Genetic improvement of rice seed vigour for dry direct-seeded conditions

Lead Research Organisation: Rothamsted Research
Department Name: Plant Sciences

Abstract

Rice is the most important staple crop in the developing world. It has been estimated that over three billion people in Asia rely on rice to provide 30 to 75% of their calories. The most widely used method for cultivating rice involves germinating the seeds in nurseries and transplanting the seedlings to flooded 'paddy' fields. This puddled transplanted rice (PTR) method accounts for ~55% of global rice cultivation and ~75% of world production. However, in many parts of South and Southeast Asia, farmers are switching to direct seeded rice (DSR) because the availability of labour and water is in decline and their associated costs are rising. A similar transition is also occurring in West Africa. To obtain a good crop using DSR it is imperative that the seeds germinate quickly in the soil and that the seedlings grow vigorously. However, because breeders have focused on developing varieties for PTR there has been little or no selection for seed vigour. Agronomists have highlighted a need for the development of new rice varieties with improved seed vigour so that they perform better in dry direct seeded conditions. The first aim of this project is to identify which genes are responsible for controlling seed vigour in rice by filming the germination of ~1000 different rice varieties that have had their genomes sequenced and identifying the key genetic differences that are associated with strong vigour. This knowledge can be used by breeder to develop new varieties. The second aim of this project is to determine whether the shoot growth of existing elite semi-dwarf rice varieties can be improved by enhancing the production of the plant growth regulator gibberellin in a specific seedling tissue called the mesocotyl. The increase in mesocotyl elongation could help seedlings to emerge from the soil when the seed has been planted at a greater depth. This is a key weakness of current varieties.

Technical Summary

Rice forms part of the staple diet of more than half the world's population. 18 of the top 20 rice producing countries are Lower and Middle Income Countries (LMICs) on the DAC list of ODA recipients. Together they account for ~95% of world production. The majority of the rice is consumed in the country of origin and only ~5% is traded internationally. The predominant method of rice production is Puddled Transplanted Rice (PTR), accounting for ~55% of cultivated area and ~75% of yield. PTR is productive but highly intensive in its requirements for water, labour and energy. It is also responsible for between 10 and 20% of global methane emissions. Dry Direct Seeded Rice (DSR) is a more sustainable alternative to PTR, particularly where low or zero tillage is employed. DSR is becoming more widely practiced in many LMICs owing to a combination of social, environmental and economic drivers. In order to achieve the best yield using DSR, strong early seedling establishment is required to overcome adverse conditions in the soil and greater weed pressure. However, current high-yielding semi-dwarf rice varieties have not been bred for seed vigour because this trait is not critical for PTR. The aim of this project is to help in the development of new DSR varieties with enhanced seed vigour. Automated high-throughput imaging technologies will be used to measure multiple component traits associated with seed vigour in a diversity set of ~1000 re-sequenced rice accessions and establishment will also be scored in small-scale field experiments. The data will be used to determine trait relationships and to perform a Genome Wide Association Study. This will identify genes and alleles that confer stronger seed vigour and can be used for molecular breeding. A targeted metabolic engineering approach will also be used to determine whether shoot growth can be enhanced in current elite semi-dwarf varieties by increasing the levels of active gibberellins in the mesocotyl.

Planned Impact

The ultimate beneficiaries of this project will be farmers in Asia and the people who rely on the food they produce. Rice is the most important staple crop in Asia, where it has been estimated that over 3 billion people rely on it to provide between 30 and 75% of their calories. The dominant method of rice cultivation is puddled transplanted rice (PTR), which accounts for about 55% of global crop area and around 75% of world production. However, many farmers are transitioning to dry direct seeded rice (DSR) owing to a combination of social, environmental and economic factors. In South Asia this transition is occurring fastest in India, Nepal and Bangladesh, and in Southeast Asia in the Philippines, Cambodia, Vietnam, Laos and Myanmar. Rice plants use between 1500-2500 litres of water to produce one kilogram of seed. The availability of water for agriculture in general (and rice cultivation in particular) is decreasing due to a shift in rainfall patterns, with less rain at the beginning of the season making irrigation for PTR less reliable. Furthermore, due to increased industrialisation and rural-urban migration, the availability of labour for agriculture has also reduced drastically in most rice-growing countries. Mechanised dry DSR is becoming a more attractive alternative to PTR. However, DSR has yet to be adopted in many countries and regions because suitable rice varieties are not available.

