“Once you’ve cloned a major gene in one crop, it is possible to find a counterpart gene that has the same function in another crop, and this is easier than finding useful genes from scratch” explains Leon Kochian, Professor in Plant Biology and Crop and Soil Sciences at Cornell University, USA, and Director of the Robert W Holley Center for Agriculture and Health, United States Department of Agriculture – Agricultural Research Service.
Aluminium toxicity and low phosphorus levels in acid soils are major factors that hinder cereal productivity worldwide, particularly in sub-Saharan Africa, South America and Southeast Asia. Globally, acid soils are outranked only by drought when it comes to stresses that threaten food security.
Tolerance to high levels of aluminium and low phosphorus is conferred by major genes, which lend themselves to cloning. Major genes are genes that by themselves have a significant and evident effect in producing a particular trait; it’s therefore easier to find and deploy a major gene associated with a desired trait than having to find and clone several minor genes.
Harvesting sorghum in Kenya.
Cloning major genes instrumental in hunt for resilient varieties
Locating a single gene within a plant’s DNA is like looking for a needle in a haystack. Instead of searching for a gene at random, geneticists need a plan for finding it.
The first step is to conduct prolonged phenotyping – that is, measuring and recording of plants’ observable characteristics in the field. By coupling and comparing this knowledge with genetic sequencing data, scientists can identify and locate quantitative trait loci (QTLs) – discrete genetic regions that contain genes associated with a desired trait, in this instance tolerance to aluminium or improved phosphorus uptake. They then dissect the QTL to single out the gene responsible for the desired trait. In the case of sorghum, researchers had identified the aluminium tolerance locus AltSB, and were looking for the gene responsible.
Once the gene has been identified, the next step is to clone it – that is, make copies of the stretch of DNA that makes up the gene. Geneticists need millions of copies of the same gene for their research: to gain information about the nucleotide sequence of the gene, create molecular markers to help identify the presence of the gene in plants and help compare versions of the gene from different species, and understand the mechanisms it controls and ways it interacts with other genes.
Drying the sorghum harvest in India.
Sorghum was one of the simpler crops to work with, according to Claudia Guimarães.
“Sorghum has a smaller genome… with clear observable traits, which are often controlled by one major gene,” she says.
The first breakthrough was the identification and cloning of SbMATE, the aluminium tolerance gene in sorghum behind the AltSB locus. The next was finding a diagnostic marker for the gene so that it could be used in breeding.
Marking genes to quickly scan plants for desired traits
Harvesting rice in The Philippines.
Once a desired gene is identified, a specific molecular marker must be found for it. This is a variation in the plant’s DNA, associated with a gene of interest, that scientists can use to flag up the gene’s presence. We can compare this process to using a text highlighter in a book, where the words represent the genes making up a genome. Each marker is like a coloured highlighter, marking sentences (genomic regions) containing particular keywords (genes) and making them easier to find.
In molecular breeding, scientists can use markers to quickly scan hundreds or thousands of DNA samples of breeding materials for a gene, or genes, that they want to incorporate into new plant varieties. This enables them to select parents for crosses more efficiently, and easily see which of the next generation have inherited the gene. This marker-assisted breeding method can save significant time and money in getting new varieties out into farmers’ fields.
Leon, who was also the Principal Investigator for various GCP-funded research projects, says that the cloning of SbMATE helped advance sorghum as a model for the further exploration of aluminium tolerance and the discovery of new molecular solutions for improving crop yields.
“This research also has environmental implications for badly needed increases in food production on marginal soils in developing countries,” says Leon. “For example, if we can increase food production on existing lands, it could limit agriculture’s encroachment into other areas.”
Rice field trials in Tanzania.
Aluminium toxicity is the result of aluminum becoming more available to plants when the soil pH is lower, and affects 38 percent of farmland in Southeast Asia, 31 percent in South America and 20 percent in East Asia, sub-Saharan Africa and North America. Meanwhile phosphorus, the second most important inorganic plant nutrient after nitrogen, becomes less available to plants in acid soils because it binds with aluminium and iron oxides. Almost half of the ricelands across the globe are currently phosphorus deficient. The research therefore has the potential for significant impact across the world.
The GCP-funded scientists used markers to search rice and maize for genes equivalent to sorghum’s SbMATE. In maize they successfully identified a similar gene, ZmMATE1, which is now being validated in Brazil, Kenya and Mali. In rice the search continues, but will become easier now that markers for ZmMATE1 have been developed.
Similarly, having validated, cloned and developed markers for PSTOL1 gene in rice, researchers at IRRI and JIRCAS then worked with researchers at EMBRAPA and Cornell University to use PSTOL1 markers to search for comparable genes in sorghum and maize. In both crops, genes similar to PSTOL1 have been identified and shown to improve grain yields under low-phosphorus soil conditions, albeit through different mechanisms.
The GCP-funded discoveries are already being used in marker-assisted selection in national breeding programmes in Brazil, Kenya, Niger, Indonesia, Japan, The Philippines and USA in sorghum, maize and rice. They have led to the release of new, aluminium-tolerant sorghum varieties in Brazil, with more currently being developed, along with phosphorus-efficient rice varieties.
Showing off freshly harvested sorghum in Kenya.
Cloning a worthwhile investment
Gene cloning was a relatively small cost in GCP’s research budget – about five percent (approximately USD 7 million) of a total research budget of USD 150 million spread over 10 years.
The gene-cloning component nonetheless yielded important genes for aluminium tolerance and phosphorus-uptake efficiency, within and across genomes. The molecular markers that have been developed are helping plant breeders across the world produce improved crop varieties.
Jean-Marcel Ribaut, Director of GCP, concludes: “The new markers developed for major genes in rice, sorghum and maize will have a significant impact on plant-breeding efficiency in developing countries.
“Breeders will be able to identify aluminium-tolerance and phosphorus-efficiency traits quicker, which, in time, will enable them to develop new varieties that will survive and thrive in the acid soils that make up more than half of the world’s arable soils.”
Rice plays a key role in global food security, particularly in Asia, where 90 percent of the world’s rice is grown and eaten. By 2050, Asia’s population is estimated to grow by one billion to 5.2 billion people, who will continue to depend on rice as their major staple food.
But with rising demand for rice has also come increasing salinity, droughts and other stresses, along with decreasing areas of land available for farming the crop.
