Combining field phenotyping and next generation genetics to uncover markers, genes and biology underlying drought tolerance in wheat.

Lead Research Organisation: University of Liverpool
Department Name: Institute of Integrative Biology

Abstract

Food security is internationally recognised as one of the major global challenges of the 21st century. By 2050, it is predicted that world food production will have to increase by 50% to meet demand. This is against the pressures of global climate change and resource limitations. Meeting this challenge is going to require the development of innovative strategies to make use of our unprecedented knowledge of modern bioscience in the post genomic era. Developing new varieties of wheat will be fundamental to meeting the 2050 goal.
For wheat growers Internationally and specifically in India one of the key issues is drought. Drought means farms cannot always guarantee a good harvest with major implications for the livelihoods and household food security of small-holder farmers. Low rainfall also reduces the land area that can be farmed. Throughout the last century improvements in drought tolerance have come through breeding crops that grow in such a way that drought and drought sensitive stages of growth do not coincide. There is however, huge potential to breed new varieties capable of maintaining stable yields in drought conditions.

In collaboration with the University of Bristol and the John Innes Centre (Norwich), work at the University of Liverpool has generated sequence data for the wheat genome. Using this information we have developed new methods to rapidly uncover the genetic variation in wheat. By combining an understanding of genetic variation with a careful study of performance under drought conditions it becomes possible to associate genetic variation with improved drought tolerance. Using this genetic information "molecular marker" tools cab be built, that can be used to rapidly select for lines that are drought tolerant. It is also possible to stack up multiple markers for different traits. As a consortium of interdisciplinary research scientists from the UK and India we plan to use this approach to identify molecular markers associated with drought tolerance in wheat and lay the foundations of an accelerated breeding program to incorporate drought tolerance into Indian wheat varieties.

The approaches we will be using will provide a blueprint for how state-of-art technologies can be applied to important food security issues. Much of the output we generate can be used to identify markers for other traits. It will also result in highly trained researchers in India and the UK capable of applying these new approaches. Through outreach work we aim to engage with other researchers and stakeholders and apply this methodology to other traits in wheat and different crops.

Technical Summary

The overall aim of this project is to combine physiological and modern molecular breeding approaches to provide tools to accelerate wheat breeding associated with abiotic stress tolerance. This project will use an enrichment platform developed in Liverpool, to genotype by sequencing a diversity panel of wheat. This will generate tens of thousands of varietal SNPs for genotyping and provide the raw materials for a SNP-based molecular breeding program in India and an international resource. Information to users will be made publicly available through a web browser interface for immediate impact. The panel of lines will be phenotypically scored for yield, and physiological traits associated with water use efficiency over two seasons under drought regimes at four sites across India. Statistical analysis will be used to associate genomic regions in specific lines, with drought tolerant phenotypes and a series of markers for yield stability identified and tested. Further methods of genotypic selection and bulk segregation will be utilised to further narrow down genomic regions with the aim of potentially identifing candidate genes conferring enhanced drought tolerance. Upon completion, this project will have generated a series of drought tolerant markers matched to drought conditions in India, thus providing important raw materials for breeding programmes aimed at achieving sustainable yield under drought conditions. Drought is also a problem in the UK, with 30 % of wheat grown on drought-prone soils and drought related losses accounting for £224-448M each year. Therefore, this proposal is also likely to have impact on UK breeding as well as in India. Finally, the aim of this project is to generate trained individuals in both India and the UK in the area of computational biology and phenotyping, specifically, how next generation genomic approaches can be applied to crop breeding.

Planned Impact

The secure supply of affordable sustainably produced wheat is of fundamental importance to global food security and to poverty alleviation. Wheat is the UK's largest cereal crop with an estimated annual production of 14 million tonnes, and total value (seed and processed products) of circa £15 billion. In India, wheat is a widely grown crop and is integral to the rice-wheat rotation of the Indo-Gangetic plains (IGP) which constitutes 10 million hectares in India and as a rotation is one of the world's largest agricultural production systems, occupying 24 million hectares of cultivated land on the IGP and in China. Wheat is a key staple essential to the household food security of 800 million people in India, many living on an income of less than $5 a day.

The key beneficiaries of this research programmes will be the wheat breeding community in India and the UK and the seed producing sector (both public and private) in both countries, and ultimately farmer co-operatives and farmers and through to consumers at large, though increased yield and through resilience in wheat production. Additional beneficiaries will be the plant and crop science community involved in crop improvement through the use of 'omic technologies.

The research will provide immediate value to wheat breeders through the provision of sets of SNPs, which can be used as molecular markers in wheat breeding. Additionally by the conclusion of the project, further impact will be achieved through the identification of molecular markers for gene pathways that confer enhanced drought tolerance in wheat. Whilst being of value to breeders in the UK and worldwide, this information will have national relevance to Indian wheat breeding programmes, being conducted on cultivars pre-adapted to agro-ecological zones in regions of the IGP where both drought stress and water logging can substantially reduce attainable yield. The impact of this research will be to provide new tools for wheat breeders to acceleate wheat breeding and enable deployment of new cultivars for small holder agriculture in a timely way to address cropping sustainability in relation to changing patterns of water resource availability. The provision of improved breeding lines for cultivar development is fundamentally important as a plank in addressing poverty alleviation.

