New Investigator Award: Rapid identification of genes and pathways that increase resistance to yellow rust disease of wheat

Lead Research Organisation: Earlham Institute
Department Name: Research Faculty

Abstract

Rust is one of the most devastating diseases of wheat, causing severe yield losses in the UK and globally. Wheat, similar to all plants, has a sophisticated immune system that is currently under-deployed in agriculture. The aim of this project is to improve cultivated wheat by isolating novel sources of rust disease resistance and making them rapidly available to wheat breeding programs. Wheat is the most prevailing plant on earth as wheat crops occupy nearly 25% of world agricultural land. With annual production at more than 650 million tons globally, wheat provides a quarter of all calories and fifth of protein supply to humanity, and yet the annual yield increases are critically below the rate required to feed the growing human population. According to the predictions from the World Bank, agricultural productivity will need to increase as much as 70% to feed 9 billion people by 2050. Growing wheat varieties resistant to diseases is an economical and environmentally friendly solution to increase yield on available agricultural land while reducing growth costs.

As a New Investigator, I am establishing a research programme focused on improving resistance of wheat to a broad range of fungal diseases. I am leveraging recent technological advances, such as cutting-age sequence technologies, for the efficient study of highly complex wheat genome. I plan to rapidly identify novel rust resistance genes derived from cultivated wheat and make these genes accessible to traditional non-transgenic breeding programmes. I have already carried out a screen for new yellow rust resistant mutants of wheat that I believe are novel and can be a new source of disease resistance. By testing resistance in our wheat lines against a variety of wheat pathogens, including mildews and Septoria leaf spot, my group will identify sources of broad-spectrum resistance. By applying new sequencing technologies in a highly efficient manner we will dramatically reduce the time of wheat gene isolation from 15-20 years to just 2-3 years. Furthermore, I am aiming to investigate the mechanisms of plant resistance and to study the evolution of these mechanisms and their diversity in wheat. Isolation of novel rust resistance genes that are derived from cultivated wheat will make these economically important traits immediately available for ongoing wheat breading programs. As our sources of resistance are derived from elite cultivars, such introduction can be achieved with conventional non-transgenic manner. Knowing the genomic locations of new disease resistance is key to accelerate this process. The gene isolation approach developed here will be applicable to any trait of interest.

The major output of my proposed project will be new disease resistance genes and the new tools that plant breeders can use to introduce resistance into the most commonly grown, high yielding wheat varieties. I foresee a great benefit from this project not only to wheat breeders and wheat growers, but also to society in general. Advanced understanding of plant defense systems and deploying it to control plant diseases is a timely economical solution to increase food supply and reduce use of pesticides.

Technical Summary

In this project I aim to isolate wheat genes responsible for gain-of-resistance to yellow rust disease in the mutant population of elite tetraploid wheat cultivar, Kronos. At The Sainsbury Laboratory, we will test our previously identified mutant lines for broad-spectrum resistance to wheat pathogens (powdery mildew, Septoria and wheat blast). We will examine the stage at which pathogen is stopped by microscopy and pre-priming of defence responses by q-PCR. Leveraging the recent advances in wheat genomics at The Genome Analysis Centre, we will establish a rapid approach for mapping wheat genes by exome capture and sequencing reducing the complexity of wheat genome (17 Gb). We will sequence the gene space of the F2 populations of resistant mutants using exome-capture design (86 Mb) and identify mutations linked to resistance with our mutation calling pipelines, providing large amount of marker data. For high-resolution mapping we will design small exome-capture to completely cover the identified chromosomal interval and sequence the region with PACBio technology. We will study natural and induced diversity in the target loci in wheat TILLING populations and 1,000 wheat cultivar and related species with published yellow rust resistance Genome Wide Association data. Specifically, I propose to:

- Adopt semi-automated assays for quantifying rust growth inside wheat leaves.
- Test mutant wheat lines for pre-priming of defence response pathways.
- Evaluate selected mutants for the broad-spectrum resistance to multiple pathogens.
- Introduce new sources of resistance to the elite UK and European wheat varieties.
- Identify markers linked to resistance using wheat exome-capture and mapping-by-sequencing approaches.
- Clone and validate gene(s) responsible for broad-spectrum resistance to yellow rust.
- Identify natural and induced diversity in the resistance loci.

