Allele Mining Based on Non-Coding Regulatory SNPs in barley germplasm:

 

- Targeted Subprogramme:  Subprogramme 1: Genetic diversity of global genetic resources.

                                               

- Principal Investigator:  Michael Baum, Ph.D., Germplasm Program, International Center for Agricultural Research in the Dry Areas, ICARDA P.O. Box 5466, Aleppo, SYRIA, Fax No. +963 (21) 2213490/ 2225105/ 2219380/ 5551860, Tel:  +963 (21) 2213433/ 2213477/ 2225112/ 2225012

e-mail:  M.Baum@cgiar.org

 

- Collaborating Scientists:

 

W. Powell, School of Agriculture and Wine, University of Adelaide, Waite Campus PMB 1, Glen Osmond  SA  5064 Australia

 

P. Langridge, Australian Centre for Plant Functional Genomics Pty Ltd, PMB 1 Glen Osmond  SA  5064  Australia

 

Mark Tester, Australian Centre for Plant Functional Genomics Pty Ltd, PMB 1 Glen Osmond  SA  5064  Australia

 

K. Eglinton, School of Agriculture and Wine, University of Adelaide, Waite Campus PMB 1 , Glen Osmond  SA  5064 Australia

 

M. Morgante Dipartimento  di Scienze Agrarie ed Ambientali Universita' di Udine Via delle Scienze 208 I-33100 Udine

ITALY Ph.: +39-0432558606 Fax: +39-0432558603

Email: michele.morgante@uniud.it

 

S. Ceccarelli, S. Grando ICARDA barley breeder,

 

S.M. Udupa, biotechnologist

Germplasm Program, International Center for Agricultural Research in the Dry Areas, ICARDA P.O. Box 5466, Aleppo, SYRIA, Fax No. +963 (21) 2213490/ 2225105/ 2219380/ 5551860, Tel:  +963 (21) 2213433/ 2213477/ 2225112/ 2225012

 

Wafaa Choumane,  Department of Basic Sciences, Faculty of Agriculture, Tishreen University, P.O.Box 2099, Lattakia, Syria.

Fax  No. +963 (41) 469040.  E mail: w.choumane@cgiar.org and wafaa627@scs-net.org.

 

 

- Participating Institutions:  International Center for Agricultural Research in the Dry Areas, ICARDA P.O. Box 5466, Aleppo, SYRIA

University of Adelaide, Adelaide  SA  5005 Australia.

 

Universita' di Udine

Via delle Scienze 208, I-33100 Udine, Italy.

University of Tishreen,  Faculty of Agriculture. Lattakia, Syria

 

 

- Submission date: Submission date: 27.8.2004

 

TABLE OF CONTENTS

 

- Cover Sheet

- TABLE OF CONTENTS.

- Executive Summary.

- Scientific Summary.

- Project Description.

- Objectives.

- Intended Specific Outcomes.

- Introduction and Rationale.

- Approach and Methods.

- References.

- Partners.

- Capacity Building.

- Management Plan.

- Critical assumptions and Contingency Plans.

- Timelines and Milestones.

- Budget

- Appendix.

- Appendix 1. Partners.

- Appendix 2 Intellectual Property Rights Statement:

- Appendix 3. CV of  PI and Co-PIs.

- Appendix 4. Letters of support

 

 

 

 

- Executive Summary:

In recent years analysis of genetic variation has focused on  the study of changes in DNA coding for proteins.  It is now becoming increasingly clear that this only accounts for one aspect of heritable  variation and for many traits, notably tolerance to environment stresses, the level of gene expression is also likely to be of great importance. If changes in gene expression underlie many evolutionary changes in phenotype, then identifying the genetic variants that regulate gene expression is a significant and important endeavor. One of the key problems in genetics is how to identify this type of variation. We propose a robust, quantitative approach to efficiently identify plant genes that harbor such regulatory variants. The approach is novel and particularly amenable to plants since it is based on monitoring gene expression in experimentally created hybrids. A successful outcome will provide a new mechanism to connect genotype to phenotype based on changes in gene expression rather than changes in the structure of an encoded protein.  This approach will be used to characterize a series of genes identified and reveal potential candidates for tolerance to drought, frost, cold and salinity stresses. The approach is generic and widely applicable. The project will also involve training researchers in Developing Countries and create a high quality collaborative network of researchers delivering new knowledge on genetic diversity and translatable outputs for the Developing World.

 

- Scientific Summary:

Heritable differences in gene expression are now considered to be a fundamental mechanism responsible for determining the genetic control of complex, multifactorial traits. Identifying naturally occurring genetic variants that regulate gene expression is an important route for connecting genotype to phenotype based on changes in gene expression rather than changes in the encoded protein. It is predicted that such mechanisms are pervasive and will also control the response of crop plants to various stresses such as those induced by limited water, salinity or high temperature. Reliable identification of genetic variants that affect gene regulation and are causatively associated with priority traits will allow the identification and isolation of mechanistically functional alleles that can be effectively deployed in breeding programs.

 

Recently methods have been developed that allow the identification of sequence polymorphisms that are linked in cis to regulatory variants and to predict which nucleotide differences are responsible for changes in gene expression.  The principle of the approach is based on the hypothesis that the relative abundance of allelic transcripts when estimated for individuals in the heterozygous condition will be devoid of trans-acting influences and environmental factors, which can confound micro-array based experiments. The approach is robust, scalable and particularly well suited to crop plants where the ability to produce sexually derived heterozygous hybrids is not rate limiting. Linkage of the current proposal to The Australian Centre for Plant Functional Genomics where genes related to abiotic stress tolerance are being identified and existing collaborative research programs at ICARDA and the University of Adelaide that examines genetic diversity for abiotic stress tolerance will facilitate the application of this technology for functional allele discovery in germplasm tolerant to salinity, Spring radiation frost, boron toxicity, or drought. This research strategy ensures that cis-acting regulatory variation detected within this project will be of maximum benefit to plant improvement and provides a novel route to unlocking the potential of a yet untapped repertoire of genetic diversity in crops.

 

 

- Project Description:

 

- Objectives:

The overall objective of this proposal is to develop a technique that allows the identification of the determinants of variation in gene expression and exploit this assay to analyze and identify novel alleles for abiotic stress tolerance in barley.  The technique will provide a robust functional assay that detects the presence of cis-acting regulatory polymorphisms.  Such polymorphisms are expected to be abundant in the wild progenitors of crop plants and be surrogates for novel alleles that control multifactorial traits such as those involved in abiotic stress. A strength of the approach is that it does not require a priori knowledge of specific regulatory polymorphism and is therefore an efficient method for both discovering and elucidating mechanisms controlling cis-acting regulatory elements.

 

Most importantly, the approach identifies a mechanism to connect genotype to phenotype based on changes in gene expression, rather than changes in the encoded protein. Such functional polymorphisms are likely to be novel and significant with respect to their impact on phenotype and therefore highly desirable for deployment in breeding programs. Although abiotic stress tolerance in barley will be the target for the development of the technique, it will have application in most crop species and a core component of the project will be to train others in the use of the technique and to develop collaborative projects that will allow investigation of this important class of variants in other species.  The specific objectives are:

• Develop and exploit genomic imbalance assays in conjunction with experimentally produced F₁ hybrids to identify cis-acting regulatory elements in barley.

• Establish novel coupling linkages between regulatory alleles and multifactorial drought and abiotic stress traits.

• Devise ‘haplotype tag’ based approaches to enable the deployment of sequence-based diagnostics in breeding programs.

Establish a training program and actively develop collaborative projects with researchers working on the molecular basis of variation in other crop species.

 

- Intended Specific Outcomes:

A key outcome will be the identification and functional characterization of candidate genes for abiotic stress related traits that exhibit heritable differences in gene expression. This will involve the isolation of novel alleles from germplasm collections together with the establishment of deployment strategies to enhance the impact of breeding programs. The proposed approach to isolate cis- acting elements is generic and applicable to all crop species.

 

A second outcome will be the enhanced ability to mine regulatory elements in monocots with the possibility to generate a conserved set of regulatory motifs with wide applicability across cereals.

 

A third outcome will be to create a network of collaborating scientists focused on delivering: scientific innovation, translatable outputs and outcomes in the form of novel, variant alleles that are associated in a causative manner with target traits and a stimulating training environment for sustainable capacity-building.

 

This proposal will establish a collaborative centre of excellence focused on the functional genomics of regulatory gene discovery. Identifying favourable mutations in non-coding regulatory regions will enhance the value of crop genetic resources. The scientific approach is innovative, timely and highly relevant to the goals of the Challenge Program. The proposal provides a distinctive framework for addressing genotype-phenotype integration issues which will allow the isolation of novel alleles and provide a platform for the delivery of superior alleles in breeding programs based on sequence designed ‘haplotype tags’ for regulatory SNPs.

