trait phenotyping: about asking the right questions to harness phenomics' progress

Trait phenotyping:

About asking the right questions

to harness phenomics’ progress

Vincent Vadez – Jana Kholova

ICRISAT

Phenodays 2013 – 16-18th October 2013

Today’s presentation

Basic considerations on trait phenotyping

Root / Water extraction

Leaf area development

Leaf conductance

Growth response to soil drying

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 traitPhenotyping should focus on the building blocks

Accurate Non accurate

Precise

Not precise

Precision / Accuracy of phenotypingIs my phenotype the right one?

Are measurements Good enough?

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




Leaf conductance


Lysimetric facility at ICRISAT

Advantages:• Gravimetric• Long term (3 Wks-maturity)• High throughput (5000 PVCs)

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 0.75D

roug

ht w

ater

ext

ract

ion

(kg

plan

t-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 1003SEQ 11CAL 96

SAB 259

RAA 21

ICA Quimbaya

SER 8

Mean: 0.56LSD0.05: 0.13

SEC 16

Mean: 6.84LSD0.05: 1.53

r = 0.08

No relation between water extraction (WS) and root length / RLD

Beans Chickpea

Post-rainy season Rainy season

0 1000 2000 3000 4000 5000 6000 700002468

10121416

Total water extracted (g plant-1)

Pod

yiel

d (g

pla

nt-

1)

0 1000 2000 3000 4000 5000 6000 70000123456789

10


Pod

yiel

d (g

kg-

1)

No relationship between total water extracted and grain yield

250030003500400045005000550060006500700002468

101214


Pod

yiel

d (g

pl

ant-

1)

Cowpea

Peanut

3500 4000 4500 5000 5500 6000 6500 70000.01.02.03.04.05.06.07.0


Pod

yiel

d (g

pl

ant-

1) Bean

Peanut

Rainy seasonRainy season

Pod yield and water extraction

Water extraction pattern (WS) in 12 tolerant / 8 sensitive chickpea

Zaman-Allah, Jenkinson, Vadez 2011 JXB

21 28 35 42 49 56 63 70 77 84 91 980123456789

Days after sowing

Cum

ulat

ed W

ater

Use

d (k

g pl

-1)

Flowering

SensitiveTolerant

Tolerant: less WU at vegetative stage, more for reproduction & grain filling


21 28 35 42 49 56 63 70 77 84 91 980123456789

Days after sowing

Wat

er u

sed

(kg

pl-1

)

SensitiveTolerant

Relationship between grain yield and water useLow early vigorLow leaf Gs


0 1 2 3 40.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Weeks after panicle emergence

WU

(kg

plan

t-1

wee

k-1)

ICMH01029

ICMH01040

ICMH01046

PRLT2/89-33

Vadez et al 2013 – Plant Soil

H77/833-2

ICMH02042Terminal drought

sensitive

Terminal drought tolerant


Water extraction pattern (WS) in pearl millet

Flowering

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.50

4

8

12

16

20

R² = 0.710763888329146

Early stress

WU in week 3 after panicle emergence

Grai

n Yi

eld

(g p

lant

-1)

Tolerant: EUW = 45 kg grain mm-1

Relationship between grain yield and water use

What platform for phenotyping?

• Outdoors / yield-based

• Water extraction at key time - Automation

• Possible IR surrogates• Staygreen




Leaf conductance


Leaf

are

a

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

Arrows indicates Re-watering

Profile of water use from flowering to maturityIn peanut

Sensitive

Tolerant

Tolerant lines develop a smaller canopy under WW conditions

Ratnakumar and Vadez 2011

Field trial

0 5 10 15 20 250

1000

2000

3000

4000

5000

6000

A = 2,91Fleur 11WW condition

R² = 0,999

Nodes number

Leaf

are

a (c

m²)

Field trial

0 5 10 15 20 250

1000

2000

3000

4000

5000

6000

A = 2,63ICG 1834WW condition

R²= 0.91

Nodes number

Leaf

are

a (c

m²)