Planned Impact

This New Investigator proposal directly addresses BBSRC's key strategic priority: "Sustainably Enhancing Agricultural Production" by providing new sources of disease resistance to yellow rust that can lower dependence on fungicides. Our approaches are underpinned by promotion of 'new ways of working' to accelerate wheat improvement.

According to the UN Food and Agriculture Organization, 30% of global wheat production is at risk due to wheat rust diseases (1). In the UK, wheat rust epidemic is exemplified by the 'Warrior' race that emerged in 2011 and pushed yellow rust to be the top threat to wheat production (2). Although effective against rust diseases, fungicides are often harmful to human health and/or environment. The cost associated with repeated application of fungicides greatly reduces farmers' profits. In developing countries, fungicides are often not affordable. In 2009 due to the harmful effects on human health, the European Parliament voted to tighten rules on pesticide use and ban 22 chemicals (3). By 2020, the EU plans to ban another 40 chemicals to reduce environmental damage. The Anderson Centre report, commissioned by the Agricultural Industries Confederation, the Crop Protection Association and the National Farmers' Union, estimated that if the pesticide control of wheat diseases was unavailable today, farmers' income would drop from £1.9 to £1.5 bn a year - 21% income loss (4). Disease control with resistant wheat varieties lowers the dependence on fungicides, and provides direct benefit to human health and sustainability of agriculture. New sources of disease resistance in wheat are urgently needed as current varieties could fall behind against newly emerging pathogen strains. These discoveries will need to be rapidly introduced to local wheat varieties to minimise crop losses.

The proposed work will provide new sources of resistance to combat yellow rust that can be deployed today in a conventional non-transgenic manner and lowering dependence on pesticides with a potential 10-20% savings to the farmers. We will also provide the tools for rapid integration of our findings into the current breeding programmes, building capacity to rapidly map, clone and breed disease resistance genes using the newest wheat genomic information. Beneficiaries include wheat breeders, wheat growers and wheat consumers. Wheat breeders and growers will benefit from the genetic material and markers we will produce as it will help them to incorporate new disease resistance loci into high yielding wheat varieties. The genetic material with improved disease resistance will be available from the beginning of this project, and the markers within the first two years. Wheat breeders will benefit immediately from our methodologies for efficient use of the wheat genomic resources in trait improvement. As our methods are generally applicable to accelerating discovery of wheat traits from forward genetic screens, they can be applied to general wheat improvement, including abiotic stress tolerance and yield improvement. Wheat breeders and researchers alike will greatly benefit from adopting these strategies. The researchers appointed on this project will benefit from cross-disciplinary training and having an outstanding opportunity to observe the translational power of their discoveries. They will have opportunities to interact with world-class researchers as well as industry, which will put them in a unique position to advance their future careers in either the public or private sector.

References: (1) FAO Wheat Rust Disease Global Programme WHEAT RUSTS - Constant threat to wheat crops around the globe, 2014 (2) AHDB Cereals and Oilseeds. Increasingly diverse UK cereal rust populations need close monitoring, 2015. (3) B. Ilbery, D. Maye, J. Ingram, R. Little, Geoforum 50, 129-137, 2013. (4) The effect of the loss of plant protection products on UK agriculture and horticulture and wider economy. The Andersons Centre, 2014.

Publications

10 25 50
 
Title Circos plot: Chinese Spring Genome 
Description Circos plot displaying features of the TGACv1 CS42 genome assembly. 
Type Of Art Image 
Year Produced 2017 
Impact This design inspired a line of (unfortunately non-dishwasher safe) collectors plates and the Circolution Collage exhibited in Office 208, Earlham Institute, Norwich, UK. 
URL https://github.com/krasileva-group/tgacv1-circos
 
Title Collage: Circolution 
Description This artwork conveys and reminds of the pain and suffering that is the basis of wheat genomics. It shows the various stages from draft to final product, with additional detours. It is exhibited in office 208, Earlham Institute, Norwich Research Park, Norwich, NR4 7UZ. Unfortunately, it is off limits to the general public. 
Type Of Art Artwork 
Year Produced 2017 
Impact This piece serves as a source of inspiration during the long days and nights of wheat genomics research. 
URL https://github.com/cschu/circolution/blob/master/circolution.jpg
 
