylene production (at harvest) • Firmness (FTA automated penetrometer, destructive) • Firmness (AWETA acoustic, non-destructive) • Ground colour (colour chart) • Chlorophyll status (DA-meter, non-destructive) • External and internal disorders (expert visual evaluation)
SensorSense ETD 300 characteristics
• Detection limit 300 ppt (0.3 ppb) • Upper limit 5 ppm (this one up to
100 ppm) • Measurement every 5 sec • Accuracy: <1% of 0.3 ppb (largest
value) • Very specific for ethylene • Calibration yearly
0 20 40 60 800
2
4
6
8
10
12
14
16
18
20
22
24
26
40
45
50
55
60
65
70
0 20 40 60 800
2
4
6
8
10
12
14
16
18
20
22
24
26
40
45
50
55
60
65
70
0 20 40 60 800
2
4
6
8
10
12
14
16
18
20
22
24
26
40
45
50
55
60
65
70
0 20 40 60 800
2
4
6
8
10
12
14
16
18
20
22
24
26
40
45
50
55
60
65
70
TA-XTplus - AED
4 mm flat head probe 5 Kg loading cell
100 mm/min Acquisition at 500 pps Compression strain: 90%
Approach Plant & Food Research
• 6 cultivars
• 10 trained panellists
• 14 sensory texture attributes
• 3 fruit per cultivar
• 3 storage times (scheduled according to softening rate)
• Air storage only
• 1 harvest (using starch and skin colour
Approaches for improved phenotyping technologies – optimising existing technologies
• Destructive
• Tensile • Single edge notch beam • Rheometer (frequency sweep) • Juicer Kinetics • Microphone
• Nondestructive • AWETA • Sinclair
Juiciness potential assoc. with larger cells, higher density and more apoplastic fluid
Scifre
sh
Sciro
s
C.P
ink
R.G
ala
Sciea
rlyCox
0.75
0.80
0.85
0.90
0.95
Cort
ical density (
g/m
L)
Scifresh Sciros RGala C.Pink0
20000
40000
60000
80000
100000
Ce
ll a
rea
m
2
Scifre
sh
Sciro
s
C.P
ink
Roy
al g
ala
0.05
0.06
0.07
0.08
Ap
opla
stic f
luid
(g
/gF
W)
List of tools
Task
Sub-
task Sub-task name Trait or step
Destructive / non Protocol / tool
Tested (1 or 2 years) Species
Conclusions on protocols
available Feasibility Reliability High-
throuput 2 2.1 Maturity
assessment Index of
differential absorbance IAD
1 apple not yet* y y y
Starch D Y 2 " y y y y
Solible solids D Y 2 " y y y y
Titratable acids D Y 2 " y y y y
Streif Index D Y 2 " y y y y
Ethylene production
N Y 1 " y y y y/n
Colour determination
N Y 2 "
y
y
y
y
Chlorophyll ? status (DA-meter)
N Y 1 "
y
y
Not sure to what
quality trait it links
y
List of tools
Task
Sub-task Sub-task name
Trait or step Sub-trait
Protocol / tool
Destructive / non
Tested (1 or 2
years) Species
Conclusions on
protocols available
Feasibility
Reliability
High-throuput
2.2 Fruit texture assessment
Physical paramet
ers
Firmness analysis by penetro measurements
Texture Analyser
D 1 apple y y y y
hand-held penetromet
er
D x " x x <y x
Firmness analysis by confined compression test
D x " x x x x
Firmness analysis by non-destructive acoustic resonance devices
AWETA AFS N 1 " y y y y
Sensor deform at impact sinclair y y y/n y
Firmness analysis combined with crispness analysis
acoustic-mechanical
N x " x x x x
Juice press/spin test D x " x x x x Color determination N 1 " y y y y Bioche
mical paramet
ers
Soluble solids D 1 " y y y y/n
Titratable acidity D 1 " y y y y/n Flavour x " x x x x Molecular characters of
texture 1(sample
s frozen) " n ? ? ?
