Detoxed grass pea: sustainable sustenance for stressful environments

Lead Research Organisation: John Innes Centre
Department Name: Metabolic Biology

Abstract

Grass pea (Lathyrus sativus) offers an excellent opportunity for sustainable agriculture and food security for the poorest of the poor, even in the face of predicted climate change, because it is a legume and performs well in marginal soils or under harsh climatic conditions. Grass pea has been grown for seed and fodder production in many countries, including large parts of India (one of the centres of its origin), Europe and China since it is a low input crop, a cheap source of protein and is particularly tolerant to drought, water logging, and moderate alkalinity. However, grass pea can cause a devastating disease called neurolathyrism believed to be due to its content of beta-N-oxalyl-l-alpha,beta-diaminopropionic acid (ODAP) This neurological disorder is irreversible and occurs when people are dependent on the crop as a sole food source. Grass pea can also inhibit growth in animals when used as feed. Grass pea, therefore, presents as a Janus-faced crop since it provides desperately needed food for those on the edges of survival, but with the concomitant danger of delivering a highly toxic compound to its consumers. Grass pea would benefit from intensified breeding efforts to remove the anti-nutritional toxin and improve its nutritional quality, enhance yields and provide resistance to key pathogens. The synthesis of the toxin is understood in part. The toxin levels in low-toxin lines developed by conventional breeding are unstable and often elevated in stressful environments. We wish to exploit a genomics route to deliver safe technologies for improving this insurance crop.
Modern genomics methods including genome sequencing, marker assisted breeding and plant transformation have revolutionised crop breeding. However, many important crops, like grass pea, have become orphaned as they are less amenable to improvement because inherently they lack some of the attributes required, like tractable genomes or transformation systems. Conventional breeding for the improvement of these crops often relies on induced mutagenesis, and in recent years, this has been linked directly to genomics via the reverse genetics tool of TILLING. TILLING facilitates the combination of forward and reverse genetics in the same programme to permit rapid screening for both phenotypes and mutation discovery. TILLING is particularly suitable for crops where there are limited or no reverse genetics resources available and hence ideal for an orphan crop.
We propose to use natural germplasm and existing mutant populations of grass pea as novel resources for the rapid identification of mutations offering improved crop characteristics via a non-GM route. A spectrophotometric method for the assay of ODAP has been adapted for high-throughput using microtitre plates suitable for screening large numbers of samples. With this assay we will screen for toxin-free lines. The acquisition of toxin-free lines will allow the true roles of these compounds in plants (e.g. in insect resistance) and in human diseases to be assessed. Simultaneously the mutagenised plant material will be used to generate DNA arrays for a TILLING platform and to screen for stress responses and other useful agricultural characteristics (e.g. branching, seed quality) both at NIAB, and in India, with BCKV. We will investigate why grass pea has improved stress tolerance compared to other legumes, with the objective of using this fundamental insight to improve stress tolerance in UK legumes such as pea. The TILLING platform will be accessible to the research community revgenuk.jic.ac.uk. To aid use of the TILLING platform we will develop transcriptome information by RNA-seq. Transcriptome data will help in gene identification. We will assess the gene complement of grass pea via the generation of RNA-seq data for different organs of the plant, including leaves and seeds where the toxin is most prevalent.

Technical Summary

The objective of the Detox project is to develop modern genomics tools and resources for the rapid domestication of grass pea (Lathyrus sativus) to provide a non-toxic, stress tolerant legume for food and fodder. Our priority is to remove the toxin, ODAP, from grass pea using a high throughput chemical screening protocol to identify mutants in a large EMS- mutagenized population. The absence of ODAP will be confirmed by mass spectrometry. ODAP-lacking mutant lines will be will be trialled for impacts on responses to biotic challenges such as insect damage, since plants often produce toxins as a defence against predators. Additional agronomic traits will be screened in the mutant population in field trials under two distinct sets of environmental conditions. Based on our expertise in legume and grass pea breeding, traits that will be sought include simultaneity of flowering, plant height, branches per plant, seed shape, 100-seed weight and yield per plant. Mutations affecting these traits will be identified, and the inheritance of each trait will be established by selfing, complementation tests and backcrossing. New phenotypes will be reported on a publically-accessible database for access by grass pea breeders.
The mutagenized population will be used to establish a TILLING platform. TILLING will be aided by the establishment of a RNA-seq transcriptome and used to identify mutations in specific target genes of importance in determining seed quality and to investigate the stress tolerance (particularly the drought tolerance) of grass pea, relative to pea and faba bean. The TILLING platform will be available through RevGenUK for future breeding activities to improve grass pea. A large collection of grass pea accessions from BCKV will be prepared for ecoTILLING. Lines selected from the toxin screen and field phenotyping will be carried forward into BCKV's breeding programmes to produce advanced materials.