Title Lab Logo designs 
Description Lab logo / crest designs which incorporate the scientific output of the lab in an artistic fashion. One of the designs was also printed onto a T-shirt. 
Type Of Art Artwork 
Year Produced 2017 
Impact Allows for added publicity of the lab along with team building among the lab members. 
URL https://twitter.com/Rust_Chaser
 
Description o We have previously identified 34 mutant lines with enhanced adult plant resistance phenotypes against yellow rust by screening T. turgidum cv Kronos EMS population in fields in the US and confirmed a subset of these lines to be resistant to UK yellow rust isolates. Our goal is to identify mutations linked to resistance and clone genes with causative mutations.
o All 34 mutant lines have been re-grown for phenotyping in the UK and a majority of lines have shown increased resistance to yellow rust in the UK.
o The collection has also been sent for in field phenotyping in Kenya (yellow rust and stem rust) through collaboration with CIMMYT and the Gates foundation. We have identified several mutant lines with increased resistance to stem rust.
o We confirmed that our wheat variety T. turgidum cv Kronos is well adapted to speed breeding having a seed to flowering time of 4 weeks and seed to seed generation time of 8 weeks.
o A total of 19 mutant lines with confirmed adult plant resistance against yellow rust have been back-crossed to Kronos wild type. Of these, 11 mutant lines have been advanced to generate mapping populations (136 F2s). For 3 mutant lines, the F3 families were already phenotyped in the field and additional populations for 7 mutant lines are planted for phenotyping this year.
o T4-620 (collaboration with Josh Hegarty and Jorge Dubcovsky, UC Davis). We have individually barcoded and sequenced with exome capture 45 homozygous F2 lines with clear phenotypes plus wt control. Applying our mapping-by-sequencing pipeline, we identified a 2.5 cM region on chromosome 1BL harbouring SNPs that co-segregate with resistance.
o Seedling resistance: We identified 1 mutant line which showed enhanced resistance also at the seedling stage. Mutant line with seedling resistance has been crossed to wild type Kronos and the population was advanced to F3 stage.
o Fast and robust mapping by sequencing in wheat: Bioinformatics processing of read obtained from wheat exome-capture sequencing is limited in terms of speed. Utilizing EI's DRAGEN Bio-IT processor (Edico Genomics), we were able to reduce the processing time from days to minutes per sample.
o Visualization of SNPs across genome: We developed vexed.js (Variant EXplorer and EDitor), a prototypical JavaScript-based visualisation and analysis tool. vexed.js can aid in identifying candidate causative mutations, recombinant regions, and is able to point out issues with misphenotyped data.
o Prediction of variant effects in un-annotated genomes: We developed scvep (super cereal VEP), which extracts the up- and downstream sequence neighbourhood around a variant position and subjects it to a blastx search against a grass-specific protein sequence database. It then translates the coordinates of the aligned amino acids (note that blastx performs six-frame translation of the input nucleic acid sequence) back into nucleic acid space and examines the codon that contains the mutation according to the alignment. Based on this examination it classifies the mutation as silent, nonsense, or missense.
Exploitation Route Our mutant lines can be crossed into any elite variety to incorporate resistance against yellow rust. We already initiated collaboration on this with one of the UK seed companies. In addition, our results show that a subset of lines also have broad spectrum resistance against stem rust in Kenya. The identified mutations co-segregating with resistance could be used directly as markers for marker assisted selection to improve resistance against both yellow rust and stem rust, two of the major pathogens of wheat.
Sectors Agriculture, Food and Drink

URL https://github.com/krasileva-group
 
Description We have collaborated with Edigo Genome to establish rapid SNP calling pipeline for wheat on their Dragen processor (http://edicogenome.com/dragen-bioit-platform/) - their protocols were previously not compatible with large genome such as wheat. This allows us to expedite data proessing >100x. We have also established collabortion with a Seed company to transfer our mutations into European and UK elite varieties.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Digital/Communication/Information Technologies (including Software),Education
Impact Types Economic