Water contribution/microstructure on texture (tissue mechanical properties): Evaluation of NMR relaxometry as a screening tool
Achieved
•Water content & compartmentalization were accessed by relaxometry but were not directly related to mechanical properties •Freezing and thawing samples exacerbate relations between mechanical properties & relaxometric variables
On going
•Relate relaxometry & mechanical data with free sugar and cell wall compositions •New assays to evaluate relaxometry of frozen samples vs mechanical/histological/chemical variables
New methods for quality screening assessment
WP5.2
INRA-Nantes Biopolymers, Interactions, Assembly
Dietary fiber content and nature : development of fast screening method by Mid IR analysis of alcohol insoluble tissue material (AIM)
Achieved
•AIM, protein and Mid-IR of 29 genotypes
On going
•DF (AOAC method TDF), cell wall sugar analysis, ash and starch contents •Chemiometric analyses
New methods for quality screening assessment
WP5.2
INRA-Nantes Biopolymers, Interactions, Assembly
Identification of genes and networks controlling major apple quality traits
Materials: 10 varieties (Golden, Gala, Elstar, Greensleave, Cox, Boskoop, Ariane, Jonagold, Fuji, Granny Smith) 4 contrasted hybrids 4 time points: Harvest, 1 month post-harvest, 2 MPH, 4 MPH 2 years of data: 2011&2012 Texture traits: Expert sensory panel (1 to 5 values): Fiber, Grain, Mealiness, Softness, Crunchiness, Juiciness, Acidity, Sugar Physical and Chemical measurements: Acidity, Sugar, Ethylene, Penetrometry, Compression Results: 2011: Phenotypic data completed (contrasted traits, differents kinetics of trait evolution) 2012: Finishing the 4MPH measurements RNA purification completed soon (112 samples) Transcriptomic results with the AryANE chip in June 2013 Bioanalayses and Network buildings during summer
WP5-3
To be completed: material exchange with DLO and PFR (comparison with RNAseq) functional validation of the candidates
R scripts developed for data treatment
24 « .pair » files Raw data
12 « .txt » files Norm. data
normalized intensities per sample, ratios, p.value, localFDR
(Limma & fdrtool packages)
RG_plot.png MA_plot.png
PCAS Clusters
Dendrograms ...
complet-lists.txt
Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf
Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf
Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf
Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf
Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf
Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf
Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqk Kjgqdkjlqkghsfhf, Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf Kjgqdkjlqk,gmqldf
Annotations.txt
MapMan
Dongxia Yao et al Genomics Volume 98, Issue 1 2011 47 - 55
Storage Stress Test Tool
• Success cultivar very much dependent on storage behaviour
• Use commercial pick and commercial storage CA, DCS, mechanical/SF
• Stress test and supporting tools to analyse this behaviour – Does it suffer in-cooling or CA stress
– Is it Low Temp Sensitive
– Firmness during storage
– Sensitive to storage disorders
Experimental setup
Five commercially grown cultivars 1. Cox O.P. 2. Elstar 3. Golden Delicious 4. Jonagold 5. Kanzi Harvested • a week before • at commercial harvest date (for long term ULO-storage) • 1 week after
regime T (°C) %O2 %CO2
1 -1 21 0
2 -1 1 5
3 10 21 0
4 10 1 5
Overview of storage stress test (Y1)
Cultivar
Max storage period
(optimal conditio
ns)
Low Temp Sensitivity flesh
browning Texture issues
Low O2/high CO2 tolerance
Disorder sensitivity
practice
test practice test practice test practice test
Cox 5-6 m +++
Can develop mealiness
before softening
- Bitter pit,
brown core, Softening
Kanzi 12 m +++ Very firm, tough peel
? bitter pit, lenticel
breakdown
Jonagold 9 m - Greasiness ++ Scald, flesh browning
Golden 8 m -
++ Scald
Elstar 7 m + Rapid softening after storage
+ Softening, skin spots
Overview of storage stress test (Y1)
Cultivar
Max storage period
(optimal conditio
ns)
Low Temp Sensitivity flesh
browning Texture issues
Low O2/high CO2 tolerance
Disorder sensitivity
practice
test practice test practice test practice test
Cox 5-6 m +++
+++
(-1°C + CA, late harvest, ≥1 month)
Can develop mealiness
before softening
Mealiness , rapid
softening (air, ≥1 month)
- - Bitter pit,
brown core, Softening
-
Kanzi 12 m +++
++++
(-1°C + CA, late harvest, 2 months)
Very firm, tough peel
Very firm indeed
?