Key ingredient in the rice research fest was GCP’s relationship with the International Rice Research Institute (IRRI), headquartered in The Philippines. GCP supported IRRI in its endeavours to use the latest molecular plant-breeding techniques, along with traditional plant-breeding tools, to develop rice crops better able to cope with various stresses and still be productive.
“These ‘super’ crops will revolutionise rice farming,” says IRRI Director General Robert Zeigler, who was also the first Director of GCP.
Climate change is one of the major threats facing rice production. As sea levels rise, salt water enters further up rivers with the high tides and affects rice production areas.
Each year in Bangladesh, during the boro rice season from November to May, salinity is so high that a white film of salt covers the country’s coastal paddy fields. For Bangladeshi farmers, this white colour is a warning sign that their land is ‘sick’. Around the world, Bangladesh, India, Myanmar and parts of Africa are most affected by increasing salinity.
Right from the beginning of GCP, salinity was a problem firmly on the rice research agenda.
Highly saline soils in India (left), and a close-up showing a surface crust of salt on afflicted soil (right).
Leading this research was IRRI plant physiologist Abdelbagi Ismail, who dreamed of the ‘super’ rice crop that could “tolerate salinity, drought and submergence”.
Abdelbagi has managed and overseen most of the progress made during the discovery of a major genetic region, or quantitative trait locus (QTL), associated with salinity tolerance and named Saltol.
Abdelbagi Ismail examines rice plants in the field in Bangladesh.
Its insertion into well-known rice varieties used by farmers in Bangladesh, Indonesia and The Philippines is part of the revolution taking place in rice research.
Abdelbagi says Saltol was mapped and markers were developed for its use in breeding more salt-tolerant rice varieties. Its salt-tolerance code is now being transferred into several varieties evaluated with IRRI partners in South and Southeast Asia.
“These projects also provided opportunities for both degree training and non-degree training to several of our partners in the countries involved,” he adds.
“Partnerships are crucial for us to build the capacity of the researchers in these countries and to ensure our outputs and outcomes reach the farmers that need them.
“All our partners benefitted from salt-tolerant varieties developed conventionally through this project, and they also provided pipelines for uptake and dissemination by farmers.”
Having developed new lines following the discovery of the Saltol QTL, Abdelbagi’s GCP-supported team trained plant breeders in country programmes to successfully breed for salt tolerance and other stresses.
In this way, Abdelbagi says, they are improving the capacity of researchers in developing countries to take up new breeding techniques, such as the use of molecular markers. “This can reduce the time it takes to breed new varieties, from six to ten years at the moment, down to two or three years,” he says.
This means that benefits to smallholder rice farmers struggling with salinity will happen sooner rather than later. And Abdelbagi credits GCP’s partnership approach, working directly with the countries in need, for the success so far.
“The salt-tolerant varieties are now being widely distributed,” he says. “Some of these varieties have doubled farmers’ productivity in affected areas.
“The work developed technologies of value to our needy farmers.
“We do believe this is the start of a second Green Revolution, especially for those who farm in less favourable areas and that missed this opportunity during the first Green Revolution.”
Partnership approach key to new rice gene for uptake of phosphorus
More than 60 percent of rainfed lowland rice is produced on poor and problem soils, including those that are naturally low in phosphorus. This is an essential for nutrient growing crops, but providing phosphorus through fertilisers is costly and unfeasible for many smallholder rice growers.
IRRI’s Sheryl Catausan prepares the roots of a rice plant for scanning as part of the work of the PSTOL1, phosphorus uptake research team at IRRI.
This problem was the focus of GCP’s rice phosphorus-uptake project led by IRRI molecular biologist Sigrid Heuer.
The project was enormously successful, with its discovery of the PSTOL1 (‘phosphorus starvation tolerance 1’) gene within the Pup1 locus, which was published in the prestigious journal Nature.
“We wanted it in Nature for a couple of reasons,” she says. “To raise awareness about phosphorus deficiency and phosphorus being a limited resource, especially in poorer countries; and to draw attention to how we do molecular breeding these days, which is a speedier, easier and more cost-effective approach to developing crops that have the potential to alleviate such problems.”
Following the PSTOL1 discovery, researchers are now working with developing-country researchers and extension agencies to help them understand how to breed local varieties of rice that can be grown in phosphorus-deficient soils. They are also collaborating with other crop breeders looking to breed similar maize, sorghum and wheat varieties.
Tobias Kretzschmar, a molecular biologist with IRRI, says that GCP’s partnership approach was the key to the project having an impact on the rice farmers who needed it most.
“For me, the collaborations that were forged through these inter-institutional projects made the difference,” he says.
Sigrid agrees: “GCP was always there supporting us and giving us confidence, even when we were not sure.”
Solving the insoluble: a gene for drought tolerance
Rice is a crop that not only needs water, but loves water. So developing a drought-tolerant rice variety is a quest to find a seemingly impossible gene.
However, GCP-supported researchers did just that: they solved the insoluble.
“They were very successful in terms of getting drought tolerance,” says Hei Leung of IRRI, who was GCP Subprogramme Leader for the Comparative Genomics Research Initiative between 2004 and 2007, and also a Principal Investigator for the Rice Research Initiative. “They’re now getting a 1.5 tonne rice yield advantage under water stress. I mean, that’s unheard of! This is a crop that needs water.
“This is one of GCP’s big success stories; that you can actually get drought tolerance is a seemingly impossible task for a water-loving rice plant.”
As Subprogramme Leader, Hei played a critical role in the creation, management, delivery and communication of a wide portfolio of research projects. He credits the nature of how GCP was set up for accelerating the breeding programme for drought-tolerant rice.
“The advantage of GCP is that it is run by a small group of people who can make fast decisions,” he says. “This means they can respond to the needs of researchers: ‘Okay, we are going to invest on that. We’re going to have a meeting on this’.
“The real advantage of GCP is its agility. Usually with other organisations you have new ideas and then have to slave away for a year to get the funding to implement them. But GCP was quick.”
Hei Leung (right) explains rice screening processes to a visiting scholar at IRRI.
IRRI and GCP deliver genetic resources to those who need them most
Tobias says one of the main objectives of GCP and IRRI is to make genetic stocks available to breeders, particularly in developing countries.