 
Title Super Cereal VEP: Annotation-free variant effect prediction for unannotated reference genomes/transcriptomes. 
Description The effects of single nucleotide polymorphisms are typically predicted using tools such as Variant Effect Predictor (VEP) or SNPEff. These tools are based on existing gene annotations and require specifically formatted databases. As such, their use is limited by the availability of high-confidence annotation data. For instance, the current T. aestivum VEP database can only be used together with TGACv1 scaffolds and not with the latest IWGSC assembly. We developed scvep (super cereal VEP), which extracts the up- and downstream sequence neighbourhood around a variant position and subjects it to a blastx search against a grass-specific protein sequence database. It then translates the coordinates of the aligned amino acids (note that blastx performs six-frame translation of the input nucleic acid sequence) back into nucleic acid space and examines the codon that contains the mutation according to the alignment. Based on this examination it classifies the mutation as silent, nonsense, or missense. In the future, we will add a HMMer scan to scvep in order to deal with sequences that did not yield a hit against the scvep protein database. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Our team were able to predict effects of mutations in unannotated portions of genomes as well as using the latest wheat genome assemblies before annotation became available. 
URL https://github.com/krasileva-group/scvep
 
Title tandem 
Description Software to find head-to-head gene pairs in a genome 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact This tool allowed to elucidate the role of head-to-head gene pairs in the formation of integrated domain fusions (cf. Bailey et al, 2018 Genome Biology) 
URL https://github.com/krasileva-group/tandem
 
Title Wheat reTILLING 
Description Rebuild of the Wheat TILLING resource using novel technologies (DRAGEN BioIT Processor + custom software) based on the new wheat reference genome (RefSeq 1.0) and TGAC/Earlham Institute cultivar-specific genome references. This is a collaboration between Earlham Institute, Rothamsted Research, John Innes Centre and UC Davis. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact When the output will be available (within 2018), then it will replace the original Wheat TILLING data and continue to be of use for researchers and breeders. Variant annotation will be hosted by EnsEMBL Plants. 
 
Description Kenya disease susceptibility screen KALRO 
Organisation Kenya Agriculture & Livestock Research Organization (KALRO)
Country Kenya 
Sector Private 
PI Contribution By supplying the seed material of the Kronos TILLING population that has shown an increase in disease resistance. Collaborative talk on disease scoring of Stripe Rust, Leaf Rust and Stem Rust, including inspecting diseased material in field environments and discussing scoring levels and techniques. Drone imaging of disease field.
Collaborator Contribution Seed material was planted in a high disease pressure field in Njoro. Collaborative talk on disease scoring of Stripe Rust, Leaf Rust and Stem Rust, including inspecting diseased material in field environments and discussing scoring levels and techniques.
Impact Disease scoring of TILLING population for Rust susceptibility
Start Year 2017
 
Title EMBER - GlutenSeq Pipeline 
Description Pipeline for processing of GlutenSeq data. 
Type Of Technology Software 
Year Produced 2017 
Open Source License? Yes  
Impact This pipeline allows the analysis and downstream processing of sequencing data obtained via targeted Gluten gene captures. 
 
Title Galaxy id_fusion pipeline 
Description Galaxy implementation of integrated domain fusion detection pipeline (cf. Sarris et al, 2016 BMC Biology) 
Type Of Technology Software 
Year Produced 2016 
Open Source License? Yes  
Impact Tool available for use on Galaxy platforms. 
URL https://github.com/krasileva-group/id_fusion
 
Title Royal Dragen 
Description Royal dragen is a software developed by Dr. Rob King (Rothamsted Research) and Dr. Christian Schudoma (Earlham Institute). The software allows to perform variant filtering on Dragen-generated variant calls. The software was designed to mimic the behaviour of MAPS in wheat tilling experiments (cf. Krasileva et al., PNAS 2017). 
Type Of Technology Software 
Year Produced 2017 
Open Source License? Yes  
Impact The software, together with the Dragen BioIT processor allows to significantly speed up tilling analyses on wheat exome capture data. 
URL https://github.com/cschu/royal_dragen
 
