- Validation of Conserved Orthologous Markers:

A proposal for extension of Commissioned Research (SP2-7) in

Subprogram 2 “Comparative Genomics” Cluster 2: Marker Development

Generation Challenge Program

- Revised Workplan For Year 2; 2005-6:

- Introduction:

GCP support is requested for consolidated experimental work on conserved markers to bring Year 1’s commissioned research in SP2C2 to a meaningful conclusion (Year 1 proposal; SP2 Progress report and Brisbane reports of Jia and Bonierbale).

Gene-based PCR markers, also called functional markers, are a third generation molecular marker, more powerful than RFLP markers (first generation) and other PCR types (second generation) for marker-assisted selection and allele discovery. However, the shortage of functional markers has become a bottleneck in a number of marker-based genetic and breeding applications. Orthology is an important aspect of functional markers, particularly for applications in comparative genomics. As a part of 2004’s commissioned research under SP2C2, 258 universal orthologous EST-SSR markers were developed based on wheat EST sequences available in public databases. Of these, 59, 59, 30, 25, 14, 9 universal markers are related to DNA synthesis/RNA transcription/protein translation, basic metabolism, signal transduction, cellular structure/development, bio/abio-stress response, transporter/others, respectively. In addition, about 100 primer pairs were designed or applied to testing tomato-Arabidopsis COS (from SGN) in a range of dicot and monocot species (legumes, grains and potato) selected for Year 1 research in the GCP, and a small number were validated by mapping and sequencing in potato. Some 50 primer pairs have been tested in Musa based on comparisons of Musa ESTs with rice, Arabidopsis and other species, including both constitutive metabolic genes and targeted genes related to abiotic and biotic stress. These are being refined, sequenced, and tested in additional species. So far COS primers for cereals and legumes were designed for 33 genes including genes for stress tolerance and some of them have been tested on a set of cool season legumes (2 genotypes each of lentils, chickpea, faba bean and lathyrus).  16 primer pairs designed for cereals and 10 primer pairs designed for legumes gave amplification products in respective crops. Further validation by sequencing is in progress. But further research and exchange of results across participating centers has not been carried out because of time limitations.

1. Objectives:

Develop and validate orthologous markers, focusing mainly on candidate genes for drought tolerance and disease resistance, extending and validating the marker sets developed in 2004 (SP2C2). There are three specific objectives:

Specific objective 1: Consolidate and expand the available set of orthologous genes by revisiting bioinformatics approaches for identifying COS.

Specific objective 2: Test and validate universal markers developed in Year 1 related to drought tolerance, disease resistance and other agronomically important traits in the monocots and dicots to enhance comparative genetic maps within and across crop families.

Specific objective 3: Develop ortholog gene-based markers universally or separately for monocots and dicots based on conserved regions of orthologous drought or disease resistance genes to enhance gene discovery (with SP2C3 and SP4). This kind of marker includes SNP and other insertion and deletion markers.

2. Deliverables:

At the end of Year 2, the research team will deliver:

1) 50 universal PCR-based markers related to drought tolerance, disease resistance and other agronomic important traits validated for use across monocots and dicots respectively and if possible between monocots and dicots.

 

2) Markers that contribute to SP1 for functional diversity measurement

 

3) Locate 20 universal markers on maps of monocots and dicots respectively.

 

4) Markers that contribute to SP2 and SP3, respectively, for gene expression studies and marker-assisted selection.

3. Experimental Approach:

1) Latest databases for respective species will be assembled on the High Performance Computing facility. Multiple bidirectional BLAST will be realized and tentative COS be re-evaluated or determined through filtering techniques taking into account additional information, e.g. from SGN, TIGR/EGO/TC and Arabidopsis intron/exon boundaries.

2) Subsets of mapping populations for the species under investigation will be prepared for validation and mapping of COS. The software used for this task will include MapPop (Vision et al. 2000) in combination with IntiMap (Simon and Bonierbale 2003).

3) Universal EST markers related to agronomically important traits will be tested and further validated through sequencing PCR products of at least 2 genotypes per crop.

4) For those markers that do not amplify sequences homologous to targeted genes revealed in 3), new primers will be designed flanking the SSR/ variable regions for other species separately and validation will be undertaken.

