Developing genetics and genomics interface to develop strategies for sustainable use of resistance to white rust in oilseed mustard (Brassica juncea)

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

Oilseed crops are important for the economy and health of people living throughout south Asia. In India, for example, annual imports of edible oil are a staggering 50% of the national consumption, costing of over £ 3.6 billion. The national average dietary intake of fat/oil in India is 9kg/person/year (kpy). In rural India, the intake is 1-7 kpy, which is well below the minimum nutritional recommendation of 14 kpy.
White blister rust caused by the oomycete Albugo candida is one of the most important diseases of oilseed mustard and other vegetable brassicas globally. The pathogen can infect all above-ground parts of the plant, but causes the most severe damage as systemic infections of the inflorescence leading to staghead formation. The disease causes yield losses in oilseed mustard of 20-60% in India, Canada and Australia. With the development of canola quality B. juncea, many countries like Australia and Canada are growing B. juncea more extensively as a crop that is better suited for areas with low rainfall than oilseed rape. However, white rust susceptibility is a major concern, so development of new B. juncea varieties that combine durable white rust resistance and drought tolerance is needed to improve productivity in south Asia and to expand global production of this important oilseed crop.
The integrated use of pathology and molecular genetics can provide an evidence-based strategy for assembling gene combinations around specific broad spectrum R-genes, and ultimately achieve sustainable disease control via anticipatory breeding of new resistant crop cultivars. This strategy is theoretically possible in any crop, including Indian mustard. However, molecular identification of matching host and pathogen determinants (R and AVR genes, respectively) are rate-limiting but nevertheless required to enable whole-genome selection of optimal gene combinations along with other essential traits.
The proposed Indo-UK partnership has unique opportunity to lead in this research of global importance to food security. This will be achieved by harnessing genomics and bioinformatics tools to extend the on-going efforts towards anticipatory breeding of disease resistance, for sustainable control of white rust in brassicas, and specifically for development of B. juncea varieties that will be adapted for cultivation in India. To date, deployment of the state-of-art genomic approaches for combating white blister rust has been minimal in India and the UK. Similarly, efforts have not yet been initiated to characterize the genome level diversity of the pathogen population. The proposed project is therefore a major opportunity to establish coordinated capability in the genomic characterization of both host and pathogen, which will extend the approaches currently being used in India towards more effective breeding and disease management strategies.
Primary outputs will be aimed at efficient identification of genes that control white blister rust in brassicas. To achieve this, three project objectives will exploit examples of white rust resistance that have already been identified in primary and secondary gene pools, and in the wild relative Arabidopsis thaliana, to enable precise genotyping of resistance in seedlings for marker-assisted breeding of R-gene combinations, and to evaluate transgenic methods for developing sustainable white rust control in B. juncea cultivars of Indian mustard.
A complementary pathology aim will advance capability of using pathogen-based information for evaluating the potential durability of targeted disease resistance genes in optimal combinations for use in Indian mustard varieties of B. juncea. Two project objectives will capitalise on the latest genomic resources available in Albugo, to define the pathotype variability of A. candida in India and to assess the potential emergence of variants that could overcome individual white rust resistance genes.

Technical Summary

White blister rust (Albugo candida) is one of the most important diseases of oilseed mustard. The disease causes yield losses of 20-60% in India, Canada and Australia. With the development of canola quality B. juncea, many countries like Australia and Canada are growing B. juncea more extensively as a crop that is better suited for areas with low rainfall than oilseed rape. Development of new B. juncea varieties that combine white rust resistance and drought tolerance is therefore needed to improve productivity in south Asia and elsewhere. The integrated use of pathology and molecular genetics can provide an evidence-based strategy for assembling gene combinations, and ultimately achieve sustainable disease control via anticipatory breeding of new resistant crop cultivars. Molecular identification of matching host and pathogen determinants is rate-limiting but nevertheless required to enable whole-genome selection of optimal gene combinations along with other essential traits.
The proposed Indo-UK partnership will lead in this research of global importance, by harnessing genomics and bioinformatics tools to extend efforts towards anticipatory breeding of white rust resistance in brassicas, and specifically for development of B. juncea varieties that will be adapted for cultivation in India. Primary outputs will be aimed at efficient identification of genes that control white rust. To achieve this, the project will exploit examples of white rust resistance that have already been identified in primary and secondary gene pools, and in Arabidopsis thaliana, to enable genotyping of resistance in seedlings for marker-assisted breeding of R-gene combinations, and to evaluate transgenic methods for white rust control in cultivars of Indian mustard. A pathology aim will advance capability of using pathogen-based information for evaluating the potential durability of targeted disease resistance genes in optimal combinations.

