2007-2008 Competitive Grants (Round 2)

Project Summaries:

1. Development of Genomics Resources for Molecular Breeding of Drought Tolerance in Cassava

2. Genetic Dissection of Drought Adaptive Mechanisms in Bread and Durum Wheat through Large Scale Phenotyping Methodologies

3. Tailoring Superior Alleles for Abiotic Stress Genes for Deployment into Breeding Programs: A Case Study Based on Association Analysis of AltSB, a Major Aluminum Tolerance Gene in Sorghum

4. Interspecific bridges that give full access to the African rice allele pool for enhancing drought tolerance of Asian rice

5. Genomic dissection of tolerance to drought stress in wild barley

6. Detecting and fine-mapping QTLs with major effects on rice yield under drought stress for deployment via marker-aided breeding


1. Project: Development of Genomics Resources for Molecular Breeding of Drought Tolerance in Cassava
Institution: The Institute for Genomics Research [TIGR]
Lead PI: Pablo Rabinowicz

Executive Summary
Cassava is one of the most important crops in unfavorable environments in developing countries, where poverty is common and severe. Because of its high productivity, even in extreme conditions, cassava constitutes a source of food and income for poor farmers in Africa, Asia and Latin America. Although cassava is fairly resistant to water stress, the molecular basis for this tolerance is poorly understood. Several traits have been associated with its drought tolerance, such as regulation of stomata activity, changing leaf expansion rates due to decrease in cell proliferation, and modifications of photosynthetic pathways to maintain high photosynthetic activity. Improving cassava’s tolerance to drought is important to help increasing yields in the semi-arid Sub Saharan African regions where cassava as an essential crop. Cassava’s natural stress tolerance can be substantially improved by breeding, especially by marker-assisted selection of key physiological traits associated with drought tolerance. In recognition of the importance of cassava improvement for dry areas in the developing world, the Generation Challenge Program (GCP) awarded a grant to study drought tolerance traits and develop molecular markers to improve cassava breeding for drought tolerance. This proposal builds on that project by offering to develop single nucleotide polymorphism (SNP) markers throughout the genome to identify favorable alleles related to drought tolerance in these mapping populations. In order to achieve this goal, a physical map of the cassava genome will be generated that will allow the development of SNP markers uniformly distributed around the genome. In this way we will be able to identify quantitative trait loci (QTL) associated with drought tolerance in a high-throughput manner. These markers will be useful for marker-assisted selection of favorable traits.

Scientific Summary
As cassava is often cultivated in dry environments, research towards improvement of drought tolerance in cassava is needed. As part of a Generation Challenge Program (GCP) project awarded to EMBRAPA/CNPMF, CIAT, IITA, and Cornell University to study traits related to drought tolerance in cassava and to develop molecular markers to improve the efficiency of breeding cassava for drought tolerance, eight mapping populations were generated from contrasting drought tolerance genotypes, which represent an important resource for the cassava community. A consensus map of markers and genes across these populations can be created to obtain universal genetic “hot spots” for genomic regions affecting drought tolerance in diverse settings. Here we propose to build on the above project by developing a panel of single nucleotide polymorphism (SNP) markers on a genome-wide basis to localize favorable alleles in these mapping populations. SNPs are also practical markers for molecular breeding in a heterozygous crop like cassava. We will identify SNPs in an existing cassava bacterial artificial chromosome (BAC) library. To do this, we propose to construct a physical map of the cassava genome by fingerprinting 70,000 BAC clones and sequencing the ends of 9,000 clones distributed throughout the genome. The availability of a physical map will constitute an important tool for map-based cloning of agronomically relevant genes in cassava. Selected low-copy sequences spread throughout the genome will be re- sequenced in a panel of 10 cassava genotypes to identify SNPs that can be used for genetic mapping. Quantitative trait loci (QTL) associated with drought tolerance will be identified by high-throughput genotyping of validated SNPs in two of the mapping populations. Additional SNP markers will be developed around the “hot spots” identified after QTL mapping to allow marker-assisted selection of desirable QTL alleles for molecular breeding for drought resistance in cassava.

