high-throughput screening protocol for salinity tolerance in lentil€¦ · lemnatec image data •...
TRANSCRIPT
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