Planned Impact

Our project has profound implications for the development of safer protein crops with low toxins for human and animal consumption. In addition, the reverse genetics platforms that will be established for this orphan crop will permit the transfer of genomics knowledge from other systems for the improvement of grass pea in the longer term. Our strategy will allow any new varieties to be released rapidly (compared to genetic modification strategies) without regulatory hurdles, and has the potential to provide a safe legume suitable for cultivation in extreme environments for food and feed. These new varieties will contribute significantly to nitrogen fertilisation and will be accessible rapidly to small farmers in developing countries. Furthermore, information from the studies on grass pea resistance to stresses will be transferrable to temperate legumes, pea and faba bean.
Who will benefit from this research?

In addition to immediate academic beneficiaries there will be numerous additional beneficiaries.
1. UK and international science base.
2. Agro-industry inc. biotechnologists and plant breeders seeking to make a safer crop, increase plant productivity and/or harvest index; metabolic engineers.
3. Pharmaceutical industry to understand the mode of action of the ODAP toxin
4. Agricultural community and advisors for advanced materials.
5. Public for general information on drought-resistant crops.

How will they benefit from this research?
1. The information from this project will enhance the knowledge base of international plant and medical science and enhance UK's profile in plant science, specifically food security.
2. The materials and knowledge from the project will help agro-industry develop approaches for removal of the toxin, improving human health and animal nutrition, and for metabolic engineering.
3. The pharmaceutical industry will have access to material that will represent the perfect control material and will enhance their ability to understand the true function of the toxin and neurotransmission in general.
4. The global agricultural community will benefit from sustainable crop improvements enabled by our research and obtain a non-toxic protein crop that can be grown more widely and one that will be suitable to meet the challenges of climate change.
5. Our research findings relate to issues of public interest and public safety including food quality, toxicity and sustainable crop production. They can potentially benefit consumers by contributing to the development of more robust, more nutritious, global protein foodstuffs produced in a more sustainable manner.

What will be done to ensure they benefit from this research?
1. Publish results in high-impact journals in a timely fashion, with open access where possible. Present research results at UK, Indian and international meetings and institutions
2. Submit data to relevant international repositories. Submit new transcriptome information to community databases e.g. EMBL, LegumeBase and the Legume Information Service. Access to materials will be from BCKV for advanced lines and ecoTILLING, and RevGenUK/IAEA for TILLING.
3. Exploit existing contacts with other UK and international organisations with relevant research interests as soon as any exploitable results/materials are generated e.g. the Pea Crop Genetic Improvement Network.
4. Make contacts with industrialists and relevant umbrella organisations using existing connections and via our collaborators, sub-contractors and partners; recognise and protect intellectual property to ensure wise and fruitful exploitation.
5. Use results as part of our regular engagement with the agricultural community through our outreach programmes and international organisations, such as FAO, CGIAR and ICARDA.
6. Use results as part of our regular engagement with non-academic audiences, e.g. local interest groups, schools, local and national shows, extension activities, science showcases, media.
 
Description We have identified enzymes encoding the final step of ODAP biosynthesis in grass pea and are searching, via reverse genetics for mutants with low toxin content in seeds. We have identified 14 low ODAP mutant lines which we are now taking forward for introgression into Ethiopian germplasm. We have generated a draft reference genome sequence of grass pea.
Exploitation Route Our findings may be used by breeders to develop sustainable low toxin lines suitable for India, Bangladesh and even Southern European countries where this resilient legume can provide a valuable source of plant protein in the face of climate change.
Sectors Agriculture, Food and Drink

 
Description Unlocking the Potential of Grasspea for Resilient Agriculture in Drought-prone Environments (UPGRADE)
Amount £1,246,884 (GBP)
Funding ID BB/R020604/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2018 
End 06/2021
 
Description 8th International Conference on Legume Genetics and Genomics (18-22 Sept 2017, Siofok, Hungary) talk 
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 8th International Conference on Legume Genetics and Genomics (18-22 Sept 2017, Siofok, Hungary) talk
Abstract
The Lathyrus sativus genome project
A. Sarkar1, P. M. F. Emmrich1, A. Edwards1, C. Martin1, T. L. Wang1.
1 The John Innes Centre, Norwich, NR4 7UH, United Kingdom.
Grasspea (Lathyrus sativus) is a hardy legume grown by poor and marginal farmers in the Indian subcontinent and Africa for animal feed, food and fodder, often on impoverished soils with minimal inputs. The presence of a neurotoxin (beta-ODAP), which can cause neurolathyrism in people subsisting on a predominantly grass pea diet for an extended length of time, is a major factor preventing wider adoption of this promising crop. It is a diploid (2n=14) with an estimated haploid genome size of 6.9 Gbp [1].
We report on the progress of sequencing the grass pea genome of a European line. A de novo shotgun sequencing strategy has been adopted based on the construction of a PCR-free library for paired end sequencing and several mate-pair libraries for sequencing on the Illumina platform. This has been supplemented by long read sequencing using MinION (Oxford Nanopore Technologies) to improve higher order assembly in the absence of good genetic or physical maps. The draft genome is being annotated using transcriptome data from this and two Indian lines, as well as data from genome and transcriptome sequences of related legumes.
The draft genome sequence will aid in the identification of the genes in the beta-ODAP biosynthesis pathway, and also of genes for various traits of interest. The data will help in the development of high quality genetic and physical maps for marker-assisted and genomic selection strategies for agronomic improvement. Additionally, the draft genome will aid in gene function analysis by TILLING, as well as enable a genome editing platform for grass pea.
References:
[1] Bennett and Leitch, 2005. Annals of Botany 95: 45-90.
Year(s) Of Engagement Activity 2017
URL http://iclgg2017.hu/program/
 