“Without that, IRRI would fail in its central mission to reduce hunger and poverty,” he says. “In order to achieve this mission, tight collaboration with our agricultural and extension partners in other countries is the key.”
In fact, this idea of protecting the future through genetic material was influential in the choice of GCP as a CGIAR Challenge Program name. Hei, who was involved from the start with GCP, talks about the meeting in a Rome pizzeria where participants came up with the name: “When we talk about ‘generation’, we are really talking about the work we do with genetic diversity; it is about the future generation,” he says.
Part of that future generation is about sharing genetic resources or stocks, but first the genetic diversity of such stocks needs to be characterised. Hei remembers that one of the first GCP projects in 2004 brought together researchers in various countries to characterise the genetic diversity of various crops, including rice.
“But everyone was using different genotyping platforms and markers, and the technology back then was not what we have now,” he recalls.
“So we spent a lot of resources getting poor quality data. In a sense, it was a failure. On the other hand, it was also a success because we alerted people to the importance of characterising diversity in every single crop. The whole concept of getting all the national partners doing genetic resource characterisation is a good one.
“We have evolved the technology together over the last 10 years of GCP. Now the country partners feel enabled. They are not scared of the technology.”
Abdelbagi agrees that characterising genetic diversity is essential, and adds that making such genetic stocks readily available to breeders is also vital.
“This has not been an issue before,” he explains. “With the new regulations of germplasm control and intellectual property issues, it became extremely difficult to exchange germplasm with some countries. One important lesson we learnt was to engage in direct discussion with our partners in efforts to influence their policies and guidelines to allow essential exchange of genetic stocks and breeding material, at least at the regional level.”
Abdelbagi adds that another big challenge has been to provide country partners with materials and DNA markers for marker-assisted selection programmes and to make sure these were properly used in breeding programmes.
“We solved this by hosting a workshop at IRRI and with continued visits with GCP collaborators,” he says.
Rice terraces in Ifugao Province in The Philippines.
‘Super’ crops: something ‘magic’ happens
Hei says that the GCP project he’s most passionate about, and one that leaves a lasting legacy, is the development of multiparent advanced generation intercross (MAGIC) populations, which will potentially yield lines that are tolerant to environmental stresses, grow well in poor soils and produce better quality grain.
“MAGIC populations will leave behind a very good resource towards improving different crop species,” says Hei. “I’m sure that they will expand on their own.”
GCP funded the development of four kinds of rice populations, including indica MAGIC, which is the most advanced of the MAGIC populations developed so far. These populations contain multiple desirable traits, including: blast and bacterial blight-resistance, salinity and submergence tolerance and grain quality.
New generation of researchers working on a new rice revolution
Rice farmer in her field in Rwanda.
Robert Zeigler’s dream of a new rice Green Revolution with ‘super crops’ is coming true, thanks in part to GCP’s focus on combining cutting-edge molecular plant-breeding techniques with conventional plant breeding.
“With all this going for us, the second Green Revolution means we can meet the great challenges ahead with unprecedented efforts that will result in unparalleled impacts,” he says. “This will range from mining the rice genomes for needed traits to developing climate change-ready rice.”
IRRI researchers like Abdelbagi agree that new plant-breeding techniques, such as those fostered by GCP, are making ‘super’ crops more likely: “I’m committed to understand how plants can be manipulated to adapt to, and better tolerate, extreme environmental stresses, which seems more feasible today than it has ever been before.
“GCP-supported work has provided mechanisms for developing varieties with multiple stress tolerances, besides the improvements in yield and quality.”
And for Hei, GCP’s 10-year legacy is not just about the technology but also about the people.
“Over ten years you have three generations of PhD students,” he says. “Many people became a ‘new generation’ scientist through this programme. Many people have benefitted. GCP is one of a kind. I’m just in love with it.”
GCP-supported researchers aimed high: they wanted to contribute to food security in the developing world by using the latest advances in crop science and plant breeding.
And with the lives of half of the world’s population directly reliant on their own agriculture, there is a lot at stake. Land degradation, salinity, pollution and excessive fertiliser use are just some of the challenges.
Rice is one of the most critical crops worldwide
Amelia Henry, drought physiology group leader at the International Rice Research Institute (IRRI), explains why rice was such a critical crop for GCP research. She says rice is grown in a diverse set of environmental settings, often characterised by severe flooding, poor soils and disease.
Cycling through rice fields in Odisha, India.
In Asia, 40 percent of rice is produced in rainfed systems with little or no water control or protection from floods and droughts – meaning rice plants are usually faced with too much or too little water, and rarely get just enough. In addition, 60 percent (29 million hectares) of the rainfed lowland rice is produced on poor and problem soils, including those that are naturally low in phosphorus.
Phosphorus deficiency and aluminium toxicity are two of the most widespread environmental causes of poor crop productivity in acidic soils, where high acid levels upset the balance of available nutrients. And drought makes these problems even worse.
Phosphorus is essential for growing crops. Its commercial use in fertilisers is due to the need to replace the phosphorus that plants have extracted from the soil as they grow. Soils lacking phosphorus are an especially big problem in Africa, and the continent is a major user of phosphate fertilisers. However, inappropriate use of fertilisers can, ironically, acidify soil further, since excess nitrogen fertiliser decreases soil pH.
Meanwhile, high levels of aluminium in soil cause damage to roots and impair crop growth, reducing their uptake both of nutrients like phosphorus and of water – making plants more vulnerable to drought. Aluminium toxicity is a major limitation on crop production for more than 30 percent of farmland in Southeast Asia and South America and approximately 20 percent in East Asia, sub-Saharan Africa and North America.
The challenge today is to tap into the genetic codes of key crops such as rice and wheat to feed a growing global population. Science plays a crucial role in identifying genes for traits that help plants tolerate more difficult environmental conditions, and producing crop varieties that contain these genes.
Plant biologists are already developing new rice lines that produce higher yields in the face of reduced water, increasingly scant fertiliser as costs rise, and unproductive soils. However, ‘super’ crops are needed that can combine these qualities and withstand climate changes such as increasing temperatures and reduced rainfall in a century when the world’s population is estimated to reach nearly 10 billion people by 2050.
Bringing the best scientific minds to improve rice varieties
Ambitious in concept, the GCP research focussed on bringing together experts to work on these critical problems of rice production for some of the world’s poorest farmers.
