Generation Challenge Program

DRAFT Concept Notes for 2006 Commissioned Projects in Subprograms 1, 2, and 3

23 September 2005

1. Strategies for allele mining and how to cope with large collections (SP1)
Germplasm management currently recognizes the value of moving from the characterization of whole collections to representative samples which contain the largest diversity possible. These samples are commonly referred to as "core" samples. They are meant as an entry to the whole collection, and certainly not a substitute to them. The GCP SP1 is implementing such a strategy with several steps: 1. Passing from a composite set (all germplasm sets accessible for a given crop) to a core set made up of one to several thousand accessions to represent the whole diversity from existing information, generally passport + gross phenotypic information. 2. Passing from the core to a reference set made up of several hundred accessions which will be the priority for all researchers to genotype further and to phenotype for as many traits as possible. The reference set is a concept that comprises some flexibility. It must help the scientists plan their characterization work in relation to a particular objective (screening? association? modeling?...) and the specific requirements in terms of standard check materials, number of accessions, etc. It comes with specific software to help the selection. This downsizing will make it possible to undertake precise phenotyping in relation to drought. However, it must be accompanied with a strategy for re-investigation of the whole collection. This change in scope and scale is of an reiterative nature that will continuously increase the diversity concentrated in the core and reference samples. Therefore there may be cases for undertaking new rounds of genotyping with markers, using the correlations just uncovered in highly studied samples for refining two types of new targets: (1) new sources of gene diversity, and (2) new genotypic combinations. If undertaken, such surveys will require selecting markers especially easy and cheap to use, and which possess the appropriate resolution power. We believe that the whole process requires definition of rationales and methods and we invite ideas for doing so. With SP1 projects, the GCP has undertaken the identification of core and reference samples for two batches of crops, internally designated as Tier 1 and Tier 2 crops, the latter being generally characterized with fewer markers and a lower proportion of the composite sets. There may already exist matrices of high quality data that enable curators and breeders to finely analyse the structure of the diversity, combining passport, pedigree, molecular and phenotypic information.

Therefore there may emerge cases for a new round of investigation leading to the characterization of new sets of materials. We invite proposals for this, provided that a strong specific justification appears in the light of the rationales and methods that are being elaborated.

A budget of up to 300 000 $ can be mobilized. This topic makes the subject of one of the parallel sessions one day 2 agenda of the ARM in Rome, on the 30th of September, 2005. It is proposed that Ruaraidh Sackville Hamilton organize the session, with support of SGRP scientists.

2. A database of candidate gene diversity in GCP crops (joint SP1 and SP2)
Justification Candidate genes are genes that are designated by convergent evidence as potentially involved in the variation of key agronomic traits. Such evidence includes: their co-localisation with QTLs, expression patterns, possible physiological roles inferred by sequence annotation, and/or their response to directional selection relative to the target traits. They are thus the collective outputs of vast coordinated research investments. Looking at allelic polymorphism of candidate genes in a particular crop, within a reference germplasm sample which also undergoes proper phenotyping, will allow testing association between observed polymorphism and the trait variability. This will enable the validation of the involvement of specific genes in a trait, and at the same time, mining of the most favorable alleles within a species. Examining the patterns of allelic diversity of orthologous loci across several crops may also define shortcuts to transfer crop-specific information to other species; throw light on a range of adaptation and polymorphism, and help resolve functionally important variations from nonfunctional ones shown by statistically associated variations.

Workplan 1. Main tasks The workplan of this project involves several tasks: - to elaborate a list of the best documented drought-tolerance-related candidate genes, with their location and sequence(s) in model plants (e.g., rice, maize, Arabidopsis, Medicago) - to assess their copy number in a range of GCP crops and to design primers for amplification of whole-length gene versions from an array of GCP crops - to investigate orthologous relationships between crops by phylogenetic analyses (sequence information) and physical mapping (microsynteny with relevant model plants) - to select a priority gene list (first 10-20) on this basis (possibly different sets between monocots and dicots) - to produce and analyse sequence diversity among accessions of GCP reference samples (e.g. 200-300 accessions per crop)

to construct a database and make the data available for further use

2. Choice of species, genes and methodologies The species under consideration should o have a genetic map and segregating progeny available for checking the location of genes (SNPs) on the map, in relation to genome colinearity and to existing QTLs for drought tolerance o have ESTs or be sufficiently close to a model species for which the target gene sequence is available o have a reference sample of some 300 accessions to be genotyped in perspective of association studies for drought tolerance The genes under consideration should o be candidates with strong evidence, or o be part of a well-understood metabolic pathway o be related by clear orthology across species, to the extent possible The methods to be used should o explore the use of consensus degenerate primers, for future applicability to other crops o preferably reveal whole-gene sequences, including upstream regions possibly displaying promoters o reveal large orthologous gene portions across all species

Expected outputs As an initial phase, (simulation) the project could include 8-10 crops with 300 accessions each, 10-20 genes known from a leading crop, and their orthologs (could be more than one copy), an average of 6 sequences per gene x accession. If fewer crops, more genes could be considered. The results are expected to be available on the GCP website before the end of 2007.

