Session 3 Breeding Methodologies II Genomics

Oral Presentation

New Insights into the Organization and Evolution of Genomes
in the Tribe Triticeae

J. Dvorak

Department of Agronomy and Range Science, University of California, Davis, CA 95616, USA,
E-mail: jdvorak@ucdavis.edu

Comparative linkage mapping suggested unexpectedly high conservation of synteny across the tribe Triticeae and the grass family, leading to suggestions that, for the purpose of gene discovery and isolation, cereal genomes can be treated as a single collinear entity. To obtain better assessment of synteny and to investigate organization of the gene repertoire in wheat, gene loci detected by 8000 EST unigenes were mapped into 159 bins covering the entire lengths of the wheat chromosomes. Recombination rates along wheat chromosome arms were shown to increase with the square of the distance from the centromere. A number of genomic parameters correlated with recombination rate, suggesting that recombination rate has played the central role in the evolution of genomes in the tribe. Synteny between the collinear wheat homoeologous chromosomes correlated with recombination rate; it was the highest in the proximal chromosome regions and declined in the distal direction. In distal regions, orthologues could be found for only 90 to 92% of loci. Locus deletions and duplications that were fixed during the evolution of wheat diploid ancestors were the principal causes of synteny erosion. The unexpectedly high rate of gene turnover and divergence of gene repertoire in otherwise collinear chromosomes argues for caution in assuming synteny for genes in the high-recombination regions, particularly for distantly related species within the tribe Triticeae and the grass family.

Current Perspectives in Barley Genomics (2004)

R. Waugh, D. G. Caldwell, N. Rostoks, A. Druka, I. Druka, D. F. Marshall,
G. Muehlbauer, J. R. Russell and L. Ramsay

Genome Dynamics, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK,
E-mail: rwaugh@scri.sari.ac.uk

Over the last three years significant advances have been made in the development of genomics tools and resources that will facilitate barley research in the coming decades. Uniquely for a major crop plant species, these resources are almost fully in the public domain and accessible to all barley researchers worldwide to explore and utilise in their own research programs. In this presentation, through contributions from several major barley genomics groups, I will summarise the advances that have been made over the last few years and give examples of how these have been and are being utilised to address a range of biological questions and to develop a fundamental understanding of the barley genome, its organisation and dynamics.

MAP-Based Cloning in Barley: Coming of Age

A. Kleinhofs

Department of Crop and Soil Sciences and School of Molecular Biosciences,
Washington State University, Pullman, WA 99164-6420, USA,
E-mail: andyk@wsu.edu

Map-based cloning in barley has a brief and recent history. The published work will be reviewed. In my laboratory, the traditional high-resolution genetic mapping and BAC contig development resulted in cloning of the stem rust resistance gene Rpg1 and the candidate genes for rpg4 and RpgQ. Synteny with rice provided valuable markers, but chromosome walking was required to close the BAC contig. A saturation mapping approach, employing low-resolution genetic mapping combined with physical mapping, was attempted. Synteny with rice was again used. While the region of interest was highly saturated with genetic markers, the identified BAC clones failed to form a contig around the gene of interest, in this case the spot blotch resistance gene Rcs5. Chromosome walking was employed to close the contig. The examples of barley genes cloned by the map-based approach indicate feasibility, but required extensive work and a bit of luck. They also illustrate the usefulness and limitations of rice synteny. A third approach was opened by the development of the 22,700-gene Affymetrix Barley1 gene chip. We used the Barley1 chip to compare gene expression levels in a fast neutron induced mutant and its parent cultivar Morex. Three candidate genes were identified by highly down-regulated gene expression. Low-resolution mapping should differentiate among genes down-regulated because of limited transcription of the gene of interest and chance mutations elsewhere in the genome or down-stream effects. Future prospects and limitations of phenotype-based cloning in barley will be discussed.

Poster Presentation

Global Gene Expression in Seed Germination
and Seedling Growth

Y.-Q. An

US Department of Agriculture, Cereal Crops Research Unit, Madison, WI 53726, USA,
E-mail: ycan@wisc.edu

Seed germination and seedling growth are important biological processes in agriculture and malting industry. A new research project was initiated to identify the genes important to the processes and related signal transduction pathways. Microarray technologies will be employed to examine global gene expression in wild type and mutated Arabidopsis and barley plants. The candidate genes will be identified by comparing gene expression profiling data from plant tissues at different physiological conditions and genetic backgrounds. The functions of the candidate genes will be further validated using genetic approaches in Arabidopsis and barley. The gene expression profiling data, selection of candidate genes and transcriptional regulation of the seed germination and seedling growth will be presented and discussed.

Construction of BAC Contig for Row Type Gene (vrs1)
in Barley

P. Azhaguvel, H. E. Congfen, B. E. Sayed-Tabatabaei, K. Tanno
and T. Komatsuda

Genetic Diversity Department, National Institute of Agrobiological Sciences, Tsukuba 305 8602, Japan, E-mail: azhaguvelu@hotmail.com

Development and fertility of lateral spikelets is mainly controlled by the vrs1 locus on the long arm of 2H chromosome, with the flanking markers of cMWG699 and MWG865. With the advent of genomic libraries with yeast artificial chromosome (YAC) or bacterial artificial chromosome (BAC), it will be possible to delimit the gene physically on the chromosome. The AFLP marker enrichment and later converted into STS, a comparative high resolution genetic map of vrs1 using six different mapping population, identified the closest flanking markers with the distance of 0.05 cM each on distal and proximal in a F2 population of Azumamugi × Golden Promise. This highly resolved mapping population made the map-based cloning of vrs1 feasible. In this study, we are constructing a contig covering the vrs1 region utilizing a barley BAC library. Our work mainly consists of PCR screening of BAC library, HindIII fingerprinting, BAC end sequencing and development of new PCR markers. Chromosome walking is a challenging task, especially considering that barley genome contains abundant repeated sequences and retrotransposans. So whenever we get the repeated sequences at BAC ends, sub-terminal HindIII-digested BAC DNA was subcloned and then used for sequencing to get new markers. Presently we had made over-lapping contig of covering AFLP3 and AFLP5 loci. We are constructing a physical map spanning 0.1 cM distance between flanking markers, and presently we aliened more than 20 bacterial artificial chromosome (BAC) clones around the vrs1 locus.

