A genetic dissection of traits required for sustainable water use in rice using Genome Wide Association Studies (GWAS)

Lead Research Organisation: Lancaster University
Department Name: Lancaster Environment Centre


Rice irrigation uses 1/3rd of the world's developed freshwater supplies, and is often unsustainable. In Bangladesh, 60% of the county's rice production is grown in the winter 'boro' season using predominantly groundwater. This results in lowering of water tables and ingress of saline water in coastal areas. The groundwater used in some parts of Bangladesh contains high concentrations of arsenic (As) which leads to dangerous concentrations of this class one human carcinogen in rice grain. Reducing irrigation demand should make water extraction more sustainable and reduce As exposure. Contributing to this change would help the UK meet its commitment to the UN Millennium Development Goals, and have direct benefit to the UK population exposed to As through consumption of rice and rice products. In Bangladesh, a new, heavily promoted irrigation scheme called alternate wetting and drying (AWD) reduces water use by 20-50% while increasing yield. But it is unknown why AWD works, and crucially if it is sustainable. Also, no work has yet been conducted to determine genetic variation for adaption (suitability) to AWD over conventional flooded crop production. This project will address these shortcomings by combining; i) a genetic screen for rice genes responsible for adaptation to AWD (exploiting advances in genome sequencing technology); ii) a chemical and physical analysis of the soil during cycles of wetting and drying; iii) a detailed physiological and transcriptomic characterisation of the changes that AWD causes in the rice plant and iv) a systems biology approach to identifying the metabolic pathways that are responsible for adaptation to AWD. Using established partnerships with the University of Calcutta, Assam Agricultural University, the Bangladesh Rice Research Institute and the International Rice Research Institute we will produce a collection of 300 rice landraces from the Bengal region suitable for boro cultivation. This will be sequenced to 1 x genome coverage to provide approx. 3 million single nucleotide polymorphisms using an innovative approach recently pioneered by Bin Han at the Chinese Academy of Science in Shanghai. This population will be screened under conventional flooding and AWD in Bangladesh in one site in the first season and three sites in the second season. Agronomic data will be collected to allow adaptation to AWD to be assessed. Shoots and grain in one site (both years) will be analysed for 17 macro and micro elements to provide a detailed description of uptake and shoot-grain translocation of the important plant and human nutrition-relevant elements. Both sets of data will be subjected to genome wide association mapping to identify the genomic regions and candidate genes associated with the traits and to identify if any specific element is related to adaptability to AWD. Detailed analysis of soil chemistry and strength will be conducted to reveal the physical/chemical changes taking place that affect plant growth. At the same time, spatial (stems and leaves) and temporal analysis of plant hormones will be measured to assess when and how plant growth is affected by AWD. A complementary transcriptomic study will show which genes, from which pathways, are being affected by AWD. The data generated will be incorporated in to a genome-scale metabolic model developed from the RiceCyc database. This will provide testable hypothesis of what metabolic pathways are important for growth in different water/soil chemistry scenarios and the most likely suitable genotypes. These hypotheses will be validated in the final year. All data will be integrated into publically accessible repositories and the panel of rice cultivars will be immensely valuable for mapping other traits such as drought, salt, cold and heat tolerance. Outputs obtained will prove valuable to researchers and breeders on rice throughout the world and to anyone concerned about aerobically-grown crops in flood-prone areas.

