Product Line 1.3. Genes and allelic diversity conferring stress tolerance and enhanced nutrition


        Tolerance of environmental stresses and enhanced nutritional value are priority traits for rice in multiple environments. In some instances, QTLs with large effects can offer solutions to major constraints. Examples of such high-value genes include some disease-resistance genes or the SUB1 gene that offers a solution to flash-flooding. With genome sequencing, mapping of QTLs for specific traits can be achieved in a relatively short time if suitable mapping populations and robust phenotyping systems are available. The question is, Which of these QTLs or major genes are worthy of investment for molecular cloning and functional validation?
        We have identified a set of genes that (1) show evidence of having large effects on the phenotypes, (2) are needed to solve a major constraint, and (3) can be deployed over a large area with potentially high impact. In some cases, markers flanking the QTL can be used directly in marker-assisted selection. However, for the potentially high-impact traits, knowing exactly which genes are responsible for the traits has multiple benefits from a breeding standpoint. First, it allows the development of functional markers that have perfect prediction of phenotypes and performance. Second, knowledge of the specific function of a gene reveals the mechanisms conferring the phenotype and possible interactions with other genes and biochemical pathways. Third, perhaps the most important benefit, is the ability to identify and deploy allelic diversity of the gene that may function in different genetic backgrounds or environments.  
        This product line is designed to focus on high-value genes with a clear pathway for impact. It has three objectives: (1) isolate genes conferring phenotypes that have a large impact for breeding programs, (2) identify allelic diversity of the high-value genes, and (3) establish an efficient pipeline to validate gene function with the expectation that increasingly more large-effect genes will become available in the near future.  


        Our current product portfolio includes genes for abiotic stresses (drought, flooding, nutrient deficiencies, extreme temperatures, and traits of grain quality that are affected by temperature extremes) and for biotic stresses (fungal, bacterial, and viral diseases, and insects). Besides stress tolerance, research efforts will be devoted to understanding root and panicle development for yield potential and nutrient uptake.    
        While the evaluation of phenotypes and genetic mapping are specific to traits, many approaches of gene isolation and functional validation are in common. By housing the gene isolation and functional validation activities under one product line, we can share resources and create synergy to support different activities. To fast-track cloning of current and future QTLs, we will establish a pipeline with technical and infrastructure support for isolating QTLs and validating functions of candidate genes. This pipeline will have the following features:  
  • Technical staff support for QTL sequencing, gene cloning, and transformation  
  • Optimized transformation systems (vector and promoter construction and design, targeted transgene integration, tissue-specific transgene expression) 
  • High-throughput production of transgenic products 
  • Genomic and molecular data sets for predicting candidate gene functions 


1.3.1 Genes for drought-tolerant and aerobic rice 
1.3.2 Genes for flood-prone environments
1.3.3 Genes for nutrient-deficient and problem soils 
1.3.4 Genes for temperature extremes and grain quality 
1.3.5 Genes for disease and insect resistance 
1.3.6 Genes for improving the architecture of rice roots and panicles
1.3.7 Transgenic prebreeding events for stress-response genes
1.3.8 Gene identification and validation pipeline