 

- Introduction and Rationale:

Alterations in gene regulation have been proposed to influence disease susceptibility in humans (1, 2) and provide the genetic basis for natural variation to antibacterial immunity in D. melanogaster. The evolutionary substrate for organismal diversity in the plant and animal kingdom (3, 4) may therefore be due to heritable differences in gene expression.  The recent identification of a causative single base pair substitution in a non-coding, regulatory region of the gene encoding IGF2 (an insulin like growth factor) that controls muscle growth in the pig, supports the long held view that regulatory mutations are important for controlling phenotypic variation (5).  Significantly, the causative allele was discovered in wild boar intercrosses, suggesting that wild ancestors of domesticated animals and plants harbor a rich collection of mutations controlling heritable differences in gene expression.

 

Identifying polymorphism in regulatory elements that influence heritable variation in gene expression is an important but challenging task and is limited by our inability to recognize the regulatory regions and more importantly to predict which nucleotide changes are responsible for changes in gene expression. This is a particular problem for complex eukaryotes where the regulatory elements can be tens or even hundreds of thousands of base pairs from the transcription unit (4, 6).  Furthermore, direct experimental evidence that gene expression is under the influence of cis-acting polymorphism is frequently confounded by the occurrence of trans-acting influences and/or environmental factors.  Recently methods have been developed for measuring allele-specific expression levels in mouse (7) and humans (8, 9, 10) that allow the influence of cis-acting effects to be identified in a robust and systematic manner.  The principle of the approach is based on the hypothesis that the relative abundance of allelic transcripts when estimated in individuals in the heterozygous condition will be devoid of trans-acting influences and environmental factors. Quantitative deviations from expected co-dominant expression of transcripts can therefore be ascribed to cis-acting regulatory variation. Experimentally, this can be achieved by creating heterozygous F₁ hybrids and comparing the expression of the two alleles derived from the donor parents (7).  In order to distinguish between transcripts derived from the parental alleles it is necessary to identify an exonic polymorphism, which can be used as a ‘copy specific tag’ (9).  The identification of SNPs in the transcript enables quantitative measures of allele expression to be applied to individual genotypes that are heterozygous for the marker polymorphism.  Quantitative methods for measuring and discriminating between alleles are available (11) and are based on RT-PCR amplification of the region surrounding the SNP followed by single base pair extension (SBE) of a primer adjacent to the variant base in the presence of fluorescently labeled nucleotides.  The detection of allelic imbalances has been used to study imprinting (12) and offers major advantages over conventional approaches for unraveling the control of regulatory variation based upon comparisons of gene expression between individuals.  These advantages are based on the fact that the expression of the two alleles are compared under identical circumstances within a single individual genotype, providing an internal control for confounding factors such as: differences in mRNA preparation and quality, environmental factors and trans-acting factors. 

 

A recent survey of genetic and epigenetic variation affecting human gene expression (10) identified 23 genes (18%) with significant allelic expression differences.  These studies suggest that cis-acting inherited variation in gene expression is relatively common and important.  Studies of changes in gene expression and regulation due to the evolution of cis-regulatory DNA sequences in plants are in their infancy but unpublished data indicate that 7 out of 12 maize genes show more than a 1.5 fold difference in expression (M. Morgante unpublished).

 

Selective breeding accompanied by domestication has led to marked phenotypic changes in our modern crop plants and cultivars. The process of domestication that resulted in changes from well adapted small, naturally dispersed seed to large seeded grain adapted to modern agricultural practices may have resulted in significant changes in the expression pattern of genes involved in adaptation to abiotic stresses. Few studies have considered these issues and our knowledge of the consequences and mechanisms of selection operating on this key developmental process is largely unexplored. An improved understanding of these processes will provide a new paradigm to identify and harness novel allelic variability based on regulatory polymorphisms. The most extensive molecular characterization of crop plant domestication has been undertaken by Doebley and colleagues (13,14) who have employed genome scans to identify signatures of selection associated with domestication. The hypothesis is that selection will increase linkage disequilbrium (LD) around selected regions of the genome relative to that observed at selectively neutral regions. The teosinte branched 1 (tb1) gene has been shown to control differences in plant architecture between maize and its wild relative, teosinte (Z. mays subsp. mexicana and subsp. parviglumis) and the 5’ region of the gene was the subject of selection during the domestication of maize. A more refined analysis (15) revealed a core region of selection operating at between 60 to 90 kb 5’ of the tb1 transcribed sequence. Predicted genes were not observed in this region, raising the possibility that selection has operated on regulatory sequences, which are distant to the presumed promoter region. Such a scenario has already been postulated by Rafalski and Morgante (16) and is consistent with studies in maize where an enhancer element for the b1 gene was localized 100 kb 5’ to the transcription start site (6). These studies raise two important points: first regulatory variants may be the targets of selection and second, such candidate genes are likely to have a significant effect on phenotype. We will therefore deploy expression-based allelic imbalance assays, in conjunction with inter-specific F₁ hybrids created from pre- and post-domesticated barley gene pools, to identify candidate, regulatory haplotype blocks that have been under selection during domestication. This will involve identifying regulatory haplotypes that are in LD with high and low expressing transcripts derived from pre- and post-domesticated barley accessions.

 

The approaches outlined in this proposal are designed to efficiently scan genes indirectly for cis-acting effects on gene expression that involve regulatory sequence variants. The innovative technological approach therefore provides new opportunities to identify regulatory polymorphisms in plants and connect genotype to phenotype based on changes in gene expression rather than changes in the encoded protein. Such functional polymorphisms are likely to be novel and significant with respect to their impact on phenotype, providing a highly desirable new class of marker for deployment in breeding programs.

 

Approach and Methods

 

In contrast to identifying variation in coding regions of the genome, characterizing the extent of cis-acting regulatory variation presents a much greater challenge since it is not possible to discern even for fully sequenced genomes, whether a particular gene harbors a polymorphism that regulates its expression. Experimentally screening for regulatory variants based on differences in transcript levels between individuals is confounded by potential trans-acting factors or environmental differences.

 

Conceptually, the identification of genes that harbor regulatory variation requires studying two alleles of a gene under identical circumstances and comparing the expression of the transcript associated with each. This can be achieved by testing individual genotypes in the heterozygous condition for differences in the expression of alleles from both parents.  The method simply requires the ability to distinguish between the transcripts derived from each of the two parental alleles based on a SNP in the transcript. The power of the approach is its simplicity which is based on measuring the relative expression levels of two alleles for a given gene in the same cellular sample and thereby eliminating variation arising from environmental or physiological, rather than genetic, factors. The basic principle is outlined in the Figure 1 (taken from Yan et al (8)).  Fluorescent Single–Base Extension (SBE) of a locus-specific RT-PCR product, using an allele-specific primer adjacent to the marker SNP of interest, will allow detection of the variant base on a fluorescence detection platform, such as the ABI377/3700/3730.

 

Figure 1.  Overview of detection of cis-acting allelic expression variation (taken from Yan et al., (8)

 

 

 

A particular strength of the approach is that it does not require a priori knowledge of specific regulatory polymorphism and is therefore an efficient method for both discovering and elucidating mechanisms controlling cis-acting regulatory elements.

 

 

The experimental approach relies on quantitative genotyping of heterozygous individuals for intragenic SNPs in RNA transcripts and comparing the observed allele ratios to corresponding genomic DNA samples.

 

The first step will therefore be to identify and utilize a set of genes containing previously validated ‘marker’ SNPs within their coding sequence.  Two data sets from our previous research will provide the foundation for this phase of experimentation.  A set of fifteen diverse EST-based SNPs, have been validated within the Oregon Wolfe Barley (OWB) population (http://barleyworld.org/owbs.html) using Pyrosequencing technology (see Table 1).

 

  

 

Table 1.Barley genes containing validated coding SNPs, indicating SCRI and international locus codes, top homologies (including accession nos.) and type of base-substitution detected. More than one validated SNP present in the same gene (eg. SNP824 and 2733) will enable confirmatory assays.

 

The second source of genes arises from the characterisation of a telomeric region of chromosome 5H of barley(17). Three overlapping BAC clones representing 300kb has been fully sequenced revealing 12 genes organised into two clusters spanning approximately 120kb, embedded in a complex collection of nested retrotransposon insertions (see figure 2).

 

Figure 2.  Fully sequenced 300 kb region of the barley genome containing genes involved in grain texture

Locus-specific primer pairs will be designed to span each validated SNP, for the set of genes outlined in Table 1, using Primer 3.0 (Whitehead Inst. Software) and standard PCR primer design parameters. Detection of cis-acting regulatory variation will be based on the fluorescent Single-Base Extension (SBE) method as outlined in section E3. Total RNA, from F₁ individuals, will be isolated using the RNeasy kit (Qiagen) from a range of tissues: (i) whole germinating seed (two day: source of genes in Table 1); (ii) seedling root (two- week); (iii) developing embryo (6-8 dpa) and; (iv) developing endosperm (6-8 dpa) and (v) leaves For expression detection, DNase-treated total RNA samples will be reverse transcribed into first-strand cDNA using Superscript (Invitrogen) with oligo d(T) random nonamer priming. Templates (gDNA, cDNA, or no-RT control) will then be amplified using locus-specific primer sets under standard PCR conditions. PCR products will be purified using MinElute columns (Qiagen) prior to SBE. Primers for SBE will be designed to anneal and terminate on the base 5’ to the SNP. SBE will be performed in the presence of fluorescently modified ddNTPs added in accordance with the polymorphism assayed, under standard cycling conditions (7). Products will be purified from unincorporated fluorescent bases by filtration columns (Qiagen) and analysed on an ABI377/3700 sequence detection system. The method will also be tested using the commercially available SNaPshot Multiplex Kit (ABI) with the ABI3700 and ABI3730 platform. Peaks of dye intensities corresponding to SBE will be determined following background subtraction and normalisation to genomic titration standards for known allelic ratios. Assimilation of intensity ratios for biological replicates will allow statistically significant differences to be determined. Multiple SNPs from the same locus will be analysed for concordant ratios from the same tissues. Biological as well as experimental (both for PCR and SBE reactions) replicates will be performed for each sample to allow statistical analysis of the data.