Peanut

Coefficients relating leaf area to node number

Need a HT method to measure the dynamics of leaf area development


21 28 35 42 49 56 63 70 77 84 91 980123456789

Days after sowing

Wat

er u

sed

(kg

pl-1

)

SensitiveTolerant

Relationship between grain yield and water useLow early vigor

6 8 10 12 14 16 18 20 22 24 260

100020003000400050006000700080009000

10000f(x) = 23.3019958 exp( 0.25624175 x )R² = 0.936669675258928

Node number on main stem

Leaf

are

a (c

m2)

6 8 10 12 14 16 18 20 220

2000

4000

6000

8000

10000

12000

f(x) = 11.99515 exp( 0.309955 x )R² = 0.960712977568497

Node number on main stem

Coefficients relating leaf area to node number

Chickpea

Need for a leaf count along with leaf area

1500 1700 1900 2100 2300 2500 2700 2900 3100 33000

5

10

15

20

25

30

R² = 0.725453254471581

Leaf area at anthesis

Grai

n yi

eld

unde

r W

ater

stre

ss

Relation between LA and grain yield in postrainy sorghum

Higher grain yield is related to lower anthesis leaf area

Variation in leaf areaVariation 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 (c

m2)

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

Canopy developmental dynamics

TPLA varying 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 = 0.66TPLAmax = 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 = 20TPLA_prod_coef - 0.018

0.33

0.66

LA development depends on:

• TPLA production coefficient and

• TPLA inflection ratio

0

500

1000

1500

2000

2500

3000

thermal time

kg ha-1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

m2 m-2

stover TR highstover TR lowgrain TR highgrain TR lowLAI TR highLAI TR lowSD TR highSD TR low

Simulation:severe terminal drought

Delayed senescence

Delayed water exhaustion

LAI

S/D ratiobiomass

water conservation

during vegetative growth and its utilization for

better grain filling = stay-green

kg h

a-1

Thermal timeYIELDflowering

~830 0dayStaygreen phenotype is a consequenceof smaller/delayed leaf development


• 3-D imaging of Leaf Area

• Measurements at early stages

• Soil moisture effects

• VPD effects (need HT measurements)




Leaf conductance


Vapor Pressure Deficit (VPD, in kPa)

Tran

spira

tion

rate

(g c

m-2 h

-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 = waterVadez et al 2013 – FPB in press

Terminal drought sensitive


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)

H77/2 833-2PRLT-2/89-33

Tran

spir

atio

n (g

cm

-2 h

-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

0 1 2 3 40.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Weeks after panicle emergence

WU

(kg

plan

t-1

wee

k-1)

ICMH01029

ICMH01040

ICMH01046

PRLT2/89-33

Vadez et al 2013 – Plant Soil

H77/833-2

ICMH02042Terminal drought

sensitive



Water extraction pattern (WS) in pearl millet

Flowering

Water saving from lower conductance

4 replications

RH & T hourly recording

Weighing:7-11am = low VPD11am-15pm = high VPD

8” pots re-saturated every daysoil evaporation minimized with plastic beads

Mapping of water saving traits

Transpiration response to high VPD - Peanut

-1 0 +33

15-30% yield decrease, especially at high latitudes (12-15ºN)

% yield increase with VPD response trait - Peanut

Mouride

If VPD < 2.09, TR = 0.0083 (VPD) – 0.002 If VPD ≥ 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)

0.025 0.030 0.035 0.040 0.045 0.050 0.055 0.060

-40-30-20-10

01020304050

f(x) = 2169.59291316378 x − 85.0377700360549R² = 0.64243052275186

Transpiration rate under high VPD

Resi

dual

tran

-sp

irati

on

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)

300 400 500 600 700 800 900 1000 1100 12000

50

100

150

200

250

f(x) = 0.150035478304917 x + 44.7065750082861R² = 0.685888174332073

Leaf area (cm2 plant-1)