The research carried out in this project is in collaboration with the International Rice Research Institute (IRRI). More than half the area of rice grown in Asia is planted to IRRI-bred varieties or their progenies. The project will therefore directly help breeders to develop better varieties for dry direct seeded conditions. These varieties require improved seed vigour so that they can germinate and emerge rapidly from deeper soil depth, where even in the event of reduced rainfall there is more moisture available. This is currently a major weakness of elite rice varieties, which have been bred for the PTR system where seed vigour is much less important. It has been estimated that PTR contributes between 10 and 20% of all methane emission and so the continued transition to dry DSR will have an environmental benefit in combating global warming. Due to the fact that DSR matures approximately 10-15 days earlier than PTR, it also provides better opportunities to farmers to grow a second crop and increase their income. DSR rice varieties with improved seed vigour will have a large-scale impact. Researchers at IRRI have estimated that they could increase rice yields by ~0.5 t/ha, reduce the need for irrigation by ~40 cm of water/ha, allow labour savings of ~25 person-days/ha, make energy savings of ~1,500 Mj/ha, reduce greenhouse gas emissions by ~1,500 kg of CO2 equivalent/ha and increased net economic returns by USD 50/ha in most of the rice growing countries in Asia.

Publications

10 25 50
 
Description The aim of this project is the genetic improvement of rice seedling vigour for dry direct-seeding. We have developed new phenotyping platforms based on live and still imaging to measure variation in rice seed germination and etiolated seedling growth and morphology and we have gathered quantitative data for >10 separate traits (e.g. seed shape, seed size, seed colour, time to 50% germination, final % germination, shoot length, coleoptile length, mesocotyl length, root length, root branching etc.) from >700 diverse rice varieties from the 3000 rice genomes project. Our collaborators at the International Rice Research Institute (IRRI) in the Philippines have also performed parallel experiments with the same varieties to measure variation in seedling growth and emergence in the field, when the seeds are planted at three different depths in the soil. All the data sets have been analysed using statistical methods to understand relationships between the many component traits and genome wide association studies have been performed to identify which regions of the rice genome (and specific genes) control seedling vigour. One of the factors that contribute to the ability of direct seeded rice to emerge from deep planting in the soil is elongation of a tissue called the mesocotyl, which account for most of the variation in shoot length. We have also carried out global gene expression analysis on mesocotyl and other reference tissues from varieties with differing mesocotyl length in order to understand how mesocotyl elongation is controlled. These data will help in future attempts to improve rice seedling vigour by increasing mesocotyl length.
Exploitation Route Our findings will help rice breeders develop new varieties of rice that perform better under direct seeded conditions. Specifically, the work carried out in this collaborative project has identified rice varieties that have superior seedling vigour under laboratory and/or field conditions and also identified molecular markers for regions of their genome that are responsible. Together these materials and data can be used as tools in rice breeding programs, such as those conducted by the International Rice Research Institute. The new phenotyping platforms, image and gene expression data sets we have produced will also provide tools and leads to help researchers improve seed vigour of other crops.
Sectors Agriculture, Food and Drink

 
Description 1) We used genome wide association studies to identify several novel candidate genes controlling traits of interest for direct seeded rice. 2) The trait/genetic marker data we have generated will form the basis of a pre-breeding pipeline to deliver better germplasm for ODA countries; this work is now being taken forward collaboration with the International Rice Research Institute and Punjab Agricultural University, supported by a GCRF GRTA grant (EP/T015551/1) led by Smita Kurup. 3) We developed and validated new high throughput laboratory phenotyping platforms for measuring early seedling traits, including a novel time-laps system to determine rates of organ growth. 4) We have generated novel transcriptome data for rice mesocotyl, this dataset will be submitted to the Rice Expression Atlas at EMBL-EBI to facilitate public access (post-publication). 5) We have generated a large data set of high resolution rice seedling images from ~730 varieties that will be submitted to GRiSP/Ricebase to facilitate public access. 6) The numerical trait data acquired within the project will be made available at the IRRI RNA SNP-Seek database. 7) Outputs were disseminated at the 5th International Rice Congress 2018 (Singapore), which is the largest gathering of 'thought leaders, scientists, policymakers, agriculture experts, and technology providers from the world of rice research'. 8) We joined the recently established UK Rice Research Consortia (http://ukrrc.org/).
First Year Of Impact 2019
Sector Agriculture, Food and Drink
 