Planned Impact

The proposed research will directly underpin the future sustainability of oilseed production in the Indian sub-continent by addressing the reduction of major crop losses due to biotic interactions. Oilseed crops are important for the economy and health of people living in throughout south Asia, as a multi-purpose crop including seeds for edible oil, leaves as a vegetable and for animal fodder, and the dried stems as domestic biofuel. Rapeseed-mustard crops comprise more than one third of the home-grown vegetable oil production in India and covers around 7.0 million hectares. B. juncea occupies more than 80% of this area. Smallholding, resource poor farmers produce most of the crop, and will be direct beneficiaries, with current average seed production of less than 0.4 tonne/ha compared with a yield potential of 2.5-3.0 tons/ha.
White blister rust is a major disease constraint on production, causing yield losses of at least 20-60% in India, Canada and Australia. Durable disease resistance would improve incomes of smallholding resource poor dry-land farmers, by stabilising mustard yields, and as a consequence boosting rural economy and enhancing nutrition of the poorest people in the Indian subcontinent. Thus, the combined aim of our Indo-UK partnership will be to genetically improve cultivars already adapted for low input production in India, particularly in the rainfed areas, as the duration of the crop is relatively short and has low water requirement compared to other crops. The white rust resistance genes will be introduced into hybrids that have been developed at Delhi University, which will already contain other useful seed quality and yield improvements. Similarly, characterization of white rust resistance will also help research programme in UK dealing with vegetable brassicas, and also with the development of canola quality B. juncea for production in dry regions of Canada and Australia.
No international institute currently coordinates research on oilseed brassica crops globally. The proposed Indo-UK partnership, however, will assemble a highly complementary team of crop scientists who will build significantly on previous DFID investment in oilseed brassica production in India, and translation of BBSRC and European Research Council investment in pathogen genomics and molecular genetics of disease resistance from Arabidopsis into brassica crops. The research partners will use knowledge gained from their coordinated effort to advance links with existing links with colleagues and research programmes in Canada, Australia and elsewhere in Asia.
Exchange visits will take place to enable research staff in both countries to develop an active and productive collaboration. This will include UK scientists visiting India in Year 1 for a project meeting and visit to field production sites; and in Year 3 for a knowledge transfer workshop. Indian scientists will visit the UK for training periods, including two PhD students from DU who will each visit for 6 months (one in Year 1 for pathology; and one in Year 2 for gene cloning and pathogen genomics), and two research scientists from AUUP and GBPUAT in Year 1 for pathology. Staff from both India and the UK will also attend the 14th International Rapeseed Congress in Saskatoon, Canada in Year 2, to jointly present results from the Indo-UK partnership to an international audience.
Knowledge gained from fundamental and strategic science will be published in international refereed journals and disseminated at international and regional conferences. The future translation of this knowledge into breeding programmes will be via the National Agricultural Research Systems of India, Pakistan, Nepal, Bhutan and Bangladesh. This will be facilitated by existing interactions amongst South Asian countries.

Publications

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Cevik V (2019) Transgressive segregation reveals mechanisms of immunity to -infecting races of white rust (). in Proceedings of the National Academy of Sciences of the United States of America

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Woods-Tör A (2018) A Suppressor/Avirulence Gene Combination in Determines Race Specificity in . in Frontiers in plant science

