**Abstracts -- Oral Presentations.**

**MAPPING:**

**Radiation Hybrid Mapping of a Species Cytoplasm Specific Gene in Durum
Wheat.**

Khwaja G. Hossain^1^, Oscar Riera-Lizarazu^2^, M. Isabel Vales^2^,
Venugopal Kalavacharal^1^, Schivcharan S. Maan^1^, and [Shahryar F.
Kianian^1^.]{.underline}

^1^Department of Plant Sciences, North Dakota State University, Fargo,
ND 58105; ^2^Department 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 *scs^ae^* 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 *scs^ae^* 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 *scs^ae^*.

**A composite molecular genetic map for wheat.**

[Rudi Appels]{.underline}

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 Buerstmayr^1^]{.underline}, Barbara Steiner^1^, Michaela
Griesser^1^, Doris Lengauer^1^, Nives Angerer^1^, Lorenz Hartl^2^ and
Marc Lemmens^1.^

^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 Ogihara]{.underline}^1^, Yasue Nemoto^1^, Koji Murai^2^,
Yukiko Yamazaki^3^, Tadasu Shin-I^3^ and Yuji Kohara^3^.

^1^Kihara Institute for Biological Research, Yokohama City University,
Japan; ^2^Department of Bioscience, Fukui Prefectural University, Japan;
^3^National 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 Fellers]{.underline}^1^, Kristi Hill-Ambroz^2^, Wanlong Li^3^, and
Bikram Gill^3^.

^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.

Number range CHAPTER**Genomic decoding of plant defense pathways**.

# [Tim Xing]{.underline} 

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 *tMEK2^MUT^* at transcription level.
**SUMMARY**Overexpression of *tMEK2^MUT^* 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.**

## Boulos Chaloub

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 Stein^1^]{.underline}, Dragan Perovic^1^, Frank Ordon^2^, Beat
Keller^3^, and Andreas Graner^1^.

^1^Institute of Plant Genetics and Crop Plant Research (IPK),
Corrensstr. 3, D-06466 Gatersleben, Germany, ^2^Institute of Crop
Science and Plant Breeding I, Justus-Liebig-University,
Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany, ^3^Institute 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.

# Genetic and morphological analysis of branching in barley.

Sheri Babb, Warren Kruger, Carla Otto and [Gary J.
Muehlbauer.]{.underline}

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. Anderson]{.underline}^1^, Diya Ren^2^, Christie Williams^1^,
Steve Goodwin^1^, Herb Ohm^3^ and Fred Regnier^2^.

^1^USDA-ARS, ^2^Chemistry Department, ^2^Agronomy 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. Beckett]{.underline}^1^, J.K.C. Rose^2^, C. Rozanas^1^ and R.
Asbury^1^.

^1^Amersham Biosciences, 800 Centennial Avenue, Piscataway, NJ 08855;
^2^Department 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.

**Number range CHAPTERProteomics-based analysis of MAPK signaling in
plant-pathogen interactions.**

[Christof Rampitsch]{.underline}

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.

### Full length cDNAs and genome analysis

[Richard Flavell]{.underline}, Max Troukan, Nickolai Alexandrov and
Kenneth Feldmann Ceres-inc., Malibu, California, 90265, USA
([RFlavell@ceres-inc.com]{.underline}).

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 Carollo^1^]{.underline}, David Matthews^2^, Gerard Lazo^1^ and
Olin Anderson^1^.

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 Matthews]{.underline}^1^ and Olin Anderson^2^

^1^Cornell University, Dept. of Plant Breeding, Ithaca, NY 14853 USA;
^2^USDA-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.