Description GCRF Impact Acceration Award- Comparative transcriptomic analysis of mesocotyl elongation in rice
Amount £38,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2017 
End 03/2017
 
Description Global Challenges Research Fund - Impact Acceleration Award Extension 2017 - 'Development and validation of new open-source phenotyping platforms for analysis of rice seedling vigour'
Amount £5,740 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2017 
End 02/2018
 
Description New varieties of direct seeded rice for farmers in Lower Middle Income Countries
Amount £602,712 (GBP)
Funding ID EP/T015551/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 09/2019 
End 03/2021
 
Title A live imaging system of measuring etiolated seedling growth 
Description We have developed a time-laps image capture system for measuring the rate of rice seed germination and seedling growth in the dark. The purpose of this technology is to quantify phenotypic variation in seed/seedling vigour in order to assist in genetic improvement for rice for direct seeding. The system can also be applied to other crops in which early vigour is important. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact The system has recently been demonstrated to our collaborators at the International Rice Research Institute. 
 
Title Etiolated rice seedling image database 
Description We are creating a database of images of etiolated rice seedling that consists of ~900 accessions. These images can be used to quantify variation in seedling morphology. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact Our data has been presented to our collaborators at the International Rice Research Institute and will be deposited in an open access database for other researchers to use. 
 
Title GWAS and gene discovery pipeline 
Description Built an in-house rapid and automated informatics pipeline using Cluster computer to run the GAPIT algorithm to perform GWAS analyses. Subsequently using R and Python scripts to discover underlying SNPs and therefore genes of interest. 
Type Of Material Computer model/algorithm 
Year Produced 2019 
Provided To Others? Yes  
Impact Facilitate rapid and accurate discovery of gene of interest controlling various agronomic traits. 
 
Title Rice mesocotyl transcriptome 
Description Mesocotyl length of etiolated rice seedlings varies greatly between accessions and this trait determines whether direct seeded rice can emerge from deep soil. To understand how mesocotyl length is controlled, we have generated a RNA-seq data set from accessions with short and long mesocotyls. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact We have presented this data to our collaborators at the International Rice Research Institute and it will be uploaded into a public database. 
 
Title rice tissue specific transcriptome 
Description There was a lack of tissue specific transcriptome data for indica rice (which would help facilitate the discovery of tissue-specific genes). Therefore we performed RNA-Seq from several tissues of rice plants (IR64). 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? No  
Impact This data will be shared with our collaborators at the International Rice Research Institute and it will be uploaded into a public database. 
 
Description Studentship 
Organisation University of Nottingham
Department School of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Co-I is involved in a new PhD studentship with new partners at the University of Nottingham titled "Understanding hypoxia tolerance mechanisms for rice improvement"
Collaborator Contribution The co-I is a co-supervisor for the studentship project. The GCRF grant enabled the establishment of new phenotyping capabilities to investigate early seedling development in rice and this platform will be utilised within the studentship. In addition RNA-Seq dataset generated within the GCRF grant is being used to identify potential genes that may play a role in explaining differences in the behaviour of germplasm during anaerobic germination.
Impact At IRRI, the student will gain experience and insight into the rice breeding pipeline, where they will grow plants both in "screenhouse" and field conditions. In addition to training in rice genome editing technology, the student will have access to IRRI training courses covering broader aspects of rice research and value chain components (https://irri.org/our-solutions/irri-education). At Nottingham, the student will be trained in state-of-the art phenotyping approaches, including sophisticated data analysis. At Rothamsted, training will be provided in: proteomics, transcriptomics, plant biochemistry and physiology, including the associated data analyses (bioinformatics and statistics, with a focus on cross-omics integration).
Start Year 2019
 
Description new collaboration 
Organisation Punjab Aricultural University
Country India 
Sector Academic/University 
PI Contribution We have set up an automated pipelines for GWAS and gene discovery in rice.
Collaborator Contribution New partnership with Dr Nitika Sandhu. There were technological restrictions to the informatics pipeline required for GWAS and gene discovery crucial to the project. With this in mind, we have created new rapid and automated pipelines in-house at Rothamsted for faster analyses and delivery on the project. We have also offered the use of this pipeline to Dr Sandhu for data analyses as part of the collaboration.
Impact Face to face meeting on 21st March, 2019 in UK. Working towards a joint publication.
Start Year 2018
 