++
(-1°C + CA, last harvest, ≥1 month)
bitter pit, lenticel
breakdown -
Jonagold 9 m - - Greasiness Greasiness (10°C, air, ≥1
month)
++
+ (-1°C + CA,
3 harvest, ≥1 month)
Scald, flesh browning
-
Golden 8 m - -
Greasiness (10°C, air, ≥1
month)
++
++
(-1°C + CA, 1+3 harvest, ≥1 month)
Scald -
Elstar 7 m + - Rapid softening after storage
Confirms practice
+ - Softening, skin spots
Skin spots (all treatm)
Samples for microarray
Cultivar
Genetic background
Max storage period
(optimal conditions)
Texture issues Low Temp
Sensitivity flesh browning
Low O2/high CO2 tolerance
Disorder sensitivity
practice test practice test practice test
Cox
5-6 m Can develop
mealiness before softening
+ +++ ++ - - Bitter pit, brown core, Softening
Golden 8 m +/- - - ++ + Scald
Kanzi
12 m Very firm, tough
peel - +++ + ++(?) ++
bitter pit, lenticel
breakdown
Task5.2 Assess fruit quality (peach) State of progress
Cultivar Texture Type Pulp Institution
Cultivar Texture Type Pulp Institution
UMIL IRTA ARO UMIL IRTA ARO
Oro A NM Pc Y √ BO 05030142 SH P G √
BO 94007020 NM Pc Y √ BO 05030149 SH P G √
Dixired M P Y √ BO 05030081 SH P G √
Iride NM Pc W √ Elegant lady M P G √
Alice Col NM Pc Y √ IFF 331 SH P B √
Ambra M N Y √ √ Sweet dream M √
Big Top SM N Y √ √ Dulcebo SM P G √
Honey Kist SM N Y √ SRG M N G √
Redhaven M P Y √ Honey Royale SM √
Rich Lady SM P Y √ Nectaross M √
Vista Rich SM P Y √ BO 10120182 NM √
Ghiaccio SH P W √ √ Bolero M P G √
Glohaven M P Y √ BO 89010005 M N B √
Durado NM Pc Y √ Fei Cheng NM Pc B √
Alipersie M P Y √ Maria delizia M P B √
BO 00020006 NM Pc Y √ Dulciva M N G √
Swelling M P W √ Summer snow SM P W √
September Snow SM P W √ Fairlaine SM N Y √
1881 SM P W √ Hermoza SM P W √
Plant material & Experimental Design
Harvest
Co
ld S
tora
ge (
21
d a
t 4
ºC)
14d
1d
3d
21+1d
21+3d
0d
21d
21+5d
5d
Postharvest trial
2d
21+2d
Classification
M1 M2 M3
2011, 2012: M1, M2, M3
Summary (I)
Trait Sub-trait Technique Years Comments
Physical parameters Firmness analysis by
penetro measurements
Texture Analyser 1&2 Differentiate between fruit with different texture types through storage
hand-held penetrometer
1&2 In some cases, differentiate between fruit with different texture types through storage
Firmness analysis by confined compression test
Texture Analyser 1 No additional information if compared to other physical firmness techniques
Firmness analysis by non-destructive acoustic sensor
AWETA AFS 1&2 Differentiate between stone fruit type but not different textures
Compression to penetration ratio
Texture Analyser 2 Interesting results in Y1 but not in Y2
Juice press & spin test Expressible juice 1&2 Differenciates fruit with different textures through storage
Color determination & Portable
spectrophotometer Minolta CR2600d 1&2
No suitable for differentiating fruit with different textures
NIR Bruker MPA Multi
Purpose FT-NIR Analyzer
0 Data currently being analyzed
Summary (II)
Trait Sub-trait Technique Years Comments
Biochemical parameters
Soluble solids
Standard
1&2 No relationship with texture
Titratable acidity 1&2
Ethylene production 1&2 Not directly related to fruit texture, some interesting results that need to be further
confirmed
Antioxidant capacity FRAP assay 1&2 No direct relationship with fruit texture
Summary (III)
Trait Sub-trait Protocol Years tested Comments
Fruit structure and imaging
TRS Instrument developed by
Politecnico of Milano 1&2
Discriminate 3 out of 4 texture type (M, Sm and SH)
Nuclear Magnetic resonance
Esaote Airis II field intensity of 0,3 Tesla
1 No difference in texture (abandoned)
Echography Multimage Aloka ssd-500
scanning frequency of 3,5-5,0-7,5 MHz
1 No difference in texture (abandoned)
Computerized tomography
Stratec Medizintechnik XCT Research SA+
1&2
Gene expression RNA seq Illumina HiSeq 2000 2 Data currently being analysed
Sensory evaluation
Firmness perception 3 texture attributes and
likeness 2
No capability to distinguish between texture types but differentiate through storage
Phenotyping tools
Standard quality (TSS, TTA)
Firmness (Penetrometry, Texture Analyser and Acoustic Firmness sensor)
Expressible juice
Objective colour (L*, a* and b*; Spectrophotometer 360-740nm)
Fruit ethylene production
Sensory Analysis (Consumer tests)
Biochemical analysis (Antioxidants, MDA…)
Fmax
F(5% Def.)