A Functional Genomics Approach to Study Phosphate Allocation
in Wheat and as a Tool for Risk Assessment of Transgenic Wheat

H. Brinch-Pedersen, P. Gregersen, G. Dionisio and P. B. Holm

Department of Plant Biology, Research Centre Flakkebjerg, Danish Institute of Agricultural Sciences,
DK-4200 Slagelse, Denmark
E-mail: henrik.brinch-Pedersen@agrsci.dk

The developing grain represents the major sink for phosphate in cereals. Seeds typically accumulate several folds more P than needed for supporting basic cellular functions in order to ensure adequate reserves for the germinating seed. About ~75% of seed P is phytic acid. It is apparent that phytic acid synthesis and deposition of phytic acid in vacuoles in the aleurone constitute the major sink pathway for P while the supply to the developing endosperm is limited. In order to study the allocation of P in developing wheat we have developed a 9k wheat cDNA microarray based on the unigene set of the Functional Genomics group at the University of Bristol (http://www.cerealsdb.uk.net/index.htm). The studies include microarray analysis of expression profiles in response to P starvation and supply. Moreover the array serves as a tool for revealing unintended side effects in transgenic wheat. Initially on transgenic wheat plants with heterologous expression of microbial phytases for improving P bioavailability in non-ruminant diets.

Segregation Analysis of SSR Markers and Two Morphological Traits in a Doubled-Haploid Population of Barley

M. Cherif, S. Hamza and M. Harrabi

Sciences of Vegetal Production Department, Institut National Agronomique de Tunisie, Tunisie,
1082 Cite Mahragene, Tunis, Tunisia,
E-mail: hamza.sonia@inat.agrinet.tn

Fifty nine doubled-haploid barley (Hordeum vulgare L) population obtained from a cross between the Tunisian cultivar Roho and the local line 90 was analysed using eighteen polymorphic simple sequence repeats (SSRs) markers localised on chromosomes 3, 4 and 6 (Ramsay et al. 2000). Thirteen SSR loci indicated a good fit to a 1:1 segregation ration. Among five distorted segregation three were localised on chromosome 3. However, this distortion may be due to small size of the population screened. The morphological traits, row number and stem colour were observed. Two-genes (3:1) and one-gene (1:1) segregation ratios were observed for the row number and stem colour respectively. No linkage was observed between SSR markers and lmorphological traits using correlation analysis. Sinteny was observed between SSRs linkage group and the mapping data obtained by Ramsay et al (2000). However, the multiloci marker Bmac 144 showed 1:1 segregation without linkage with the SRR markers mapped on chromosome 6.This is a preliminary genetic map result for QTL identification of resistance and agronomic traits for which the parents differentiated.

Ac/Ds Transposon-Mediated Gene Tagging in Barley

L. D. Cooper1, L. Marquez-Cedillo1, J. Singh2, A. K. Sturbaum3, S. Zhang2, V. Edwards3,
K. Johnson4, A. Kleinhofs4, S. Rangel2, V. Carollo5, P. Bregitzer3, P. G. Lemaux2
and P. M. Hayes1

1Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA,
E-mail: patrick.m.hayes@oregonstate.edu; 2Department of Plant and Microbial Biology, University
of California, Berkeley, CA 94720, USA; 3National Small Grains Germplasm Research Facility,
USDAARS, Aberdeen, ID 83210, USA; 4Department of Crop and Soil Sciences and School of Molecular Biosciences, Washington State University, Pullman, WA 99164-6420, USA; 4Western Regional Research Center, USDAARS, Albany, CA 94710, USA

A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley (Hordeum vulgare subsp. vulgare). The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics. AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation. Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location. The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR. Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 17 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions. The principal strategy was to identify and map sequence polymorphisms in the region corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population. The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines. In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex genomic BAC library inserts, thus integrating barley genetic and physical maps. BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences. Transposon tagging in barley using Ac/Ds promises to provide a useful tool for Triticeae functional genomics.

Comparison of the Waxy Locus Sequence from a Non-Waxy Strain and Two Waxy Mutants in Barley and SNP Detection by PCR-CTPP Procedure

E. Domon1, T. Yanagisawa2, A. Saito1 and K. Takeda3

1National Agricultural Research Organization, KONARC, 2421 Suya, Nishigoshi, Kikuchi,
Kumamoto 861-1192, Japan, E-mail: domon@affrc.go.jp; 2National Agricultural Research Organization, WeNARC, 1-3-1 Senyu, Zentsuji, Kagawa 765-8508, Japan; 3Research Institute for Bioresources,
Okayama University, 2-20-1, Chuo, Kurashiki, Okayama 710-0046, Japan

Three alleles of the barley waxy gene from a non-waxy strain, an indigenous waxy strain and an artificially induced waxy mutant strain were compared via a PCR direct sequencing. Compared to a non-waxy gene sequence in the public DNA database, the 5,190-bp region of a non-waxy strain had 110 polymorphic sites. The indigenous waxy and non-waxy allele were substantially identical but for a 418-bp deletion in the 5 non-coding sequence of the former allele. Induced waxy mutant had a base substitution of a C to T in the exon 5, which converted Gln-89 into a stop codon. The non-waxy and induced waxy mutant alleles with single nucleotide polymorphisms (SNP) could be correctly identified using PCR with confronting two-pair primers (PCR-CTPP). The PCR-CTPP is a simple and timesaving SNP genotyping procedure that produces allele-specific PCR products with different size, which inherited in a co-dominant manner. Segregation of the SNP was also detected by PCR-CTPP in an F2 population, which fitted the expected 1:2:1 ratio. The PCR-CTPP procedure could provide an alternative to the derived cleaved amplified polymorphic sequence in marker-assisted selection.