The programme was rolled out in two phases that sought to explore the genetic diversity of key crops and use the most important genes for valuable traits, such as Sigrid’s discovery made in a rice variety that is tolerant of phosphorus-poor soils. Each phase involved dedicated teams in partner countries.
GCP Principal Investigator Hei Leung, from IRRI, says GCP is unique, one of kind: “I love it.” He says GCP has enabled rice researchers and breeders to embrace cutting-edge science through partnerships focussed on improving crop yields in areas previously deemed unproductive.
Hei says GCP wanted to target research during its second phase on those crops that most poor people depend upon. “We wanted to have a programme that is what we call ‘pro-poor’, meaning the majority of the world’s people depends on those crops,” he says.
Rice is the ‘chosen one’ of GCP’s cereal crop research and development, with the biggest slice of GCP’s research activities dedicated to this, the most widely consumed staple food.
It is crucial to increase rice supplies by applying research and development such as that carried out by GCP researchers over the past 10 years, Hei says.
Relying on rice’s small genome in the hunt for drought-tolerance genes
Researchers had been trying to map the genomes of key cereal crops for over two decades. Rice’s genome was mapped in 2004, just as GCP started.
Rice has a relatively small genome, one-sixth the size of the maize genome and 40 times smaller than the wheat genome. This makes it a useful ‘model’ crop for researchers to compare with other crops.
“People like to compare with rice because wheat and maize have very big genomes, and they don’t have the resources,” explains Hei.
After the rice genome had been sequenced, the next step was to focus down to a more detailed level: the individual genes that give rice plants traits such as drought tolerance. Identifying useful genes, and markers that act as genetic ‘tags’ to point them out, gives scientists an efficient way to choose which plants to use in breeding.
One of GCP’s Principal Investigators for rice was Marie-Noëlle Ndjiondjop, a senior molecular scientist with the Africa Rice Center.
“Rice is becoming a very important crop in Africa,” she says. “Production has been reduced by a lot of constraints, and drought is one of the most important constraints that we face in Africa.”
Meet Marie-Noëlle below (or on YouTube), in our series of Q&A videos on rice research in Africa.
Marie-Noëlle’s team recognised that drought tolerance was likely to be a complex trait in rice, involving many genes, due to the mix of physiological, genetic and environmental components that affect how well a plant can tolerate drought conditions. To help discover the rice varieties likely to have improved drought tolerance, Marie-Noëlle’s team used an innovative approach known as bi-parental marker-assisted recurrent selection (MARS).
“With such a complex trait, you really need to have all the tools and infrastructure necessary; through GCP we were able to buy the necessary equipment and put in the infrastructure needed to find and test the drought trait in rice lines.
“By using the MARS approach we identified the genetic regions associated with drought and are moving towards developing new rice lines that the African breeder and farmer will be using in the next decade to grow crops that are better able to withstand drought conditions.”
Likewise, Amelia Henry’s IRRI team also developed drought-tolerant lines, particularly for drought-prone areas of South Asia. She says many of the promising deep-rooted or generally drought-tolerant varieties identified in the early decades after IRRI’s foundation in 1960 are still used today as ‘drought donors’.
“Since the strength of our project was the compilation of results from many different sites, this work couldn’t have been done without the GCP partners,” she says. “They taught me a lot about how rice grows in different countries and what problems rice farmers face.”
Hei agrees that GCP partnerships have been crucial, including in the successful breeding of rice with drought tolerance: “They’re getting a 1.5-tonne rice yield advantage under water stress. I mean, that’s unheard of! This is a crop that needs water.”
A rice farmer in Rwanda.
But the researchers could not rest with just one of rice’s problems solved.
Hei says GCP’s initial focus on drought was a good one but then, “I remember saying, ‘We cannot just go for drought. Rice, like all crops, needs packages of traits’.”
He knows that drought is just one problem facing rice farmers, noting “this broadened our research portfolio to include seeking to breed rice varieties with traits of tolerance to aluminium toxicity, salt and poor soils.”
The scope widens: phosphorus-hungry rice and a huge success
Sigrid Heuer was in The Philippines working for IRRI when she became involved in the ground-breaking phosphorus-uptake project for rice.
She took over the project being headed by Matthias Wissuwa. Much earlier, Matthias had noted that Kasalath – a traditional northern Indian rice variety that grew successfully in low-phosphorus soil – must contain advantageous genes. His postdoctoral supervisor, Noriharu Ae, thought that longer roots were likely to be the secret to some rice varieties being able to tolerate phosphorus-deficient soils.
Screening for phosphorus-efficient rice, able to make the best of low levels of available phosphorus, on an IRRI experimental plot in The Philippines. Some types of rice have visibly done much better than others.
Sigrid Heuer used her background in molecular breeding to take up the challenge with GCP to find the genes responsible for the Kasalath variety’s long roots.
“I spent years looking for the gene,” Sigrid says. “It was like trying to find a needle in a haystack; the genomic region where the gene is located is very complex.
“We had little biogenomics support at the time and I had three jobs and two kids; I was spending all my nights trying to find this gene.”
Sigrid Heuer in the field at IRRI.
But one day, Sigrid’s postdoctoral student Rico Gamuyao excitedly called her downstairs to the transgenic greenhouses. “Rico had used transgenic plants to see whether this gene had any effect. He was digging out plants from experimental pods.”
Sigrid says that moment in the Manila labs was the turning point for the project’s researchers.
Matthias’ team had previously identified a genomic region, or locus, named Pup1 (‘phosphorus uptake 1’) that was linked to phosphorus uptake in lines of traditional rice growing in poor soils. However, its functional mechanism remained elusive until the breakthrough GCP-funded project sequenced the locus, showing the presence of a Pup1-specific protein kinase gene, which was named PSTOL1 (‘phosphorus starvation tolerance 1’). The discovery was reported in the prestigious scientific journal Nature on 23 August 2012 and picked up by media around the world.
The gene instructs the plant to grow larger and longer roots, increasing its surface area – which Sigrid compares to having a bigger sponge to absorb more water and nutrients in the soil.
“Plants growing longer roots have more uptake of phosphorus – and PSTOL1 is responsible for this.
“GCP was always there, supporting us and giving us confidence, even when we weren’t sure we were going to succeed,” she recalls. “They really wanted us to succeed, so, financially and from a motivational point of view, this gave us more enthusiasm.”