Strategically the proposed research will contribute to elevating the wheat yield potential in the face of global climate change, which in the UK in high input systems is targeted at 20 ha-1, through improvements in plant breeding and crop management. The potential economic impact of a 50% increase in UK wheat production (current attainable yield on average 8 t ha-1) is in excess of £7 billion pa to the UK economy. Whilst Indian agriculture is characterised by dominance of small holder agriculture, wheat yields vary from ~4 t ha-1 on productive larger farms in the north-eastern plains zones to 0.7 t ha-1 in the southern hills zone. Irrigated regions are under threat of reduced water availability and the emphasis of governmental agricultural policy is to improve productivity in rainfed systems. Matching the increasing demographic demand for wheat in India by 2020 will require a 2.2% annual increase in production which in rainfed systems will require abiotic stress tolerant germplasm. This research will directly contribute to attaining that goal.

The research project will provide capacity building at the cutting-edge of molecular plant breeding and phenotypic trait analysis for drought tolerance, through training of Indian research fellows and through knowledge exchange amongst partners at annual workshops. By implementation of a clearly defined impact pathway, knowledge will be disseminated widely across plant breeding groups in India and the UK and through networked collaborations with other international wheat improvement programmes coordinated through CIMMYT and ICARDA.

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/L011786/1 30/11/2014 30/09/2016 £763,833
BB/L011786/2 Transfer BB/L011786/1 01/10/2016 30/11/2018 £436,305
 
Description Objective 1. Establishing and genotyping the diversity panel: To date a diversity panel comprising of approximately 220 wheat accessions has been assembled from collections established over the last 30 years at ICAR-Indian Institute of Wheat and Barley Research (IIWBR) that includes rain-fed wheat genotypes as well as elite lines, and lines from CIMMYT and ICARDA pre-screened for drought and heat tolerance. After considerable difficulty in shipping this material from India, we finally received DNA of these lines in March 2017. In May 2017, these 220 lines were genotyped using the 35K Wheat Breeders array, and initial analysis indicates that over 20,000 informative polymorphic SNPs have been detected between the different lines.
The Watkins collection is a genetically and phenotypically diverse collection of bread wheat landrace cultivars that captures a snapshot of genetic diversity from before the start of modern breeding. A subset of this collection, comprises 119 lines that are thought to represent 96-98% of the total genetic diversity of the collection was analysed using a 12Mb capture probe set. An average of 48Mb of the wheat genome was captured for each sample to a minimum depth of 10x, identifying 1.2M SNPs shared between 95% of the collection (Gardiner, unpublished, https://grassroots.tools/data/under_license/toronto/Gardiner_2018-01-29_Watkins-diversity-12Mb/ ).
To date we have: 1) Established a diversity panel made up of Indian, CIMMYT and international wheat collections; 2) Genotyped the Indian diversity material (made up of CIMMYT, Indian and international lines) and the Watkins collection; 3) Analysed the population structure and produced a genome specific haplotype map and 4) Developed the first part of a shared bioinformatics resource.
Remaining: Extending the utility of shared bioinformatics resources, to develop it beyond a data store into a shared informatics resource (http://cyverseuk.org/applications/gwasser/) and including the final field phenotyping data set.
Objective 2
Full phenotypic characterisation of the diversity panel material: Phenotyping of the 220 lines of the Indian diversity panel has been carried out under two conditions (irrigated, drought) across four locations in India (Baramati, Durgapura, Vijapur and Karnal). Traits associated with drought stress were measured - germination rate, days to heading, days to physiological maturity, number of tillers, height, canopy temperature and chlorophyll fluorescence. After significant delays, we received year one phenotyping data for these lines at the end of Dec 2017 and our collaborators at ICAR-IIWBR are currently compiling year two data, which will be ready at the end of Feb 2018.
Collaborators at The University of Lancaster have also phenotyped the 119 Watkins Core Collection for water use efficiency (WUE). The capture based genotyping (objective 1) along with this preliminary phenotyping indicates that they will provide a useful source of genetic variation for traits associated with drought tolerance. A subset of 9 of these Watkins lines that demonstrate similar phenology but exhibit different WUE/biomass measurements have been selected for precision phenotyping. This precision phenotyping included detailed assessments of root characteristics and immunoassays to measure drought associated hormone concentrations (ABA, ACC, cytokinins and auxins) enabling us to further characterise the drought stress response.
To date we have: Completed the fine phenotyping of the Watkins collection. We have analysed the first year of Indian field phenotyping data and the second year's data has already been collected.
Remaining: Integrate year 1 and 2 field data (we are due to receive year two data at the end of Feb 2018).
Objective 3
Identifying markers and underlying genes responsible for enhanced drought tolerance:Now we have received the initial phenotyping data of the Indian material, and have the results of these lines interrogated with the 35K SNP array, we are in a position to carry out the GWAS and uncover markers linked to the drought tolerance phenotype. Extending this research grant a further five months, will enable us to deliver on this final objective. Completing the GWAS using two years of extensive phenotypic characterisation of the Indian drought tolerance diversity panel will allow us to identify QTLs for drought tolerance and their associated SNP markers. From this we will be able to deliver a robust set of SNP markers, linked to drought tolerance that we can pass to our Indian collaborators for validation in the field environment. We will also investigate and potentially identify candidate genes within these identified genomic regions that contribute to the ability of wheat to maintain yield under drought stress. Completion of Objective 3 will also provide us with enough experimental data to write a joint manuscript with our collaborators at the University of Lancaster and IACR-IIWBR, which we would submit within the five month extension. It will also allow us to deliver all the tools and resources necessary for our Indian collaborators to integrate into their breeding programmes.
To date: All the data is in place with the last field trial data set arriving before the end of February 2018, this has been a huge investment in time and energy and it is now critical we have the time to fully exploit these comprehensive datasets.