Parallel Gene Expression Analysis of Barley and Wheat Development

A. Druka1, G. Muehlbauer2, R. Wise3, T. Close4, A. Kleinhofs5, P. M. Hayes6, A. Graner7,
A. H. Schulman8, P. Langridge9, K. Sato10 and R. Waugh1

1Genome Dynamics, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland,
E-mail: adruka@scri.sari.ac.uk; 2Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA; 3Corn Insects and Crop Genetics Research, USDA-ARS, and Department of Plant Pathology, Iowa State University, Ames, Iowa 50110-1120, USA; 4Department of Botany and Plant Sciences, University of California, Riverside, CA 92521-0124, USA; 5Department of Crop and Soil Sciences and Genetics and Cell Biology, Washington State University, Pullman, WA 99164-6420, USA; 6Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA; 7Institute
of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Germany; 8Department Crops
and Biotechnology, MTT Agrifood Research Finland, Plant Production Research Unit, FIN-31600 Jokioinen, Finland; 9University of Adelaide, Plant Science, Waite Campus, Glen Osmond, SA 5064, Australia; 10Research Institute for Bioresources, Okayama University, Okayama 710-0046, Japan

Transcriptional profiles of 22,786 barley genes at 15 developmental stages throughout the barley and wheat life cycle will be presented. The main goal of this experiment is to provide reference data set for future parallel gene expression analysis experiments. Additionally, identification of immediate genetic targets for developmental and tissue-specific studies, hypothesis generation for functions of unknown genes and comparative expression profiling of barley and wheat is also addressed. The implications of the experimental design, technical procedures and the methodology of the data analysis will be discussed.

Allele-Specific Markers and Molecular Diversity at the Bmy1 Locus Determining Enzyme Thermostability

P. E. Eckstein1, C. D. Li2, D. Hay1, B. G. Rossnagel1, R. C. M. Lance2 and G. J. Scoles1

1Department of Plant Sciences/Crop Development Centre, University of Saskatchewan, Saskatoon,
SK S7N 5A8, Canada, E-mail: peter.eckstein@usask.ca;
2Western Australia Department of Agriculture, South Perth, WA 6151, Australia

The Bmy1 locus on chromosome 4H determines enzyme activity, thermostability, isoenzyme form, and free/bound enzyme ratio. The enzyme can be classified into types Sd1 and Sd2, with a sub-group (Sd2-H) being more thermostable. Based on DNA sequence comparisons of the beta-amylase gene from the Canadian Sd1 cultivar Harrington and sequences (Genebank) from several other Sd1 and Sd2 cultivars, allele-specific PCR based markers were constructed for each of six amino acid (AA) substitutions. PCR markers that identify variation in a microsatellite region, a 126bp palindromic insertion/deletion (I/D), and a 19bp I/D, all located in intron III of the gene, were also developed. Analysis of 107 cultivars from Australia, Canada, China, Europe, Mexico (CIMMYT), and the USA, revealed that the Sd1 and Sd2 isoenzyme types are determined by 4 of 6 AA substitution markers which co-segregate with the 19bp I/D. Only one AA substitution marker and the microsatellite marker show variability within Sd1 types. Divergence within Sd2 types was greater with 4 of 9 markers showing variability. All known Sd2-H cultivars could be clustered according to the amino acid present at position M527 and the absence of the 126bp I/D, suggesting that isoenzyme type and thermostability are influenced by regions within intron III. In addition, several Canadian cultivars have the same Bmy1 sequence as thermostable cultivars.

A Simple and Effective Procedure for Molecular Marker-Assisted Screening

P. E. Eckstein, D. Hay, B. G. Rossnagel and G. J. Scoles

Department of Plant Sciences/Crop Development Centre, University of Saskatchewan, Saskatoon,
SK S7N 5A8, Canada,
E-mail: peter.eckstein@usask.ca

Molecular markers are tools for the indirect selection of traits in breeding populations. To be relevant, markers must demonstrate benefits in accuracy or cost over conventional screening methods. We present a simple, accurate, and cost effective MMAS approach that can be adapted to breeding programs of various sizes and capabilities. DNA is isolated from seedlings germinated in an 8 × 12, 96-well format, that is preserved throughout the process. Template preparations are carried out in 96-well low-profile microplates sealed with a pierceable foil to allow for multi-channel pipetting. The preparations involve the addition of sodium hydroxide, hydrochloric acid, and Tris solutions with two brief incubations. The released DNA is of sufficient quality for repeatable analysis with SCAR, RAPD, or microsatellite markers, and can be stored for several weeks for re-analysis or analysis with other markers. PCR reactions are carried out in 96-well, heated lid thermal cyclers, and PCR products are visualized on agarose gels with 96 wells spaced suitably for multi-channel pipetting. Cost per sample is estimated at less than $ 2.00 (CDN) including all costs for materials (including Taq polymerase), and labour. These costs are further reduced when screening the same DNA with additional markers either in separate or multiplexed PCR reactions. The procedure allows for the screening of 1,000 breeding lines per technician week.

Advanced Backcross QTL Analysis in Barley

J. K. Eglinton1, S. J. Coventry1, D. E. Mather2, J. Kretschmer1, G. L. McMichael1
and K. J. Chalmers1

1School of Agriculture and Wine, University of Adelaide, Glen Osmond, SA 5064, Australia,
E-mail: jason.eglinton@adelaide.edu.au;
2Department of Plant Science, McGill University, Ste-Anne-de-Bellevue, QC H9X 3V9, Canada

An advanced backcross population was constructed from H. spontaneum (accession CPI 71284-48) and the well-adapted Australian feed variety Barque as the recurrent parent. 19 BC1F1 individuals were each backcrossed to Barque. The subsequent 19 BC2F1 individuals were used to generate separate doubled haploid populations. The final combined population comprised 370 DH lines, which was reduced to 325 after selection against brittle rachis. An F2 population was also developed from the H. spontaneum × Barque F1. Genotyping was completed with 122 SSR markers. The F2 derived population was genotyped with the same markers to provide an accurate linkage map, and the BC1 and BC2 generations were also genotyped to examine recombination of H. spontaneum linkage blocks through the backcrossing process. An analysis of the genetic structure of this population will be presented and the implications for introgression from H. spontaneum discussed. The AB-QTL population was evaluated in summer nursery rows in 2001/02, in yield trials at 2 low rainfall sites in southern Australia in 2002 and 2003. A range of agronomic and production traits, and NIR predicted malting quality traits were assessed. Markers linked to deleterious wild barley traits were identified, including head shattering, long peduncle, and free threshing ability. QTL analysis identified H. spontaneum alleles associated with improved grain yield and disease resistance. The application of AB-QTL analysis in genetic analysis and barley breeding will be discussed.

