High-Throughput Screening Protocol For Salinity Tolerance in Lentil

Hossein V Kahrood

Background• First grown commercially in Australia during the early 1990s• Mainly grown in the semi-arid regions of Victoria and South Australia• Export crop

Salinity Toxicity – A Problem for Crop Expansion • A major abiotic stress

• Global problem in arid and semi-arid regions due to water scarcity and poor drainage of irrigated lands

• In Australia, 32 million hectares of cropland affected by salt

• Lentil is significantly more sensitive compared to commonly cultivated Australian broad-acre crops such as barley, wheat and canola

• Salinity is a concern in all states especially in NSW and WA, affecting the crop expansion into new areas

Salinity Tolerance in Lentil vs Other Crops

Lentil: 20% yield loss at EC = 2 dS/mLentil: 90–100 % loss at EC = 3 dS/m

Compared to commonly cultivated Australian broad-acre crops such as;

Barley: 10% yield loss at EC = 6.6 dS/mWheat: 10% yield loss at EC = 4.9 dS/mCanola: 10% yield loss at EC = 7.3 dS/m

Low salinity threshold level in lentil.

Rationale and Aims• Pulse Breeding Australia (PBA) Lentil Breeding Program continuously focuses on

breeding varieties with improved salt tolerance• Conventional screening- laborious, expensive and less reliable • Aim – to develop cost-effective and reliable high-throughput screening protocols

that can be deployed in the breeding project

Pilot Experiment

Development of HTP Screening Method• Six lentil varieties with most discriminatory salinity

scores– ILL2024, CIPAL 1522 - Tolerant – PBA Bolt, PBA Hurricane - Medium tolerant/intolerant– PBA Ace, PBA Jumbo2 - Intolerant

• Four salt levels - 0, 42.5, 85 and 127.5 mmol NaCl as optimised within lentil breeding program

• Four replicates per salt treatment (total 96 plants) to choose most discriminatory concentration for screening unknown genotypes

• Imaging at 0, 90⁰ and top view before the start of salt treatment, then every second day for up to 6 weeks (early growth).

• Measurements– Total pixels; a proxy for shoot area– Fresh biomass– Dry biomass

Development of HTP Screening Method-Measurements

https://youtu.be/8pAaBDznmh0

Side view image Processed image

From Image to Data

Response of Different Salt Concentrations on Projected Shoot Area

Intolerant MI/MT Tolerant

Intolerant MI/MT Tolerant

Response of Different Salt Concentrations on Above-ground Biomass

Intolerant MI/MT Tolerant

Fresh Weight Dry Weight

Df Sum sq Mean sq F value Pr(>F)Treatment 3 40697 13566 117.974 < 2E-16

Line 5 3765 753 6.548 4.50E-05Treatment:Line 15 5336 356 3.094 0.000673

Residuals 72 8279 115

Correlation Between Above-ground Weights and LemnaTecMeasurements

R2 = 0.88p-value < 0.001

R2 = 0.92p-value < 0.001

Fresh Weight Dry Weight

Projected Shoot Area Over Time

Boxplot for projected shoot area under salt treatments.

Determination of Salt Concentration for Screening Unknown Genotypes

Comparisons diff lwr upr p adjT2-T1 -0.00041 -0.00991 0.009088 0.999T3-T1 0.00295 -0.00655 0.012447 0.820T4-T1 0.016366 0.00687 0.025863 0.001T3-T2 0.003359 -0.00614 0.012856 0.757T4-T2 0.016776 0.007279 0.026273 0.000T4-T3 0.013417 0.00392 0.022913 0.004

Selected concentration = 100 mmol NaCl

Implementation of HTP Screening Method into PBA Lentil Breeding Program

Experimental Design• 191 advanced breeding lines

– including 12 check varieties

• Two salt levels – 0 and 100 mmol NaCl

• Partial replicated design – 20% of the lines 4x replicated

Data AnalysisLemnaTec image data• Shoot area

– The rate of biomass accumulation under treatment

• Linear mixed model

• Best Linear Unbiased Prediction (BLUE)

• Salinity Susceptibility Index (SSI)

Elemental detection • Plasma spectrometry

• Sodium (Na+) and Potassium (K+)

• Roots and leaves

Rate for Biomass Reduction and Senescence

Rate growth / biomass reduction under control and salt treatment

Biomass reduction Senescence

Rate of senescence under control and salt treatment

Ranking Based on Salt Susceptibility Index (SSI)

* Fischer and Mauer 1978.

SSI = (1 − 𝑇𝑇𝑡𝑡𝑇𝑇𝑐𝑐

)/ (1 − 𝜇𝜇𝑡𝑡𝜇𝜇𝑐𝑐

)*

Salt Susceptibility Index (SSI) for Check Varieties

* Fischer and Mauer 1978.

Conventional Scoring for Salt Resistance

Na+ and K+ MeasurementsIn Roots and Shoots

Chemical Elements in Roots and Shoots

Inductively Coupled Plasma optical Emission Spectrometry (ICP-OES)

Accumulation of Na inroots and leaves

Accumulation of K inroots and leaves

Chemical Elements in Roots and Shoots

K/Na ratio

Correlation between Chemical and HTP Imaging Measurements

Na K K_Na

Root -0.03 -0.40 0.42

L Leaves 0.81 0.44 0.44

U Leaves 0.87 0.16 0.82

ICP-OES: More Elements

Remarks:

• Phenotyping by imaging: • reliable, cost-effective and HTP

• Provides a number of traits: • shoot area, plant height, chlorosis/necrosis …

• Introducing drought and soils properties (similar to the field)

Thanks to …

Molecular Genetics: Sukhjiwan KaurGarry Rosewarne

Brittney CaruanaRuwani DissanayakeArun Shunmugam

PPVB:Adam DimechKatrina Hill

Linear mixed model was used to calculate Best Linear Unbiased Estimates (BLUE).

y = Xβ + Zu + e

y response vector of values for the trait being analysed; β vector of fixed effects; u vector of random effects; e vector of residual effects;X, and Z are the design matrices corresponding to β and u, respectively.

Complete randomised block design with pairing of a salt treated and untreated plant from same variety

Given that there was a good relationship between the aboveground biomass and sum of pixel area, the following model can be used to combine image data from the three different angles (0, 90⁰ and TV):

𝑥𝑥 = ((∠0 + ∠90)/2)2 + (∠𝑇𝑇𝑇𝑇)2

Data Summarisation

Ca

Zn

B

Mg

Fe

Al

Mn

Cu

P

S

ICP-OES: More Elements