In interpreting the data generated from this study, it will be extremely important to determine whether the variation detected is cis-regulated or imprinted, i.e. parent- of origin specific, therefore reciprocal F₁ hybrids will also be analyzed. Where imprinting is demonstrated, imbalance will be observed between allelic expression ratios and reciprocal lines, in the same tissues. It should be noted that barley endosperm is triploid, which will also cause imbalance in the allelic expression ratios observed.

 

Reciprocal F₁ hybrid development.

Initially, the allelic expression imbalance assay will be optimized using F₁ reciprocal hybrids derived from the OWB dominant and recessive lines (2 x F₁). In order to infer the ratio of detected expressed alleles of each locus, mixtures of parental genomic DNAs in known proportions will be used as reference standards for each primer set.  With suitable biological replication, such an assay should allow detection of minimum expressed allelic variation of 20% (8).

The survey will then be extended to include reciprocal hybrids derived from an interspecific cross between Hordeum spontaneum (CPI71284-48) and cultivated variety Barque-73 (2 x F₁). This will enable any allelic expression imbalance to be assessed with respect to cis-acting regulation or imprinting during domestication, and polymorphisms will be genetically mapped in the F₁ derived doubled haploid population. An Advanced Backcross population has also been developed from this germplasm and is undergoing detailed screening for abiotic stress tolerance as part of an existing UA/ICARDA collaborative project, allowing analysis of association between regulatory variation and phenotype. 

Following database analysis of the initial F₁ hybrids, a defined set of genes will be selected to screen a full diallel (including reciprocals) mating design.  The following parents will be included: OWB dominant and recessive stocks, Tadmor, Alexis, H.spontaneum 41-1, CPI71284-48, Arta, Sloop and WI3408.  Excluding ‘selfs’ this will create 72 F₁ hybrids. Co-dominant microsatellite analysis will be used to confirm the hybrid origin of all F₁s.

 

Survey of cis-regulated control of genes involved in abiotic stress

Following successful assay development utilizing the validated SNPs from the set of genes listed in Table 1, a further set of SNPs from up to eighty additional genes will be targeted, potentially involved in abiotic stress.  Such genes will include those encoding: transcription factors such as drought responsive element binding proteins (e.g. BU998093); chaperones, such as the osmotins (e.g. BQ471491) and dehydrins (e.g. CAA58875); enzymes involved in compatible solute synthesis and breakdown, such as ornithine delta-aminotransferase (e.g. AV925372); enzymes involved in sucrose accumulation, such as sucrose-phosphate synthase (e.g. CAB45558); enzymes involved in ABA synthesis and breakdown, such as 9-cis-epoxycarotenoid dioxygenase (e.g. BF065327); aquaporins (e.g. HvPIP1;5, BAA23746); nonselective cation channels, such as the glutamate receptors (e.g. BJ471622; plasma membrane and vacuolar Na⁺:H⁺ antiporters (e.g. HvNHX1: AB089197); plasma membrane H⁺-extruding ATPases (e.g. AJ310846); the vacuolar H⁺-pumping pyrophosphatase, AVP1 (e.g. BAA02717); and a gene important for root-to-shoot allocation of Na⁺, HKT1 (e.g. AV936990) Immediately, potential polymorphic regions can be identified from the vast publicly available barley EST collection (~350,000 ESTs) by eSNP analysis using contig viewing software, such as HarvEST (www.harvest.ucr.edu ), or AutoSNP dedicated SNP identification software (18).  In addition, preliminary SNP data will be available from Single-Feature Polymorphism (SFP) assays (19) developed in our lab for barley, which will allow direct detection of expressed polymorphic loci in the mapping population parents

Following marker SNP validation in parental lines by re-sequencing, the allele imbalance assay will be applied to these genes using the reciprocal F₁ panel. This will identify those genes under cis-regulated control and also those that may undergo parental imprinting or exhibit parental imbalance.

 

Meiotically mapping cis-acting regulatory variation in an inter-specific doubled haploid population of barley.

Candidate, regulatory haplotype blocks that have been under selection during domestication provide a template for de novo discovery of candidate genes that act as surrogates for allelic variation in gene transcription. The wild barley accession H. spontaneum 41-1 and the North African landrace CI3576 have been key donors of adaptation for the ICARDA and University of Adelaide barley germplasm pools respectively. A set of advanced lines derived from these parents is currently undergoing phenotypic screening and conserved linkage block analysis, providing the germplasm base for analysis of regulatory haplotype blocks. Regulatory haplotypes that are in LD with high and low expressing transcripts derived from pre- and post-domesticated barley accessions will be meiotically mapped in a doubled haploid (DH) population derived from an inter-specific F₁ hybrid (CPI71284-48 x Barque-73) of barley. Segregation data will be amalgamated with existing data in a range of highly characterized populations and Joinmap software used to determine map location. Assignment to the barley bin-framework (http://barleygenomics.wsu.edu/db1/db1-searchframes.html) will allow alignment with putative orthologous regions of the rice genome, providing opportunities to identify conserved regulatory motifs.

Depending on progress and if time permits the regulatory haplotype linkage disequilibrium blocks identified in the mapping population will be tested in different genetic backgrounds to evaluate the spectrum of genetic variability available for manipulation in breeding programs.

 

References

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2. Peltonen L, McKusick VA. 2001 Dissecting human disease in the postgenomic era. Science. 16;291(5507):1224-9.

 

3. King, M.C. & Wilson, A.C. 1975 Evolution at two levels in humans and chimpanzees. Science 188, 107-116. 

 

4. Levine, M. & Tjian, R.2003 Transcriptional regulation and animal diversity. Nature 424, 147-151. 

 

5. Van Laere, A-S., Nguyen, M., Braunschweig, M., Nezer, C., Collette, C., Moreau, L., Archibald, A.L., Haley, C.S., Buys, N., Tally, M., Andersson, G., Georges, M. & Andersson, L.  2003.  A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig.  Nature 425: 832-836. 

 

6. Stam M, et al 2002  The Regulatory Regions Required for B' Paramutation and Expression Are Located Far Upstream of the Maize b1 Transcribed Sequences .Genetics  162: 917-930. 

 

7. Cowles, C.R., Hirschhorn, J.N., Altshuler, D. & Lander, E.S.  2002.  Detection of regulatory variation in mouse genes.  Nature Genetics 32: 432-437 

 

8. Yan, H. Yuan, W., Velculescu, V.E., Vogelstein, B. & Kinzler, K.W.  2002.  Allelic variation in human gene expression.  Science 297(5584): 1143 

 

9. Bray, N.J., Buckland, P.R., Owen, M.J. & O’Donovan, M.C.  2003.  Cis-acting variation in the expression of a high proportion of genes in the human brain.  Hum. Gen. 113: 149-153.  

 

10. Pastinen, T.  et al 2003 A survey of genetic and epigenetic variation affecting human gene expression. Physiological genomics 10: 1152- 1165. 

 

11. Sham, P., Bader, J.S., Craig, I., O’Donovan, M. & Owen, M.  2002.  DNA pooling: a tool for large-scale association studies.  Nature Genetics 3: 862-871. 

 

12. Singer-Sam, J., Chapman, V., LeBon, J.M., Riggs, A.D.  1992.  Parental imprinting studied by allele-specific primer extension after RCR: paternal X chromosome-linked genes are transcribed prior to preferential parental X chromosome inactivation.  Proceedings of the National Academy of Sciences, USA 89: 10469

 

13. White S.E. & Doebley J.F. 1999. The Molecular Evolution of terminal ear1, a Regulatory gene in the genus Zea. Genetics153:1455-1462. 

 

14. Vigouroux, Y., McMullen, M., Hittinger, C.T., Houchins, K., Schulz, L., Kresovich, S., Matsuoka, Y. & Doebley, J.  2002.  Identifying genes of agronomic importance for evidence of selection during domestication.  Proceedings of the National Academy of Sciences 99: 9650-9655. 

 

15. Clark, R.M., Linton, E., Messing, J. & Doebley, J.F.  2004.  Pattern of diversity in the genomic region near the maize domestication gene tb1.  Proceedings of the National Academy of Sciences, USA 101: 700-707. 