Changes in SSR Alleles Frequency and Diversity Index
in a Decentralized-Participatory Barley Breeding Program

F. Fufa1, M. Baum2, S. Grando2 and S. Ceccarelli2

1Sinana Agricultural Research Center, Bale-Robe, Ethiopia; 2Germplasm Program, International
Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria,
E-mail: m.baum@cgiar.org

A total of 181 barley entries, 105 fixed genotypes and 76 heterogeneous populations, were grown in seven locations in trials planted in farmers fields in Jordan. The changes in allele type, allele frequency and genetic diversity due to selection by individual farmers and breeders were assessed using Simple Sequence Repeats (SSRs) during one cycle of selection in a program of Decentralized Participatory Barley Breeding (DPBB).The average number of alleles retained per locus after the one cycle of selection at almost all of the locations was significantly lower than the number of alleles in the original population of entries. The reduction in diversity indices was not as high as the reduction in the number of alleles. The allelic composition and the diversity level of the populations were maintained after one cycle of decentralized-participatory selection regardless whether breeders or farmers conducted the selection. This study demonstrates the importance of decentralized participatory plant breeding in maintaining genetic diversity.

QTLs for Straw Traits Identified in Recombinant Inbred Lines
of the Cross Arta × Hordeum spontaneum 41-1

S. Grando1, M. Baum1, S. Ceccarelli1, A. Goodchild2, F. Jaby El-Haramein1, A. Jahoor3
and G. Backes3

1Germplasm Improvement Program, ICARDA, Aleppo, Syria, E-mail: m.baum@cgiar.org;
2Natural Resources Management Program, ICARDA, Aleppo, Syria; 3Plant Biology and Biogeochemistry Department, Risoe National Laboratory, DK-4000, Roskilde, Denmark

A genetic linkage map has been developed for recombinant inbred lines (RILs) derived from the cross Arta × Hordeum spontaneum 41-1.194 RILs randomly chosen from a population of 494 RILs were mapped with 189 markers including one morphological trait (btr = brittle rachis locus). The linkage map extended to 890 cM. Straw samples from RILs grown at Tel Hadya and Breda ICARDAs research stations in the years 1996/97 and 1997/98 were analysed by NIRS for acid detergent fiber, neutral detergent fiber, lignin, dry organic matter digestibility, voluntary intake, and crude protein. QTL analysis was performed using QTL cartographer software package and QTLs were localised. For acid detergent fiber, none of the identified QTLs was common between the environments. However, for neutral detergent fiber, lignin, dry organic matter digestibility, and crude protein one of the identified QTLs each was common between the two environments. There were four main locations within the barley map where a number of QTLs were clustered. The 1H-5-8 interval had 7 QTLs, the 2H-3-6 interval 6 QTLs, the 7H-5-8 interval 5 QTLs, and the 5H-5 interval 6 QTLs. The identified QTL locations could be used to initiate marker-assisted selection for straw quality traits.

AB-QTL Analysis in Spring Barley

M. v. Korff, H. Wang, J. Leon and K. Pillen

Department of Crop Science and Plant Breeding, University of Bonn, D-53115 Bonn, Germany,
E-mail: k.pillen@uni-bonn.de

The advanced backcross QTL analysis (Tanksley & Nelson 1996) was proposed as a method of combining QTL analysis with variety development. It is tailored for the discovery and transfer of valuable QTL alleles from unadapted donor lines into elite cultivars. The present AB-QTL analysis utilizes exotic germplasm of Hordeum vulgare ssp. spontaneum (Hsp) for the genetic localization of QTL in barley. QTL-analysis is followed by selection and transfer of favourable Hsp alleles into QTL-NILs. Two spring barley populations with altogether 400 BC2DH lines resulting from crosses of the two malting varieties Scarlett and Thuringia with the Hsp accession ISR42-8 from Israel are genotyped with approximately 100 SSR markers. QTL analysis is carried out for agronomic traits, malting quality parameters, and pathogen resistance evaluated under two nitrogen regimes, at four locations in Germany and in two consecutive years. Candidate lines for the development of QTL-NILs are selected based on the presence of the donor interval and a low remaining proportion of other donor segments. A QTL-NIL library will be constructed where each line carries a different introgressed region of the donor genome. These QTL-NILs serve for the verification of QTL effects and provide a valuable resource for the unravelling of gene function, e.g. by expression profiling or map-based cloning.

Effect of Vrn-H2 Vernalization Response Locus (4H) on Plant Development in Winter Barley Mapping Population

I. Karsai1, K. Meszaros1, P. Szuecs1, T. FilichkIn2, P. M. Hayes2, L. Lang1 and Z. Bedoe1

1Agricultural Research Institute of the Hungarian Academy of Sciences, H-2462 Martonvásár, Hungary,
E-mail: karsai@mail.mgki.hu;
2Department of Crop and Soil Sciences, Oregon State University, Corvallis, OR 97331, USA

A significant effect of alleles at the Sh locus on plant development was identified in the Dicktoo (D) × Kompolti korai (KK) winter barley mapping population in controlled environment tests. This locus explained more than 60% of the phenotypic variance in first node appearance and heading date. Lines carrying the KK allele headed 99.7 and 29.4 days later in unvernalized and vernalized treatments, respectively. In addition to the vernalization response, photoperiod sensitivity was also mapped to this locus with a LOD score of 14.3. Allele effects became significant at photoperiods longer than 12 hours and reached their highest levels at photoperiods of 16 and 18 hours: the D allele accelerated plant development. Alleles at the Sh locus influenced other plant developmental and agronomic traits. Effects on plant height, node number and spikelet number were similar to thos characteristic of photoperiod response loci, and the effects on frost tolerance and tillering were similar to those described for the Sh2 vernalization response locus. Under autumn sown field conditions this locus was not a primary determinant of heading date as it explained 16.5% of the phenotypic variance; lines with KK alleles were 2.5 days earlier to head than lines with D alleles.

