Presentation on Trait phenotyping: About asking the right questions to harness phenomics' progress. Phenodays 2013 - 17 Oct 2013

1. Trait phenotyping:
About asking the right questions
to harness phenomics’ progress
Vincent Vadez – Jana Kholova
ICRISAT
Phenodays 2013 – 16-18th October 2013

2. Today’s presentation
Basic considerations on trait phenotyping
Root / Water extraction
Leaf area development
Leaf conductance
Growth response to soil drying

3. Grain Yield
Grain Number Grain Size & N
 Biomass RADN
TE T RUE Rint
vpd
kl LAISLNRoots k

TN LNo
A >A
APSIM Generic Crop Template, from Graeme Hammer
Yield and its determinants
Yield is not a trait
Phenotyping should focus on the building blocks

4. Accurate Non accurate
Precise
Not precise
Precision / Accuracy of phenotyping
Is my phenotype
the right one?
Are measurements
Good enough?

5. What is a “drought tolerant” plant?
A plant with:
• enough water to fill up grains
• no more water after grain filling
Hypotheses:
• Tap water?
• Save/manage water?
Focus on traits affecting plant water budget

6. Basic considerations on trait phenotyping
Root / Water extraction
Leaf area development
Leaf conductance
Growth response to soil drying

7. Lysimetric facility at ICRISAT
Advantages:
• Gravimetric
• Long term (3 Wks-maturity)
• High throughput (5000 PVCs)

8. Root length density and water extraction
Drought root length density (cm cm-3)
0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75
Droughtwaterextraction(kgplant-1)
5.5
6.0
6.5
7.0
7.5
8.0
8.5
BRB 191
PAN 127
SUG 131
VAX 1
BAT 477
DOR 364
CAL 143
VAX 3
RCW
SEA 5
SEA 15
SER 16
SEQ 1003
SEQ 11CAL 96
SAB 259
RAA 21
ICA Quimbaya
SER 8
Mean: 0.56
LSD0.05: 0.13
SEC 16
Mean: 6.84
LSD0.05: 1.53
r = 0.08
No relation between water extraction (WS)
and root length / RLD
Beans Chickpea

9. Post-rainy season Rainy season
0
5
10
15
0 1000 2000 3000 4000 5000 6000 7000
Podyield(gplant-1)
Total water extracted (g plant-1)
0
2
4
6
8
10
0 1000 2000 3000 4000 5000 6000 7000Podyield(gkg-1)
Total water extracted (g plant-1)
No relationship between total
water extracted and grain yield
0
2
4
6
8
10
12
14
0 1000 2000 3000 4000 5000 6000 7000
Podyield(gplant-1)
Total water extracted (g plant-1)
Cowpea
Peanut
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
0 1000 2000 3000 4000 5000 6000 7000
Podyield(gplant-1)
Total water extracted (g plant-1)
Bean
Peanut
Rainy seasonRainy season
Pod yield and water extraction

10. Water extraction pattern (WS)
in 12 tolerant / 8 sensitive chickpea
Zaman-Allah, Jenkinson, Vadez 2011 JXB
0
1
2
3
4
5
6
7
8
9
10
21 28 35 42 49 56 63 70 77 84 91 98
CumulatedWaterUsed
(kgpl-1)
Days after sowing
Flowering
Sensitive
Tolerant
Tolerant: less WU at vegetative stage,
more for reproduction & grain filling

11. Zaman-Allah, Jenkinson, Vadez 2011 JXB
0
1
2
3
4
5
6
7
8
9
10
21 28 35 42 49 56 63 70 77 84 91 98
Waterused(kgpl-1)
Days after sowing
Sensitive
Tolerant
Relationship between grain yield and water use
Low early vigor
Low leaf Gs
Tolerant: less WU at vegetative stage,
more for reproduction & grain filling

12. 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 1 2 3 4
WU(kgplant-1week-1)
Weeks after panicle emergence
ICMH01029
ICMH01040
ICMH01046
PRLT2/89-33
Vadez et al 2013 – Plant Soil
H77/833-2
ICMH02042
Terminal drought
sensitive
Terminal drought
tolerant
Tolerant: less WU at vegetative stage,
more for reproduction & grain filling
Water extraction pattern (WS) in pearl millet
Flowering