 
Description The overall purpose of this project has been to develop an anticipatory breeding strategy for sustainable deployment of white rust resistance (WRR) in oilseed mustard (Brassica juncea) with advances that are now possible using genetic analyses of genomic data (i.e, reference genomes and high-resolution SNP-based genotyping).
Primary aims have been to develop 1) molecular marker technology to mobilize the use of WRR alleles from primary (B. juncea), secondary (B. oleracea and B. rapa) and tertiary (Arabidopsis thaliana) gene pools of related host species; and 2) pathogenomics for decision-support to enable identification of optimal R-alleles combinations for use in Indian adapted varieties of B. juncea. In June of Year 1, white rust in UK production of English mustard (B. juncea and Sinapis alba) was a key discovery which provided experimental material that substantially improved our capability to advance the project aims (described below), and established an important link in our collaborative outlook for future impact on food production in both India and the UK.
Three specific objectives of Aim 1 have focused on target genes that individually offer broad spectrum resistance to variants of the white rust pathogen Albugo candida. Identifying the causal genes provides the most precise molecular markers to assist recombination of complementary alleles by conventional breeding and/or to be used more flexibly as a transgenic stack of alleles. From the primary gene pool (Obj 1), the Dehli University group extended their previous mapping work to identify a gene (WRR-Don) encoding a canonical NBS-LRR cytoplasmic R-protein that confers dominant resistance in a European cultivar of B. juncea (Donskaja). Transgenic 'gain-of-function' testing is now underway to confirm function of the causal allele. From the secondary gene pool (Obj 2), our group at Warwick have successfully tested genotyping-by-sequencing to map a locus for recessive resistance from a landrace of B. oleracea. We then used gene enrichment sequencing (RenSeq, developed at TSL-Norwich) to refine the mapping interval and exclude NB-LRR genes as candidates. CRISPR-Cas9 editing of a single candidate gene that encodes an extracellular GDSL lipase is now underway to test the predicted dominant susceptibility allele by knock-out mutation. A technician funded on the project in 2016 re-joined the group as a BBSRC funded food security student in July 2017 and will complete testing of knock-out mutants as part of his thesis research. And finally, from the tertiary gene pool (Obj 3), the TSL-Norwich group conducted an extensive screen for WRR genes from wild A. thaliana accessions and confirmed that the WRR4 cluster is the best source for resistance alleles against a broad spectrum of available pathotypes. Isolates virulent in accession Columbia (breaking the broad-spectrum resistance allele WRR4A-Col) were discovered in Aim 2, and used to identify a new WRR4A allele from accession Oy-0 which confers resistance to all isolates available in our UK collection. Resistance at other loci were isolate specific and in several cases were non-functional as transgenes in B. oleracea and B. juncea, and therefore strategically redundant. To initiate transgenic experiments in India, single and combined constructs of WRR4A and WRR4B alleles were provided in 2016 to the Amity University group for them to test against Indian isolates of A. candida in a standard Indian cultivar of B. juncea.
Expectation: gene stacking of WRR4A-Col, WRR4A-Oy0 and WRR-Don in a knock-out mutant of the GDSL lipase may potentially be a durable combination for controlling white rust in B. juncea. We have included this as an objective to continue as part of a proposal pending in the BBSRC-DBT Pulse and Oilseed Research initiative (PORI).
Two specific objectives of Aim 2 were focused on identifying pathotypes of A. candida that could break individual R alleles, and combining genome-wide effectorome data from available pathotypes to generate a spreadsheet-based pathogenomics tool (AlbugoSEEK) and to then use for association mapping of avirulence (AVR) elicitors. AlbugoSEEK v1 was assembled (Obj 4) from eighteen genomes including seven Indian isolates of race 2 (from B. juncea), single Canadian isolates of race 2 and 7 (from B. rapa), five UK isolates of race 4 (from wild host species), and four UK isolates of race 9 (from B. oleracea). Three of the race 4 isolates were collected in Obj 5 from wild accessions of Arabidopsis (A. halleri, A. lyrata and A. thaliana) and confirmed as being Col-virulent (i.e., natural mutants breaking WRR4 resistance). We used AlbugoSEEK v1 in June 2016 for a training visit of co-Is from Amity and GBPUAT universities (Pankaj Sharma and Anand Tewari). From this, as proof-of-concept, we identified a single candidate AVR-WRR4 elicitor that has a conserved allele (as predicted) in isolates of all four races except the three Col-virulent race 4 isolates. Transient co-expression of the conserved effector allele with WRR4-Col in tobacco elicited cell death, confirming its avirulence function and establishing our first pathogen diagnostic marker.
In June 2015, we discovered white rust in an English mustard field of B. juncea near Peterborough. We established cultures of two isolates (AcBj12 and AcBjDC) and confirmed in 2017 that they are different pathotypes; most importantly, AcBjDC is virulent in Donskaja (i.e., breaking WRR-Don resistance). Effectorome sequence from both isolates is currently being added to AlbugoSEEK v2. A short list of 38 effectors are conserved across eight Donskaja-avirulent isolates of race 2, and we are predicting that at least one of these will be conserved in AcBj12 (avirulent) but altered by loss-of-function mutation in AcBjDC (virulent). If this is the case, we will have diagnostic marker(s) for surveying white rust epidemics in the UK and India, with an objective of capturing isolates that have mutant allele(s) and then confirming the avirulence elicitor by association with interaction in Donskaja (i.e., predictive phenotyping by reverse genetics). We have initiated testing of minION sequencing technology of Albugo DNA as a potential tool for future sequence-based diagnostics. William Crowther will continue this work in collaboration with Elsoms and English mustard growers as part of his PhD thesis research; and has been included as a strategic objective for Indian colleagues in our PORI proposal (described above).
In January 2017, we launched a revised objective to identify WRR genes directly from Genebank accessions of brassicas, building on progress in Aims 1 & 2 and collection of race 2 isolates from UK mustard production. We screened a sample of 15 B. rapa accessions for white rust resistance. These have been maintained as small multi-parent populations in the Warwick Vegetable Genebank. Ten were heterogenous for resistance/susceptibility to AcBj12; and seven similarly for AcBjDC. Leaf samples from resistant individuals and bulk samples from at least ten susceptible individuals were collected from each accession x isolate experiment, and have been sent out for genotyping-by-sequencing. This data will be used to test feasibility of coarse mapping WRR loci directly from genebank accessions, as a first step in selecting new target loci for resistance (e.g., for anticipated control against WRR-Don virulent pathotypes).
Long-read sequencing (PacBio technology) of AcBJ12 and Hyaloperonospora brassicae (Brassica downy mildew; Objective 6) has been generated to combine with shortread and RNA sequence data and improve reference genomes of the important brassica pathogens.
Exploitation Route Work is in progress for the Indian partners to take the material and methods forward beyond the duration of the project. Their aim will be field testing of stacked genes from A. thaliana in transgenic B. juncea. This has been included as a specific objective in a proposal submitted for Newton-BBSRC/India-DBT funding in a 'Pulse and Oilseed Research Initiative.'