P&D ratio
F
Stiffness Frecuency Impact Force
Phenotyping tools
Time Resolve Reflectance Spectroscopy (TRS)
Computerised tomography (CT)
Near Infrared Spectroscopy (NIR)
Echography
Nuclear Magnetic resonance(NMR)
Gene expression analysis
Transcriptomics
cytokinesis cell stretching
time
Fase I Fase II Fase III Fase IV
t0 t1
t2
t3
Results Fruit firmness: penetration
Days at 20ºC
0 1 2 3 4 5 6
Fir
mn
es
s (
Kg
)
0
1
2
3
4
5
6
7
Days at 20ºC
0 1 2 3 4 5 6
Fir
mn
es
s (
Kg
)
0
1
2
3
4
5
6
7
Fir
mn
es
s (
Kg
)
0
1
2
3
4
5
6
7
Fir
mn
es
s (
Kg
)
0
1
2
3
4
5
6
7
Ambra (M)F
irm
ne
ss
(K
g)
0
1
2
3
4
5
6
7
8
After CS
Before CS
Fir
mn
es
s (
Kg
)
0
2
4
6
8
Big Top (SM)
Honey Royale (SM) Nectaross (M)
Rome Star (M) Sweet Dream (SM)
S
M
-> No clear differences among different texture types -> Values from IRTA much lower than those from ARO (Impact of Agroclimatic conditions?)
Am
bra
Big
Top
Honey
Roya
le
Nec
taro
ss
Rom
e Sta
r
Swee
t Dre
am
Oded
Swel
ling
1881
Septe
mber
Snow
Fairla
ne (N
ecta
rine)
Her
moza
Summ
er S
now
P/D
ra
tio
0
1
2
3
4
IRTA ARO
Slow melting
Melting
Results Fruit firmness: deformation
*
*
*
a a
0
10
20
30
40
50
60
70
80
0 1 2 3 4 5
Exp
ress
ible
Ju
ice
(%
)
Day at 20°C
Melting
Non Melting
Slow Melting
a
a
a
b b b
b
b ab
c
0
10
20
30
40
50
60
70
80
0 1 2 3 4 5
Exp
ress
ible
Ju
ice
(%
)
Day at 20°C
Melting
Non Melting
Stony Hard
Slow Melting
a a
a a a ab
bc
ab
b b
c b
c
b
c
2012
2011
Results Expressible juice
Mie theory
b
s a )('
Stony Hard Slow Melting Melting
Results TRS
Mie theory
b
s a )('
Stony Hard Slow Melting Melting
Results TRS
Mie theory
b
s a )('
Stony Hard Slow Melting Melting
Results TRS
Mie theory
b
s a )('
Stony Hard Slow Melting Melting
Results TRS
Days at 20ºC
0 1 2 3 4 5
Aco
usti
c F
irm
ness
0
10
20
30
40
50
Days at 20ºC
0 1 2 3 4 5
Aco
usti
c F
irm
ness
0
10
20
30
40
50
Aco
usti
c F
irm
ness
0
5
10
15
20
25
30
Aco
usti
c F
irm
ness
0
5
10
15
20
25
30
Aco
usti
c F
irm
ness
0
5
10
15
20
25
30
35
40
Aco
usti
c F
irm
ness
0
5
10
15
20
25
30
35
40
After CS
Before CS
Ambra Big Top
Honey Royale Nectaross
Rome Star Sweet Dream
Acoustic firmness changes did not reveal different softening behaviors among the different cultivars investigated Significant differences in the fruit acoustic firmness were observed between different stone fruit types (Nectarine vs Peach)
Results Acoustic firmness
Before cold storage
After cold storage (21 d 4°C)
(M)
(M)
(M) (SM)
(SM)
(SM)
Results CT
Results CT
Big Top Oro A
Redhaven Alipersie
Rich Lady
IFF 331
BO 94007020
Ghiaccio
SM
M
NM
SH
0
50
100
150
200
250
300
Canning peach Peach Nectarine
De
nsi
ty g
cm
-3
Texture within peach group
Preliminary results average density
0
50
100
150
200
250
300
SM M SH
De
nsi
ty g
cm
-3
NGS raw data
Quality trimming and filtering (erne-filter)
Alignment to reference genome (Bowtie2)
Hits raw count
T1 T2
M SM SH NM M SM SH NM
T1
M – Redhaven – Rep 1 1 0 0 0 0 0 0 0
M – Bolero – Rep 2 1 0 0 0 0 0 0 0
SM - Big Top – Rep 1 0 1 0 0 0 0 0 0
SM - Rich Lady – Rep 2 0 1 0 0 0 0 0 0
SH - IFF 331 – Rep 1 0 0 1 0 0 0 0 0
SH - BO05030081 – Rep 2 0 0 1 0 0 0 0 0
NM - Oro A – Rep 1 0 0 0 1 0 0 0 0
NM - BO010120182 – Rep 2 0 0 0 1 0 0 0 0
T2
M – Redhaven – Rep 1 0 0 0 0 1 0 0 0
M – Bolero – Rep 2 0 0 0 0 1 0 0 0
