Translating mechanisms of biological nitrogen response from Brachypodium to cereal crops

Dr Stephanie Smith (CINTRIN PDRA) (SLCU)Dr Matt Milner (NIAB), Dr Alison Bentley (NIAB), Prof Ottoline Leyser (SLCU)

Nitrogen use in India

• Nitrate application rate in India can be double that of UK (>240 kg/Ha)

• Heavy subsidies - little incentive to reduce

• Pressure of food security – 1.2 billion inhabitants

• Severe pollution, including agricultural air pollution

Good & Beatty 2011

Cambridge-India Network for Translational Research in Nitrogen

A translational pipeline

• WP1: ‘Interdisciplinary research to prime the translation of biological N use’

• Two models: Arabidopsis and Brachypodium

• Four target crops: wheat, sorghum, pearl millet, foxtail millet

• Aim: use genetic approaches to identify targets for breeding new crop ideotypes

Brachypodium distachyon as a model

• Minimum generation time: 8 weeks

• Diploid, self-fertilising, very high degree of homozygosity, few repeating elements, no population bottleneck, transformable

• Sequenced lines and RILs available

• Good synteny with rice, maize, sorghum, millet and other cereals

• Similar anatomy and developmental staging to wheat

Neil Harris, University of Alberta

A developmental difference to nitrate

• Previous observations: Arabidopsis displays two contrasting growth strategies when nitrate limited

• Shoot branching (tillering) is one key area of difference (along with flowering time, seed production, root:shootratios)

• Question of ‘sensitivity’ to nitrate status

‘James Dean’ Strategy: Live fast and die young!- Nitrate insensitive

Alternative strategy - ‘wait in hope’ - it might be worth it!- Nitrate sensitive

Brachypodium accessions differ in shoot branching variation and plasticity

• Different accessions of Brachypodium have different inherent levels of branchiness – ‘variation’

• But they also have different levels of shoot branching in response to nitrate – ‘plasticity’

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16% increase

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40% increase

All 1.8 mM NO3- : 0.2 mM vs 1.8 mM NO3- :

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Genetic strategies to uncover N use targets

• CRISPR/Cas9: Targeting members of strigolactone synthesis and signalling pathway – increased branching in Arabidopsis

• MAX3, MAX4, D14, TB1 (BRC1)

• Analyse if same response happens in Brachypodium – if so, promise for cereals in future (for both concept and technique)

• GWAS of natural variation in accessions, OR mapping within a RIL population to find the underlying basis of N sensitivity and tillering

max2

Bd21 (bg)

Translating N use targets into wheat

• WP3: Translational N germplasm for pre-breeding and exploitation

• Along with defining optimal N use in key cereal germplasm (WP2), findings can be taken into genetics-led pre breeding/transgenic approaches

• Use of the NIAB Elite wheat MAGIC population, wheat introgression lines, tetraploid and hexaploid wheat TILLING mutants

Dr Matthew Milner

Overall future aims

• Identify genetic targets involved in shoot branching/N sensitivity

• Feed research findings into pre-breeding programs for target crops

• Contribute to CINTRIN’s integrated duel strategy for simultaneous improvement of (i) agronomic and (ii) biological N use

• Foster interdisciplinary, international collaborations

Acknowledgements

• CINTRIN network

• Ottoline Leyser lab, SLCU

• Devin O’Connor lab, SLCU

• Sam Elton

• All collaborators & funding bodies

Funded by:

Additional info

• WP1. Interdisciplinary research to prime the translation of biological N Previous work at SLCU has identified fundamental variation in Arabidopsis thaliana relating to shoot branching plasticity correlated with biomass allocation. We have developed a screening system for Brachypodium distachyon and Setaria italicaallowing precise control of nitrate supply. CRISPR/Cas9 mutagenesis is also underway in Brachypodium to target key candidate genes.

• WP2. Defining optimal N use in key cereal germplasm

• In order to maximize yield potential and grain quality, there is a pressing need to develop a better mechanistic understanding of the physiological basis to N and carbon (C) partitioning. This WP is developing new methodologies for application of stable isotopes to evaluate genetic variation in N use in key cereal crops and coupling these experiments with screening in WP1 and WP3.

WP2

WP3

• WP3. Translational N germplasm for pre-breeding and exploitation• This WP is focussed on the translation of new understanding of

variation in biological N use from model plants (WP1) and new physiological N knowledge (WP2) via genomics-led pre-breeding. We are using and developing next generation germplasm resources and genomic tools to allow widespread uptake of WP1 and WP2 findings into breeding and production. This includes the use of the NIAB Elite wheat MAGIC population, wheat introgression lines, tetraploid and hexaploid wheat TILLING mutants, the ICRISAT sorghum BC-NAM population and finger millet ICRISAT variety collections. In addition, a transgenic approach is being used to modify key targets in wheat (linking to WP1).

WP4

• WP4. Field-scale evaluation of optimal N allocation to identify HYLO lines

• The knowledge and germplasm developed or identified in WP1, WP2 and WP3 is being assessed in “Optiplot” wheat field trials with six ramped N levels in the UK and India. This will extend knowledge of biological N use at the field scale and several important agronomic characters are being assessed for hypothesis testing in relation to biological N use. We are comparing the performance of elite varieties and germplasm from WP3 for “N requirement” and to identify High Yield Low Optimum (HYLO) traits. The N requirement or optimal N rate is the rate at which any further increase in N application will result in greater N fertiliser costs than the value of the additional grain produced.

Feed Composition

9mM NO3 1.8mM NO3

Standard ATS stock solutions ml ml

1M KNO3 50 10

1M K...PO4 buffer (pH 5.5) 25 25

1M MgSO4 20 20

1M Ca(NO3)2 20 4

20mM Fe-EDTA 25 25

micronutrients 10 10

deionised water 9850 9850

Additional solutions

1M KCl 0 40

1M CaCL2 0 16

10 Liter