13. R² = 0.7108
0
4
8
12
16
20
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Early stress
WU in week 3 after panicle emergence
GrainYield(gplant-1)
Tolerant: EUW = 45 kg grain mm-1
Relationship between grain yield and water use

14. What platform for phenotyping?
• Outdoors / yield-based
• Water extraction at key time - Automation
• Possible IR surrogates
• Staygreen

15. Basic considerations on trait phenotyping
Root / Water extraction
Leaf area development
Leaf conductance
Growth response to soil drying

16. Leafarea
Thermal time
A – Fast early LA
B – Slow early LA
C – Fast early LA / small max LA
D – Slow early LA / small max LA
Leaf area development dynamics
Speed of development / size of canopy = water

17. Arrows indicates Re-watering
Profile of water use from flowering to maturity
In peanut
Sensitive
Tolerant
Tolerant lines develop a smaller canopy
under WW conditions
Ratnakumar and Vadez 2011

18. Field trial
0 5 10 15 20 25
0
1000
2000
3000
4000
5000
6000
A = 2,91
Fleur 11
WW condition
R² = 0,999
Nodes number
Leafarea(cm²)
Field trial
0 5 10 15 20 25
0
1000
2000
3000
4000
5000
6000
A = 2,63
ICG 1834
WW condition
R²= 0.91
Nodes number
Leafarea(cm²)
Peanut
Coefficients relating leaf area to node
number
Need a HT method to measure
the dynamics of leaf area development

19. Zaman-Allah, Jenkinson, Vadez 2011 JXB
0
1
2
3
4
5
6
7
8
9
10
21 28 35 42 49 56 63 70 77 84 91 98
Waterused(kgpl-1)
Days after sowing
Sensitive
Tolerant
Relationship between grain yield and water use
Low early vigor

20. y = 23.302e0.2562x
R² = 0.9367
0
2000
4000
6000
8000
10000
12000
0 5 10 15 20 25
Leafarea(cm2)
Node number on main stem
y = 11.995e0.31x
R² = 0.9607
0
2000
4000
6000
8000
10000
12000
0 5 10 15 20 25
Node number on main stem
Coefficients relating leaf area to node
number
Chickpea
Need for a leaf count along with leaf area

21. R² = 0.7255
0
5
10
15
20
25
30
1500 1700 1900 2100 2300 2500 2700 2900 3100 3300
Grainyieldunder
Waterstress
Leaf area at anthesis
Relation between LA and grain yield
in postrainy sorghum
Higher grain yield is related to
lower anthesis leaf area

22. Variation in leaf area
Variation affected by water stress
WW&WS- LA development
0
500
1000
1500
2000
2500
3000
18-Nov 23-Nov 28-Nov 3-Dec 8-Dec 13-Dec 18-Dec 23-Dec
LA(cm2)
6008 WW
6016 WW
6026 WW
7001 WW
S35 WW
6008 WS
6016 WS
6026 WS
7001 WS
S35 WS
Progressing drought
Need to account for: - soil moisture effect
- VPD effects
Leaf area development
response to drought
WW
WS

23. Canopy developmental dynamics
TPLAvarying TPLA_prod_coef
0
5
10
15
20
25
0 200 400 600 800
TTemerg_to_flag
TPLA
-0.01
-0.018
-0.026
TPLA_inflection_ratio = 0.66
TPLAmax = 20
0.01
TPLA varying TPLA_inflection_ratio
0
5
10
15
20
25
0 200 400 600 800
TTemerg_to_flag
TPLA
0.66
0.5
0.33
TPLAmax = 20
TPLA_prod_coef - 0.018
0.33
0.66
LA development depends
on:
•TPLA production
coefficient and
•TPLA inflection ratio

24. 0
500
1000
1500
2000
2500
3000
02.4823.914
5.266.5847.9969.44410.7220.36275.504141.84212.9281.47346.74413.21465.36521.08566.23612.08654.97694.29731.48761.5782.41807.44858.98920.38977.521040.4
thermal time
kgha-1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
m2m-2
stover TR high
stover TR low
grain TR high
grain TR low
LAITR high
LAITR low
SD TR high
SD TR low
Simulation:severe terminal drought
Delayed
senescen
ce
Delayed
water
exhaustion
LAI
S/D ratio
biomass
water
conservation
during vegetative
growth and its
utilization for
better grain filling
= stay-green
kgha-1
Thermal time
YIELD
flowering
~830 0dayStaygreen phenotype is a consequence
of smaller/delayed leaf development