Molecular Analysis of T-DNA Integration into the Barley Genome

M. Lange, M. G. Moeller, E. Vincze and P. B. Holm

Department of Plant Biology, Danish Institute of Agricultural Sciences, DK-4200 Slagelse, Denmark,
E-mail: mette.lange@agrsci.dk

Several studies have shown that barley can be transformed routinely with Agrobacterium. However, the technique still has a number of shortcomings. The transformation frequencies are moderate, often more than one T-DNA copy is inserted and vector backbone is integrated into the barley genome on a regular basis. In our experiments barley immature embryos were transformed with pVec8-GFP, using hygromycin as selectable marker and the gene for green fluorescent protein (GFP) as reporter. The transformation frequency ranged from 222%. It was apparent that the growth conditions of the donor plants were major determinants for the transformation frequencies. Integration of T-DNA and non-T-DNA parts into the barley genome was investigated. Southern blot analysis of GFP expressing lines showed that 66% had single copy integrations while two to five copies were found in the remaining 34% of the lines. We also found examples of that one embryo could give rise to several transformants with different integration patterns, indicating multiple transformation events in a single embryo. PCR screening for the presence of the vector backbone spectinomycin gene in the barley genome was performed on 191 hygromycin-resistent independent transgenic plants. Twenty-six percent of the lines were positive in this screen, but preliminary southern blot analyses indicate a lower frequency.

Cloning of a WRKY Transcription Activator Involved
in Cold and Drought Response in Barley

C. Mare, E. Mazzucotelli, G. Dalfino and L. Cattivelli

Experimental Institute for Cereal Research, 29017 Fiorenzuola dArda (PC), Italy,
E-mail: l.cattivelli@iol.it

A cDNA clone encoding a WRKY transcription factor was isolated in barley in response to low temperature exposure. This transcript was characterised by an early and rapid expression in comparison with other known barley cor genes usually detectable only after 24 h of cold treatment. This gene encodes for a 38kDa protein homologous to Avena fatua ABF2 a member of the large WRKY (pronounced worky) family of plant transcription factors. The barley gene, named corWRKY38, is characterised by the WRKY domain together with a zinc-finger-like motif and a leucine zipper sequence. During hardening at +2°C the corWRKY38 expression is rapid and transient and achieves its highest level after 12 hours in the leaves and 5 hours in the roots. corWRKY38 mRNA accumulation is promoted by drought treatment but not affected by ABA. Gel mobility shift assay experiments demonstrated that an in vitro synthesised WRKY38 protein binds oligonucleotides containing two W-box motifs suggesting that corWRKY38 can potentially act as transcription factors also in vivo. This work provides the first evidence of involvement of WRKY transcription factors in cold-and drought-stress response.

Use of SSR Marker Data to Study Linkage Disequilibrium
and Population Structure in Hordeum vulgare: Prospects
for Association Mapping in Barley

D. E. Mather1, P. M. Hayes2, K. J. Chalmers3, J. K. Eglinton3, I. Matus4, K. L. Richardson2,
J. von Zitzewitz2, L. Marquez-Cedillo2, P. Hearnden3 and N. Pal1

1Department of Plant Science, McGill University, Ste-Anne-de-Bellevue, QC H9X 3V9 Canada,
E-mail: diane.mather@mcgill.ca;
2Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA;
3Molecular Plant Breeding CRC and School of Agriculture and Wine,
University of Adelaide, Glen Osmond, SA 5064, Australia; 4Instituto de Investigaciones Agropecuarias,
CRI Quilamapu, 426 Chillán, Chile

Random samples of lines developed for genetic mapping are in extreme linkage disequilibrium (LD) and have no population structure. In contrast, non-random sets of existing cultivars, breeding lines or accessions have unknown LD and may have complex structure. We examined simple-sequence repeat (SSR) marker information for several such germplasm sets. LD was high among cultivars and lines of cultivated barley (H. vulgare ssp. vulgare) and low among accessions of H. vulgare ssp. spontaneum. Within diverse sets of barley germplasm, there was frequent LD even among non-linked loci, suggesting that, in such sets, association mapping would have a high rate of Type-I error. Among subsets representing known germplasm groups, LD between non-linked loci was greatly reduced while LD among closely linked loci was largely maintained. Similar effects were obtained using clusters identified by model-based analysis of population structure. With sufficient marker density and control of population structure, patterns of LD among loci in barley may be appropriate for association mapping of trait loci.

Genetic Study of Adaptational Traits in a Set of Winter Barley Varieties (Hordeum vulgare L.) Using Molecular Markers

K. Meszaros1, I. Karsai1, P. M. Hayes2, L. Lang1 and Z. Bedoe1

1Agricultural Research Institute of the Hungarian Academy of Sciences, H-2462 Martonvásár, Hungary,
E-mail: klara@mail.mgki.hu;
2Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA

Molecular markers linked to genes determining heading date, vernalization response and photoperiod sensitivity were identified based on controlled environment phenotyping tests in a set of 17 winter barley varieties. The genomic locations of 25 significant markers, as identified via marker locus-phenotype regression analysis, were determined by linkage mapping in two populations. Loci explaining more than 40% of the phenotypic variation among varieties were located on chromosomes 1H, 3H, 5H and 7H. Photoperiod sensitivity loci were identified on chromosomes 1H, 3H and 7H, with the largest single marker effect on chromosome 1H (R2 = 0.542). Vernalization response showed significant correlations with markers on chromosomes 1H, 3H, 5H and 7H with the largest single marker effect on chromosome 3H (R2 = 0.637). Varieties representing special adaptational features carried rare marker alleles with higher frequencies. The three photoperiod insensitive Eastern European varieties with large vernalization responses (Rex, Rodnik, Kompolti korai) carried unique marker combinations on chromosomes 1H and 5H. The photoperiod sensitive varieties that lacked a vernalization response (Scio and Dicktoo) differed significantly from the other varieties in the marker alleles at loci on chromosome 3H.

AB-QTL Analysis in Spring Barley and Three Strategies
of QTL Verification

K. Pillen, A. Zacharias, A. Taheri and J. Leon

Department of Crop Science and Plant Breeding, University of Bonn, D-53115 Bonn, Germany,
E-mail: k.pillen@uni-bonn.de

The advanced backcross quantitative trait locus (AB-QTL) strategy, introduced by Tanksley and Nelson (1996), combines the two processes of (1) the localization of exotic polygenes which exhibit effects on quantitative traits like yield or quality and (2) the utilization of the QTL information to select new, improved breeding lines. In barley, we finished two AB-QTL analyses, so far. For this, two BC2F2 populations were investigated which resulted from crosses of the Israelian wild barley accession ISR101-23 with the German spring barley varieties Apex (A × 101) and Harry (H × 101), respectively (Pillen et al. 2003 and Pillen et al. in prep.). Our goals were to 1. localize QTLs for the expression of quantitative traits in spring barley, 2. identify favorable exotic QTL alleles which improve agronomic traits and 3. compare the genetic effects of donor QTL alleles between the two related backcrosses. The genotype data were collected from 45 and 50 informative SSR (simple sequence repeat) markers and the phenotype data were compiled for 13 quantitative agronomic traits measured in a maximum of six environments. In the A × 101 and H × 101 backcross populations, 86 and, respectively, 108 putative QTLs were identified by means of a two-factorial ANOVA. At 29 and, respectively, 52 putative QTLs favorable effects of the exotic QTL allele were identified which resulted in an improvement of the trait under investigation. The individual QTLs will be presented and its common or contrasting effects in both populations will be compared.

