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