5) For those orthologous genes conferring drought and disease resistance but not containing SSRs, primers will be designed from the conserved domain flanking polymorphic regions, exons, containing SNPs or other Indel markers.

6) Final validation of the primers will be done by sequencing the sequences amplified by each primer pair, where appropriate in two mapping populations (6 genotypes: 2 x (2 parents + 1 off spring) in each crop.

7) The universal EST or any other PCR-based markers that are confirmed to be homologous to the targeted genes will be localized on the existing maps in each crop respectively, then comparative analysis will be done. The markers will be evaluated by testing in a set of germplasm for each crop.

8) Primers demonstrated to amplify homologous products in at least one new target species (potato, Musa, wheat, maize, lentil, chickpea) will be databased, and aliquots exchanged among collaborators for testing and reciprocal validation across crops within this set, and to other (tier 1/ tier 2) GCP species. The sequences of COS that are validated across monocots, dicots, or both will be assessed phylogenetically across source and target species. Template DNA (6) and appropriate documentation for comparability of procedures across labs will be provided with primer aliquots.

4. Task Assignment: Monocot Group

• Dr. Jizeng Jia, CAAS, EST-SSR marker development and validation in wheat, barley, maize and rice; integration and links with SP4

• Dr.Lifeng Gao, CAAS, comparative map development

• Qingming Sun, Ph.D. student, CAAS, in silico sequence analysis and diversity detection

• Subcontractor to INIBAP (Dr Nicolas Roux) within the framework of the Global Musa Genomics Consortium: Prof. Pat Heslop-Harrison, U. Leicester, COS primer design and testing based on rice-musa-arabidopsis-others, with cloning and sequencing of Musa products, following by testing on other species

• Mathieu Rouard, INIBAP, in silico sequence analysis and diversity detection and link with SP4

Dicot Group

• Drs. Merideth Bonierbale and Roland Schafleitner: CIP, candidate gene/contiging in dicots; potato and cross – species COS testing and validation; comparative map development.

• Mr. Reinhard Simon: CIP, Potato/Dicot genome annotation, in silico analysis of multiple genome sequences: integration for dicots and links with SP4

• Drs Michael Baum, Sripada M. Udupa  ICARDA, in silico sequence analysis, cloning and sequencing legume products and application to aligning maps of chickpeas and lentils

5. Project Linkages:

This project will be linked with other commissioned projects under SP4 that involve mapping and display tools for markers and stress gene orthologs. Also the team will continue to work with partners (IRRI, Cornell) participating in marker development in Year 1 commissioned work. (IRRI can continue to help with testing the primers without channeling funds from this project)

6. Capacity Building:

A Ph.D. student will be employed to develop thesis research in bioinformatics applied to marker development. Fellowship grants will be awarded for a researcher at the University of Leicester for programme of training through research. A Bioinformatics trainee will assemble a database of putative COS and cross-crop sequences.

7. In-kind Contributions:

Each participating institution will contribute research infrastructure and staff time for the commissioned research. The following facilities or special projects have generated or will generate outputs that are relevant to, and that may be used by the Challenge Program; counterparts directly relevant to the Year 2 workplan are as follows:

CAAS - 90% and 50% of two senior scientists respectively, 90% of one Ph.D student’s time. High throughput robotics and genotyping. Bioinformatics platform. Dataset of EST-SSRs derived from wheat known to be orthologous across wheat, maize and rice.

CIP: 10 % of two senior scientists, HPC platform management.

INIBAP: will contribute in kind contribution of 5% of the Global Musa Genomics Consortium coordinator.

ICARDA: 10% each of the two senior staff.

References:

Simon, R., Bonierbale, M. 2003. IntiMap - A program for comparing and publishing genomic maps. PAG XI, San Diego, Jan 11-15, 2003.

Vision, T. J., Brown, D.G., Shmoys, D.B., Durrett, R.T., and Tanksley, S.D. 2000. Selective Mapping: A strategy for optimizing the construction of high-density linkage maps. Genetics 155:407-420.

Work Plan Budget Tables (Year 1, 2005-2006)

Continuation Subprogram 2 Cluster 2 Marker Development

Total Budget x Institution