Technical Summary

Alternate Wetting and Drying (AWD) is a promising water-saving method that is being widely adopted in Bangladesh but it is not known why it improves crop water-use efficiency, if it is sustainable, if its adoption will reduce the exposure of the population to arsenic (As) poisoning or if genetic variation for adaptation to this new regime exists in rice. A panel of 300 rice landraces will be produced and sequenced using next generation sequencing to produce approx. 3 million SNP markers. This will be grown in Bangladesh in field experiments comparing AWD to conventional flooding. Shoot and grain samples will be analysed for 17 elements including all macro nutrients plus important micro nutrients and elements As, Fe, Zn, Se and Cd. Genome wide association studies (GWAS) will identify quantitative trait loci (QTLs) and candidate genes for agronomic traits associated with adaptation to AWD and nutrient uptake and translocation to grain. At the same time, detailed soil chemistry, plant hormones and gene expression will be assessed during the wetting/drying cycle to provide understanding of the likely chemical limitations to the sustainability of the method and the underlying plant physiology and genetics that determines adaptation and improved water use efficiency. All of the data gathered will be employed to develop a genome-scale metabolic model based on the RiceCyc database that will identify biochemical pathways and individual enzymes implicated in adaptation to AWD and nutrient uptake. The applicants form a multi-disciplinary team of world-leading experts who have the scientific knowledge and connections to get the work done and pipeline to maximise the impact of the findings. The project will produce a genomic tool with great potential for the identification of QTLs and genes for tolerance to a range of constraints (drought, heat) and the findings will have application in maximising nutrient and water use efficiency in all crops.

Planned Impact

The principle beneficiaries from the research will be; Plant biologists interested the consequences and adaptation to contrasting soil water conditions, and the mechanisms of nutrient accumulation into shoots and grain Rice breeders, biotechnologists and agronomists interested in natural allelic variation in rice, and in improving water use efficiency and nutritional value of rice production Rice scientists in the Bengal area interesting in wider aspects of adaptation to biotic and abiotic stress for rice improvement The people of Bangladesh who suffer the consequences of unsustainable groundwater extraction; costs of pumping, power cuts due to electrical demand for irrigation, salinisation, arsenic poisoning UK rice consumers whose exposure to arsenic will be reduced The short-term beneficiaries will be scientists interested in allelic variation in rice and in the optimisation of the alternate wetting and drying (AWD) method of water saving in rice production. Crucially, it will; i) established if AWD is sustainable or if it depletes limited nutrient resources in the soil; ii) establish if it reduces the problem of arsenic accumulation in soils and rice grain; and iii) identify the degree of genetic variation for adaptation to the method. This information will guide agricultural policy in Bangladesh and probably in the bordering parts of India with similar climate, geochemistry and rice cultivars. Reducing water use in the winter season in Bangladesh will ease the critical electricity shortages that currently result in daily power cuts throughout the country. For a medium term impact (5 years), the project will identify best cultivars, quantitative trait loci (QTLs) and candidate genes for adaptation to AWD which can be used throughout the Bengal region in breeding better cultivars. The results on soil chemistry and plant nutrient uptake will provide strategies to explore maximising the sustainability of AWD (i.e. identify a difference in farm inputs) which can be tested by agronomists. Confirming that water-saving strategies also reduce grain arsenic will enable rice producers worldwide to reduce the grain arsenic in local and exported rice and rice products, benefiting rice consumers worldwide including UK. The results will also provided strategies to ensure cadmium in rice is minimised. In the longer term (5+ years) the effect of individual candidate genes can be fully explored and strategies to utilise them in wider plant breeding (including orthologues in other cereals) can be evaluated. The hormone studies will identify the role of root signalling in adaptation to soil chemistry and matric potential providing hypothesis for wider agronomic practice (e.g. design of root systems to match predicted soil water content). The panel of 200-300 sequenced aus and boro cultivars will provide an immensely valuable tool for researchers interested in identifying candidate genes related to climate change (drought, salinity, heat and cold tolerance) and be a most welcome resource to the poorly funded researchers in the Bengal area who the applicants plan to work with in future. Ultimately, by contributing to understanding options to reduce water use in agriculture, this research will help the UK meet its commitment to the UN Millennium Development Goals on environmental sustainability and the eradication of poverty and hunger.
Description The project is part of a multi-disciplinary (Aberdeen-Lancaster-Bangladesh-IRR) I consortium programme that aim to integrate cutting edge genomics, soils chemistry, plant physiology and systems biology to pressing concerns regarding rice cultivation practices. Lancaster university was allocated a part-time (0.5) technician for the project. The main involvement of Lancaster University was Activity 3 "The physiology, transcriptomics and soil chemistry of adaptation to AWD".