She adds, jokingly, “With so many people having expectations about the project, it was better not to disappoint.”
For some insight straight from the source, listen to Matthias in our podcosts below. In these two bitesized chunks of wisdom he discusses the importance of phosphorus deficiency and of incorporating PSTOL1 into national breeding programmes; his work in Africa and the possibility of uncovering an African ‘Pup2’; what the PSTOL1 discovery has meant for him; and the essential contribution of international partnerships and GCP’s support.
Members of the IRRI PSTOL1, phosphorus uptake research team chat in the field in 2012. From left to right they are are: Sigrid Heuer, Cheryl Dalid, Rico Gamuyao, Matthias Wissuwa and Joong Hyoun Chin.
Phosphorus-uptake gene not all it seemed – an imposter?
But PSTOL1 was definitely not what it seemed. “It was identified under phosphorus-deficient conditions and the original screen was set up for that,” says Sigrid.
Researchers eventually discovered that Pup1 and the PSTOL1 gene within it were not really all about phosphorus at all: “It turns out it is actually a root-growth gene, which just happens to enhance uptake of phosphorus and other nutrients such as nitrogen and potassium.
“The result is big root growth and maintenance of that growth under stress. If you have improved root growth, there is more access to soil resources, as a plant can explore more soil area with more root fingers.”
Her team showed that overexpression of PSTOL1 gene significantly improves grain yield in varieties growing in phosphorus-deficient soil – by up to 60 percent compared to rice varieties that did not have the gene.
In field tests in Indonesia and The Philippines, rice with the PSTOL1 gene produced about 20 percent more grain than rice without the gene. This is important in countries where rice is grown in poor soils.
A farmer harvests rice in South Sulawesi, Indonesia.
Sigrid, now based in Adelaide at the Australian Centre for Plant Functional Genomics, says the introduction of the new gene into locally adapted rice varieties in different locations across Asia and Africa is expected to boost productivity under low-phosphorus conditions.
“The ultimate measure for these kinds of projects is whether a gene works in different environments. I think we have a lot of evidence that says it does,” she says.
The discovery of PSTOL1 promises to improve the food security of rice farmers on phosphorus-deficient land though assisting them to grow more rice and earn more.
Titbits of further research successes: aluminium tolerance and MAGIC genes
Drought, low-phosphorus soils, aluminium toxicity, diseases, acid soils, climate change… the list seems never-ending for challenges to growing rice. Apart from the successes with drought and phosphorus that GCP scientists achieved, there was to be much more in the works from other GCP researchers.
In Phase II, they worked towards breeding aluminium-tolerant sorghum lines for sub-Saharan Africa, as well as applying what they learnt to discover similar genes in rice and maize.
Hei Leung says GCP leaves a lasting legacy in the development of multiparent advanced generation intercross (MAGIC) populations. These help breeders to identify valuable genes, and from among the populations they can also select lines to use in breeding that have favourable traits, such as being tolerant to environmental stresses, having an ability to grow well in poor soils or being able to produce better quality grain.
“MAGIC populations will leave behind a very good resource towards improving different crop species,” says Hei. “I’m sure that they will expand on their own.”
GCP funded the development of four different MAGIC populations for rice, including both indica and japonica types. And the idea of developing MAGIC populations has spread to other crops, including chickpeas, cowpeas and sorghum.
Meeting the challenges and delivering outcomes to farmers
But with success come the frustrations of getting there, according to Nourollah Ahmadi, GCP Product Delivery Coordinator for rice across Africa. “This is because things are not always going as well as you want.”
Project Delivery Coordinators monitor projects first-hand, conducting on-site visits, advising project leaders and partners and helping them implement delivery plans.
“One of the problems was the overall level of basic education of people who were involved in the project,” Nourollah says.
Rice cultivation in Mali is on the rise.
His work with GCP has opened up new prospects for some of the poorest farmers in the world: “For five years, I have been coordinating one of the rice initiatives implemented by the Africa Rice Center and involving three African countries.” These are Burkina Faso, Mali and Nigeria.
He says GCP has brought much-needed expertise and technical skills to countries which can now use genetic insights to produce improved crops tolerant of drought conditions and poor soils and resistant to diseases. Using new molecular-breeding techniques has provided a more effective way to move forward, still firmly focussed on helping the world’s poorest farmers achieve food security.
“We don’t change direction, we change tools – sometimes you have a bicycle, sometimes you have a car,” Nourollah says.
Hei agrees there have been challenges: “It’s been a bumpy road to get to this point. But the whole concept of getting all the national partners doing genetic resource characterisation is a very good one.
Right now they are enabled; they are not scared about the technology. They can apply it.”
Sigrid says applied research is judged on two scales: “One is the publications and science you’re doing. The other is whether the work has any impact in the field, whether it works in the field. Bringing these two together is sometimes a challenge.”
GCP has managed to meet both challenges. New crop varieties have been released to farmers, and more than 450 scientifically reviewed papers have been published since 2004.
Building on the rice success story and leaving a lasting legacy
The work that GCP-supported researchers have done for rice is also being used in other crops. For example, researchers used comparative genomics to determine if genes the same as or similar to those found in rice are present and operating in the same manner in sorghum and maize.
The GCP team found sorghum and maize varieties that contained genes, similar to rice’s PSTOL1, that also confer tolerance of phosphorus-deficient soil with an enhanced root system. They were then able to develop markers to help breeders in Brazil and Africa identify phosphorus-efficient lines.
The knowledge that GCP-supported rice researchers have generated is shared through communities of practice, through websites, publications, research meetings and the Integrated Breeding Platform.
As Amelia Henry notes, GCP’s achievements will be defined by “the spirit of dedication to openness with research data, results and germplasm and giving credit and support to partners in developing countries.” The work in rice in many ways exemplifies GCP’s collaborative approach, commitment to capacity building and deeply held belief that together we can go so much further in helping farmers.
Unlocking genetic diversity in crops for the resource-poor was at the heart of GCP’s mission, which in 2003 promised ‘a new, unique public platform for accessing and developing new genetic resources using new molecular technologies and traditional means’.
Certainly for poor rice farmers in Asia and Africa, the work that GCP has supported in applying the latest molecular-breeding techniques will lead to rice varieties that will help them produce better crops on poor soils in a changing climate.
