Abstracts – Oral Presentations.
MAPPING:
Radiation Hybrid Mapping of a Species Cytoplasm Specific Gene in Durum Wheat.
Khwaja G. Hossain1, Oscar Riera-Lizarazu2, M. Isabel Vales2, Venugopal Kalavacharal1, Schivcharan S. Maan1, and Shahryar F. Kianian1.
1Department of Plant Sciences, North Dakota State University, Fargo, ND 58105; 2Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331. (s.kianian@ndsu.nodak.edu).
Species cytoplasm specific (scs) genes restore compatibility between the cytoplasm of Aegilops longissimum (lo) and the nucleus of durum wheat. A compatible interaction results in plump seeds and male sterile progeny while an incompatible interaction results in shriveled non-viable seeds. One scsae gene located on the long arm of the 1D (1DL) chromosome of Triticum aestivum has been transferred to a durum alloplasmic line [(lo) durum]. This compatible alloplasmic line believed to contain a 1AL.1DL translocation was characterized by genomic in situ hybridization (GISH) and 27 genomic, cDNA, EST, and STS markers mapped to different regions of chromosome 1D. The absence of 8 markers suggested that the distal region of 1DL was not present in the (lo) 1AL.1DL durum line. GISH analysis suggested that the 1AL.1DL chromosome was instead a 1DS.1DL-1AL. The presence of the 1DS arm and the proximal region of 1DL were confirmed with DNA-based marker analyses. Furthermore, our analyses suggested that the 1D chromosome in (lo) 1DS.1DL-1AL might have resulted from homoeologous recombination between the distal ends of the long arms of chromosomes 1D and 1A. Because of the limited numbers of deletion stocks for the long arm of chromosome 1D and because a conventional mapping approach is not suitable for detailed analysis, localization, and cloning of the scs gene due to absence of a homologue and the very limited, if any, pairing between homoeologues, radiation hybrid mapping methodology is being used to further localize the scsae gene on the long arm of chromosome 1D. So far, characterization of 100 radiation hybrid lines with over 23 markers revealed 57 radiation-induced chromosome breaks. Radiation-based chromosome dissection coupled with increased marker saturation will allow the localization of scsae.
A composite molecular genetic map for wheat.
Rudi Appels
WA Agriculture/PMB-CRC, Murdoch University, Sth Perth, WA, Australia. (rappels@central.murdoch.edu.au).
A summary of an extensive collaboration between groups in Canada, France, Australia, USA and CIMMYT to establish a detailed comparison between the molecular genetic maps generated for 7 different wheat crosses will be provided. A significant finding for the wider use of microsatellites was the consistency in the relative order in each of the 21 chromosomes and some examples of this will be presented. Some inconsistencies were found but these were relatively few and some examples of these will also be discussed with the view to providing a focus for how best to develop a single map for wider distribution. Finally the value of a single map will also be discussed with respect to providing sources of alternate markers for a region of interest, in order to define markers that are polymorphic in a cross that may be utilised in a breeding program.
Molecular mapping of resistance to Fusarium head blight in wheat.
Hermann Buerstmayr1, Barbara Steiner1, Michaela Griesser1, Doris Lengauer1, Nives Angerer1, Lorenz Hartl2 and Marc Lemmens1.
1 IFA-Tulln, Institute for Agrobiotechnology, Department of Biotechnology in Plant Production, Konrad Lorenz Strasse 20, A-3430 Tulln, Austria; 2 Bayerische Landesanstalt für Bodenkultur und Pflanzenbau, Vöttingerstrasse 38, D-85354 Freising, Germany. (buerst@ifa-tulln.ac.at).
QTL mapping was performed in two doubled haploid populations derived from the crosses CM-82036/Remus and Frontana/Remus. The lines were evaluated for the expression of Fusarium head blight resistance traits in artificially inoculated field trials in 1999 and 2001. Both populations were genotyped with more than 350 DNA markers (SSR, AFLP, RFLP). In the CM-82036/Remus population the most significant QTL effects were detected on chromosomes 5A and 3B (Buerstmayr et al. 2002) and in Frontana/Remus on chromosomes 3A, 2B and 7A (Table 1). Our results confirm the robust QTL effect on chromosome 3BS derived from Sumai#3 (Anderson et al. 2001).