Description Discussions with IRRI scientists 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Discussions with Nese Sreenivasulu (IRRI), Outcome Theme Leader - Facilitating rice research through Enabling Technologies regarding new collaboration in rice seed research.
Year(s) Of Engagement Activity 2018
 
Description Engagement with industry 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact We are using the phenotyping platform generated as part of the project to test on crop species of interest to Elsoms.
Year(s) Of Engagement Activity 2019
 
Description Engagement with industry/stakeholder 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Discussions with Rice Tech regarding current research and possibilities regarding future collaborations using technologies created within this project.
Year(s) Of Engagement Activity 2019
 
Description Engagement with industry/stakeholder 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Discussions with Elsoms regarding new technologies developed within this project (time-lapse system) for use in the industry.
Year(s) Of Engagement Activity 2018
 
Description Lecture to undergraduate students 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact A lecture on this project and related research and its impact in general terms was presented to 2nd year undergraduate students specialising in Plant Sciences module from Oxford Brookes University.
Year(s) Of Engagement Activity 2019
 
Description Member of UK Rice Consortium 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact The UK Rice Research consortium (UKRRC) has been established to highlight the breadth and quality of research in UK's based research institutions on rice, and to provide a focal point for building new networks both within the UK and with international partners. We have joined the consortium and also given a brief description of our research activities within this project.
Year(s) Of Engagement Activity 2018
URL http://ukrrc.org/
 
Description Poster presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation at the International Rice Congress, Singapore, 2018

A shoot growth force phenotyping platform of rice seedling vigour improvement

In areas of South and Southeast Asia, rice production systems are gradually changing from puddled transplanted rice to (dry) direct seeded rice (DSR). Shortages in labour and irrigation are among the reasons why farmers are switching to DSR. To obtain a good crop using DSR it is imperative that the seed germinate quickly in a wide range of seedbed conditions and that the seedlings grow rapidly from depth through soil of increasing impedance to emerge. Historically, there has been relatively little selection for seed vigour in modern rice varieties, which have been bred for transplanting, and agronomists have highlighted a need for improvement in this trait for DSR. Considerable genetic variation exists in rice seedling growth rate and morphology. However, it is not know whether variation also exists in the forces exerted by the shoot and root, which are crucial to penetrate strong soil. We have therefore developed an apparatus for measuring shoot growth force of etiolated rice seedlings. This apparatus uses an array of cheap micro strain gauge sensors, capable of measuring the rate of increase and the maximum force exerted by the shoots. This innovative system will be used to screen for phenotypic variation within a subset of accessions from the 3000 Rice Genomes Project. This poster will describe the apparatus and the first results obtained by screening rice accessions.
Year(s) Of Engagement Activity 2018
 
Description Poster presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation at the International Rice Congress, Singapore, 2018

Comparative transcriptomic analysis of mesocotyl elongation in rice
Rice is the most important staple crop in South and Southeast Asia. Some farmers in these regions are switching their cultivation method from puddled transplanted rice to dry direct seeded rice because the availability of labour and water is in decline and their associated costs are rising. Agronomists have identified several traits that are necessary for rice to perform well in dry direct-seeded conditions. Among these traits, mesocotyl elongation is thought to be important for seed vigour because a long mesocotyl enables seedlings to emergence from planting deeper in the soil. The availability of moisture is more consistent at greater soil depth and seed is less vulnerable to pests. Modern semi-dwarf varieties of rice have been bred for transplanting and tend to have relatively short mesocotyls. Therefore, increasing mesocotyl length is an important target for breeding dry direct-seeded rice. Substantial genetic variation in mesocotyl length exists in rice. Mesocotyl elongation is also known to be driven by cell expansion and is repressed by light, and modulated by several growth regulators, including gibberellins (GAs). Although there is a wealth of transcriptomic data available for rice tissues, there appears to be none for mesocotyl. We have therefore use RNA-Seq to carry out a comparative transcriptomic analysis of rice mesocotyl elongation in long and short mesocotyl varieties, and in the presence and absence of exogenous GAs. We will present the outcome of this work, which will improve our molecular understanding of mesocotyl elongation and may assist in the development of dry direct seeded rice varieties.
Year(s) Of Engagement Activity 2018
 