 

16. Rafalski, J-A. & Morgante, M.  2004.  Corn and humans: recombination and linkage disequilibrium in two genomes of similar size. Trends in Genetics, Volume 20, Issue  2, February 2004, Pages 103-111. 

 

17. Caldwell, K.S., Langridge, P. & Powell, W. (2004) Comparative sequence analysis of the region harbouring the hardness locus (Ha) in barley and its collinear region in rice.  Plant Physiology (in press).

 

18. Barker, G., Batley, J., O' Sullivan, H., Edwards, K.J. & Edwards, D.  2003  Redundancy based detection of sequence polymorphisms in expressed sequence tag data using autoSNP.  Bioinformatics. 12;19(3):421-2. 

 

19. Borevitz, J.O., Liang, D., Plouffe, D., Chang, H.S., Zhu, T., Weigel, D., Berry, C.C., Winzeler, E., Chory, J.  2003.  Large-scale identification of single-feature polymorphisms in complex genomes.  Genome Research 13(3):513-23.

 

- Partners:

This proposal seeks to create a collaborative centre of excellence focused on the functional genomics of regulatory gene discovery. Identifying favorable mutations in non-coding regulatory regions will enhance the value of crop genetic resources. The scientific approach is innovative, timely and highly relevant to the goals of the Challenge Program. The proposal provides a distinctive framework for addressing genotype-phenotype integration issues which will allow the isolation of novel alleles and provide a platform for the delivery of superior alleles in breeding programs based on sequence designed ‘haplotype tags’ for regulatory SNPs. The proposal takes advantage of existing research programs examining the genetic basis of abiotic stress tolerance, and current collaborative projects between ICARDA and the University of Adelaide. Diverse germplasm and detailed phenotypic data will be provided to the project through current programs on frost tolerance, salinity tolerance, boron toxicity tolerance, and adaptation to low rainfall environments. A total of 16 mapping populations, including two advanced backcross populations derived from H. spontaneum, are also available to the project. The GRDC/MPB funded program “Collaborative barley breeding for low rainfall environments” is a joint initiative between ICARDA and the University of Adelaide, and provides the resources to validate the adaptive significance of cis-acting regulatory variation identified through this project.

 

- Capacity Building:

A major goal of the project is to train personnel at ICARDA, Udine and Adelaide and to initiate an outreach program for partner institutions in developing countries. This will be achieved through close interaction and alignment with subprogram 5 (Capacity Building) of the Generation Challenge Programme and will be guided be the principles and priorities articulated in the Medium-Term Plan (2005-2007). ICARDA already offers a number of training activities for plant breeders and germplasm specialists from several National Programs. Individual degree and non-degree training can be conducted in the laboratory facilities in Aleppo year round.  Furthermore, training of ICARDA scientists in Adelaide will enable technology transfer and the integration of the technology into ongoing ICARDA training

 

Adelaide University offers a number of degree and training programs ranging from undergraduate training to doctoral programs and advanced scientific training.  In particular, a new Masters in Plant Breeding will be available from 2006 and several scholarships will be available from the University Adelaide to students from developing countries. This course will have a strong focus on marker-assisted selection in plant breeding.  The University is exploring options to attracting additional masters and PhD scholarships from sources in Australia. 

 

In addition, the Australian Centre for Plant Functional Genomics will provide two six month fellowships for students or scientists from developing countries to come to Adelaide to learn the analytical techniques and take part in this program.  ACPFG will provide travel expenses and a living allowance. The University of Adelaide and the University of Udine will actively support and facilitate sabbatical appointments, internships and other types of exchanges to empower national program scientists to actively participate in this program. The University Udine will seek support from the EU to facilitate staff exchanges and visits by national program scientists. Annual workshops will be organized; specific courses will be designed to facilitate training and on-line distance learning mechanism established. The project will also provide a mechanism to allow the participants to better understand the practical challenges and needs of the National Agricultural Research System.

 

- Management Plan.

The project co-coordinator is Dr Michael Baum who has overall responsibility for the implementation and co-ordination of the project. For each collaborating center a team leader will be identified to assume responsibility for monitoring progress, report writing and to support the project co-coordinator in delivering the outcomes of the project to the Challenge Program leadership. The participating scientists have extensive experience of managing trans-national research programs in both the public and private sector.

 

- Critical assumptions and Contingency Plans.

The proposed assay measures relative steady state levels of transcripts for the two alleles present in a heterozygous individual. The differences in relative levels for the two alleles may therefore be ascribed either to different transcription rates or to different transcript stability (degradation rate). We will not be able to distinguish between the two situations but will assume that in most cases the observed differences will depend upon differential gene regulation rather than stability (Garcia-Martinez J, Aranda A, Perez-Ortin JE.  Genomic run-on evaluates transcription rates for all yeast genes and identifies gene regulatory mechanisms. Mol Cell. 2004 Jul 23; 15(2):303-13). Furthermore, differences in transcription rates may either be due to variation in regulatory elements that affect transcription rate and timing or to variation in elements that may cause differential epigenetic silencing (e.g. via methylation or through formation of antisense transcripts) of one of the two alleles. The main thrust of this proposal will focus on detecting cis acting regulatory elements rather than unraveling mechanistic processes. However, resources in the labs of Morgante and Powell are focusing on complementary areas of research and will therefore provide a strong experimental framework to support the current proposal. At present the most efficient assay for allelic imbalance is the SBE assay followed by detection on fluorescent sequencers. We will continue to review the human and mammalian genetics literature to keep abreast of any future technical developments and move promptly to ensure that the work described in this proposal is contemporary and benefits from any technological breakthroughs.

 

 

Timelines and Milestones.

2005

2006

2007

Location

Milestones

1st

2nd

3rd

4th

1st

2nd

3rd

4th

1st

2nd

3rd

4th

Activity 1: Establish robust protocol to identify and quantify frequency of cis-acting regulatory elements

X

X

X

X

X

X

X

X

X

X

X

X

Adelaide Udine

Develop genomic, expression imbalance assays Assembly of candidate genes for evaluation

Activity 2: Reciprocal F1 hybrid development & hybridity confirmed with SSRs

X

X

X

X

Adelaide ICARDA

72 F1 hydrids will be developed in year 1 in two locations

Activity 3: Design and execute factorial experiments to test the influence of various stress treatments on expression of alleles in heterozygous condition.

x

x

x

x

X

X

X

X

X

X

X

X

Adelaide Udine

Allelic imbalance assay in F1 on 24 F1s in year 1 48 in year 2,  72 in year 3.

Activity 4: Meiotically mapping cis-acting regulatory variation in doubled haploid population of barley a) CPI71284-48 x Barque-73

X

X

X

X

X

X

X

X

X

X

Adelaide

Mapping of expression profiles of 15 SNPs each year

b) Arta x H. spontaneum

X

X

X

X

X

X

X

X

X

X

ICARDA

Mapping of expression profiles of 15 SNPs each year

Activity 5: MAS for  agronomic important traits based on allelic imbalance assays.

X

X

X

X

Adelaide ICARDA

Assays developed for deployment in MAS prior to end of project.

Activty 6: Training: Allele imbalance and MAS

X

X

X

X

X

X

X

X

X

ICARDA

Annual workshop end of year 1; training course in the second year attended by 10 participants, 2 Individual trainees every year,

X

X

X

X

X

X

X

X

X

Adelaide

2 NARS scientists are enrolled in Masters course at University Adelaide

 

  Budget

 

- Budget notes:

 

- Personel:

Post-doctoral Fellow(1)  at ICARDA:  The Post-doctoral Fellow will be responsible for mapping  Budgeted according to ICARDA’s standard  budget for  a Research Associate at level RA II with 4% inflation.

 

Post-doctoral Fellow (2)  at Adelaide:  The Post-doctoral Fellow will be responsible for SNP discovery, primer design and develop genomic, expression imbalance assays togther with the Post-doc in Udine. Budgeted according to Adelaide’s standard  budget for  a Research Associate at level RA II with 4% inflation.

 

Post-doctoral Fellow(3)  at Udine:  The Post-doctoral Fellow will be responsible for SNP discovery, primer design and develop genomic, expression imbalance assays togther with the Post-doc in Adelaide. Budgeted according to Adelaide’s standard  budget for  a Research Associate at level RA II with 4% inflation.