GENE EXPRESSION:
Wheat transcriptome project in Japan.
Yasunari Ogihara1, Yasue Nemoto1, Koji Murai2, Yukiko Yamazaki3, Tadasu Shin-I3 and Yuji Kohara3.
1Kihara Institute for Biological Research, Yokohama City University, Japan; 2Department of Bioscience, Fukui Prefectural University, Japan; 3National Institute of Genetics, Japan. (ogihara@yokohama-cu.ac.jp).
In order to develop functional genomics of wheat, we carried out large scale analysis of ESTs (transcriptome analysis) in common wheat. Total RNAs were extracted from ten wheat tissues, i.e., crowns of 14 days old seedlings and roots at the same stage, spikelets at early flowering and flower differentiation stages, spikes at booting stage, heading date and flowering date, pistils at flowering date, and seeds 10 and 30 days after pollination. The cDNA libraries were constructed from these RNAs. The cDNA clones were randomly picked up from these libraries, and sequenced from both 5’ and 3’ ends. At present, 115,714 sequences were accumulated. According to the PHRAP method, these sequences were classified into 25971 contigs, about one third of which stood as singletons. These contigs were, furthermore, grouped into 14,096 gene groups with the BLAST method. Since total number of genes in Arabidopsis was estimated to be ca. 27,000, about half of genes could be captured even in common wheat. About 75 % of gene groups had significant similarities to known genes. The compositions of the contigs showed library-specific distributions, reflecting gene frequencies expressed in each tissue. In these ESTs, approximately six thousands of novel genes that had not been deposited to the DNA data base, were captured. Additionally, a number of cDNA libraries which are stress-induced and/or specific tissues, are now being constructed. These data provide useful information for SNPs analysis and cDNA microarray systems of transcriptome analysis in wheat.
Gene expression in wheat spikes infected with Fusarium and Karnal Bunt.
John Fellers1, Kristi Hill-Ambroz2, Wanlong Li3, and Bikram Gill3.
1 USDA-ARS Plant Science and Entomology Unit, Manhattan, KS; 2 Li-Cor, Lincoln NE; 3 Department of Plant Pathology, Kansas State University, Manhattan, KS. (jpf@alfalfa.ksu.edu)
Expressed sequence tags have become a rapidly growing area for the identification of genes and characterization of their expression in organisms with large genomes. Gene expression can be studied at various stages of development and environmental response. We have utilized two cDNA libriaries to evaluate gene expression during infection of wheat spikes with either Fusarium graminearum or Karnal bunt. One cDNA library was made from wheat spikes of the variety ‘Sumai 3’, 24 h after inoculation with Fusarium graminearum. cDNA membrane arrays consisting of the 580 unigenes, were used to evaluate host gene expression after infection by Fusarium. Expression analysis determined that 80 of the unigenes were induced. Of these unigenes, ten are involved in defense response, nine in gene expression and regulation, 29 in other cell functions, and 32 are without a known function. The induced genes catalyze key steps in the formation of lignin, energy production, and production of phytoalexins suggesting that resistance in wheat to Fusarium is provided by a different pathway than that of the hypersensitive response. This library was combined with a second that was a subtraction library enriched for expressed genes of the resistant parent, ‘HD29’. The combined clones total 1085 unigenes. Karnal bunt infected spikes from both resistant and susceptible lines were evaluated and the results will be presented.
1Genomic decoding of plant defense pathways.
Agriculture and Agri-Food Canada, Cereal Research Centre, Winnipeg, Canada. (XingT@em.agr.ca).
Mitogen-activated protein kinase (MAPK) pathway is a key pathway in plant defense. With genomics and proteomics approaches, we studied this pathway in tomato and wheat. An array of pathogenesis-related (PR) genes were activated by tMEK2MUT at transcription level. SUMMARYOverexpression of tMEK2MUT in tomato and wheat enhanced resistance against attacks by bacterial and fungal pathogens. Two MAP kinase genes were mined out from our wheat EST databases. When wheat was challenged by wheat leaf rust, both were activated but differentially. Programmed cell death (PCD) is an active process of cellular suicide triggered by a variety of physiological and stress stimuli. We identified several PCD components in a wheat EST database. Although we found no caspases or poly(ADP-ribose) polymerases (PARPs), there are accumulative evidence that support the existence of caspase-like proteins (CLPs) and PARP-like proteins in plants. DNA laddering and terminal transferase-mediated dUTP nick-end labeling (TUNEL) analysis have revealed detailed progress of PCD during the infection. We will use the identified proteins as anchors to study protein-protein interactions.