EST-Derived Markers: a Resource for Comparative Mapping between Barley and Rice

M. Prasad, H. Zhang, R. Kota, R. K. Varshney, D. Perovic, T. Thiel, N. Stein and A. Graner

Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Germany,
E-mail: rajeev@ipk-gatersleben.de

In context of the barley genomics program so far more than 110,981 ESTs from 22 different cDNA libraries were generated. Cluster analysis yielded a tentative unigene set comprising of 25,224 genes. To facilitate the integration of EST data with mapped traits and to open a gateway for comparative mapping with rice, the non-redundant set of ESTs has been exploited for construction of a high density transcript map. The transcript map contains more than 1,000 genes including 571 RFLP loci, 257 SNP loci and 191 SSR loci. BLASTN analysis of 894 non-redundant mapped barley ESTs against the rice BAC/PAC database yielded in a total a number of 650 hits (E value: < 1E-5, sequence identity: > 80%). Of these, 414 (63%) ESTs displayed a syntenic relationship at the chromosomal level and 245 ESTs showed co-linearity of markers between barley and rice. The number of co-linear markers is a strong underestimate resulting from minor inaccuracies in the consensus map construction. In addition to the known syntenic relationships between barley and rice, two small coherent stretches of rice chromosome 1 were found to be syntenous with barley chromosome 1H (5-10-1-5) and 5H (12-11-9-3-1-3). In conclusion, the transcript map enables barley geneticists to rapidly identify target regions in rice, for an efficient marker saturation of defined regions in the barley genome.

The Semi-Dwarfing Gene sdw1 in European Spring Barley

L. Ramsay, M. Macaulay, W. T. B. Thomas, A. Druka, D. F. Marshall and R. Waugh

Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK,
E-mail: lramsa@scri.sari.ac.uk

The semi dwarf mutation sdw1 is present in almost all spring barley cultivars grown in North-west Europe, e.g. in all but one of the spring varieties on the current UK recommended list. Most current varieties carry the gene from one particular source, the mutation in the Czech variety Valticky that produced Diamant. Whilst sdw1 has been widely adopted, it does carry a number of associated agronomic defects that limit its wider deployment. For instance, its late maturity limits its market potential in areas with a restricted growing season and its small grain size means that it is unsuitable for deployment in winter barley. The map position of sdw1 on the long arm of 3H(3) allows the genomic sequence of the broadly homologous rice chromosome 1 to be used to provide molecular markers that delineate the sdw1 region in barley. These markers can be used to determine the size of this region that derives from Valticky and has survived into the many varieties which can be traced backed to Diamant and its derivative Trumpf (Triumph) and that probably also affects the plants biological and agronomic performance. Work will be presented that utilises molecular markers to characterise this region in a range of germplasm and also reports on the progress made in the more detailed characterisation of the sdw1 gene.

Linkage Disequilibrium in European Barley

L. Ramsay, J. R. Russell, M. Macaulay, W. T. B. Thomas, W. Powell and R. Waugh

Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK,
E-mail: lramsa@scri.sari.ac.uk

Linkage Disequilibrium (LD) is the non-random association of alleles at different loci although it can be caused by other factors such as population substructure and selection. LD is of interest as it relates directly to the underlying patterns of polymorphism and therefore affects at a basic level the reliability of diagnostics and the general application of molecular markers to the characterisation of germplasm. LD studies in plants have to date been mainly limited to outbreeders such as maize. The applicability of the LD in barley is at present unclear because, as an inbreeding species, it would be expected to show higher levels of spurious associations due to population structure and the relative lack of recombination. At SCRI germplasm surveys of barley cultivars using SSRs (simple sequence repeats) have shown significant LD across the whole genome with complexities due to population structure that could be attributed to past breeding practice. However this work has indicated that LD studies are feasible in cultivated barley although care is needed in the choice of material and that the use of unlinked marker loci is necessary to estimate population substructure and give empirical thresholds for the appropriate levels of statistical significance. Details of the pattern of LD found and results of more detailed studies at particular loci of agronomic interest will be presented.

Functioning of Maize Ac/Ds Transposons in Barley Gene
Tagging Populations

J. Singh1, S. Zhang1, L. Cooper2, S. Rangel1, P. M. Hayes2 and P. G. Lemaux1

1University of California, Berkeley, CA 94720, USA; 2Department of Crop
and Soil Science,, Oregon State University, Corvallis, OR 97331, USA,
E-mail:patrick.m.hayes@oregonstate.edu

The Ac/Ds transposon system, which causes heritable mutations in maize, is now used for gene tagging in other plant species. The use of Ac/Ds in barley, a diploid, provides an ideal platform for functional genomics since it is closely related to important cereal species. Here, we report the reactivation characteristics of single- or low-copy transposed Ds insertions after crossing with transposase-expressing lines. Analyses of 20 transposed events were conducted using iPCR/TAIL-PCR to generate 5- and 3- flanking sequences adjacent to Ds. The results indicate that Ds tends to insert in parallel with regard to gene orientation. In most lines, characteristic 8 base pair duplications were observed adjacent to the Ac inverted repeat ends. Approximately 20% of lines had defective inverted repeats, always associated with incomplete 8 base pair duplications. Analysis of flanking sequences using BLAST searches against DNA and protein databases indicates that approximately 82% of flanking sequences were from either known or putative genes, which include wall-associated kinases, cytochrome P450 and several ESTs from barley and wheat. Preliminary sequence analyses confirm the preferential insertion of Ds into gene-rich regions, providing an ideal tool for functional genomics. It also provides a platform for selective sequencing of gene-rich regions in barley and wheat, which have large genomes with a high percentage of repetitive DNA.