SM - Big Top – Rep 1 0 0 0 0 0 1 0 0
SM - Rich Lady – Rep 2 0 0 0 0 0 1 0 0
SH - IFF 331 – Rep 1 0 0 0 0 0 0 1 0
SH - BO05030081 – Rep 2 0 0 0 0 0 0 1 0
NM - Oro A – Rep 1 0 0 0 0 0 0 0 1
NM - BO010120182 – Rep 2 0 0 0 0 0 0 0 1
Differential expression analysis (R, Limma and EdgeR)
Results Transcriptomic
For each type, the two time points group together.
Only SH flesh type groups tightly together
NM groups together at least in one axis, while M and SM samples group separately in both dimensions
Results Transcriptomic
Conclusions and prespectives
It is feasible to differentiate between fruit with different textures using time-course postharvest experiments with certain techniques.
Task5.3 Adaptative traits
State of progress
INRA Montpellier and Bordeaux UNIBO
Objectives
• Is it a genetic adaptation to ongoing climatic changes?
• Setting protocols easy to perform on populations for genetic studies and to be duplicated in different sites
• Chilling and heating requirements – Changes in temperature (during winter and spring) influences tree
phenology – Can we phenotype for selecting cultivars with desired chilling and heating
requirement ?
• Water scarcity – Identification of physiological parameters, potential candidates for
phenotyping tolerance and/or resilience to root water stress
Planned actions: CR et HR apple and peach
• In apple: 3 cultivars (Golden Delicious, Gala, Granny Smith)
• In peach: 9 cultivars (Fantasia, Ferjalou Jalousia, Flavorcrest, Mayglo, Redhaven, Summergrand, Summer Lady, Sunred, Tasty Free).
chosen from bibliographical data on their respective temperature requirements (contrasting CR from 200 to 1000 CH).
• Common methods performed in autumn 2011 and 2012 to estimate dates of dormancy release for floral and vegetative buds
• Samples of shoots collected from October year n to february year n+1 (collection every week at each site from december to february)
• Prospect other tests, search for new descriptors
Two biological tests:
forcing of ‘one-bud cuttings’ (vegetative buds)
forcing of floral primordia (Tabuenca’s test) from paradormancy period (summer in year n-1) to ecodormancy period (winter year n)
Characterization of genetic and annual influences both in Southern France and Southern Brazil (bilateral Project Capes Cofecub)
Observation of ‘green-tip’ stage: average time and percentage of budburst
VEGETATIVE BUD FORCING
FLORAL PRIMORDIA FORCING
(within floral bud)
Weighting of primordia before (in orchard ) and after forcing (fresh and dry weights)
Results: CR et HR apple
Results CR in apple
1 – Apple: Soil water restriction characterised by FTSW (Fraction of Total Soil Water)
- Bologne: Same protocol than in 2011: Leaf T° and fluorescence - Mtp: with a volumetric control of water in the soil (Volumetric Humidity assessment experiment);
* with mild root water restriction, applied during 3 weeks at 50% FTSW followed by 3 weeks at 20% FTSW, at morphological Measurements: (leaf area, shoot length and number of nodes) and eco-physiological (stomatal conductance) levels, • the ability to resume growth after a severe root water restriction, ie
provoking apex growth arrest, and possibly death, for most genotypes, applied during 3, 4 and 5 weeks (resilience).