25. What platform for phenotyping?
• 3-D imaging of Leaf Area
• Measurements at early stages
• Soil moisture effects
• VPD effects (need HT measurements)

26. Basic considerations on trait phenotyping
Root / Water extraction
Leaf area development
Leaf conductance
Growth response to soil drying

27. Vapor Pressure Deficit (VPD, in kPa)
Transpirationrate(gcm-2h-1)
0.0 2.0 4.0
0.0
1.0
A – Insensitive to VPD – High rate at low VPD
B – Sensitive to VPD – High rate at low VPD
C – Sensitive to VPD – Low rate at low VPD
D – Insensitive to VPD – Low rate at low/high VPD
Main types of Tr response to VPD
Water use
difference
Leaf conductance differences = water
Vadez et al 2013 – FPB in press

28. Terminal drought
sensitive
Terminal drought
tolerant
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
0.50 1.00 1.50 2.00 2.50 3.00 3.50
VPD (kPa)
H77/2 833-2
PRLT-2/89-33
Transpiration(gcm-2h-1)
From Kholova et al 2010b
2 mechanisms of water saving:
•Low Tr at low VPD
•Further restriction of Tr at high VPD
Transpiration response to high VPD –
Pearl millet

29. 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 1 2 3 4
WU(kgplant-1week-1)
Weeks after panicle emergence
ICMH01029
ICMH01040
ICMH01046
PRLT2/89-33
Vadez et al 2013 – Plant Soil
H77/833-2
ICMH02042
Terminal drought
sensitive
Terminal drought
tolerant
Tolerant: less WU at vegetative stage,
more for reproduction & grain filling
Water extraction pattern (WS) in pearl millet
Flowering
Water saving from
lower conductance

30. 4 replications
RH & T hourly recording
Weighing:
7-11am = low VPD
11am-15pm = high VPD
8” pots re-saturated every day
soil evaporation minimized with plastic beads
Mapping of water saving traits

31. Transpiration response to high VPD -
Peanut

32. -1 0 +33
15-30% yield decrease, especially at
high latitudes (12-15ºN)
% yield increase with VPD
response trait - Peanut

33. Mouride
IfVPD<2.09,TR=0.0083(VPD)–0.002
IfVPD≥ 2.09,TR=0.0013(VPD)+0.015
R²=0.97
B UC-CB46
TR=0.0119(VPD)-0.0016
R²=0.97
D
Transpiration response to VPD
- cowpea
Tolerant lines have a breakpoint
(water saving)
Tolerant Sensitive
Belko et al – 2012 (Plant Biology)

34. R² = 0.64
-40
-20
0
20
40
60
0.000 0.010 0.020 0.030 0.040 0.050 0.060
Residualtranspiration
Transpiration rate under high VPD
What drives transpiration in that population??
Leaf area
(69%)
Conductance
at high VPD
(64% of residual)
Phenotype both these traits
(LA – Transpiration response to VPD)
R² = 0.69
0
50
100
150
200
250
0 200 400 600 800 1000 1200
Totaltranspiration
(gplant-1)
Leaf area (cm2 plant-1)

35. QTLs from ICI Mapping – Drought
tolerance traits
TraitName
Chromo
some
Position
(cM)
Flanking
markers LOD PVE(%)
Additive
effect
Positive
allele
Plt DW 2 4 1_0113 - 1_0021 3.1 15.5 0.3 CB46
SLA 2 31 1_1139 - 1_1061 3.6 14.4 -11.5 IT93K-503-1
LA 2 85 1_0834 - 1_0297 4.0 18.5 57.0 CB46
Leaf DW 2 85 1_0834 - 1_0297 2.8 13.4 0.2 CB46
Plant transp Total 6h 2 85 1_0834 - 1_0297 2.9 13.1 8.9 CB46
Conductance High VPD 5 19 1_0806 - 1_0557 3.2 16.3 0.0 IT93K-503-1
Conductance Low VPD 5 20 1_0806 - 1_0557 2.8 13.3 0.0 IT93K-503-1
Conductance Low VPD 5 23 1_0806 - 1_0557 3.3 14.0 0.0 IT93K-503-1
Conductance Low VPD 7 13 1_0279 - 1_1482 3.6 15.0 0.0 IT93K-503-1
SLA 9 25 1_0051 - 1_0048 4.9 19.7 13.5 CB46
Conductance high VPD 9 52 1_0425 - 1_1337 2.6 11.5 0.0 IT93K-503-1

