Digital image analysis for high throughput phenotyping of powdery mildew resistance in oats

Transcription

1 Digital image analysis for high throughput phenotyping of powdery mildew resistance in oats L. Gallego 1 G. Montilla 1 E. Prats 1 CSIC Institute for Sustainable Agriculture, Cordoba - Spain

2 Oat Powdery mildew Causal agent Ascomycete Blumeria graminis (syn. Erysiphe graminis) forma special avenae

3 Vegetative life cicle Blumeria graminis Wind dispersed Mainly by asexual, haploid conidia (about 200K conidia per colony). Pathogenicity fixed rapidly, the selection pressure is high Sexual phase is mainly for overwintering / oversummering. Allows recombination of desirable characteristics. EVOLUTIONARY RISK IS VERY HIGH

4 Timing of infection process 2 hours 18 hours 24 hours 24 hours 2 days 5 days 7 days Late stages

5 Resistance mechanisms against Blumeria graminis Penetration resistance Hypersensitive Response Usually more durable Usually easily overcome by new emerging isolates

6 Screening for disease resistance MACROSCOPIC Faster than microscopic BUT also time consuming Subjective (depends on the visual evaluation) Does not discern the particular resistance mechanisms underlying the resistance response MICROSCOPIC Slower than macroscopic Objective Allow to discern among the difference resistance mechanism. This is crucial to breed resistance plants that show durable resistance in the field.

7 Digital assesment of disease symptoms may be the solution? Camera Sample Fiji Free Software distributed by ImageJ 1st sample analysis 2nd sample analysis

8 Experimental settings PLANT GROWTH Until 40 days aprox. INOCULATION 5th leaf FIXATION MICROSCOPIC ASSESMENT At 48h TAKING PICTURES MACROSCOPIC ASSESMENT After 7 days

9 Macroscopic assesment SCRIPT TRANSFORM SCRIPT PIXELS

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13 SCRIPT PIXELS

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15 SCRIPT PIXELS 40% DISEASE SEVERITY

16 Microscopic assesment Genotipo 129 Genotipo 180 REP 1 REP 2 REP 3 REP 1 REP 2 REP 3 A+HR A+HR A A A+HR H A+HR A+HR A H A+HR H A+HR A A+HR A H A A A+HR A+HR H H A+HR A+HR A A+HR A+HR H A+HR A A A A A+HR H A+HR A+HR A A+HR A+HR A+HR A+HR A+HR H+HR A+HR A+HR A A+HR A+HR A+HR H A+HR H A+HR A+HR A H H H A A+HR A+HR H H H A+HR A+HR A+HR H A H A+HR A+HR A A+HR H H A+HR A A+HR H H A+HR A+HR A A H H A A+HR A+HR A+HR A A H A A A+HR A H H A+HR A A+HR H H A+HR A+HR A+HR A+HR A+HR H A+HR A+HR A+HR A+HR A+HR A+HR A+HR A A+HR A+HR A H+HR A+HR A+HR A A+HR A H A A+HR A+HR A+HR A A+HR A+HR A+HR A+HR A+HR H A+HR H A+HR A A+HR A+HR A+HR H A+HR A+HR A+HR A A A A A+HR A+HR H A+HR A A A+HR A+HR A+HR A+HR A A A A+HR A H A A+HR A A+HR A H H A A A A A+HR A+HR A A H A+HR A+HR A+HR A+HR A A A A A A A+HR A H H H A+HR A+HR A + HR A H A Penetration resistance Haustoria formation and colony development Early Hypersentitve Response Late Hypersensitive Response

17 The main GOAL To make association analysis (GWAS) to look for markers related to specific resistance mechanisms, we would need to analyze microscopically thousand of samples which is not feasible. Is it possible to reduce the number of samples for microscopic assessment (to the resistant ones) and assign a number of haustoria to susceptible accessions based on macrocopic assesment? We are validating a posible correlation between macroscopic disease and percentage of haustoria for evaluating faster susceptible accessions This would reduce the number of samples to assess microscopically

18 Macro and Micro CORRELATION

19 Macro and Micro CORRELATION Post-haustorial mechanisms??

20 Resistant and moderately resistant Accessions <50 % disease severity Population (80 genotypes) GWAS Susceptible and moderately susceptible Accessions >50 % DISEASE SEVERITY Papilla Early H.R. Haustoria Late H.R. Markers associated to particular resistance mechanism i.e. papilla formation or HR Evaluated by Fiji + CORRELATION

21 THANKS FOR YOUR ATTENTION =)