Microsatellite Genotyping of Latvian Barley Varieties
and Related European Ancestors

T. G. Sjakste1, M. S. RoEder2, M. Ganal3 and I. Rashal1

1Laboratory of Plant Genetics, Institute of Biology, Salaspils, LV 2169, Latvia,
E-mail: tanjasjakste@email.lubi.edu.lv;
2Gene and Genome Mapping, Institute of Plant Genetics and Crop Research (IPK),
D-06466 Gatersleben, Germany; 3Trait Genetics, D-06466 Gatersleben, Germany

Genetic diversity, parental inheritance and transmission of 65 microsatellite loci were analyzed in a set of 55 barley accessions composed of 21 Latvian and 29 European and 1 Indian commercial varieties, Himalaya landrace Brachytic and three Hordeum spontaneum lines. Graphical genotypes of the chromosomes revealed the genomic regions of similarity and dissimilarity between the genotypes as well as conserved linkage blocks transmitted through the generations.

Regions of the Genome Affecting Hull Peeling
in Two-Row Barley and Malt

D. J. Somers1, W. G. Legge2, B. G. Rossnagel3 and J. S. Noll1

1Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, R3T 2M9 Canada;
2Brandon Research Centre, Agriculture and Agri-Food Canada, Brandon, R7A 5Y3 Canada;
3Crop Development Centre, University of Saskatchewan, Saskatoon, S7N 5A8 Canada,
E-mail: blegge@agr.gc.ca

Hull peeling resistance in barley and malt is desired for malting and brewing. To investigate the inheritance of these traits in Canadian two-row malting barley, a molecular map consisting of 60 microsatellites and 120 AFLPs was constructed for the Harrington (susceptible)/Manley (resistant) cross using 160 F2-derived F9 recombinant inbred lines (RILs). Barley samples from standard yield test plots grown at two sites in each of Manitoba and Saskatchewan during 1999 and 2000 were evaluated for percentage hull peeling on a weight basis direct from the plot combine (RPWB) and after inducing peeling with an air-blast de-huller (APWB). Micromalted samples were evaluated for percentage hull peeling of the malt as is (RPWM) and after air-blast de-hulling (APWM). Two QTL were identified for RPWB, three for APWB, two for RPWM, and two for APWM. Both RPWB QTL were coincident with two for APWB. The two QTL for APWM and RPWM were coincident, and one of these may be coincident with a QTL for RPWB and APWB. The QTL were consistent over locations and years, indicating they could be candidate targets for molecular breeding.

Array Analysis of Cold Acclimation in Barley Albino Mutants Reveals the Key Role of the Chloroplast during Adaptation
to Low Temperature

J. T. Svensson1, C. Crosatti2, C. Campoli2, R. Bassi3, A. M. Stanca2, T. J. Close1
and L. Cattivelli2

1Department of Botany and Plant Sciences, University of California, Riverside, CA 92521-0124, USA;
2Experimental Institute for Cereal Research, 29017 Fiorenzuola dArda (PC), Italy,
E-mail: l.cattivelli@iol.it; 3Université Aix-Marseille II, Département de Biologie-Case 901-163,
13288 Marseille Cedex 09, France

Previously, we have shown that barley plants carrying a mutation preventing chloroplast development, beside the expected albino phenotype, are completely frost susceptible as well as impaired in the expression of several cor (cold-regulated) genes. Barley genetic stocks offer a unique collection of chloroplast deficient mutants, most of which have been characterised at genetic and biochemical levels. Although these mutations are generally lethal, the large endosperm of barley seeds supports plant growth for several weeks. We investigated four albino and xantha barley mutants representing successive steps in chloroplast biogenesis and the corresponding wild type with the Affymetrix barley1 GeneChip to assess the effect of the chloroplast on the expression of cold-regulated genes. First, by comparing control vs cold hardened WT plants a set of about 2.7 thousand cor genes have been identified, then the expression of these cor genes have been followed in control and cold hardened mutants. The data analysis allowed us to identify two main classes of cor genes: chloroplast independent (sequences cold regulated in all genotypes); and chloroplast dependent (sequences cold regulated only in WT and not regulated by cold in any mutants). Notably, about 65% of cor genes showed a tight chloroplast control, being cold-regulated only in WT. Among these are several hundred genes involved in protein synthesis machinery, but, surprising, none of previously known stress-induced genes.

EST-Derived Markers and Transcript Map of Barley: a Resource for Interspecific Transferability and Comparative Mapping in Cereals

R. K. Varshney, M. Prasad, H. Zhang, R. Kota, R. Sigmund, U. Scholz, N. Stein
and A. Graner

Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Germany
E-mail: rajeev@ipk-gatersleben.de

The availability of sequence data from large-scale EST (expressed sequence tag) projects has made it possible to develop markers directly from the genes. In order to develop functional markers and preparation of transcript map (integrating of genes/transcripts to genetic map) of barley, more than 1,000 ESTs/cDNAs including 589 RFLP (Restriction Fragment Length Polymorphism), 255 SNP (Single Nucleotide Polymorphism) and 185 SSR (Simple Sequence Repeat or microsatellite) markers have been developed. These markers provide a good resource for a variety of purposes like interspecific transferability and comparative mapping in other cereals, pedigree analysis, marker-trait association etc. A computational study suggests a theoretical transferability of barley markers to wheat (95.4%), maize (69.7%), sorghum (66.2%), rye (38.7%) and even to dicot species (~15%). Furthermore, comparative mapping of barley ESTs in rice showed that a total of 311 markers exhibit collinearity between barley and rice with an average of 40 syntenic barley ESTs for each rice chromosome. The transcript map enables for a rapid identification of target regions in rice, for efficient marker saturation of defined regions of the barley genome and also serves as a resource of mapped candidate genes for agronomic traits.