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2. Project: Genetic Dissection of Drought Adaptive Mechanisms in Bread and Durum Wheat through Large Scale Phenotyping Methodologies
Institution: CIMMYT
Lead PI: Matthew Reynolds

Executive Summary
Declining water resources and unpredictable rainfall are serious threats to crop productivity throughout the world. Although wheat is relatively well adapted to moisture stress, and breeding progress using conventional approaches has resulted in significant improvements in productivity in rain-fed areas, there is considerable scope to improve the scale and pace of progress through exploiting the genetic diversity that exists in wheat genomes. Through a combination of precision phenotyping on well designed populations grown at key field locations in conjunction with deployment of the latest molecular marker technologies, it is anticipated that genetic markers associated with drought adaptive traits will be identified or confirmed. Such markers will then permit targeted molecular screening of genetic resources within wheat and related genomes thus identifying new parental sources and markers for progeny selection. The collaborative model proposed combines partners with expertise in genetics, breeding and physiology thus facilitating the design of agronomic and genetically relevant mapping populations, a realistic and rigorous approach to phenotyping, and application of the most appropriate biotechnologies. The proposed research material (bread wheat and durum wheat mapping populations) offers a unique ability to dissect the genomic effects of drought tolerance (particularly for the D genome). The collaborators work in three major wheat producing countries (India, Mexico and Australia) where the crop is either rain-fed or grown with restricted irrigation. The project will provide selection tools and methodologies including genetic and physiological markers that can be applied in breeding programs worldwide and well characterized experimental populations that can be used to develop similar tools in other stress prone environments. This proposal also addresses the considerable methodological challenges associated with determining the genetic basis of drought adaptation in that it will validate high throughput screening protocols in controlled environments and develop more optimal parents for a subsequent generation of molecular mapping populations.

Scientific Summary
Conventional linkage mapping of QTL influencing complex traits such as drought tolerance has been confounded by several biological, logistical and statistical factors. In this project we will address these problems in wheat by combining the forces of scale and relevance (multiple large mapping populations from well adapted parental genotypes and selective analysis of mega-populations), marker density (intensive DArT and multiplex SSR genotyping), precision phenotyping (improved approaches to phenotyping in well characterized environments), comparative genomics (applying genome-specific markers on mapping populations with different genome compositions) and new biometrical approaches (that help to deal with genotype by environment interaction and epistasis). There will be a number of significant outputs from this process beyond the precision mapping of drought tolerance including: (i) optimized high throughput precision phenotyping protocols with good correlation to drought tolerance field screening and international trial performance, (ii) better understanding of the role of the D genome in drought tolerance, (iii) optimized biometrical approaches for mapping drought tolerance QTL. An additional output will be the identification of parental genotypes for future generations of mapping populations that will show negligible segregation for confounding factors associated with phenology and height; this will be achieved by genotyping potential parental genotypes contrasting in drought adaptation with available genetic markers for Ppd, Vrn, and Rht as well as phenotyping them in diverse environments to evaluate genotype by environment interaction for phenology. It is also expected that material will be identified for subsequent use in drought tolerance breeding programs across the world in combination with the markers identified in this project.

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3. Project: Tailoring Superior Alleles for Abiotic Stress Genes for Deployment into Breeding Programs: A Case Study Based on Association Analysis of AltSB, a Major Aluminum Tolerance Gene in Sorghum
Institution: Embrapa Maize and Sorghum
Lead PI: Jurandir Vieira Magalhaes