Tota

l tra

nspi

-ra

tion

(g p

lant

-1)

QTLs from ICI Mapping – Drought tolerance traits

TraitNameChromo

somePosition

(cM)Flanking markers LOD PVE(%)

Additive effect

Positive allele

Plt DW 2 4 1_0113 - 1_0021 3.1 15.5 0.3 CB46SLA 2 31 1_1139 - 1_1061 3.6 14.4 -11.5 IT93K-503-1LA 2 85 1_0834 - 1_0297 4.0 18.5 57.0 CB46Leaf DW 2 85 1_0834 - 1_0297 2.8 13.4 0.2 CB46Plant transp Total 6h 2 85 1_0834 - 1_0297 2.9 13.1 8.9 CB46Conductance High VPD 5 19 1_0806 - 1_0557 3.2 16.3 0.0 IT93K-503-1Conductance Low VPD 5 20 1_0806 - 1_0557 2.8 13.3 0.0 IT93K-503-1Conductance Low VPD 5 23 1_0806 - 1_0557 3.3 14.0 0.0 IT93K-503-1Conductance Low VPD 7 13 1_0279 - 1_1482 3.6 15.0 0.0 IT93K-503-1SLA 9 25 1_0051 - 1_0048 4.9 19.7 13.5 CB46Conductance high VPD 9 52 1_0425 - 1_1337 2.6 11.5 0.0 IT93K-503-1


• 3-D imaging of Leaf Area

• Leaf area + Transpiration together

• VPD effects




Leaf conductance


Fraction of Transpirable Soil Water (FTSW)

Nor

rmal

ized

tran

spira

tion

1.0 0.5 0.00.25

0.0

1.0

Growth response to water stress

0.75


Nor

rmal

ized

tran

spira

tion

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


Nor

rmal

ized

tran

spira

tion

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 rainGrowth response to water stress

Water saving, but…

0.75

0.00.20.40.60.81.00.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Fraction of transpirable soil water

Nor

mal

ized

tran

spir

atio

n ra

te

---- ---- Bambey 21: FTSW = 0.63

0.00.20.40.60.81.00.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4


Nor

mal

ized

tran

spir

atio

n ra

te

Mouride: FTSW = 0.44(A)Glasshouse

0.00.20.40.60.81.00.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4


Nor

mal

ized

tran

spir

atio

n ra

te

---- ---- IT82E-18: FTSW = 0.62

0.00.20.40.60.81.00.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4


Nor

mal

ized

tran

spir

atio

n ra

te

IT84S-2049: FTSW = 0.47(B)Glasshouse

0.00.20.40.60.81.00.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4


Nor

mal

ized

tran

spir

atio

n ra

te

---- ---- Bambey 21: FTSW = 0.69

0.00.20.40.60.81.00.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4


Nor

mal

ized

tran

spir

atio

n ra

te

Mouride: FTSW = 0.48(C)Outdoors

0.00.20.40.60.81.00.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4


Nor

mal

ized

tran

spir

atio

n ra

te

---- ---- IT82E-18: FTSW = 0.71

0.00.20.40.60.81.00.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4


Nor

mal

ized

tran

spir

atio

n ra

te

IT84S-2049: FTSW = 0.49(D)Outdoors

Soil moisture thresholds for transpiration decline - cowpea

(A)

(B)

Soil moisture thresholds for transpiration decline - chickpea


• Plant transpiration

• Automatic re-watering to set weight

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

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 FoundationGCPACIARDFIDICRISAT

Technicians / Data analyst:Srikanth MalayeeRekha BadhamM AnjaiahN Pentaiah

Students:M TharanyaS SakthiT RajiniN Belko

Colleagues:KK Sharma / T Shah / F HamidouHD Upadhyaya / R Srivastava / Bhasker RajSP Deshpande / PM Gaur

trait phenotyping: about asking the right questions to harness phenomics' progress

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