PHYSICAL MAPPING AND LARGE INSERT LIBRARIES:
Preparation of BAC library resources for studying genome organization and cloning of interesting genes and QTLs in hexaploid wheat.
Unité de Recherches en Génomique Végétale (URGV-INRA), 2, rue Gaston Crémieux , CP 5708 , 91057 Évry cedex, France (chalhoub@evry.inra.fr)
The hexaploid wheat (Triticum aestivum L) has a very large genome (16000 Mb) that is 127 times that of the plant model A. thaliana. One of the main challenges that we have fixed is the preparation of BAC libraries resources of the huge genome of hexaploid wheat.
A BAC library of 1 000 320 clones has now been constructed (Genoplante project) using the INRA-French cultivar ‘Renan’. This library represents the largest BAC library constructed to date. Insert size ranges from 70 to 250 Kb with an average of 140 Kb (7 genome-equivalent).
In a recent INRA/John Innes Center project (collaboration with Graham Moore), another Hexaploid BAC library is now being constructed with cv Chinese Spring (500 000 from each side). Strategies and methods that have been developed for 'rapid' construction of these libraries as well as their characteristics will be presented.
We are now focusing on developing original methods and approaches for an intelligent exploitation of these resources. These libraries are open for collaborations and will be available for the scientific Community.
We believe that wheat genome organization programs as well as cloning of important genes and QTLs will be facilitated by access to these resources.
Map-based cloning in the Triticeae genomes: case studies of wheat and barley
Nils Stein1, Dragan Perovic1, Frank Ordon2, Beat Keller3, and Andreas Graner1.
1Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany, 2Institute of Crop Science and Plant Breeding I, Justus-Liebig-University, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany, 3Institute of Plant Biology, Zollikerstr. 107, CH-8008 Zürich, Switzerland. (stein@ipk-gatersleben.de).
Gene isolation in Triticeae species still depends mainly on the success and the advances in map-based cloning strategies. Since the availability of a number of different large insert libraries from barley and diploid, tetraploid and hexaploid wheats map-based cloning was proven to be a realistic goal towards the isolation of genes for agronomically interesting traits.Map-based cloning in hexaploid wheat was recently achieved by following a subgenome-specific chromosome walking strategy. A 450 kb BAC contig of the orthologous region of the wheat leaf rust resistance locus Lr10 on chromosome 1AS was established in T. monococcum - a close relative to the wheat A-genome progenitor. Candidate genes were identified and are currently tested for their biological function.The successful isolation of genes from barley involved in disease resistance has been repeatedly performed by map-based cloning or chromosome landing. We are interested in the positional cloning of the virus resistance locus ym4/5 from chromosome 3H conferring resistance to the barley yellow mosaic virus-complex. Data will be presented on the progress of the high resolution genetic and physical mapping of this target locus.
Sheri Babb, Warren Kruger, Carla Otto and Gary J. Muehlbauer.
Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108. (muehl003@tc.umn.edu).