Title scvep - super cereal variant effect predictor 
Description This software allows effect prediction of single nucleotide polymorphisms without a reference annotation. 
Type Of Technology Software 
Year Produced 2017 
Open Source License? Yes  
Impact This software was used to filter wheat candidate lines for (virus?) resistance in a collaboration with IPK Gatersleben, Germany. 
URL https://github.com/krasileva-group/scvep
 
Title tandem 
Description Software to detect (head-to-head) gene pairs (tandems) in a genome. 
Type Of Technology Software 
Year Produced 2017 
Impact This software allowed to analyse whether gene tandems might play a role in the formation of integrated domain fusions (cf. Bailey et al, 2018, Genome Biology) 
 
Description Conference talk at Galaxy Community Conference 2016, Indiana University, Bloomington, IN, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented Galaxy implementation of Integrated Domain Fusion pipeline to Galaxy community.
Year(s) Of Engagement Activity 2016
 
Description International wheat congress (Frankfurt) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Conference presentation organized by German Federal Office for Agriculture and Food (BLE)
Year(s) Of Engagement Activity 2016
URL http://www.wheatinitiative.org/events/international-wheat-congress
 
Description Invited Talk, Institute of Evolution, University of Haifa, Israel 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of advances in Wheat Yellow Rust research
Year(s) Of Engagement Activity 2016
 
Description Invited talk: From tracking Arabidopsis RNAs to exploring the wheat genome - A journey through plant bioinformatics. Evolution Seminars IOE and EEB joint seminar program, University of Haifa, Israel, April 18, 2016. Hosted by Prof. Tzion Fahima 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact People were introduced into the complex and versatile world of plant bioinformatics, into how methods from other bioinformatics areas can be used for problems in plant-pathogen and crop bioinformatics.
Year(s) Of Engagement Activity 2016
 
Description Monogram 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I presented a scientific poster describing the background information about my work with the TILLING population about how it was created and then how it was to be used in our future experiments. Also included a section on incorporating future drone technologies to aid in phenotyping plant populations
Year(s) Of Engagement Activity 2017
 
Description Plant and Animal Genome Conference 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented a scientific poster on ongoing work and future directions at the Plant and Animal Genome Conference (PAG) 2018. The poster was on show throughout the event for viewing with an allocated afternoon for questions to be asked about the selected posters. During which I was able to engage and talk with both scientists and industry persons about the ongoing work and how this could be applicable with developing technologies and other research projects.
Year(s) Of Engagement Activity 2018
 
Description Poster Presentation Schudoma C, Clark M, and Krasileva KV. It's a grass, grass, grass - Profiling plant gluten genes with targeted resequencing and bioinformatics, Genome 10K and Genome Science Conference, Earlham Institute, Norwich, UK, August 29-31, 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact People were introduced to the discovery of the "dark glutenome", i.e. non-canonical gluten loci found in hexaploid wheat.
Year(s) Of Engagement Activity 2017
 
Description Poster Presentation Schudoma C, Raats D, Bailey P, Gordon L, Stitt T, and Krasileva KV. Taming the beast - Overcoming the challenges in working with complex plant genomes, ISMB 2016, Orlando, Florida, USA, July 8-12, 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact People were made aware that large plant genomes are a challenge and that this can be tackled by better algorithms, use of unconventional hardware (first use of DRAGEN BioIT processor, which was originally developed for human diagnostics, for plant genomics).
Year(s) Of Engagement Activity 2016
 
Description Talk at The 13th International Wheat Genetic Symposium, Tulln, Austria 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of research in wheat yellow rust resistance.
Year(s) Of Engagement Activity 2017
 
Description Talk at The I International Workshop "Plant Genetics and Genomics for Food Security", Novosibirsk, Russia 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of the search for resistance against wheat yellow rust.
Year(s) Of Engagement Activity 2016
 
Description Talk at Wild Wheat Relatives Conference, Tel-Aviv, Israel 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of wheat Yellow Rust resistance research through exome capture based mapping by sequencing experiments.
Year(s) Of Engagement Activity 2016
 
Description Wheat as a model organism 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact A one day workshop highlighting most recent tools that enable high impact wheat research
Year(s) Of Engagement Activity 2016