 

 

- Capital equipment, supplies and services:

 

1) Provided by ICARDA: greenhouses, growth chamber facilities, experimental fields, ABI377 sequencer, ABI3100 sequencer, -80^oC freezer, agarose and polyacrylamid gel electrophoresis, bench top and microfuge centrifuges,

  

    Requested by the project: replacement of laser for ABI sequencer,

Supplies: commercial kits for PCR analysis, sequencing, meiotic mapping,

 

2) Provided by Adelaide: : greenhouses, growth chamber facilities, experimental fields, ABI3730 sequencer, -80^oC freezer, agarose and polyacrylamid gel electrophoresis, bench top and microfuge centrifuges,

    

     Requested by the project: Supplies: commercial kits imbalance assays, fluorescent Single-Base Extension (SBE),  (the RNeasy kit (Qiagen), first-strand cDNA using Superscript (Invitrogen), oligo d(T) random nonamer priming, MinElute columns (Qiagen),  filtration columns (Qiagen), kits for ABI377/3700/3730 sequencer,  SNaPshot Multiplex Kit (ABI)

 

3) Provided by Udine : ABI3730 sequencer, -80^oC freezer, agarose and polyacrylamid gel electrophoresis, bench top and microfuge centrifuges,

 

     Requested by the project: Supplies: commercial kits imbalance assays, fluorescent Single-Base Extension (SBE),  (the RNeasy kit (Qiagen), first-strand cDNA using Superscript (Invitrogen), oligo d(T) random nonamer priming, MinElute columns (Qiagen),  filtration columns (Qiagen), kits for ABI377/3700/3730 sequencer,  SNaPshot Multiplex Kit (ABI)

 

- International travel:

1) Provisions are  made for the travel of the ICARDA scientists to Adelaide once a year to discuss, coordinate and evaluate research activities.

 

2) Provisions are  made for the travel of 2 Adelaide scientists once a year to Udine or ICARDA to discuss, coordinate and evaluate research activities.

 

3) Provisions are  made for the travel to Adelaide once a year to Udine or ICARDA to discuss, coordinate and evaluate research activities.

 

- Appendix: Appendix  SEQ Appendix \* ARABIC 1. Partners

 

- International Centre for Agricultural Research in the Dry Areas:

 

The International Center for Agricultural Research in the Dry Areas (ICARDA), based at Aleppo, Syria is one of the 16 Centers supported by the Consultattive Group on International Agricultural Research (CGIAR). ICARDA’s research provides global benefits of poverty alleviation through production improvement integrated with sustainable natural resource management practices. ICARDA serves the entire developing world for the improvement of barley.

 

ICARDA has a long history of planning and implementing research in cooperation with its NARS partners throughout West Asia and North Africa (WANA). In addition to collaborative research and training, ICARDA has also assisted numerous NARS in administering and technically back-stopping a series of sub-regional research. ICARDA has established collaborative research programs with NARS. Germplasm development and distribution has been very successful and has led to the release of many varieties. The barley improvement project at ICARDA aims at a sustainable increase in barley productivity by adapting the crop to the different farming systems and uses in developing countries with special emphasis in those areas where the crop is grown by resource-poor farmers, thus contributing to alleviation of poverty.  To achieve its objective the project has adopted a strategy which has evolved over the last 20 years based on:

1. Conducting selection, from the early segregating populations, under farmers' conditions, i.e. without use of fertiliser, pesticides and weed control, and under strict rainfed conditions.

 

2. Using locally adapted germplasm such as landraces and Hordeum vulgare ssp. spontaneum (the wild progenitor of cultivated barley).

 

3. Testing the value of mixtures to cope with the unpredictable year-to-year variation of abiotic stresses such as low winter temperatures, low and irregularly distributed rainfall, and high temperature during grain filling.

Work conducted at ICARDA, aimed at exploiting landraces in a conventional barley breeding program for marginal environments, has shown that barley landraces from the Fertile Crescent carry useful genes to improve barley yields. At the moment three lines selected from Syrian landraces have been adopted by farmers on small scale. Within this project ICARDA will be subcontracted to conduct a series of contrasting trials in Syria and for the multiplication and distribution of seed for the Consortium.

 

- Biotech: ICARDA has a fully established molecular marker laboratory. Multifluorophore fragment analysis can be carried out on an ABI prism ^TM 377 DNA Sequencer and analyzed with GeneScan^TM analysis software version 2.0.2 and Genotyper ^TM analysis software version 2.0 (PE Applied Biosystems). A Genescan 5000 microarray reader allows microarray analysis.  ICARDA has also purchased an ABI 3100 capillary sequencer and ABI7500 real time PCR that should be in operation before the end of the year. 

 

 

- University of Adelaide:

The School of Agriculture and Wine is the center piece of the Southern Hemisphere’s largest collection of expertise in plant genomics, crop improvement, sustainable agriculture and  dryland farming,. Major research facilities embedded in the school include the CRC for Molecular Plant Breeding and the Australian Centre for Plant Functional Genomics, a node of the Australian Genome Research facility, the Australian Grain Technologies Pty Ltd, the largest wheat breeding company in the world. As such the school represents a world-class concentration of scientific research, education and infrastructure and functions across the Waite and Roseworthy campuses of the University of Adelaide.

 

The Waite Campus has historically been the centre of excellence for barley breeding in Australia. Since its inception in 1960, the UA Barley Program has released Australia’s most successful malting and feed varieties (Clipper, Schooner, Galleon, Sloop and Keel). The UA Barley Program is one of the largest integrated barley breeding and research groups in the world, and is internationally recognised as a leader in the application of new technology to plant breeding outcomes.

 

- University of Udine:

The University of Udine is a research and educational institution with approximately 15000 students, providing both undergraduate as well as graduate degrees in a variety of subjects. The Faculty of Agriculture has degrees both in Agronomy as well as in Biotechnology, Food Science and Environmental Sciences. The Department of Crop and Environmental Sciences at the University of Udine is a leader in plant science research in Italy and is active in plant genomics, plant breeding, agronomy, crop ecology and physiology, plant and soil biochemistry. In the plant genomics area the laboratory facilities include liquid handling robots, two capillary DNA sequencers, PCR machines, bioinformatics facilites. The research group of Prof. Michele Morgante is composed of 12 between graduate students and postdocs and has been active in plant genomics for quite some time, with considerable experience gained in sequence diversity analysis, physical mapping, genome evolution, repetitive DNA analysis and bioinformatics.

 

University of Tishreen

The laboratory of molecular genetic is a recent establishment created in the faculty of Agriculture at Tishreen University on 2000 with the initiative of  Prof. Wafaa Choumane, Head of the department of basic sciences, and  the support of Dr. Michael Baum from the International Center for Agriculture Research in the Dry Areas (ICARDA). Prof. Choumane, is involved in the following projects:

1- Evaluation of a barley core collection which was assembled from landraces from the West Asia North Africa (WANA) regions.

 

2- Development of microsatellite markers and their application in chickpea, lentil and pea.

 

3- Identification of dehydrin genes in barley. This approach should lead to the identification of new genes involved in drought tolerance in barley.

 

- Appendix  SEQ Appendix \* ARABIC 2 Intellectual Property Rights Statement:

 

  All Co-PIs and collaborators have agreed to the IP principles of the CP program.

 

 

- Appendix  SEQ Appendix \* ARABIC 3. CV of  PI and Co-PIs

 

 

 

- MICHAEL BAUM:

ICARDA, P.O. Box 5466 Aleppo, Syria Tel. 00963-21-2213477     e-mail: M.Baum@cgiar.org

 

Education:

         1985    Diploma in Agriculture at University of Göttingen, Germany.

         1988    Dr. agr., University of Göttingen, Germany

         1989    Post-doctoral fellow,  CSIRO, Division of Plant Industry, Canberra, Australia.

         1991    Lecturer, University of Göttingen, Plant Breeding and Cytogenetics.

         1992    Legume-Biotechnologist, ICARDA.               

         1997    to date,  ICARDA biotechnologist.

 

Awards:

         1989    Post-doctoral fellowship from German Academic Exchange Service “DAAD”.

         2000    “Frosty Hill Fellowship”, Cornell University, 20.1.2000-20.8.2000.

 

Recent refereed journal articles:

Sayed H., G. Backes, H. Kayyal, A. Yahyaoui, S. Ceccarelli, S. Grando, A. Jahoor, M. Baum (2004). New molecular markers linked to qualitative and quantitative powdery mildew and scald resistance genes in barley for dry areas. Euphytica 135: 225–228, 2004.

 

Udupa SM, Malhotra RS Baum M (2004). Tightly linked di-and trinucleotide microsatellites do not evolve in complete independence. Theor Appl Genet. 108(3):550-7.

 

Baum M, Grando S, Backes G, Jahoor A, Sabbagh A, Ceccarelli S (2003) QTLs for agronomic traits in the Mediterranean environment identified in recombinant inbred lines of the cross ‘Arta’ x H. spontaneum 41-1. (Theor Appl Genet  107:1215–1225

 

Russell JR, Booth A, Fuller JD, Baum M, Ceccarelli S, Grando S, Powell W (2003) Patterns of polymorphism detected in the chloroplast and nuclear genomes of barley landraces sampled from Syria and Jordan. Theor Appl Genet : 107: 413 – 421

 

Eujayl I, Sorrells M, Baum M, Wolters P, Powel W (2002) Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat. Theor Appl Genet (2002) 104:399–407.

 

Research interest:

doubled haploid breeding in cereals, molecular marker application: disease screening, mapping and MAS; transformation technology: in lentil and chickpeas;  identification of stress induced genes, their cloning and sequencing, Training: headquarter and in country training courses for NARES scientists.

 

 

 

- WAYNE POWELL:

School of Agriculture and Wine, University of Adelaide, Australia.