Plant architecture is governed by the activity of meristems. Shoot apical meristems lead to the development of all above-ground structures including nodes, internodes, leaves, axillary meristems and the inflorescence. Vegetative axillary meristems form from the base of the plant and differentiate into tillers. We have collected a set of ten recessive mutations that affect vegetative axillary development. These mutants fall into three classes including: (1) mutants that do not tiller; (2) mutants that produce tillers but they are fewer in number than wildtype; and (3) mutants that produce many tillers. In addition to the vegetative phenotypes, these mutants exhibit a wide range of inflorescence phenotypes. Two recessive mutations, uniculm2 (cul2) and uniculm4 (cul4) exhibit zero and very few tillers, respectively. Histological analysis of these mutants shows that the cul2 mutant develops an axillary meristem that does not differentiate into a tiller, whereas the cul4 mutant forms abnormal axillary meristems that occasionally develop into tillers. Double mutant analysis of the cul2 mutant with the other tillering mutants shows that cul2 is epistatic to all of these mutants for vegetative axillary development, all double mutants are uniculm. However, cul2 in combination with granum (gra), a high tillering mutant, occasionally produced multiple shoots. In addition, we have observed synergistic interactions between these mutants for inflorescence development, indicating that these genes are involved in combinatorial regulation of inflorescence development. Our morphological and genetic analysis shows that vegetative and inflorescence axillary meristem development is controlled by partial overlapping functions. We have mapped cul2 with RFLPs to chromosome 6H. The map position places this gene in the vicinity of a high recombination (~0.1Mb/cM) region on the barley map, possibly facilitating positional cloning of the gene. We are using the barley BAC library and ESTs in combination with the rice genomic sequence to begin to develop a physical map of this region.
PROTEOMICS:
Combining Liquid Chromatography with Mass Spectrometry to Detect Differential Expression of Proteins in Plant-Pathogen Interactions.
Joseph M. Anderson1, Diya Ren2, Christie Williams1, Steve Goodwin1, Herb Ohm3 and Fred Regnier2.
1USDA-ARS, 2Chemistry Department, 2Agronomy Department, Purdue University, 1150 Lilly Hall, West Lafayette, IN 47907. (janderson@purdue.edu).
The reversible phosphorylation of specific proteins are involved in the regulation of many aspects of cell physiology, plant development and defense responses. However, detection of phosphorylated peptides in a complex mixture of other peptides is challenging due to their low abundance and poor MALDI-TOF ionization efficiency. In this study, by Ga(III) immobilized metal affinity chromatography is optimized, phosphopeptides are enriched and then separated by reverse phase chromatography. Identification and mapping of the phosphorylation sites on the peptides are done by MALDI or ESI/MS with database searching based on the mass and sequence comparison. The combination of global isotope stable tagging by differential labeling with acetate and trideuteroacetate and mass spectrometric peptide mass mapping can identify the up-down regulation of the phosphoproteins in pathogen treated samples. This protocol has been used to study the changes in phosphoproteins in wheat leaves treated by the leaf pathogen Septoria tritici.
A proteomic study of tomato fruit ripening based on 2D - DIGE analysis.
P. Beckett1, J.K.C. Rose2, C. Rozanas1 and R. Asbury1.
1Amersham Biosciences, 800 Centennial Avenue, Piscataway, NJ 08855; 2Department of Plant Biology, Cornell University, Ithaca. NY 14853.
Tomato fruit ripening serves as an excellent model system to understand fruit development for many reasons, including the availability of non-ripening mutants such as rin (ripening inhibitor). The rin gene acts as a key regulatory switch that coordinates a number of ripening-related signal transduction and metabolic pathways. The work presented here is the initial phase of a proteomic study using Differential In Gel Electrophoresis (DIGE) coupled with 2-D gel analysis using the DeCyder software package, to compare and quantify protein populations from the fruit of wild type and non-ripening rin tomatoes. This global study will provide insights into the regulation of fruit development and ripening as well as suggesting strategies for enhancing fruit quality traits.
1Proteomics-based analysis of MAPK signaling in plant-pathogen interactions.
Christof Rampitsch
Agriculture and Agri-Food Canada, Cereal Research Centre, 195 Dafoe Road, Winnipeg, MB, R3T 2M9, Canada. (crampitsch@em.agr.ca).
Signal transduction pathways link pathogen perception with the final execution of defence action. We are taking a proteomics-based approach to explore these complex key defence pathways and their control mechanisms systematically. Tomato (Lycopersicon esculentum) and wheat (Triticum aestivum cv AC Fielder) transformed with a constitutively expressed, modified tomato mitogen-activated protein kinase kinase (MAPKK) showed increased resistance to Pseudomonas syringae pv tomato and Puccinia triticina (leaf rust) respectively. To explore biochemical changes due to the modified MAPKK at the protein level we are using two-dimensional electrophoresis to detect expression pattern changes and western blotting with anti-phosphoserine and anti phospho-p44/42 kinase antibodies to detect phosphorylation events. Mass spectrometry is used to help identify responding proteins. Protein phosphorylation in response to the transgene is of particular interest because this is a common intracellular signal which cannot be detected by a DNA-based approach. This pathway dissection and component identification will facilitate designs to enhance the defence mechanism and the control of gene activation in biological and cellular contexts.