It’s a cruel feature of some of the most populous areas of the world, particularly in the tropics and subtropics: acid soils. They cover a third of the world’s total land area – including significant swathes of Africa, Asia and Latin America – and 60 percent of land we could use for growing food. Today around 30 percent of all arable land reaches levels of acidity that are toxic to crops.
Soil acidity occurs naturally in higher rainfall areas and varies according to the landscape and soil. But we also make the problem worse through intensive agricultural practices. The main cause of soil acidification is the overuse of nitrogen fertilisers, which farmers apply to crops to increase production. Ironically, the inefficient use of nitrogen fertiliser can instead make matters worse by decreasing the soil pH.
60 percent of the world’s potential crop-growing land is highly acidic. Map courtesy of Leon Kochian.
Acidity prevents crops from accessing the right balance of nutrients in the soil, limiting farmers’ yields. Its negative effect on world yield is second only to drought and is particularly hard felt by subsistent and smallholder farmers who cannot afford to correct soil pH using calcium-rich lime. As a result, these farmers are forced to grow less profitable, acid-tolerant crops like millet, or suffer huge yield losses when growing more popular cereal crops like wheat, rice or maize.
“In Kenya, acidic soils cover almost 90 percent of the maize-growing areas and can reduce yields by almost 60 percent,” says Samuel Gudu, Professor and Deputy Vice-Chancellor (Planning & Development) at Moi University in Kenya. “Farmers know that the soil affects their yields, but they still grow maize because it is so popular.”
As is true in many other sub-Saharan countries, maize is a staple of the Kenyan diet: the average Kenyan consumes 98 kilograms of it each year. But maize prices in Kenya are among the highest in Africa, which directly affects the poorest quarter of the population, who spend 28 percent of their income on the crop.
“Yield losses play a big part in this economic imbalance and are why we need affordable agronomic options to help our farmers improve yields,” says Samuel, who was a Principal Investigator of a GCP comparative genomics project which sought to provide some of these options.
A Kenyan farmer prepares her maize plot for planting. Acid soils cover almost 90 percent of Kenya’s maize-growing area, and can more than halve yields.
Aluminium toxicity and phosphorus deficiency: Public enemies number one and two in the fight against acidic soils
Between 2004 and 2014, crop researchers and plant breeders across five continents collaborated on several GCP projects to develop local varieties of maize, rice and sorghum that can withstand phosphorus deficiency and aluminium toxicity – two of the most widespread constraints leading to poor crop productivity in acidic soils.
Aluminium toxicity is the primary limitation on crop production for more than 30 percent of farmland in Southeast Asia and Latin America and approximately 20 percent in East Asia, sub-Saharan Africa and North America. Aluminium becomes more soluble in acid souls, creating a toxic glut of aluminium ions that damage roots and impair their growth and function. This results in reduced nutrient and water uptake, which in turn depresses yield.
Phosphorus deficiency is the next biggest soil deficiency after nitrogen to limit plant production. In acid soils, phosphorus is stuck (fixed) in forms that plants cannot take up. All plants need phosphorus to survive and thrive; it is a key element in plant metabolism, root growth, maturity and yield. Plants deficient in phosphorus are often stunted.
In a double whammy, the damage that aluminium toxicity causes to roots means that plants cannot efficiently access native soil phosphorus or even added phosphorus fertiliser – and adding phosphorus is an option that is rapidly becoming less viable.
“The world is running out of phosphorus as quickly as it is running out of oil,” says Leon Kochian, a Professor in the Departments of Plant Biology and Crop and Soil Science at Cornell University in the USA. “This is making its application a more expensive and less sustainable option for all farmers wanting to improve yields on acidic soils.” Indeed, the price of rock phosphate has more than doubled since 2007.
For 30 years, Leon has combined lecturing and supervising duties at Cornell University and the United States Department of Agriculture with his scientific quest to understand the genetic and physiological mechanisms that allow some cereals to tolerate acidic soils while others wither. And for the last 10 years, he has played an important leading role in GCP’s effort to develop new, higher yielding varieties of maize, rice and sorghum that tolerate acidic soils.
GCP builds on past crop breeding successes
The rationale behind GCP’s efforts stems from two independent and concurrent projects, which had been flourishing on different sides of the Pacific well before GCP was created.
One of those projects was co-led by Leon at Cornell University in collaboration with a previous PhD student of his, Jurandir Magalhães, at the Brazilian Corporation of Agricultural Research (EMBRAPA) Maize & Sorghum research centre.
Working on the understanding that the cells in grasses like barley and wheat use ‘membrane transporters’ to insulate themselves against excessive subsoil aluminium, Leon and Jurandir searched for a similar transporter in the cells of sorghum varieties that were known to tolerate aluminium.
“In wheat, when aluminium levels are high, these membrane transporters prompt organic acid release from the tip of the root,” explains Jurandir. “The organic acid binds with the aluminium ion, preventing it from entering the root.” Jurandir’s team found that in certain sorghum varieties, the gene SbMATE encodes a specialised organic acid transport protein, which stimulates the release of citric acid. They cloned the gene and found it was very active in aluminium-tolerant sorghum varieties. They also discovered that the activity of SbMATE increases the longer the plant is exposed to high levels of aluminium.
The rice variety on the left (IR-74) has the the gene locus Pup1, conferring phosphorus-efficient longer roots, while that on the right does not.
The other project, co-led by Matthias Wissuwa at Japan International Research Centre for Agricultural Sciences (JIRCAS) and Sigrid Heur at the International Rice Research Institute (IRRI) in The Philippines, was looking for genes that could improve rice yields in phosphorus-deficient soils. They had already identified a gene locus (a section of the genome containing a collection of genes) that produced a protein which allowed rice varieties with to grow successfully in low-phosphorous conditions. The locus was termed ‘phosphorus uptake 1’ or Pup1 for short. With GCP support, the team were able to make the breakthrough of discovering the protein kinase gene responsible, PSTOL1 (‘phosphorus starvation tolerance 1’), and understanding its mechanism.
“In phosphorus-poor soils, this protein instructs the plant to grow larger, longer roots, which are able to forage through more soil to absorb and store more nutrients,” explains Sigrid, a plant geneticist at IRRI and a GCP Principal Investigator. “By having a larger root surface area, plants can explore a greater area in the soil and find more phosphorus than usual. It’s like having a larger sponge to absorb more water.”