We have now completed the remaining objectives, we have a final conclusion and test to make before writing up and publishing the results. With luck we hope to confirm a QTL and mark which we are testing for association with water use efficiency that can be used across Indian material
Exploitation Route We are currently testing a marker for water use efficiency which hopefully could be used by our Indian partners in their breeding programs.
Sectors Agriculture, Food and Drink

 
Description International Wheat Yield Partnership (IWYP).
Amount $2,000,000 (USD)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2016 
End 01/2019
 
Description SEQUENCING THE GENIC PORTION OF SEEDS OF DISCOVERY ADVANCE PRE- BREEDING GERMPLASM TO UNCOVER THE GENETIC VARIATION
Amount £330,000 (GBP)
Funding ID BBS/OS/NW/000017 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 10/2019
 
Title promoter exon capture platform 
Description Whole genome shotgun re-sequencing of wheat is expensive because of its large, repetitive genome. Moreover, sequence data can fail to map uniquely to the reference genome making it difficult to unambiguously assign variation. Re-sequencing using target capture enables sequencing of large numbers of individuals at high coverage to reliably identify variants associated with important agronomic traits. Previous studies have implemented cDNA/exon or gene-based probe sets where promoter and intron sequence is largely missing alongside newly characterized genes from the recent improved reference sequences. Results We present and validate two gold standard capture probe sets for hexaploid bread wheat, a gene and a putative promoter capture, which are designed using recently developed genome sequence and annotation resources. The captures can be combined or used independently. We demonstrate that the capture probe sets effectively enrich the high confidence genes and putative promoter regions that were identified in the genome alongside a large proportion of the low confidence genes and associated promoters. Finally, we demonstrate successful sample multiplexing that allows generation of adequate sequence coverage for SNP calling while significantly reducing cost per sample for gene and putative promoter capture. Conclusions We show that a capture design employing an 'island strategy' can enable analysis of the large gene/putative promoter space of wheat with only 2x160 Mb probe sets. Furthermore, these assays extend the regions of the wheat genome that are amenable to analyses beyond its exome, providing tools for detailed characterization of these regulatory regions in large populations. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Currently being used by DFW, CIMMYT and US CAPS projects 
 
Title Watkins core collection re-sequencing data 
Description Re-sequence data for the Watkins collection 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact International collabration 
URL https://grassroots.tools/data/under_license/toronto/
 
Description CIMMYT seeds of discovery 
Organisation Australian National University (ANU)
Country Australia 
Sector Academic/University 
PI Contribution Collaboration to provide Advance genetic approaches
Collaborator Contribution Access to wheat material and phenotyping data
Impact None yet
Start Year 2016
 
Description CIMMYT seeds of discovery 
Organisation International Centre for Maize and Wheat Improvement (CIMMYT)
Country Mexico 
Sector Charity/Non Profit 
PI Contribution Collaboration to provide Advance genetic approaches
Collaborator Contribution Access to wheat material and phenotyping data
Impact None yet
Start Year 2016
 
Description Combining field phenotyping and next generation genetics to uncover markers, genes and biology underlying drought tolerance in wheat. 
Organisation Indian Institute of Wheat and Barley Research
Country India 
Sector Academic/University 
PI Contribution We are providing next generation genetic approaches to identity markers for Indian wheat
Collaborator Contribution Access to material and feild phenotyping
Impact Not yet
Start Year 2014
 
Description Agro-Biodiverstiy conference -India 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker at "Present and Future Opportunities for Using Biodiversity for Wheat Improvement"
Year(s) Of Engagement Activity 2016
 
Description CyVerse Workshop (University of York) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Contributed to a CyVerse UK workshop (20-21 March 2017), delivering a session on RNAseq analysis. Workshop participants were guided in real-time through an established analysis pipeline in a cloud computing environment.
Year(s) Of Engagement Activity 2017
 
Description Earlham Institute Open Evening as part of Agri-Tech Week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
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Year(s) Of Engagement Activity 2018
 
Description RNA-seq workshop in Nairobi, Kenya (BecILRI Hub) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
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Year(s) Of Engagement Activity 2018
 
Description Royal Norfolk Show 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
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Year(s) Of Engagement Activity 2017