2 – Peach: 2 cvs grafted on a F1 rootstock progeny 2012 was the first year of water stress (field experiment)
OBJECTIVES FOR WATER SCARCITY
2. Identification of a cluster of physiological parameters, potential candidates for phenotyping
Scatterplot (DATASHEET FATTORI pca STRESSED.sta 10v*17c)
F2FLUOTL = -8.8167E-16+0.6582*x
7S
23S
26S35S
37S
38S
40S
41S
48S
54S
70S
96S
117S
121S
125S
gsS
stkS
-4 -3 -2 -1 0 1 2 3 4
F2ALL
-3
-2
-1
0
1
2
3
F2
FL
UO
TL
F2ALL:F2FLUOTL:
r = 0.8153; p = 0.00007;
y = -8.4004E-16 + 0.6582*x
Scatterplot (DATASHEET FATTORI pca STRESSED.sta 10v*17c)
F1FLUOTL = 6.1837E-16+0.6678*x
7S
23S
26S
35S
37S
38S
40S
41S
48S54S
70S96S
117S
121S
125S
gsS
stkS
-6 -4 -2 0 2 4 6 8 10
F1ALL
-4
-3
-2
-1
0
1
2
3
4
5
F1
FL
UO
TL
F1ALL:F1FLUOTL:
r = 0.9387; p = 0.00000002;
y = 4.3311E-16 + 0.6678*x
PCA ALL vs. PCA FLUO_TL (2011)
2. Identification of a cluster of physiological parameters, potential candidates for phenotyping
PCA ALL vs. PCA FLUO_TL (2012)
Scatterplot: F1_ALL vs. F1_FLUO_TL (Casewise MD deletion)
F1_FLUO_TL = 0.0000 + .61268 * F1_ALL
Correlation: r = .91001
7
23
26
35
37
38
40
41
48
54
57
70
96
106
111
117
121125
gs
stk
-8 -6 -4 -2 0 2 4
F1_ALL
-5
-4
-3
-2
-1
0
1
2
3
4
F1
_F
LU
O_
TL
95% confidence
Scatterplot: F2_ALL vs. F2_FLUO_TL (Casewise MD deletion)
F2_FLUO_TL = 0.0000 + .86070 * F2_ALL
Correlation: r = .92362
7
2326
35
37
3840
41
48
5457
70
96
106111
117
121
125
gs
stk
-3 -2 -1 0 1 2 3 4
F2_ALL
-2
-1
0
1
2
3
4
F2
_F
LU
O_
TL
95% confidence
Projection of the cases on the factor-plane ( 1 x 2)
Cases with sum of cosine square >= 0.00
Labelling variable: trt
Active
7s
23s26s
35s
37s
38s40s
41s
48s
54s57s
70s
96s
106s111s
117s
121s
125s
GSs
STKs
-6 -5 -4 -3 -2 -1 0 1 2 3 4 5
Factor 1: 53.19%
-3
-2
-1
0
1
2
3
4
5
Fa
cto
r 2: 3
1.5
5%
Projection of the cases on the factor-plane ( 1 x 2)
Cases with sum of cosine square >= 0.00
Labelling variable: trt
Active
7S
23S
26S35S
37S
38S
40S
41S
48S54S
70S
96S
117S
121S
125S
gsS
stkS
-8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5
Factor 1: 65.47%
-5
-4
-3
-2
-1
0
1
2
3
4
Fa
cto
r 2: 2
4.9
3%
41 Stk 38 26 35 54 48
41 Gs 106 96 111 54
Gs 23 96 125 70 40 7 117 37 121
37 38 Stk 121 70 40 26 48 125 35 117 57 7 23
3. A tentative discrimination
2012
2011
As expected gs of 50% FTSW WS plants was higher than gs of 20% FTSW WS plants, with a decrease of ca. 80% and 35% compared to gs of WW plants, respectively.