Screening for phosphorus-efficient rice, able to make the best of low levels of available phosphorus, on an International Rice Research Institute (IRRI) experimental plot in the Philippines. Some types of rice have visibly done much better than others.
Leon clarifies that both projects were fairly advanced before they became part of the GCP fold. “Our team had already identified the gene SbMATE and were in the process of cloning it for breeding purposes. The IRRI and JIRCAS team had also identified Pup1 and were in the process of identifying and cloning the gene.”
The purpose of cloning these genes was to create molecular markers to help breeders identify whether the genes were present in the varieties they were working with. As an analogy, think of ‘reading’ a plant’s genome as you would read a story: the story’s words are the plant’s genes, and a molecular marker works as a text highlighter. Different markers can highlight or tag different keywords in the story. Tagging the location of beneficial genes in the DNA of plant genomes allows scientists to see which of the plants or seeds they are interested in – perhaps only a few out of hundreds or thousands – contain these genes. This forms the basis of marker-assisted breeding, which can help plant breeders halve the time it takes them to breed new high-yielding varieties for acidic soil conditions.
Leon says that GCP provided both projects with the opportunity to validate their discoveries and to use what they had found to develop new aluminium-tolerant sorghum varieties and phosphorus-efficient rice varieties for farmers. But it’s what happened next that made this GCP initiative unique.
Finding the best genes within the crop family
Sorghum, rice, maize and wheat are all part of the Poaceae (true grasses) family, evolving from a common grass ancestor 65 million years ago. Over this time, they have become very different from each other. However, at the genetic level they still have a lot in common.
Over the last 20 years, genetic researchers all over the world have been mapping these cereals’ genomes. These maps are now being used by geneticists and plant breeders to identify similarities and differences between the genes of different cereal species. This process is termed ‘comparative genomics’ and was a fundamental research theme for GCP during its second phase (2008–2014).
“The objective during GCP Phase I (2004-2007) was to study the genomes of important crops and identify genes conferring resistance or tolerance to various stresses, such as drought,” says Rajeev Varshney, Director of the Center of Excellence in Genomics at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). “This research was long and intensive, but it set a firm foundation for the work in GCP’s second phase, which sought to use what we have learnt in the laboratory and apply it to breed better varieties of crops.”
Rajeev oversaw GCP’s comparative genomics research projects on aluminium tolerance and phosphorus deficiency in sorghum, maize and rice, as part of his GCP role as Leader of the Comparative and Applied Genomics Research Theme.
“The idea behind the sorghum, maize and rice initiative was to use the discoveries we had independently made in sorghum and rice to see if we could find the same genes in the other crop,” explains Rajeev. “In other words, we wanted to see if we could find PSTOL1 in sorghum and SbMATE in rice.”
Working together through a number of comparative genomics projects, the researchers were highly successful in reaching this goal, discovering valuable sister genes and beginning to introduce them into new improved crop varieties for farmers.
Extending research in sorghum and rice to maize
Researchers at Cornell and EMBRAPA had already been using similar comparative techniques to look for SbMATE in maize because of its close familial connection to sorghum. This research was overseen by Leon and another EMBRAPA researcher, Claudia Guimarães.
“We used the knowledge that Jurandir and Leon’s SbMATE project produced to prove that we had a major aluminium-tolerance gene,” reflects Claudia.
The SbMATE gene in sorghum explains about 80 percent of its aluminium tolerance, but Claudia says that in maize it explains only about 20 per cent, making it harder for researchers to find without a little help knowing what to look for. “So we had to dig a little deeper for other similar genes that confer aluminium tolerance, and we found ZmMATE.”
Maize trials in the field at EMBRAPA. The maize plants on the left are aluminium-tolerant while those on the right are not.
ZmMATE1 has a similar genetic sequence to SbMATE and encodes a similar protein membrane transporter that releases citric acid from the roots. Just as in sorghum, citric acid binds to aluminium in the soil, making it difficult for it to enter plant roots. The team have also discovered related gene ZmMATE2, which also encodes a transporter protein, but appears to confer aluminium tolerance via a different mechanism, as yet unclear.
Claudia has developed a number of molecular markers for ZmMATE, which have been successfully used by breeders at EMBRAPA as well as by African partners in Niger and Kenya, such as Samuel Gudu, to identify maize breeding lines that have the gene.
“We used aluminium-tolerant maize varieties sourced locally and from Brazil to develop a range of potential new varieties,” says Samuel. “The goal is to develop varieties that are suited to our environment and not too dissimilar to varieties that Kenyan farmers like to grow, except they have a higher tolerance to aluminium toxicity.”
Left to right (foreground): Leon Kochian, Jurandir Magalhães and Samuel Gudu examine crosses between Kenyan and Brazilian maize, at the Kenya Agricultural Research Institute (KARI), Kitale, in May 2010.
Involving farmers in the crop breeding process is an important part of such programs being successful, explains Samuel. “They help us identify maize varieties that they have observed have higher tolerance to acidic soils. We also try to incorporate other features that they want, such as disease resistance and higher yield. By incorporating their feedback into the breeding process they are more likely to grow any new varieties, as they have played a part in their development.”
Samuel says they have developed some local aluminium-tolerant varieties, which rank among the best for aluminium tolerance. Interestingly, these varieties seem to have a different aluminium-tolerance mechanism to the Brazilian varieties.
“From the work Samuel has done, we’ve possibly identified a novel source of aluminium tolerance in Kenyan maize varieties,” says Claudia. “We are now working together with Leon to identify the genes that are conferring this tolerance so we can develop markers to help Kenyan maize breeders also identify these varieties more efficiently.”
To help in the process, Samuel and his team are developing single-cross hybrids with a combination of both the novel Kenyan sources of aluminium tolerance and ZmMATE from Brazil, which will be even more tolerant to acidic soils.
Breeding for multiple stresses is a step-by-step process
Suradiyo, a farmer from Bojong Village near Yogyakarta, Indonesia, harvests rice.
In Asia, about 60 percent of rainfed rice is grown on soils that are affected by multiple stresses. These typically include phosphorus deficiency as well as aluminium toxicity, salinity and drought.
These stresses are particularly hard felt in Indonesia, which is the world’s third-largest rice producer. Joko Prasetiyono is a molecular rice breeder at the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD). His team have been collaborating with IRRI and JIRCAS for many years and contributed to validating the effect of Pup1 by embedding it into three popular local rice varieties – Dodokan, Situ Bagendit and Batur – which were then able to tolerate phosphorus-deficient conditions.