Richard Flavell, Max Troukan, Nickolai Alexandrov and Kenneth Feldmann Ceres-inc., Malibu, California, 90265, USA (RFlavell@ceres-inc.com).
Deriving the structure of genes from genomic sequences is problematic because of the difficulties of determining the intron/exon boundaries, the transcription start sites and the transcription termination sites. ESTs help identify expressed genes but do not reveal the full encoded protein
structures helpful in defining gene function. We have therefore made cDNA libraries from several plant species in which the proportion of full-length cDNAs is very high. Sequencing very large numbers of the 5'ends of the constituent clones enabled the longest clones to be selected for full sequencing that was done using primer walking strategies. Using this approach, large collections of full length cDNA sequences have been obtained for arabidopsis and corn, amongst other species. These cDNAs have helped annotate genomic DNA correctly, build algorithms for annotating genomic DNA better and have enabled extensive comparative gene and protein analyses between species. They have also been useful to design expression chips carrying non redundant gene features. The full length cDNAs also facilitate the rapid construction of genes in vectors for transformation of plants. Some of the results of this approach and their application to the triticeae genomes will be described.
BIOINFORMATICS:
GrainGenes and beyond: Bioinformatics tools for the wheat genome project.
Victoria Carollo1, David Matthews2, Gerard Lazo1 and Olin Anderson1.
1 USDA-ARS-WRRC, 800 Buchanan Street, Albany, CA 94710 USA; 2 Cornell University, Dept. of Plant Breeding, Ithaca, NY 14853 USA. (vcarollo@pw.usda.gov).
Sequencing efforts in the last few years have yielded an enormous wealth of data for Triticeae genomics. Although sequences are deposited in GenBank and are available to the public, further efforts by the bioinformaticists at the USDA-ARS have improved the accessibility and offer unprecedented ways to view and manipulate these data. GrainGenes, the Triticeae genome database (http://wheat.pw.usda.gov) sponsored by the USDA-ARS serves as a common portal by linking to sequence information of expressed sequence tags (ESTs), contig clustering results using textual and graphical displays of assembled ESTs, and access to the raw data in the form of trace files (electropherograms) as generated by high-throughput sequencers. This is in addition to other features provided by the GrainGenes project. Sequence records in GrainGenes are also linked to external databases including GenBank, links to the NCBI UniGene cluster sets assembled for Triticeae genera, links to TIGR tentative consensus sequences and links to the “Genome View” from Gramene. The wEST database project (http://wheat.pw.usda.gov/wEST) serves the sequence data prior to submission into GrainGenes and includes map data generated by the efforts to assign 10,000 wheat ESTs to bin maps as part of an NSF-sponsored project. The wEST-SQL database is a relational database accessible from this site that holds the data for the BLAST alignments to wheat EST sequences. An overview of the ARS-sponsored databases and tips on their utilization will be presented.
Public databases for Triticeae genomics.
Dave Matthews1 and Olin Anderson2
1Cornell University, Dept. of Plant Breeding, Ithaca, NY 14853 USA; 2USDA-ARS-WRRC, 800 Buchanan Street, Albany, CA 94710 USA (matthews@greengenes.cit.cornell.edu)
Now that wheat and barley have become a power in the world of plant genomics, there has been a little explosion of publically available sources of Triticeae data, analyzed in various ways. These include general-purpose data resources (NCBI, TIGR, Gramene, GrainGenes) and the websites of individual research projects. In the interest of helping coordinate the flow of information, these data sources will be overviewed. New developments since the last ITMI meeting in September include the Triticeae Repeat Sequence Database, several new EST assemblies, and consortia for SNP and SSR characterization. There is also a need to coordinate the EST mapping projects worldwide to avoid duplication of effort.