Description Presentation at International Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Flash presentation at the International Rice Congress, Singapore, Oct 14-17 2018.
Time-lapse for Rice: A live-imaging phenotyping platform for rice seedling vigour improvement.
Guillaume Menard, Peter Eastmond, Smita Kurup, Daniel Anderson, Jason Lim, Paul Compton, Arvind Kumar

Abstract
In areas of South and Southeast Asia, rice production systems are shifting from puddled transplanted rice to (dry) direct seeded rice (DSR). Shortages in labour and irrigation are among the reasons why farmers are switching to DSR. To obtain a good crop using DSR it is imperative that the seed germinate quickly in a wide range of seedbed conditions and that the etiolated seedlings grow rapidly through strong soils and emerge. Historically, there has been comparatively little selection for seed vigour in modern rice varieties, which have been bred for transplanting, and agronomists have highlighted a need for improvement in this trait for DSR. One of the factors that limits seed vigour in modern rice varieties is elongation of the mesocotyl, which is important to allow emergence from deeper soil. Rapid development of a long and thick root system is also important for DSR to promote water and mineral uptake under the variable soil moisture conditions. It is relatively easy to make 'end-point' measurements on tissues of etiolated rice seedlings that have been grown in vitro. However, no method currently exists to measure dynamic changes in the growth rates of these tissues over time. Such measurements may help breeders to develop better rice varieties for direct seeding. We have recently developed an open source phenotyping platform for live imaging of etiolated seedling growth from germination up to ~14 days after seed imbibition. This platform uses a DSLR camera for HD image capture, equipped with a low aperture macro lens and an adapted flashgun with a green light filter to allow repeated image capture, without inhibiting skotomorphogenesis (e.g. mesocotyl elongation). The platform is currently being used to phenotype a subset of accessions from the 3000 Rice Genomes Project. The system, the first results and further applications will be discussed in this presentation.
Year(s) Of Engagement Activity 2018
 
Description Presentation at International Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Attendance and presentation at the International Rice Congress, Singapore, Oct 14-17 2018.
Genetic improvement of rice seedling vigour for dry direct-seeded conditions
Guillaume Menard, Peter Eastmond, Smita Kurup, Daniel Anderson, Steve Penfield, Lorelei Bilham, Arvind Kumar, Nitika Sandhu
Abstract
Rice is the most important staple crop in South and Southeast Asia. Some farmers in these regions are switching their cultivation method from puddled transplanted rice to dry direct seeded rice (DSR), primarily because the availability of labour and water is in decline and their associated costs are rising. Agronomists have identified several traits that are necessary for rice to perform well in dry direct-seeded conditions and among these is seed/seedling vigour. For DSR, seeds must be able to germinate rapidly at deeper soil depth using available soil moisture and mesocotyl elongation must be sufficiently strong in both rate and duration to break through strong soil. Rapid development of a long and thick root system is also important for DSR to promote water and mineral uptake under the variable soil moisture conditions. Modern semi-dwarf varieties of rice have been bred for transplanting and there has been relatively little selection for seed/seedling vigour. Through a collaborative project between researchers at Rothamsted Research and the John Innes Centre in the UK and the International Rice Research Institute (IRRI) in the Philippines, we have used new lab-based high-throughput live-imaging phenotyping platforms to measure germination rate and skotomorphogenic seeding growth and morphology of >700 accessions from the 3000 Rice Genomes Project. The performance of these accessions under dry direct-seeded conditions has also been assessed in parallel in field experiments, where seed has been drilled to different depths. The new phenotyping platforms will be described and the results of the data analysis (inc. GWAS) will be presented. This comprehensive phenomic analysis of seed/seedling vigour will identify useful germplasm and molecular markers to accelerate breeding of DSR varieties.
Year(s) Of Engagement Activity 2018
 
Description Presentation at UK Rice Consortium meeting for Early Career Researchers 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact This short meeting was focussed on providing a forum for all postdocs and PhD students involved in rice research projects to come together and exchange ideas and experience on working with rice in the UK. The meeting was very much led by the researchers themselves, providing them the opportunity to both network (find out about the range of rice research being performed across the country) but with a focus on the nuts-and-bolts of rice research in the lab and field.
Year(s) Of Engagement Activity 2020
URL https://www.dur.ac.uk/dccit/events/?eventno=46060