“The aim [with GCP research] was to breed varieties identical to those that farmers already know and trust, except that they have PSTOL1 and an improved ability to take up soil phosphorus,” says Joko.
Joko says that these varieties – which will be available in one to two years – will yield as well as, if not better than, traditional varieties, and will need 30–50 percent less fertiliser.
But the work is only partly finished for Joko and his Asian partners. They are now building on previous work done at Cornell and EMBRAPA to include the SbMATE gene in their varieties. “Higher yields will only be possible if the plant can also tolerate excess aluminium, which severely inhibits root growth and thereby water and nutrient uptake,” explains Joko. “We are also looking at incorporating salt-tolerance and drought-tolerance genes. It’s a step-by-step process where we hope to build tolerance to the multiple stresses that afflict most rice-growing areas throughout Asia and the world.”
Introducing PSTOL1 into maize and sorghum
At EMBRAPA, Claudia is also interested in building up tolerance to multiple stresses and was involved in the project to look for genes similar to PSTOL1 in maize. “As soon as IRRI and JIRCAS had cloned the gene and created markers, we started using the markers to search for the gene in maize, as Jurandir did in sorghum,” she says.
Women farmers in India bring home their sorghum harvest.
Finding genes that confer phosphorus-efficiency traits in maize and sorghum has been a more challenging project, according to Leon. “From the rice work, we knew a big part of phosphorus efficiency was to do with root architecture – you want to have shallow horizontal roots instead of roots that grow down, which is often the case in maize and sorghum,” he explains. “This is because there is less accessible phosphorus further down the soil profile.”
Observing root architecture is difficult in ordinary soil, so the team had to develop new ways to visualise the plants’ roots. They grew plants in a transparent nutrient gel, which they then photographed to create three-dimensional images of the root structure.
The team found sorghum and maize varieties that contained genes similar to PSTOL1 in rice, but which also have longer root systems that radiated outwards rather than downwards in gels with higher concentration of aluminium. “These observations helped validate multiple PSTOL1 regions in sorghum and maize, which we’ve been able to develop markers for to help breeders identify these traits more easily,” says Leon.
These markers have successfully been used by sorghum breeders in Brazil and Africa to identify phosphorus-efficient varieties. Maize breeders in both Brazil and Africa are expected to use similar markers to validate their varieties in 2015.
New sorghum varieties prove their worth in the field
Eva Weltzien is one Africa-based sorghum breeder who has benefited from these PSTOL1 and SbMATE markers. Based in Mali at ICRISAT, Eva and her team have been using the markers to select for aluminium-tolerant and phosphorus-efficient varieties and validating their performance in field trials across 29 environments in three countries in West Africa.
She says the markers have helped evolve the way they do their breeding. “Using molecular markers, we are able to identify whether the lines we are breeding have genes that confer the traits that we want,” explains Eva. “It has really revolutionised our breeding program and helped it make great progress in the past three to four years.”
In Mali, sorghum is an important staple crop. It is used to make tô (a thick porridge), couscous, and local beers. Part of its popularity is its adaptability to various climates – in Mali it is grown in very dry environments as well as in forest/rainforest zones. However, it is widely affected by acidic soils.
Sorghum farmers at work in the field in Mali.
“Low phosphorus availability is a key problem for farmers on the coast of West Africa, and breeding phosphorus-efficient crops to cope with these conditions has been a main objective of ICRISAT in West Africa for some time,” says Eva.
“We’ve had good results in terms of field trials. We have at least 20 lines we are field testing at the moment, which we selected from 1,100 lines that we tested under high and low phosphorous conditions.” Eva says that some of these lines could be released as new varieties as early as next year.
“Overall, we feel the GCP partnership with EMBRAPA and Cornell is enhancing our capacity here in Mali, and that we are closer to delivering more robust sorghum varieties that will help farmers and feed the ever-growing population in West Africa.”
Leon notes that the work by Eva in Mali and by other African partners in Niger and Kenya is imperative for the research. “Just because plants have these genes, doesn’t mean they will all display aluminium tolerance or phosphorus efficiency. You still need to test and observe for these traits in the field and determine what other factors might affect plants grown in acidic soils.”
One surprising observation that has Leon intrigued is a local sorghum variety with a phosphorus-efficiency gene that is close to where the SbMATE gene resides in the sorghum genome. “This suggests that SbMATE, which aids with aluminium tolerance, may also improve phosphorus efficiency. This means we could use SbMATE markers to look for both phosphorus efficiency and aluminium tolerance,” he says. Leon and Jurandir will continue to validate this result post-GCP.
Working together to improve food security worldwide
GCP’s comparative genomics projects have laid a significant foundation for further research into and breeding for tolerance to multiple plant stresses.
A Kenyan farmer in her maize field.
“We’re in a golden age of biology where we are learning more and more about the complexities and commonalities of plants, which is allowing us to manipulate them ever so slightly to help them tolerate multiple environmental stresses,” says Leon. “As a geneticist, I am extremely proud to be part of this, particularly seeing the potential impact that the basic research we do in the laboratory can have on crop improvement and the lives of people in poorer countries.”
Although not all projects produced new and improved varieties ready for release, they are well and truly in the pipeline. Each partner institute is committed to work together and source new funding to continue on their quest to produce further products.
“GCP has really installed in us a spirit to see this work through and expand on it,” says Leon. “I mean, we are now working with other countries and institutes to share what we have learnt with them and help them make the discoveries that we have. It’s a credit to GCP for bringing us all together; that was a key to the success of the project. Each partner has brought their expertise to the table – genomics, molecular biology, plant breeding – and it has been great to see the impact filter into Africa and Asia.”
In Kenya, Samuel agrees with Leon’s assessment. “GCP gave us an opportunity to build our expertise and start interacting with the rest of the world,” he says. “But more importantly, it means that we’re contributing to food security in Kenya, and that makes us really proud.”
Although the sun is setting on GCP, work on comparative genomics projects is still in progress, with all parties still working towards delivering important new acid-beating varieties to farmers.
A boy rides his bicycle next to a rice field in the Philippines. With acid soils affecting half the world’s current arable land, acid-beating crop varieties will help farmers feed their families – and the world – into the future.