Extension of the AlbugoSEEK database is already underway, specifically for brassica crops by initiating reference genome sequencing of Hyaloperonospora brassciae (causing downy mildew and a major co-disease agent in combination with white rust). We have also adapted the concept for bacterial pathogens including Pseudomonas and Xanthomonas of brassica and haricot beans (with development funding from GCRF-IAA projects). These tools will be particularly useful for application with breeders and pathologists in sub-Saharan Africa.
Sectors Agriculture, Food and Drink,Education

 
Description BB/L011646/1 was a collaborative grant with crop scientists from three universities in India. The project advanced complementary genetic techniques (transgenic 'gene-stacking' and pathogen genomics) for use in a multi-purpose crop (Indian oilseed mustard) to provide smallholding farmers with potential durable control of a major disease constraint (white blister rust). Translating the research approach across other traditional oilseed and pulse crops was proposed with a broader consortium including a cohort of young scientist from five regions of India; unfortunately, additional India/UK funding was declined. Nevertheless, the cross-cultural exchange elevated the UK PIs interest in food systems, specifically from understanding how the traditional plant-based diet in India is intrinsically dependent on the countries self-sufficiency from smallholder production of a wide diversity of food crops. Ideas were stimulated for transforming our food system at home, and initiated with BBSRC-IAA proof-of-concept funding (BB/S506783/1) to launch a food hub platform called '2BHealthyGB'. The USDA working definition of a food hub is "a business management structure facilitating the aggregation, storage, processing, distribution, and/or marketing of regionally produced food products." An important aim is to provide "wider access to institutional and retail markets for small to mid-sized producers, and increasing access of fresh healthy food for consumers." '2BHealthyGB' (www.2BHealthyGB.com) was launched using this working definition as a development platform for a food hub featuring haricot beans as a novel British crop to generate transformative thinking about self-sufficiency of our future food culture. It aims to connect farmers with consumers who are seeking a healthier diet. And it's timely, in light of the EAT-Lancet Commission report published in 2019 which envisions a future sustainable planet from a global shift to eating more plant-based food (particularly legumes as a dietary fibre-rich alternative to meat-based food). Impact funding from Warwick Ventures enabled National Listing of 'Capulet' (a UK adapted white haricot bean) in 2018. BBSRC-IAA funding in turn supported: 1) breeder seed production needed to establish agreement with an industry partner (Agrii Ltd) to begin scaling-up Capulet for commercial production; 2) seed production of the next two varieties for DUS testing (Godiva, a blonde kidney-type; and Olivia, a black cannellini-type); 3) first farmer-grown crop of Capulet and Godiva for food, which in turn enabled links to begin developing with community food providers (e.g., Feeding Coventry). This project provided a key KE component of NURTURE (a £4 million demonstration project for InnovateUK Transforming Food Production; submitted 25 Feb 2021; start date 26 Aug 27 mo).
First Year Of Impact 2019
Sector Agriculture, Food and Drink
Impact Types Cultural,Societal

 
Description BBSRC IAA University of Warwick
Amount £300,000 (GBP)
Funding ID BB/S506783/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 03/2021
 
Description Establishing an India-Africa-UK innovation hub for seed and root health in brassica and bean crops
Amount £23,516 (GBP)
Funding ID BB/GCRF-IAA/17/22 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2017 
End 02/2018
 