Academic Qualifications:

1974    BSc (Hons) Agricultural Botany, University College of Wales, Aberystwyth

1975 Postgraduate Certificate in Education, University College of Wales, Aberystwyth

1980    MSc (Distinction) Genetics & Plant Breeding, University College of Wales, Aberystwyth

1985    PhD Quantitative Genetics ~ University of Birmingham

1993    DSc Plant Genetic Manipulation ~ University of Birmingham

Professional Experience:

1987 – 1998    Head of Cell & Molecular Genetics Department, SCRI, Dundee, UK

1991 – 1998    Individual Merit Promotion based on research productivity

1993 – 1994    Senior Fullbright Scholar

1998 – 2000    DuPont Company, Wilmington, Delaware, USA ~ Wheat Genomics

2000 – 2004    Deputy Director & Director Post-graduate studies SCRI Dundee, UK

April 2004 -     Head, School of Agriculture and Wine, University of Adelaide, Australia

Professional responsibilities include:  Managing Director of the International Triticeae Mapping Initiative (ITMI) Office, based at SCRI: Board member of the BBSRC Initiative on Gene Function (IGF); Advisory Board member for Scottish Informatics Mathematics Biology & Statistics (SIMBIOS) Centre; member of the Programme Management Committee for the DEFRA Sustainable Arable Link Programme and Associate Editor of ‘Molecular Ecology’. Reviewing research sponsored by the Consultative Group for International Agricultural Research (CGIAR) in various centers. 

 

Research Interest:

Cereal genetics and DNA based diagnostics for trait introgression in breeding programs.

 

Selected Recent Publications:  From a total of  220 refereed publications.

1.Caldwell, K.S., Dvorak, J., Lagudah, E.S., Akhunov, E., Luo, M-C., Wolters, P. & Powell, W.  (2004). Haplotype based sequence variation at starch biosynthesis genes provides evidence for recurrent origin of wheat and its relative Aegilops cylindrica.  Genetics 167: 941-947.

 

2.Powell, W. & Langridge, P.  (2004) Unfashionable crop species flourish in the 21^st century.  Genome Biology 5: 233-240.

 

3.Caldwell, K.S., Langridge, P. & Powell, W. (2004) Comparative sequence analysis of the region harbouring the hardness locus (Ha) in barley and its collinear region in rice.  Plant Physiology (in press).

 

4.Russell, J.R., Booth, A. Fuller, J. Harrower, B. Hedley, P.E. Machray, G.C. Powell, W.  (2004). A comparison of sequence-based polymorphism and haplotype content detected in transcribed and anonymous regions of the barley genome.  Genome 47 (2): 389-398

 

5.Morgante, M., Hanafey, M., Powell, W.  (2002).  Microsatellites are preferentially associated with the non-repetitive DNA in plant genomes.  Nature Genetics 30: 194-200.

 

 

- PETER LANGRIDGE:

 

Academic Qualifications

         1974-1977           Bachelor of Science, Australian National University.

         1978-1980           Ph.D., CSIRO Plant Industry, Australian National University

 

Recent Employment History

         2003 – present     CEO, Australian Centre for Plant Functional Genomics

         1998 - present     Professor in Plant Science, University of Adelaide, Australia

         1996 - 1998         Associate Professor in Plant Science, University of Adelaide, Australia

         1990 - 1995         Senior Lecturer in Plant Science, University of Adelaide, Australia

 

Professional Activities

Editorial Boards: Theoretical and Applied Genetics (1996-present); Chromosome Research (1995-2000); Faculty 1000 (2001-present).

• 32 PhD students supervised to completion;

• Chair, ARC Biological Sciences Expert Advisory Committee (2001-present);

• Member, Gene Technology Technical Advisory Committee to OGTR (2001-present);

• Overall Planning Committee, International Triticeae Mapping Initiative (2000);

• Convener, Steering Committee of International Triticeae EST Cooperative (1999-2000);

• Chair, Organising Committee 11^th Aust. Plant Breeding Conf. (1999);

• Member Advisory Group, USA, NSF Wheat Genomics Program (1999-present);

• Research Director, CRC for Molecular Plant Breeding (1998-2002);

• Coordinator National Wheat and Barley Molecular Marker Programs (1998-2001);

• Interim Director, CRC for Molecular Plant Breeding (1997-98);

• Director, Australian Research Council's Special Research Centre for Basic and Applied Plant Molecular Biology (1995-2000);

• Member, Genetic Manipulation Advisory Committee, Planned Release and Scientific Sub-Committees (1994-2001);

Prizes and Awards:   Australian Technology Award for Excellence in a Technology Developed by a University (1998); Australian Technology Commendation for Excellence in Agrobiotechnology (1998); Steven Cole The Elder Prize for Scholarship (1997); Prize of the American Society for Enology and Viticulture (1996); Alexander von Humboldt Foundation Research Prize (1995);  Alexander von Humboldt Research Fellowship, (1994-5)

 

Selected recent publications. Total peer reviewed 52 from 1999

Huang, C., Barker, S.J., Langridge, P., Smith, F.W. Graham, R.G. (2000) Zinc deficiency up-regulates expression of high-affinity phosphate transporter genes in both phosphate-sufficient and –deficient barley roots.  Plant Physiol. 124, 415-422

 

Pallotta M.A., Graham R.D., Langridge P., Sparrow D.H.B., Barker S.J. (2000) RFLP mapping of manganese efficiency in barley. Theor. Appl. Genet. 101, 1100-1108

 

Langridge P. Lagudah E. Holton T. Appels R. Sharp P., Chalmers K. (2001) Trends in genetic and genome analyses in wheat: a review. Aust. J. Agric. Res. 52; 1043-1077

 

Ma Y.F., Evans D.E., Logue S.J., Langridge P. (2001) Mutations of barley β-amylase that improve its thermostability and substrate-binding affinity. Mol. Gen. Genet. 266; 345-352

 

Sutton T, Whitford R, Baumann U, Dong C, Able JA, Langridge P (2003) The Ph2 pairing homoeologous locus of wheat (Triticum aestivum): identification of candidate meiotic genes using a comparative genetics approach. Plant J 36, 443-456

 

 

- MICHELE MORGANTE:

Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Universita' di Udine

Via delle Scienze 208, I-33100 Udine, Italy

Ph.: +39-0432558606  Fax: +39-0432558603

Email: michele.morgante@uniud.it

 

Education:

         1987 Ph.D.  Universita' di Padova, Italy

 

Professional Experience:

         1992 – 1994   Visiting Scientist DuPont

         1994 – 1998   Assistant Professor, Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Universita' di Udine, Italy

         1998 – 2002   Senior Scientist Genomics DuPont

         2002 –           Associate Professor Dip. Scienze Agrarie E Ambientali, Universita' di Udine, Italy

 

Recent Publications:

Morgante M., Hanafey M., Powell W. 2002. Microsatellites are preferentially associated with non repetitive DNA in plant genomes. Nature Genetics, 30:194-200.

 

Hoth S., Morgante M., Sanchez J-P., Hanafey M., Tingey S.V., Chua N-H 2002. Genome-wide gene expression profiling in Arabidopsis thaliana revealed new targets of abscisic acid and largely impaired gene regulation in the abi1-1 mutant. Journal of Cell Science, 115:4891-4900.

 

Palaisa K., Morgante M., Williams M., Rafalski A. 2003 Contrasting effects of selection on sequence diversity and linkage disequilibrium at two phytoene synthase loci. Plant Cell 15:1795-806.

 

Meyers B.C., Scalabrin S., Morgante M. 2004 Mapping and sequencing complex genomes: let's get physical! Nat. Rev. Genet. 5: 578-588.]

 

Palaisa K., Morgante M., Tingey S., Rafalski A. Long-range patterns of diversity and linkage disequilibrium surrounding the maize Y1 gene are indicative of an asymmetric selective sweep. Proc. Natl. Acad. Sci. U.S.A. 101:9885-9890.

 

Research Interests:

Prof. Michele Morgante has a large experience in the development of molecular tools for genetic analysis in plants and in plant genomics.  Most recently his group in Udine has focused on genome analysis, characterization of repetitive sequences, physical mapping,  analysis of genetic variation both in coding as well as regulatory regions, association mapping. He has also been in charge of the physical mapping efforts in the genomics group at DuPont (Wilmington, USA).

 

 

 

- JASON K. EGLINTON: 

School of Agriculture and Wine, University of Adelaide – Waite Campus, PMB 1, Glen Osmond, 5064, Australia.

Tel +61 8 8303 6553  e-mail: jason.eglinton@adelaide.edu.au

 Education:

         1989-1993       B.Sc. (Hons), Flinders University, Australia.

         1994-1998       Ph.D., Department of Plant Science, University of Adelaide.

 

Professional Experience:

         1999              PostDoctoral Fellow, University of Adelaide

         2001              Barley Breeder, University of Adelaide

         2003              Barley Program Leader, University of Adelaide

 

Recent Publications:

Coventry S.J., McDonald G.K., Barr A.R., Eglinton J.K. (2003) The determinants and genome locations influencing grain weight and size in barley (Hordeum vulgare L.). Aust.J.Agric.Res. 54 (11-12): 1103-1115.