7
2326
35
37
38
4041
48
54
57
7096
97
106
111
117
121
125
GS
SK
7
23
26
35
37
3840 41
48
54
57
7096
97106
111
117121
125
GS
SK
y = 0.1361x + 276.38R² = 0.0464
ns
y = 0.09x + 102.6R² = 0.1802
P=0.05
0
50
100
150
200
250
300
350
400
450
0 100 200 300 400 500 600
20
12
-gs
in W
S p
lan
ts (
mm
ol.
m-2
.s-1
)
2012- gs in WW plants (mmol.m-2.s-1)
X12_gs_WS50
X12_gs_WS20
2012 results – Apple Mtp
BUT the ranking of genotypes relative to each other for the three traits in either WW or WS between the two periods was different (example of the number of nodes). This would suggest that apart from the proper effect of the water regime, a plant development effect (growth dynamics varying along the growing season) and/or other environmental factors likely different between the two measurement periods, affected the development of the shoot.
Number of nodes developed during the period, either 50% or 20%
Pearson coefficient / Rank
correlation (Kendall’s t) WW - 50% FTSW vs WW - 20% FTSW R=0.32, ns; t=0.17, ns
WS - 50% FTSW vs WS - 20% FTSW R=0.21, ns; t=0.15, ns
2012 results – Apple Mtp
The combination leaf temperature and fluorescence provides a very good compromise between rapid and effective assessment of the drought resistance of a given phenotype.
WS protocol seems to be effective, simple for Leaf T° and fluorescence (and duplicable ??)
Summary-APPLE
Ranking was different between the two years and between stress conditions.
Ongoing discussions and analyses
0,0
5,0
10,0
15,0
20,0
25,0
0,0
20,0
40,0
60,0
80,0
100,0
120,0
140,0
1 2 3 4 5 6 7 8 9 10 11 12
tem
pe
ratu
re (°
C)
Rai
nfa
ll (m
m)
month 2012
rainfall temperature
2012 Results on peach
Harvest date
Surprised: 19 and 23 July 2012
Summergrand: 31 July 2012
No rootstock effect on fruit growth
But, fruit weight impacted …
In the same year or from n-1 year effect?
Publications
• Water scarcity
– Unibo communication at ISHS symposium on « orchard management »in South Africa, Dec. 2012
– Joint statistical analyses between Unibo & Mtp:
1 common publication planned in 2013
• CR:
– Statistical analyses of 2011 and 2012 results currently carriet out and publication planned, at least for apple results
linked to other projects
technical improvements on forcing tests, and application to a segregant progeny (genetic determinism of chilling requirement trait)
Validation of chilling models previously selected in apple by AFEF Team, based on the forcing test results obtained in the two hemispheres
Apply of ‘one-bud cuttings’ test Research of new alternative methods as NIRS Technology, based on the forcing test results in France (French Project Perpheclim ACCAF)
Main challenges for 2013 (CR, Apple)
Apple: 1 – Joint analyses between Bologna and Montpellier 2 - Same protocoles and observations in 2013, as in 2011 and 2012. No resilience experiment. Need to better study the ranking of genotypes in the various water conditions and years 3 –Comparison of 1YO shoots between plants in a greenhouse and plants in nearby outside conditions in Montpellier + possibly, at least for some of the variables, comparison with adult-fruiting trees of the same genotypes in the field. Peach: New experiment in 2013 in Bordeaux (2nd year) and in Bologna (1st year)
Main challenges for 2013 (Water scarcity)
Interactions WP5.3 and the rest of the project
• Interactions with other WPs of the project:
– Apply of ‘one-bud cuttings’ test to an apple segregant progeny (genetic determinism of chilling requirement trait)
– Apply water restriction to an apple core collection from WP4
Publications
• Communications in different meetings in 2012 and planned for 2013
• Publication plan has to be discussed during this meeting
Main challenges for 2013
• Complete sample analyses
• Analyze all data acquired
• Make synthetic analyses between years
• Merge results between partners
• Give conclusions on methods and tools
• Complete D5.2 (due February 2013)
Interactions between WP5 and the rest of the project
• From WP5 to WP2, WP3, WP4 – phenotyping tools for WP2, WP3 and WP4 Ex: Monilinia resistance on peach ; crispiness and water scarcity
on apple
– provide interesting genitors to WP2
• Interactions planned with the stakeholders of the project Test the phenotyping protocoles when available
Action Plan for 2013
To be discussed during this meeting
Should include many exchanges between partners via mels, phone calls, visio conf or workshop meetings
WP5 workshops
This afternoon:
• Task 5.2 _ 15:30 to 18:30 _ sala delegacions
• Task 5.3 _ 17:00 to 18:30 _ room 2.06
Tomorrow morning:
8:30 to 10 : 3 tasks separately