Executive Summary
One of the most important factors limiting agriculture in developing countries involves the large areas of acid soils found in these countries. On acid soils, toxic levels of aluminum (Al) ions are released into soil solution, where they damage roots and impair their growth and function. This results in reduced nutrient and water uptake, with concomitant reductions in crop yield. There is considerable natural variation in Al tolerance both within and between plant species, and we have assembled an interdisciplinary team of scientists to take advantage of this variation to improve crop tolerance to Al toxicity, building upon our recent success in isolating a novel Al tolerance gene in sorghum. Thus, as we have been able to identify at least one apparently improved version of this gene, we will now apply association mapping to undertake a comprehensive scan for even better versions of this gene for deployment into sorghum breeding programs. The research group we have assembled has considerable expertise in the genetics, molecular biology and physiology of aluminum tolerance, and has the necessary genetic resources to ensure the success of this project. Through the use of cutting edge genomics and statistical genetics approaches, this research will bridge the gap between basic research on Al tolerance and applied breeding programs, to develop the tools that plant breeders can use to efficiently and effectively breed for improved acid soil tolerance. The long-term goals of this research are to generate sorghum genotypes expressing improved Al tolerance that ultimately can be distributed to farmers who till acid soils in Africa and other developing regions, thus exploiting a wide range of still hidden genetic variation for Al tolerance. Increasing the Al tolerance of staple crops, such as sorghum, will help increase yields and thus food security worldwide.

Scientific Summary
Our research consortium has recently cloned, via high resolution mapping, a major aluminum tolerance gene in sorghum. This gene, AltSB, encodes the root citrate efflux transporter that underlies an important physiological mechanism of sorghum aluminum tolerance. We have conducted a preliminary diversity analysis of the AltSB locus, and have identified a number of superior alleles in different sorghum lines. From this material, we are in the process of generating near-isogenic lines in a common, elite background so that we can begin to assess the breeding utility of these new AltSB alleles. We are now proposing to unify three sorghum germplasm sets assembled by different institutes into a large and diverse panel that will enable us to use association mapping to identify and tag “ideal” AltSB haplotypes. In this proposal, we will build upon this progress and use traditional near-isogenic lines, biparental mapping, and association analysis together with physiological and molecular investigations to:

1) Survey AltSB alleles across diverse sorghum germplasm; 2) Identify superior AltSB haplotypes by association mapping; 3) Develop and use haplotype-specific markers for introgression into pre-breeding near-isogenic lines; and 4) Survey sorghum homologs of novel candidate Al tolerance genes we have identified from complementary work in maize, wheat and Arabidopsis.

Expected Outputs. 1) A diverse sorghum collection that has been phenotyped for aluminum tolerance and genotyped at AltSB. 2) (A) Identification of superior AltSB haplotypes. (B). Identification of causal polymorphisms for aluminum tolerance (if the rate of linkage disequilibrium decay is rapid around the gene). 3) (A) A set of AltSB haplotype-specific markers. (B) A set of pre-breeding BC3F3 near-isogenic lines (NILs) carrying different AltSB haplotypes. (C) Development of a low-cost, easy to use SNP genotyping assays. (4) Identification of novel Al tolerance genes in sorghum.

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5. Project: Genomic dissection of tolerance to drought stress in wild barley
Institution: SCRI
Lead PI: Robbie Waugh