Description GCRF-IAA (University of Warwick)
Amount £23,225 (GBP)
Funding ID BB/GCRF-IAA/22 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2016 
End 03/2017
 
Description Access to pathogen material from breeder trial located on an English mustard growers farm 
Organisation Elsoms Seeds
Country United Kingdom 
Sector Private 
PI Contribution Identified white rust as an emerging disease in UK production of English mustard (brown-Brassica juncea, and white-Sinapis alba). Three distinct pathotypes were collected from the breeder trial in 2015. One pathotype breaks a resistance gene that Indian partners have been using to develop Indian mustard varieties, with the expectation of broad spectrum disease control. Resistance to this UK pathotype has been identified from GeneBank collections of B.juncea and B.rapa.
Collaborator Contribution Provided access to field populations of the pathogen, and has hosted visits for Indian partners in June 2016 and 2017. This included opportunity to discuss research with a lead mustard grower.
Impact Indian partners can now anticipate the need for combining at least two R-genes. The same R-gene combination can also be recommended for use in breeding mustard varieties adapted for English production. Tightly linked markers and possibly candidate genes are expected by the end of the project. Sequence data from the UK isolates has been added to the pathogen genomic database (AlbugoSEEK), and used to identify a candidate AVIRULENCE genes for development of pathotype-specific diagnostic markers.
Start Year 2015
 
Description 'Fascination in Plants' public event at Warwick Crop Centre 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Series of short presentations and displays featuring soil health and vegetable research at Warwick. A talk on 'Science for Closed-loop eating' and two display featured 1) crop variation and genetics research, and 2) brassica diseases from this project.
Year(s) Of Engagement Activity 2017
 
Description 14th International Oilseed Rape Conference, Saskatoon, Canada 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Opportunity to meet with Indian partners from our collaborative project, to introduce a postgraduate student (member of PIs group) to international stage of oilseed research community, and to re-connect with Canadian collaborators.
Year(s) Of Engagement Activity 2015
 
Description British Council-Newton exchange workshop for early career scientists in Turkey and UK on 'Latest Technologies for Crop Improvement' 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact This workshop highlighted current applications of new technologies for crop improvement. The workshop brought together leading researchers as mentors, young early career research scientists and industrialists from the plant breeding sectors to exchange knowledge and share best practice in crop improvement.
Presentations from experts in the field provided early career delegates with an overview of the new genomic technologies including next generation sequencing and quantitative and statistical genetic techniques applied to crop breeding. Researcher and mentor-led discussion sessions provided an opportunity for delegates to identify and exploit best practice across the plant breeding industry. Networking sessions at both the beginning and end of the workshop supported understanding of the research areas and skills of each other and identified potential links with potential partners for future collaborations.
Year(s) Of Engagement Activity 2015
 
Description Evening tour for local farmers of field trials at Warwick Crop Centre 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact Local farmers (organized by NFU) were interested in talking about developments at the Wellesbourne Innovation Campus and touring field trials, which could potentially impact on food production in Warwickshire.
Year(s) Of Engagement Activity 2018
 
Description Public Evening: Keep Plant Pathogens at Bay 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talks, displays and tour featuring research in School of Life Sciences on modern approaches for combating disease and pests that affect food production.
Year(s) Of Engagement Activity 2017
 
Description Second international conference on Wild Plant Pathosystems 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Small gathering of researchers from complementary disciplines of ecology, molecular genetics and pathology, applying research to natural and cultivated systems. Half of the audience were early career scientists. Aim was to bridge the gap between a body of historical information with latest approaches to investigate how plants and microbes interact at whole systems level.
Year(s) Of Engagement Activity 2016
 
Description Seed company (Elsoms Ltd) open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Event sponsored by the seed company for knowledge transfer and sales. Tent provided for us to display research activity at Warwick Crop Centre including our work on white rust in brassicas. Conversation with the mustard breeder in 2014 identified the disease as a potential threat to English mustard growers. Plans were set for summer 2015 to observe disease in their field trial, and to collect samples of pathogen for use in our research project. Follow-up visit to field trials in summer 2016 included participation from two of our Indian research partners. Current view is that the disease may be increasing in significance, and effort should be made to anticipate use of disease resistance in English mustard, using knowledge being gained and developed to support smallholder producers of Indian mustard.
Year(s) Of Engagement Activity 2014,2015,2016,2017
 
Description Warwick Think Tank, Invited lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact 'Warwick Think Tank' is a cross university interest group run by undergraduate and postgraduate students. They invite speakers to present topics for discussion. In this case, the topic was 'The Future of Food, with or without GM'
Year(s) Of Engagement Activity 2015