 

Eglinton, J.K., Langridge, P. and Evans, D.E. (1998) Thermostability variation in alleles of barley beta-amylase. J.Cer.Sci. 28: 301-309.

 

Ma, Y., Eglinton, J.K., Evans, D.E., Logue, S.J. and Langridge, P. (2000). Removal of the four C-terminal glycine-rich repeats enhances the thermostability and substrate binding affinity of barley ß-amylase. Biochemistry 39 (44): 13350-13355.

 

Paris, M., Jones, M.G.K., Eglinton, J.K. (2002). Genotyping single nucleotide polymorphisms for selection of barley beta-amylase alleles. Pl.Mol.Biol.Rep. 20 (2): 149-159.

 

Reinheimer, J.L., Barr, A.R., and Eglinton, J.K. QTL mapping of chromosomal regions conferring reproductive frost tolerance in barley (Hordeum vulgare L.) Theor.Appl.Genet. (In Press)

 

Research Interests:

The genetics and physiology of adaptation to abiotic stress, the application of wild relatives to crop improvement, novel molecular marker assisted selection strategies.

 

 

- Mark TESTER:

Australian Centre for Plant Functional Genomics                    Fax: + 61 8 8303 7102

University of Adelaide                                                      Tel:  + 61 8 8303 7368

Adelaide, Australia                                                          Email: mark.tester@acpfg.com.au

Education

         B.Sc.(Hons), 1984       University of Adelaide, Australia

         Ph.D., 1988                Plant Sciences, Univesity of Cambridge, UK

 

Employment

         1988-1990       Glaxo Junior Research Fellow at Churchill College, Cambridge

         1990-1992       Lecturer, Department of Botany, University of Adelaide, Australia

         1993-2000       Lecturer, Department of Plant Sciences, University of Cambridge, UK

         1994-2003       Fellow, Churchill College, Cambridge, UK

         2000-2003       Senior Lecturer, Department of Plant Sciences, University of Cambridge, UK

         2001-2003       BBSRC Research Development Fellow

         2004-               Australian Research Council Federation Fellow

 

Research Interests

Ion transport, salinity tolerance in higher plants, plant nutrition, molecular genetic control of shoot nutrient accumulation

 

Collaborative research

            Joint project with E Guiderdoni (CIRAD) developing enhancer trap rice lines

            EU RTN, ‘Novel ion channels in plants’ (with B Müller-Röber et al.)

            Lead PI, ARC Research Network, on The Australian Plant Nutriomics Network

 

Competitive Funding (1999 Onwards)  (i) ARC research network seed funding, $40,000 (ii) ARC FF, $2,700,000; (iii) BBSRC research grants, £1,190,000 ($Aus2.9m); (iv) EU collaborative grant of €961,443 ($Aus1.7m).

 

Training

PhD students: 8 past, 8 present; Postdocs: 7 past, 6 present

 

Recent relevant publications. (Total peer reviewed=63)

Demidchik, V., Essah, P. & Tester, M. (2004) Glutamate activates sodium and calcium currents in the plasma membrane of Arabidopsis root cells. Planta, DOI: 10.1007/s00425-004-1207-8

 

Tester, M. & Davenport, R.J. (2003) Na⁺ transport and Na⁺ tolerance in higher plants. Annals of Botany 91, 503-527

Essah, P.A., Davenport, R.J. & Tester, M. (2003) Sodium influx and accumulation in Arabidopsis thaliana. Plant Physiology 133: 307-318

 

Demidchik, V., Shabala, S.N., Coutts, K.B., Tester, M.A. & Davies, J.M. (2003) Free oxygen radicals regulate plasma membrane Ca²⁺- and K⁺-permeable channels in plant root cells activation by hydroxyl radicals mediates early plant stress responses. Journal of Cell Science 116: 81-88

 

Berthomieu, et al. (2003) Functional analysis of AtHKT1 in Arabidopsis shows that Na⁺ recirculation by the phloem is crucial for salt tolerance. EMBO Journal 22: 2004-2014

 

- STEFANIA GRANDO:

ICARDA, P.O. Box 5466 Aleppo, Syria, Tel. +963-21-2213477     e-mail: s.grando@cgiar.org

Education:

- MSc 1980 Agricultural Science, Institute of Plant Breeding, Faculty of Agriculture, University of Perugia, Italy.

- PhD 1986 Productivity of Crop Plants (Plant Breeding), Institute of Plant Breeding, University of Perugia, Italy.

 

Professional Experience:

Over 20 years experience in barley breeding of which the last 15 in international breeding.

1998-2004 Barley Breeder, Germplasm Program, International Center for Agricultura Research in the Dry Areas (ICARDA), Aleppo, Syria.

- 1987-1998  Research Scientist, Germplasm Program, ICARDA.

 

- 1986          Visiting Scientist, Cereal Improvement Program, ICARDA.

 

- 1985          Visiting Scientist, Cereal Improvement Program, ICARDA.

 

- 1984-1986  Lecturer in Genetics, Plant Breeding and Plant Genetic Resources, Institute of Plant Breeding, Faculty of Agriculture, University of Perugia, Perugia, Italy.

 

- 1980-1983 Accademia Nazionale dei Lincei Fellowship, Lecturer in Plant Breeding and Applied Genetics, Institute of Plant Breeding, Faculty of Agriculture, University of Perugia, Perugia, Italy.

 

Recent Publications:

Baum M, Grando S, Backes G, Jahoor A, Sabbagh A, Ceccarelli S (2003) QTLs for agronomic traits in the Mediterranean environment identified in recombinant inbred lines of the cross ‘Arta’ x H. spontaneum 41-1. (Theor Appl Genet  107:1215–1225

 

Russell JR, Booth A, Fuller JD, Baum M, Ceccarelli S, Grando S, Powell W (2003) Patterns of polymorphism detected in the chloroplast and nuclear genomes of barley landraces sampled from Syria and Jordan. Theor Appl Genet : 107: 413 – 421

 

Lakew, B., Semeane, Y., Alemayehu, F., Gebre, H., Grando, S., van Leur, J.A.G. and Ceccarelli, S., 1997. Exploiting the diversity of barley landraces in Ethiopia. Genetic Resources and Crop Evolution, 44: 109-116.

 

Ceccarelli, S. and Grando, S., 1996. Drought as a challenge for the plant breeder. Plant Growth Regulation, 20: 149-155.

 

Grando, S. and Ceccarelli, S., 1995. Seminal root morphology and coleoptile length in wild (Hordeum vulgare ssp. spontaneum) and cultivated (Hordeum vulgare ssp. vulgare) barley. Euphytica, 86: 73-80.

 

 

Research Interests:

Utilization of landraces and wild relatives in barley improvement, breeding for drought tolerance/resistance, grain quality in relation to improved nutritional value.

 

 

SALVATORE CECCARELLI:

ICARDA, P.O. Box 5466 Aleppo, Syria,  Tel. +963-21-2213477     e-mail: s.ceccarelli@cgiar.org

Education:

        - M.A.   (1964)           Plant Breeding, Inst. of Plant Breeding, Faculty of Agriculture, U. Perugia, Italy

        - Ph.D. (1967)           Applied Genetics Institute of Genetics, Faculty of Agriculture, University of Milano, Italy

 

Professional Experience: Over 30 years experience in barley breeding of which nearly 20 in international breeding.

         1970-1973  Assistant Professor of Plant Breeding, Institute of Plant Breeding, U. Perugia, Italy

         1973-1974  Research Fellow, Dept. Genetics, North Carolina State University, Raleigh, U.S.A.

         1974-1980  Assistant Professor of Plant Breeding, Inst. of Plant Breeding, U. of Perugia, Italy

         1980-1982  Forage Breeder, Pasture and Forage Improvement Program, ICARDA

         1983          Associate Professor in Plant Genetic Resources, Inst. Plant Breeding, U. Perugia, Italy  

         1984-1995  Barley Breeder, ICARDA

         1986          Full Professor in Agricultural Genetics, Inst. of Plant Breeding, U. Perugia, Italy.

         1984-1996  Barley Breeder (Project Manager) and Acting Leader of the Germplasm Enhancement Program at ICARDA

         1996 to date Barley Breeder, ICARDA

 

Recent Publications (total >140):

 

Ceccarelli, S. and Grando, S., 1996. Drought as a Challenge for the Plant Breeder. Plant Growth Regulation, 20: 149-155.

 

Akem, C., Ceccarelli, S. Erskine, W. and J. Lenné, 2000. Using Genetic Diversity for Disease Resistance in Agricultural production. Outlook on Agriculture, 29: 25-30.

 

J.R. Russell, A. Booth, J. D. Fuller, M. Baum, S. Ceccarelli, S. Grando and W. Powell. 2003. Patterns of polymorphism detected in the chloroplast and nuclear genomes of barley landraces sampled from Syria and Jordan. Theor. Appl. Genet. 107: 413-421.