36. What platform for phenotyping?
• 3-D imaging of Leaf Area
• Leaf area + Transpiration together
• VPD effects

37. Basic considerations on trait phenotyping
Root / Water extraction
Leaf area development
Leaf conductance
Growth response to soil drying

38. Fraction of Transpirable Soil Water (FTSW)
Norrmalizedtranspiration
1.0 0.5 0.00.25
0.0
1.0
Growth response to water stress
0.75

39. Fraction of Transpirable Soil Water (FTSW)
Norrmalizedtranspiration
1.0 0.5 0.00.25
A – Standard genotype
B – Early stomata closure
C – Late stomata closure
0.0
1.0
Growth response to water stress
Early growth decline = water saving
0.75

40. Fraction of Transpirable Soil Water (FTSW)
Norrmalizedtranspiration
1.0 0.5 0.00.25
A – Standard genotype
B – Early stomata closure
C – Late stomata closure
0.0
1.0
B’ – Early stomata closure + stress relief
C’ – Late stomata closure + stress relief
Irrigation or rain
Growth response to water stress
Water saving, but…
0.75

41. 0.00.20.40.60.81.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Fraction of transpirable soil water
Normalizedtranspirationrate
---- ---- Bambey 21:FTSW = 0.63
0.00.20.40.60.81.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Fraction of transpirable soil water
Normalizedtranspirationrate
Mouride:FTSW = 0.44
(A)
Glasshouse
0.00.20.40.60.81.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Fraction of transpirable soil water
Normalizedtranspirationrate
---- ---- IT82E-18:FTSW = 0.62
0.00.20.40.60.81.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Fraction of transpirable soil water
Normalizedtranspirationrate
IT84S-2049:FTSW = 0.47
(B)
Glasshouse
0.00.20.40.60.81.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Fraction of transpirable soil water
Normalizedtranspirationrate
---- ---- Bambey 21:FTSW = 0.69
0.00.20.40.60.81.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Fraction of transpirable soil water
Normalizedtranspirationrate
Mouride:FTSW = 0.48
(C)
Outdoors
0.00.20.40.60.81.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Fraction of transpirable soil water
Normalizedtranspirationrate
---- ---- IT82E-18:FTSW = 0.71
0.00.20.40.60.81.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Fraction of transpirable soil water
Normalizedtranspirationrate
IT84S-2049:FTSW = 0.49
(D)
Outdoors
Soil moisture thresholds
for transpiration decline - cowpea

42. (A)
(B)
Soil moisture thresholds
for transpiration decline - chickpea

43. What platform for phenotyping?
• Plant transpiration
• Automatic re-watering to set weight

44. In summary
New technologies offer great opportunities
Platform development driven by research questions
Traits: building block or emerging consequence??
Link of traits to yield
Trait response to environmental cues

45. Thank you
Collaborators:
F. Chaumont (Univ. Louvain)
G. Hammer / A. Borrell / G McLean /
E van Oosterom (Univ. Queensland)
B Sine / N Belko / Ndiaga Cisse (CERAAS)
C Messina (Pioneer)
Donors:
B&MG Foundation
GCP
ACIAR
DFID
ICRISAT
Technicians / Data analyst:
Srikanth Malayee
Rekha Badham
M Anjaiah
N Pentaiah
Students:
M Tharanya
S Sakthi
T Rajini
N Belko
Colleagues:
KK Sharma / T Shah / F Hamidou
HD Upadhyaya / R Srivastava / Bhasker Raj
SP Deshpande / PM Gaur