Indicative budget The project will involve the production of some 230,000 sequences, which represents a large part of the total cost. This can be outsourced or negociated within collaborative agreements with high capacity partners. The French National Genotyping Center (CNG, Evry) has indicated its interest and could produce some 230 000 sequences and basic bioinformatic treatment with a GCP contribution of approximately $400,000. Should this option be considered, the projet should be formally submitted to the CNG. Each participating crop center should receive $10,000 for contributing DNA samples and integrating crop diversity information. The Lead Principal Investigator should be strong in bio-analysis and will receive support for a Postdoc for the preparatory work in coordination with the sequencing partners. The GCP would also fund two postdoctoral fellows for a year to contribute part of the preparatory work in two other sites. In total, the indicative budget is: Subcontract (e.g. CNG) Crop centers (for 10 crops) Lead PI - personnel Other partners - personnel

$400,000 $100,000 $70,000 $140,000

- workshop, travel, ... $30,000 - overheads $60,000 The anticipated total budget is $800,000, to be distributed over 2006 ($580,000) and 2007 ($220,000). This project idea will be at the center of a discussion on "Strategies for identifying candidate genes and how to cope with complex traits" in one of the parallel sessions of the ARM in Rome on the 30th of September.

3. SNP analysis of the genetic diversity along the rice genome (SP1)
Justification The analysis of the genetic diversity of a crop has considerable and well recognised benefits. The availability of whole-genome sequences in some pioneer plants has released an unprecedented wealth of polymorphism information, notably Single Nucleotide Polymorphisms (SNP). The progress in high-throughput SNP analysis in specialised laboratories makes it possible to undertake whole-genome molecular diversity characterization with hundreds or thousands of markers in order to localize favorable genes and alleles through linkage disequilibrium (LD). This approach, which is now widely applied in human genetics, when applied to relevant plant populations will open up opportunities unprecedented in crop research and improvement, The use of SNPs for undertaking LD mapping in a crop plant is yet a new undertaking. The work on maize has a different rationale (very low LD) and rests on candidate genes. Many of the CGIAR crops are self-pollinators and represent a different category. Work on Arabidopsis provides information on LD in a self-pollinating plant, which, however, has not gone through the bottlenecks associated with domestication. We still need assessment of feasability in a crop and development of proper know-how. The crop of choice is rice, for which finished genome sequence is available for a variety, and several shot gun sequences exist for several other varieties. A first work has been completed, recording numerous potentially useable SNPs along the rice genome (Feltus et al, Genome Research 14:1812-1819, 2004). Another work is ongoing with participation of the GCP, for adding more sequence from a larger panel of varieties. The objective of this project is to produce a first set of SNP data in a large sample of rice accessions, in order to prepare full dimension whole-genome LD scans in rice and develop generic know-how for LD mapping in crops.

Workplan The GCP has already characterized with SSR markers a sample of more than 1500 accessions identified at IRRI and representative of the diversity. This sample is undergoing phenotyping for various types of traits, including 600 accessions for drought tolerance . Selection of materials should be done largely within this sample.

The project should involve the production of a large number of SNP datapoints, which represents a major part of the total cost. This can be outsourced or negociated within agreements with high capacity partners. The SNP study should include tightly linked SNPs along a significant length on the genome, in order to assess the pattern of LD, in particular in relation to the indica-japonica differntiation. Preparatory work is necessary, in order to select the SNPs to be surveyed, according to the quality of the sequence, the adequacy of SNP revelation and the distribution along the genome. Indicative budget The indicative budget is $150,000.

4. Phenotyping in the field: global capacity accessible to the GCP (SP1)

Accurate phenotyping is currently becoming the bottleneck for identifying sources of favourable alleles and gene combinations in genetic resources with the view to supporting breeding drought tolerant varieties, i.e. the main goal of the GCP. At a workshop on phenotyping for drought tolerance held in 2004 in Montpellier, 50 scientists from IARC, ARI and NARS consulted and identified priorities: - Identifying a minimum set of environmental descriptors to characterize trials and allow comparability; optimal use of test genotypes - Comparing genotypes with comparable adaptation (phenology, reaction to biotic stresses, ) - Identifying traits with high heritability, ecophysiological background and clear relation to drought tolerance - Undertaking whole-plant modelling to support breeding approaches and refine phenotyping protocols. The GCP has decided to help the emergence of phenotyping networks for this complex feature. It is supporting a network at Embrapa in Brazil, that gives access to a group of environments, facilities and expertise with a global institutional coordination. Simultaneously, the GCP supports a project for developing whole-plant modeling with three components: - characterization of the environments with their spatial and temporal variations - quantification of the impact of environment variations on phenotypic traits - integration of trait variation in whole plant and crop functioning. This project works in concertation with the Embrapa network. Other possibilities may exist for the GCP but they are not listed and characterized. A comprehensive survey of the "phenotyping offer" is needed given the forthcoming characterization of the reference germplasm samples which will be required shortly. The CGIAR centers may have their networks and partners, and additional potential partners may exist.

The GCP wishes to have an inventory of the resources, capacities and facilities that are acessible to it, in order to plan properly future phenotyping experiments with optimised returns within identified budgets. SP1 is willing to fund such an inventory. This could rest on the knowledge present in the SGRP network, assisted by non-CG experts who may have complementary views and knowledge, and possibly are already involved in whole-plant modeling activities in relation to the GCP. An indicative budget is $100 000 for this project.