Description Bridging the Gap between Agriculture and Human Health (Workshop) 19 - 22 September 2016, Cape Town, South Africa. Keynote lecture. 
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 Bridging the Gap between Agriculture and Human Health (Workshop); 19 - 22 September 2016, Cape Town, South Africa.
Theme 02: Crops improvement (Chair: Prof Sagadevan G. Mundree)
Keynote lecture Title: Grasspea: a legume for food and nutritional security in the 21st century (Invited Speaker: Dr Abhimanyu Sarkar, The John Innes Centre, UK)

Abstract
"Grasspea: a legume for food and nutritional security in the 21st century"
Abhimanyu Sarkar, Peter Emmrich, Di Yang, Cathie Martin and Trevor Wang.
The John Innes Centre, Norwich, NR4 7UH, United Kingdom.

Sustainable, low-input agriculture is an important strategy to feed a rapidly increasing world population and achieving food security. This is especially important in developing countries, to minimize the environmental impact of increased food production and mitigate the effects of climate change. Legumes are vital to this strategy as they not only fix atmospheric nitrogen, reducing artificial nitrogen fertilizer usage, but also provide nutritional security through protein production at a much lower input usage as compared to animal protein production.
Grasspea (Lathyrus sativus), a hardy grain and fodder legume, offers outstanding potential for reducing energy inputs in farming. Its drought and flooding tolerance is unparalleled among legume crops. The plant provides food as a grain crop and leafy vegetable; it is used as feed for livestock and as a green manure. One of the major drawbacks of this crop is the presence of the neurotoxin ß-ODAP in the seeds and vegetative tissues of the plant. The toxin is believed to cause lathyrism, a paralysis of the lower limbs in humans, usually occurring in epidemics in people subsisting on a diet heavily reliant on grasspea over 2-3 months.
We have identified several low-ODAP grasspea mutants created by EMS mutagenesis and are now performing the crosses necessary to develop an entirely toxin-free variety. This approach has the added benefit over a GM approach that improved varieties can be rapidly transferred to the farmer without regulatory hurdles. We are currently sequencing the genome of grasspea and are in the process of identifying key genes required for ODAP biosynthesis as well as stress tolerance mechanisms using a variety of approaches including transcriptomics and comparative bioinformatics.
Year(s) Of Engagement Activity 2016
 
Description Meeting with Detox partners 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Meeting of project partners
Year(s) Of Engagement Activity 2016
 
Description The Second International Legume Society Conference "2016: Legumes for a sustainable World" , Tróia, Portugal (12-14 October, 2016) poster and flash talk 
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 The Second International Legume Society Conference "2016: Legumes for a sustainable World" , Tróia, Portugal (12-14 October, 2016) poster and flash talk
Poster and Flash talk : The Lathyrus sativus genome project
Abstract
P74 - S8
The Lathyrus sativus genome project.
Sarkar A., Edwards A., Emmrich P. M. F., Martin C., Wang T. L.
The John Innes Centre, Norwich, NR4 7UH, United Kingdom.
Grass pea (Lathyrus sativus) is a hardy, drought tolerant legume grown
for animal feed, food and fodder on poor soils with minimal inputs. It
serves as an insurance crop for marginal farmers in the Indian
subcontinent and Africa. It is a diploid (2n=14) with an estimated
genome size of 6.9 Gbp (Bennett and Leitch, 2005. Annals of Botany 95:
45-90). Its main drawback is the presence of a neurotoxin (beta-ODAP),
which can cause neurolathyrism in people subsisting on a
predominantly grass pea diet for an extended length of time.
We are currently sequencing the grass pea genome of a European line
(LS007). A TILLING population is also being created for this line. A de
novo shotgun sequencing strategy has been adopted based on the
construction of a PCR-free library (using the Discovar protocol) and
mate-pair libraries for Illumina sequencing. The draft genome will be
annotated using ours and others' transcriptome data and data from
completed genome sequences of related legumes.
The genome sequence will be valuable for the identification of the
genes in the beta-ODAP biosynthesis pathway, as well as for traits of
agronomic importance. The data will also provide insights into
comparative genomics across legumes, help in the development of high
quality genetic and physical maps for marker-assisted and genomic
selection strategies and enable a genome-editing platform for grass
pea.
Year(s) Of Engagement Activity 2016
URL http://www.itqb.unl.pt/meetings-and-courses/legumes-for-a-sustainable-world/programme#content