Executive Summary
Through an existing collaboration we have developed a unique segregating population of 140 barley lines composed of an advanced elite genetic background containing introduced chromosomal segments from a wild barley accession that comes from the Fertile Crescent. The wild species, the donor of the introduced genomic segments, is genetically distant from the cultivated line and is both adapted to, and tolerant of, drought and salt stresses. Using genetic tools that allow us to follow the inheritance of the genomic segments from the donor into the recipient line we have been able to show that in this unique population we have representative segments covering the entire genome of the donor in each of the different lines. In genetical terms we call these lines recombinant chromosome substitution lines or RCSL’s. Evolution by natural selection, domestication and plant breeding has resulted in each of the paired genomic segments from the wild species and elite line having subtly to strikingly different versions of the same genes. This variation will affect the growth and/or performance characteristics of each of the RCSLs compared to each other and to their parents. For example, if the introduced segment contained a version of a gene that conferred resistance to salinity that was absent in the elite line, then we expect all of the individual RCSLs that contain that segment also to become resistant to salinity. The unique feature of RCSLs that is different from standard bi-parental cross populations is that by breaking the donor genome up into many small segments and having these segments in an otherwise identical genetic background, it becomes possible to precisely dissect even complex characteristics into a series of genetically tractable parts. We know that we have been successful in doing this as we have already examined the effects of the introgressed wild species genome segments on a range of phenotypes (Matus et al, 2003). In the interim, we have also developed a technology (we call it an oligo pool assay or OPA) that allows us to very precisely characterise the genomes of each of the RCSLs and identify the genes that are present on the introduced donor segments. In this project we propose to combine the power of our OPA genome characterisation technology with relevant phenotypic trait information on the unique RCSL genetic resource to identify segments of the donor genome that confer increased (or decreased) drought tolerance to the recipient. Although these characteristics are considered to be controlled by many genes, by isolating a small number (sometimes individual) donor genome segments in an identical genetic background, RCSLs effectively fragment the genetic contributions of many loci into individual component loci that can be subsequently analysed in detail by simple genetic analysis. Once we have identified specific target regions of the wild species genome that confer increased drought tolerance, for the most clear cut examples, we will use the model rice genome sequence to provide a putative barley regional gene content and a list of candidate stress tolerance genes. We have successfully used this approach in the past for winter hardiness. We will then pursue the objective of characterising the DNA sequence of a selection of the genes in this region from both parents to develop the tools that will allow us to accurately associate the drought tolerant character with specific genes. We will extend these studies to a broad selection of agro-ecologically adapted landraces where we will use both the genes identified in the RCSL studies and, in a pilot study, the genes on the OPA, to validate observed, and identify new associations between genes and drought tolerant phenotypes. Finally, we will initiate crosses to mobilise favourable alleles from the landrace germplasm into a common elite genetic background for further testing and validation of their impact on stress tolerance.

Scientific Summary
Through an existing collaboration we have developed a full genome set of barley recombinant chromosome substitution lines (RCSL’s) using the advanced backcross strategy of Tanksley and Nelson (1996). We used the Canadian malting barley Harrington as recurrent parent and a genetically distant salt and drought tolerant Hordeum vulgare ssp. spontaneum accession (from the Fertile Crescent) as donor (Matus and Hayes, 2002). We established the genomic architecture of the RCSLs using Simple Sequence Repeats (SSRs), and the effects of the introgressed H. spontaneum genome segments on a range of phenotypes (Matus et al, 2003). In the interim, we also developed a Pilot Illumina Oligo Pool Assay (OPA) platform for high resolution genotyping in barley. We used genes that are transcriptionally responsive to abiotic stress (1524 Single Nucleotide Polymorphisms (SNPs) in each assay). Here, we want to combine the power of OPA genotyping with relevant phenotypic information on this unique (for barley) genetic resource, to identify segments of the donor genome that confer drought tolerance and morphological variation. Although these phenotypes are considered quantitative, by isolating a restricted number of individual donor genome segments in an isogenic background, RCSL’s move towards effectively ‘Mendelising’ the individual genetic contributions to the overall phenotype. Single recombinant chromosome substitution lines – either present in the current RCSL collection or derived from a further cross in which we would deploy a combination of both ‘offensive’ and ‘defensive’ MAS - represent a genetically simplified platform from which the genes underpinning individual QTL can be isolated and characterized. Once we have identified specific target regions of this sample of the H. spontaneum genome that confer increased drought tolerance, for the most clearcut examples, we will identify the orthologous regions in the model rice genome sequence in order to provide a putative barley regional gene content and the identity of candidate stress tolerance genes. We have already successfully used this approach for genes conferring winter hardiness (Skinner et al., 2005, 2006). This process is however simplified enormously by the OPA platform that assays variation in gene sequences and makes alignment with the rice genome relatively straightforward. We will then pursue the parallel objectives of regionally resequencing alleles to develop markers for further haplotype analysis and phenotypic association studies in a set of 340 lines from the Syrian and Jordanian Landrace Collection (SJLC). These lines, sampled from a gradient of agroecological conditions, showed a wide range of responses to drought stress as established by extensive field trials. In addition, analysis of genetic diversity and population structure with SSR markers has already indicated that changes in allele frequency may be driven by a drought gradient. Using new genotypic data from the Illumina Oligo Pool Assay (OPA) platform and the available phenotypic data for the SJLC, we will investigate the use of whole genome association scans to identify and validate genes and markers linked to performance under drought stress. Finally we will initiate the process of validating diverse alleles (more probably linked ‘regional haplotypes’) by introgressing them into a common genetic background.