 

Baum, M., Grando, S., Backes, G., Jahoor, A., Sabbagh, A. Ceccarelli, S., 2003. QTLs for agronomic traits in the Mediterranean environment identified in recombinant inbred lines of the cross 'Arta' x H. spontaneum 41 1. Theor. Appl. Genet., 107: 1215-1225.

 

Ceccarelli, S., Grando, S., Baum, M. and Udupa, S.M., 2004. Breeding for Drought Resistance in a Changing Climate. pp……….Rao and J. Ryan (ed.) 2004. Challenges and Strategies for Dryland Agriculture. CSSA Spec. Publ. 32. ASA and CSSA, Madison, WI (in press).

 

Research Interests:

Genotype x Environment Interactions, Breeding for drought resistance, stress physiology.

 

- SRIPADA M. UDUPA:

Biotechnologist

Germplasm Program, International Center for Agricultural Research in the Dry Areas (ICARDA), P.O. Box 5466, Aleppo, Syria. E-mail: s.udupa@cgiar.org

Tel:  (963-21) 2213433 / 2213477

USA direct (Tel): 1-650-833-6680

USA direct (Fax): 1-650-833-6681

 

Education:

        - B.Sc.  (1980-84) Agriculture, University of Agricultural Sciences, Bangalore, India

        - M.Sc. (1984-86) Agriculture, University of Agricultural Sciences, Bangalore, India

        - Ph.D.(1987-93)  Molecular Biology and Biotechnology, Indian Agricultural Research Institute, New Delhi, India.

 

Research Experience:

• Research Associate at the Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi - 110012, India, from Nov. 1992 to May 1993.

• Post-Doctoral Fellow (University of Frankfurt) from June 29, 1993 to June 28, 1996,

• Consultant in Molecular Biology for one month (July 1996) at ICARDA, Aleppo, Syria 

• Post-Doctoral Fellow (Molecular Biology) at ICARDA, Aleppo, Syria from Aug. 1, 1996 to Jan. 31, 1999.

• JIRCAS Visiting Fellow (molecular biology of drought stress), working in Dr K. Yamaguchi-Shinozaki’s laboratory, Japan International Center for Agricultural Sciences, Tsukuba, Japan (from Nov. 2001 to March 2002).

• Biotechnologist at ICARDA, Aleppo, Syria, since Feb. 1999

Selected publications

Udupa SM, Malhotra RS, Baum M. (2004) Tightly linked di- and tri-nucleotide microsatellites do not evolve in complete independence: evidence from linked (TA)_n and (TAA)_n microsatellites of chickpea (Cicer arietinum L.). Theoretical and Applied Genetics, 108: 550-557

 

Udupa S.M. and Baum M. (2003) Genetic dissection of pathotype-specific resistance to ascochyta blight disease in chickpea (Cicer arietinum L.) using microsatellite markers. Theoretical and Applied Genetics, 106:1196-1202

 

Udupa S.M. and Baum M. (2001) High mutation rate and mutational bias at (TAA)_n microsatellite loci of chickpea (Cicer arietinum L.). Molecular Genetics and Genomics, 265: 1097-1103.

 

Udupa, S.M., Robertson, L.D., Weigand, F., Baum, M. and Kahl, G. (1999) Allelic variation at (TAA)_n microsatellite loci in a world collection of chickpea (Cicer arietinum L.) germplasm. Molecular and General Genetics, 261: 354-363.

 

Winter, P., Pfaff, T., Udupa, S.M., Hüttel, B., Sharma, P.C., Sahi, S., Arreguin-Espinoza, R., Weigand, F., Muehlbauer, F.J. and Kahl, G. (1999) Characterization and mapping of sequence-tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome. Molecular and General Genetics, 262: 90-101.

 

Research Interests:  Application of molecular biology tools for crop improvement especially for abiotic and biotic stress tolerance.

 

 

Wafa Choumane

Department of Basic Sciences, Faculty of Agriculture, Tishreen University, P.O.Box 2099, Lattakia, Syria.Fax  No. +963 (41) 469040.  E mail: w.choumane@cgiar.org and wafaa627@scs-net.org.

 

Education:

• 1978    B.Sc. in Agriculture Sciences, Faculty of Agriculture, Tishreen University, Lattakia, Syria

• 1982    Diploma , Faculty of Agriculture, Tishreen University, Lattakia, Syria

• 1985    Diploma (D.E.A.) in Molec. Cellular Biology, U. of Claud Bernard –, Lyon, France

• 1988    Ph.D. in Molecular Biology, University of Claud Bernard – Lyon I, Lyon, France.

• 1988 - 1994: Associate professor at Tishreen University, Faculty of   Agriculture, Lattakia, Syria

• 1992 - 1993: Visiting scientist at ICARDA, Aleppo, Syria

• 1993 - 1996 : Consultant in Biotechnology , Germplasm program at ICARDA, Aleppo, Syria

• 1996 -1998: Post Doctoral Fellow in Biotechnology , at ICARDA, Aleppo, Syria

• 1997 - 1997: Visiting scientist for six month in the laboratory of molecular biology, in the Biocenter, University of Frankfurt, Germany

• 1998  - 1999: Assistant Professor in  the department of Fundamental Sciences in the  Faculty of Agriculture, Tishreen University, Lattakia, and  Consultant at ICARDA, Aleppo, Syria

• 1999 – until now: Professor and Head of the Department of Basic Sciences in the  Faculty of Agriculture, Tishreen University, Lattakia, and  Consultant at ICARDA, Aleppo,

 

Research experience:

Evaluation of a barley core collection which was assembled from landraces from the West Asia North Africa (WANA) regions, Development of microsatellite markers and their application in chickpea, lentil and pea., Identification of dehydrin genes in barley, Genetic Characterization of  a Syrian-Jordanian barley landraces collection with SSR markers.

 

Relevant publications:

Ashtar S., Choumane W., Ghazal H., and Baum M. (1999).  Evaluation of genetic variability in a barley core collection by using DNA markers. J Aleppo University. Vol: 33

 

Huttel B., Winter P., Weising K., Choumane W. Weigand F. and Kahl G. (1999) Sequence-tagged microsatellite site markers for chickpea (Cicer arietinu m L.). Genome 42: 1-8

 

Choumane W., Ashtar S. Valkoun J. and Weigand F. (1999).  The use of DNA markers in the study of biodiversity in barley. The Third International Triticeae Symposium, IPGRI, May 4-8. Aleppo, Syria.

 

Choumane W., Winter P. Weigand F. and Kahl G. (2000) .  Conservation and Variability of Sequences Tagged Microsatellite Sites from Chickpea (Cicer arietinum) within the genus Cicer. Theor Appl Genet . 101: 269-278

 

Choumane W. , P. Winter,  M.  Baum & G. Kahl (2004). Conservation of microsatellite flanking sequences in different taxa of Leguminosae. (Submitted)

 

Research interest:

Evaluation of genetic resources by molecular markers, Identification of markers involved in the drought resistance.

 

 

- Appendix  SEQ Appendix \* ARABIC 4. Letters of support:

 

 TIME \@ "d MMMM yyyy" 10 March 2005

 

Dr. Robert S. Zeigler

Program Director

The Generation Challenge Program

C/o CIMMYT

Apartado # 370

PO Box 60326

Houston, TX 77205

USA

                                                           

 

Dear Dr. Ziegler,

 

This letter certifies our institutional intent to participate with ICARDA in the pre-proposal for submission to the Competitive Grants Programme of the GENERATION Challenge Program.

 

We believe the proposed project contributes to the objectives of the Challenge Program to use plant genetic resources to improve livelihoods and increase food security in developing countries, and to broaden partnerships to efficiently and effectively utilize genetic resources in the improvement of staple crops in the developing world. 

 

We accept that the Challenge Program will be managed according to the terms set forth in a CP Consortium Agreement, and understand that full details will be made available at the time full proposals are invited.

 

In signing this letter, we understand that it forms the basis for a Memorandum of Understanding between the parties of the project proposal, should the proposal be successful. 

 

We look forward to your consideration of the proposal.

 

Yours sincerely

Professor Wayne powell

Head, School of Agriculture & Wine

 

May 10, 2004

 

 

Dr. Robert S. Zeigler

Program Director

The Generation Challenge Program

C/o CIMMYT

Apartado # 370

PO Box 60326

Houston, TX 77205

USA

                                                  

 

 

 

Dear Dr. Ziegler,

 

This letter certifies our institutional intent to participate with ICARDA in the pre-proposal for submission to the Competitive Grants Programme of the GENERATION Challenge Program.

 

We believe the proposed project contributes to the objectives of the Challenge Program to use plant genetic resources to improve livelihoods and increase food security in developing countries, and to broaden partnerships to efficiently and effectively utilize genetic resources in the improvement of staple crops in the developing world. 

 

We accept that the Challenge Program will be managed according to the terms set forth in a CP Consortium Agreement, and understand that full details will be made available at the time full proposals are invited.

 

In signing this letter, we understand that it forms the basis for a Memorandum of Understanding between the parties of the project proposal, should the proposal be successful. 

 

We look forward to your consideration of the proposal.

 

Yours sincerely,

Professor Peter Langridge

CEO, Australian Centre for Plant Functional Genomics