        The diversity of the traits that will be addressed in this product line will require a large number of partners with specialized expertise. For each product, a unique composition of partners will be formed depending on the traits and target genes. All collaborative projects involve multiple CG centers, NARES, and ARIs that together have the needed expertise at different levels and enable the best use of available infrastructure. Whereas ARIs contribute the highest level of expertise in specialized research areas supported by up-to-date technologies, CG centers and NARES provide strengths in genetic analysis of germplasm, QTL identification, and field evaluation capacity to ensure that the selected genes are of high value and relevant to farmers. Below, we provide several illustrative examples of this collaboration model.
        Research on drought-tolerance genes will involve IRRI, AfricaRice, Cirad, CIAT, and NIAS-Japan, in which field-proven, drought-tolerant breeding materials will be used for gene discovery to ensure agronomic relevance of the target genes. For analysis of genes for flood-prone environments, our key partners are UC-Riverside and UC-Davis, which provide the expertise in molecular biology and physiology. Molecular work on genes with tolerance of flood-prone areas will be channeled to breeding programs in theme 2. For example, submergence-tolerant (Sub1) varieties are being tested in farmers’ fields in collaboration with AfricaRice and NARES. The upstream work on the phosphorus uptake gene (Pup1) is mainly conducted in a collaborative project between IRRI and JIRCAS, with contributions from MPI-Golm. In parallel, Pup1 breeding lines are being developed at ICABIOGRAD in Indonesia. A similar project is being pursued by Embrapa to isolate the Pup1 gene in sorghum. Further out-scaling of Pup1 breeding now involves AfricaRice and various NARES in Asia. 
        The identification of novel genes and processes leading to improved roots and panicles (1.3.6) will be approached using different strategies and starting materials depending on the partner institute. For instance, Cirad and IRD in collaboration with CIAT and other partners will mine and characterize transcription factors and miRNAs through forward and reverse genetic approaches using expression arrays and insertion mutants. Other partners will contribute in proteomics (UWA and NIPGR), metabolomics (IPK and MFU), and glycomics (NU) analyses to identify genes for improved root traits under drought and genes for spikelet development to increase yield potential. At IRRI, genetic diversity in root development will be explored to identify novel QTLs and processes that enhance stress tolerance. Detailed biochemical analyses will be conducted at MPI-Golm. For work on disease and insect resistance, we will engage partners with traditional strengths in research on host-plant resistance. These include Kyushu University, Ohio State University, and Colorado State University for their expertise in molecular cloning of host resistance genes. 
For producing transgenic prebreeding events for stress-response genes, IRRI in collaboration with CIAT, JIRCAS, and RIKEN (Japan) has formed a collaborative network for high-throughput transgene production to evaluate candidate genes for tolerance of abiotic stresses. JIRCAS and RIKEN provide gene constructs, whereas IRRI incorporates the genes into lowland indica lines and CIAT incorporates the genes into upland rice. Lead transgenic events will be evaluated under stress conditions and promising materials will be transferred to NARES and FLAR for further evaluation. 
        To establish an efficient gene validation platform, we will engage laboratories with strong expertise in vector construction and chromosome engineering. IRRI has developed a high-throughput transformation system to overexpress and down-regulate gene expression in indica rice. Partners such as the University of Minnesota, Meijo University, University of Arkansas, University of Wisconsin, Cirad, and IRD will provide state-of-the-art technology for multigene transformation and gene targeting using zinc finger nuclease, homologous recombination, and cre/lox recombinase. In parallel, the University of Düsseldorf and other partners will work toward identifying tissue-specific, inducible, and developmental stage-specific promoters. This technology development will be crucial for studying and incorporating high-value genes for yield, quality, and biotic stresses, and engineering metabolic pathways in rice. 

 Uptake and impact pathway

        The immediate users of the products will be national system and private-sector breeders with responsibility for incorporating target traits in their breeding programs. The eventual users will be farmers using improved varieties. For effective uptake, there must be close interaction among molecular biologists, physiologists, breeders, and agronomists to ensure that the criteria of high-value genes are met and that there is good integration of molecular data and tools into breeding projects to ensure that the cloned genes are agronomically relevant.  
        Product Line 1.3 is the logical extension of Product Line 1.2; large-scale genetic characterization of germplasm will lead to the prediction of QTLs and gene-phenotype relationships. Investments made in sequencing the genebanks (Product 1.2.3) will directly benefit the search for allelic diversity of the target genes. This product line will receive analytical support from the bioinformatics team established under Product Line 1.2.
        Linkage with theme 2 will be in two ways. Functionally validated genes can be used for transgenic products and gene-based markers for marker-assisted selection in breeding programs. From theme 2, breeding lines with proven performance can be used as starting materials for QTL cloning. This approach has been exemplified by the work on large-effect QTLs for drought tolerance originally identified from drought breeding programs.    
        We expect strong interest in co-investment in activities under Product Line 1.3, particularly with research institutions interested in the biology of the target traits. The involvement of academic institutions to jointly investigate practical problems has yielded many successful results. A number of research projects supported by the USAID Linkage Programs exemplify this type of linkage that leads to impact. 

 Financing strategy

        Currently, a majority of the products are supported by specific grants targeting a breeding objective. Additional investment in a gene-validation pipeline that serves multiple gene-cloning activities will improve efficiency by sharing resources for common activities. Such investment should consider both infrastructure and continuous training of technical staff.  
Because of the high-impact nature of the targeted genes, the prospect of attracting new funding for Product Line 1.3 is good. Early success in any of the products in demonstrating impact will attract further funding. This is also an area of considerable interest to the private sector, which may provide funding. However, this will require careful IP management to ensure public access to the products.