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6. Project: Detecting and fine-mapping QTLs with major effects on rice yield under drought stress for deployment via marker-aided breeding
Institution: IRRI
Lead PI: Arvind Kumar

Executive Summary
Rice production losses due to drought are a risk on more than 20 million ha, and primarily affect the poorest communities. Drought risk depresses productivity even in favorable years because risk of crop failure drives farmers to limit investment in fertilizer.

Varieties with improved tolerance could reduce risk and help alleviate poverty, but progress in their development has been slow because few rice breeding programs screen directly for grain yield under drought stress, assuming that the trait is too complex for conventional breeding approaches. However, research by IRRI and collaborators has shown that, when stress is carefully imposed in the field, large differences in the yield of tolerant and susceptible varieties can be reliably detected. Recent experiments also show that much of the difference between tolerant and susceptible cultivars appears to result from the effects of a small number of genes. Several such genes have been identified at IRRI, but they must be precisely “tagged” by DNA markers to be used in developing improved varieties. The proposed project will tag (or fine-map) four genes that have been shown to reliably affect yield under both artificially imposed and natural drought. The physiological basis for their effects on tolerance will be studied, and their effects in farmers’ environments in India and southern China will be confirmed. Many such genes probably exist in rice genebanks, but have not been identified because conventional mapping requires that large populations derived from crosses between tolerant and susceptible parents be subjected to expensive DNA analysis. However, only genes with large effects on stress tolerance are likely to be useful in breeding; these can be detected by “quick and dirty” methods that involve DNA testing of only the most tolerant and susceptible progeny of a cross. This approach, known as selective genotyping, will be optimized for rice drought gene detection. Lines developed by introducing genes that improve drought tolerance into elite varieties will be disseminated in collaboration with NARES partners.

Scientific Summary
As many as 20 million ha of rainfed rice production area are regularly affected by drought. Progress in breeding drought-tolerant rice cultivars has been slow because of the difficulty of reliably imposing stress. Much previous work focused on genetic dissection of secondary traits putatively associated with drought tolerance, on the assumption that they are more heritable and more easily mapped than stress yield per se. However, recent research has shown that this assumption is untrue, and that yield under drought stress is more heritable than most component physiological traits. Quantitative trait loci (QTLs) with large effects on yield under stress have been detected in new populations derived from parents differing greatly in yield under drought, and analyzed using selective genotyping. In Vandana/Way Rarem, a QTL on chromosome 12 explained 40% of the genetic variance for yield under stress through effects on both biomass and harvest index. This QTL also affects yield under upland stress in populations derived from drought-tolerant upland cultivar Apo and susceptible lowland varieties IR64 and IR72. Another QTL on chromosome 5 affects yield under reproductive-stage stress in both IR55419-04/Way Rarem, where it explained over 30% of genetic variance, and Vandana/Way Rarem, where it explained 15%. Other QTLs consistently affecting stress yield in Apo/IR64 were detected on chromosomes 7 and 8 and confirmed in more than one environment. The proposed project will fine-map these genes, clarify their physiological effects, and confirm them under natural stress in India and China.

Fine-mapped QTLs will be introgressed into elite varieties via marker-aided selection and disseminated. More alleles with large effects on drought tolerance likely exist in rice germplasm, but their detection has been constrained by the cost of genotyping. A high-throughput, low-cost approach based on bulk segregant analysis and suited to detecting drought-tolerance QTLs with large additive effects will therefore also be optimised. Lines developed by introgressing QTLs for drought tolerance into elite varieties will be disseminated with NARES collaborators.

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