The AWD regime profoundly affects the redox chemistry of soils, with metals in porewaters varying dramatically, both temporally and spatially. The new techniques of diffusive equilibration/gradients in thin films (DET/DGT) that can be readily deployed in situ to give precise information on metal concentrations and dynamics in porewaters have been used for the project. DET measures porewater concentrations of metals and nutrients while DGT provides information of fluxes and rates of those elements that can be readily released from the solid phase. For both DGT and DET (gel constrained to compartments) the metal is captured, allowing probes to be transported and stored prior to analysis. ICP-MS technique has the sensitivity and low sample volume requirements for analysing the eluents of the small gel sections. A comprehensive set of trace metal cations and oxyanions have been measured, including Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd, Pb, P, V, As, Se, Mo, Sb, W.

The highly dynamic soil environment during AWD (decreased soil oxygen concentrations during flooding and decreased matric potential during drying) will produce dramatic fluctuations in the root synthesis of chemical signals and their transport to the shoot. Understanding the effects of AWD on the relationships between plant hormone status and instantaneous soil conditions requires repeated sampling over AWD cycles (this is important for determining when transcriptomic samples should be taken).

Specific objectives and achievements to date:

1) Monitor chemical concentrations in soils and soil waters in field during different AWD schemes and different rice growing stages. Two field campaigns have been successfully conducted in 2014 and 2015 as planned. Deployments of DET and DGT were carried out in 3 field locations orientated to the irrigation inlet (at inlet, distant to inlet and centre) at one field site over two years, with two replications at three time points (vegetative growth, heading and harvest) for two water regime treatments (continuous flooded and AWD). Sample treatments have been undertaken at Lancaster clean room conditions to avoid any contaminations. Samples for 2014 and some samples for 2015 have been analysed by ICPMS. Rhizon samplers for extracting porewaters were also deployed to measure S and N and analysis has been carried out by Aberdeen.

2) Monitoring plant growth hormones in field during different AWD schemes and different rice growing stages.
A physiological study has been conducted on four cultivars. In 2014 we have selected to cover known suitability for AWD and genetic and phenological diversity and in 2015 comprising two pairs with very different adaptabilities to AWD, and contrasting flowering times. Stem and flag leaf tissue were removed hourly (to determine diurnal variations in hormone status) every two days, rapidly frozen in liquid nitrogen, then freeze-dried for subsequent analysis of ABA status (using a high throughput immunoassay available at Lancaster). Four cultivars differing in flowering time were sampled in 2014 field campaign to distinguish phenological and soil effects on the relationships between ABA and soil oxygen concentrations and matric potential. The rate of grain filling were monitored by tagging flowers at panicle emergence and harvesting grain to record weight every two days. Grain yield and its components have been determined at harvest. In 2015, a more complete hormonal profiling (ABA, ACC, cytokinins) have been carried out at representative times during the AWD cycles (guided by temporal effects on plant hormone status identified in 2014) to coincide with leaf transcriptomic analysis.

3) Understanding biogeochemical processes of metals and nutrients at AWD conditions and the chemical effect on the growth and physiological behaviours of rice plants.
From the data obtained by Aberdeen group, we have identified cultivars which display very different adaptabilities to AWD yet have rather similar genetic structure. Results from Lancaster will provide the reason for these differences, if this is because of different abilities to take up soil nutrients or respond to plant growth hormones, and the degree to which these are related to root growth. Results from 2014 samples show extremely dynamic system with big reservoir of Fe-oxide, controlled by organic matters. Limiting factor is Mn-oxide, need OM too, easily reduced, oxidation is slow, stay in Mn2+ form longer and flashed out. As displayed its own redox chemistry, but strong association with Fe. Co is also controlled by its own redox chemistry, but strong association with Mn. Trace metals such as Zn, Cd, Pb, Ni, Cu, have nothing to do with redox. They are controlled by adsorption on fresh Fe-oxide surfaces.
Exploitation Route Further researches are needed to understand the heterogeneity and the dynamic biogeochemical processes of metals and nutrients in paddy soils. Findings from the project may be taken forward by other scientists.
Sectors Agriculture, Food and Drink,Environment