Genetic Conversion of Feed Barley Varieties to Malting Types

E. J. Vassos1, A. R. Barr2 and J. K. Eglinton3

1Molecular Plant Breeding CRC, 2Australian Grains Technology (AGT) and 3School of Agriculture and Wine, University of Adelaide, Glen Osmond, SA 5064, Australia,
E-mail: elysia.vassos@adelaide.edu.au

Malting barley breeding programs have typically applied conservative strategies for the introgression of traits from non-malting germplasm, and this traditional approach can be considered as recognition for the genetic complexity of malt quality. However, these strategies intrinsically limit the rate of genetic gain for adaptation and the drought conditions in Australia have recently highlighted the superior adaptation of feed varieties, particularly in low rainfall environments. This paper presents preliminary results from a novel strategy that tests a new paradigm for breeding malting barley. The project aims to introduce key malting quality genes from a range of elite international malting varieties into the well adaptated feed variety Keel, while maintaining the superior adaptation and agronomic profile of the feed barley. This strategy implies that the genetics of malting quality is now better understood than the genetics of adaptation. Keel was used as the recurrent parent to produce backcross lines containing the key quality genes from Alexis, Haruna nijo and AC Metcalfe. The BC1 and BC2 generations for each introgression stream were screened using molecular markers for a range of malt quality loci. The subsequent populations were evaluated in double row trials in the 2002 and 2003 season. Agronomic selection was applied to identify individuals exhibiting the Keel phenotype, the subsequent grain samples were evaluated for grain size and NIR predicted malt quality. Populations derived from intercrosses between the introgression streams have also been developed, to pyramid the malt quality genes from the three international varieties into a Keel genetic background. The agronomic and malting quality profiles of this germplasm will be presented, and the prospects for developing malting quality barley using this novel breeding strategy will be discussed. Selected individuals were screened as BC1F2 single plants to identify individuals homozygous for the target loci. Elite lines were identified and promoted for further agronomic evaluation in yield plots in the 2003 season. Selected BC1F2 individuals were used to develop BC2 generations for each of the introgression streams. This germplasm is currently in a double row trial for agronomic and predicted malt quality evaluation.

AB-QTL Analysis in Winter Barley

H. Wang, M. v. Korff, J. Leon and K. Pillen

Department of Crop Science and Plant Breeding, University of Bonn, D-53115 Bonn, Germany,
E-mail: k.pillen@uni-bonn.de

The AB-QTL (advanced backcross QTL analysis) strategy was developed by Tanksley and Nelson (1996) in order to detect and to introgress favorable genes from unadapted donors into elite cultivars. By means of the AB-QTL strategy, the direct and specific utilization of exotic germplasm for improvement of quantitative characters like yield parameters or quality components seems feasible for plant breeding. Two BC2DH populations, resulting from crosses of the two winter barley varieties Carola and Theresa (Hordeum vulgare) with the wild barley (H. vulgare ssp. spontaneum Hsp) accession ISR101-23
from Israel, are genotyped with approximately 100 SSR markers. QTL analysis is carried out for agronomic traits and pathogen resistance evaluated under two nitrogen levels at four test locations in Germany in two consecutive years. Meanwhile, candidate lines for development of QTL-NILs are selected based on the results of genotyping. Each line includes a short and different introgressed segment of the donor genome. A QTL-NIL library will be constructed, which will be made available to (i) barley breeders as improved breeding material and to (ii) plant molecular biologist as a genetic resource tool for future studies on the molecular regulation of quantitative traits and as a starting tool for succeeding projects on map-based cloning of the underlying genes for quantitative traits.

Construction of a Barley Recombinant Chromosome
Substitution Library

G. R. Young, P. E. Lawrence, R. Waugh and W. T. B. Thomas

Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK,
E-mail: rwaugh@scri.sari.ac.uk

Four barley genotypes cvs. Haruna Nijo, Morex, a land race from Western Syria and a Hordeum spontaneum were used in a marker assisted backcrossing programme with cv. Chime to produce a series of isolines possessing different segments of donor genome in an otherwise uniform genetic background. Analysis of SSR data from over the whole genome for the BC1F1 showed that the Chime contribution was approximately 75% but there were regions of the genome where the donor contribution was either less or greater than expected. The SSR data was augmented by AFLP data for the Hordeum spontaneum BC1 and the data sets combined to produce a composite map. Using GENEFLOW to analyse the SSR data, we were generally able to detect BC1F1 individuals that were heterozygous at a marker locus but carried entirely recipient alleles at the remaining loci on the chromosome and these were used in BC2 construction. Where such lines were not detected, lines that carried donor alleles in regions remote from the marker in question were chosen for BC2 construction. Because we do not know exactly where cross-overs have occurred, we also used lines that carried donor alleles for two adjacent markers in the BC2 construction. This strategy minimised the chances of missing donor segments of the genome. On average, prior genotyping of the population reduced the amount of crossing by 67%.

SSR Marker Tagging of Dwarfing Gene uz
in Barley (Hordeum vulgare L.)

Zhang Jing

Institute of Crop Germplasm Resources, Chinese Academy of Agricultural Sciences,
Beijing 100081, P. R. China,
E-mail: jingzhang@95777.com

Dwarfing gene uz exists widely in barley land-races and bred cultivars in China, Japan and Korea. It has been extensively used in barley breeding in China and is carried by the long arm of 3H chromosome. To tag the gene uz molecularly, simple sequence repeats (SSR) were employed. Two SSR markers HVM33 and HVM60, which were known to be on the long arm and close to the centromere of 3H chromosome previously, were found to link to uz by 3.4 and 10.6 cM respectively in the cross of CZLDM × HZLM. The dwarfing gene uz was located between the two SSR markers. Both HVM33 and HVM60 could be used as diagnostic markers for uz. A linkage map of dwarfing gene uz to SSR markers was created.

Development of a Two-Component Ac/Ds System
for Functional Genomics in Barley

T. Zhao and T. Koprek

Max Planck Institute for Plant Breeding Research, D-50829 Köln, Germany,
E-mail: tzhao@mpiz-koeln.mpg.de

The main challenge of the genomic era is to identify the function of the predicted 25,00040,000 genes present in plant genomes. Transposon tagging is one of the widely-used methods for the generation of insertional mutants for the functional analysis of plant genes. In order to develop a two-element system for targeted insertional mutagenesis in barley we introduced a stabilised Ac-Transposase gene and different constructs containing the non-autonomous Ds-element from maize into barley using Agrobacterium mediated gene delivery. Several hundred single-copy Ds lines have been generated. After genetic mapping of their insertion sites they will be used as launch pads for targeted gene tagging in barley. Mapped Ds elements in vicinity of a locus of interest have been activated by crossing the Ds plant with a plant expressing functional AcTransposase. Due to the preference of Ds elements to reinsert into genetically closely linked sites, the activation of Ds results in a high degree of saturation with independent Ds insertions close to the original Ds launch pad. F2 plants are being analyzed for Ds insertions in the target gene.