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1.  Genetic Diversity and Population Structure in a Legacy Collection of Spring Barley Landraces Adapted to a Wide Range of Climates 
PLoS ONE  2014;9(12):e116164.
Global environmental change and increasing human population emphasize the urgent need for higher yielding and better adapted crop plants. One strategy to achieve this aim is to exploit the wealth of so called landraces of crop species, representing diverse traditional domesticated populations of locally adapted genotypes. In this study, we investigated a comprehensive set of 1485 spring barley landraces (Lrc1485) adapted to a wide range of climates, which were selected from one of the largest genebanks worldwide. The landraces originated from 5° to 62.5° N and 16° to 71° E. The whole collection was genotyped using 42 SSR markers to assess the genetic diversity and population structure. With an average allelic richness of 5.74 and 372 alleles, Lrc1485 harbours considerably more genetic diversity than the most polymorphic current GWAS panel for barley. Ten major clusters defined most of the population structure based on geographical origin, row type of the ear and caryopsis type – and were assigned to specific climate zones. The legacy core reference set Lrc648 established in this study will provide a long-lasting resource and a very valuable tool for the scientific community. Lrc648 is best suited for multi-environmental field testing to identify candidate genes underlying quantitative traits but also for allele mining approaches.
PMCID: PMC4277474  PMID: 25541702
2.  Genetic Dissection of Photoperiod Response Based on GWAS of Pre-Anthesis Phase Duration in Spring Barley 
PLoS ONE  2014;9(11):e113120.
Heading time is a complex trait, and natural variation in photoperiod responses is a major factor controlling time to heading, adaptation and grain yield. In barley, previous heading time studies have been mainly conducted under field conditions to measure total days to heading. We followed a novel approach and studied the natural variation of time to heading in a world-wide spring barley collection (218 accessions), comprising of 95 photoperiod-sensitive (Ppd-H1) and 123 accessions with reduced photoperiod sensitivity (ppd-H1) to long-day (LD) through dissecting pre-anthesis development into four major stages and sub-phases. The study was conducted under greenhouse (GH) conditions (LD; 16/8 h; ∼20/∼16°C day/night). Genotyping was performed using a genome-wide high density 9K single nucleotide polymorphisms (SNPs) chip which assayed 7842 SNPs. We used the barley physical map to identify candidate genes underlying genome-wide association scans (GWAS). GWAS for pre-anthesis stages/sub-phases in each photoperiod group provided great power for partitioning genetic effects on floral initiation and heading time. In addition to major genes known to regulate heading time under field conditions, several novel QTL with medium to high effects, including new QTL having major effects on developmental stages/sub-phases were found to be associated in this study. For example, highly associated SNPs tagged the physical regions around HvCO1 (barley CONSTANS1) and BFL (BARLEY FLORICAULA/LEAFY) genes. Based upon our GWAS analysis, we propose a new genetic network model for each photoperiod group, which includes several newly identified genes, such as several HvCO-like genes, belonging to different heading time pathways in barley.
PMCID: PMC4242610  PMID: 25420105
3.  Separating the wheat from the chaff – a strategy to utilize plant genetic resources from ex situ genebanks 
Scientific Reports  2014;4:5231.
The need for higher yielding and better-adapted crop plants for feeding the world's rapidly growing population has raised the question of how to systematically utilize large genebank collections with their wide range of largely untouched genetic diversity. Phenotypic data that has been recorded for decades during various rounds of seed multiplication provides a rich source of information. Their usefulness has remained limited though, due to various biases induced by conservation management over time or changing environmental conditions. Here, we present a powerful procedure that permits an unbiased trait-based selection of plant samples based on such phenotypic data. Applying this technique to the wheat collection of one of the largest genebanks worldwide, we identified groups of plant samples displaying contrasting phenotypes for selected traits. As a proof of concept for our discovery pipeline, we resequenced the entire major but conserved flowering time locus Ppd-D1 in just a few such selected wheat samples – and nearly doubled the number of hitherto known alleles.
PMCID: PMC4050481  PMID: 24912875
4.  Mapping-by-sequencing accelerates forward genetics in barley 
Genome Biology  2014;15(6):R78.
Mapping-by-sequencing has emerged as a powerful technique for genetic mapping in several plant and animal species. As this resequencing-based method requires a reference genome, its application to complex plant genomes with incomplete and fragmented sequence resources remains challenging. We perform exome sequencing of phenotypic bulks of a mapping population of barley segregating for a mutant phenotype that increases the rate of leaf initiation. Read depth analysis identifies a candidate gene, which is confirmed by the analysis of independent mutant alleles. Our method illustrates how the genomic resources of barley together with exome resequencing can underpin mapping-by-sequencing.
PMCID: PMC4073093  PMID: 24917130
5.  Genome-wide association studies for agronomical traits in a world wide spring barley collection 
BMC Plant Biology  2012;12:16.
Genome-wide association studies (GWAS) based on linkage disequilibrium (LD) provide a promising tool for the detection and fine mapping of quantitative trait loci (QTL) underlying complex agronomic traits. In this study we explored the genetic basis of variation for the traits heading date, plant height, thousand grain weight, starch content and crude protein content in a diverse collection of 224 spring barleys of worldwide origin. The whole panel was genotyped with a customized oligonucleotide pool assay containing 1536 SNPs using Illumina's GoldenGate technology resulting in 957 successful SNPs covering all chromosomes. The morphological trait "row type" (two-rowed spike vs. six-rowed spike) was used to confirm the high level of selectivity and sensitivity of the approach. This study describes the detection of QTL for the above mentioned agronomic traits by GWAS.
Population structure in the panel was investigated by various methods and six subgroups that are mainly based on their spike morphology and region of origin. We explored the patterns of linkage disequilibrium (LD) among the whole panel for all seven barley chromosomes. Average LD was observed to decay below a critical level (r2-value 0.2) within a map distance of 5-10 cM. Phenotypic variation within the panel was reasonably large for all the traits. The heritabilities calculated for each trait over multi-environment experiments ranged between 0.90-0.95. Different statistical models were tested to control spurious LD caused by population structure and to calculate the P-value of marker-trait associations. Using a mixed linear model with kinship for controlling spurious LD effects, we found a total of 171 significant marker trait associations, which delineate into 107 QTL regions. Across all traits these can be grouped into 57 novel QTL and 50 QTL that are congruent with previously mapped QTL positions.
Our results demonstrate that the described diverse barley panel can be efficiently used for GWAS of various quantitative traits, provided that population structure is appropriately taken into account. The observed significant marker trait associations provide a refined insight into the genetic architecture of important agronomic traits in barley. However, individual QTL account only for a small portion of phenotypic variation, which may be due to insufficient marker coverage and/or the elimination of rare alleles prior to analysis. The fact that the combined SNP effects fall short of explaining the complete phenotypic variance may support the hypothesis that the expression of a quantitative trait is caused by a large number of very small effects that escape detection. Notwithstanding these limitations, the integration of GWAS with biparental linkage mapping and an ever increasing body of genomic sequence information will facilitate the systematic isolation of agronomically important genes and subsequent analysis of their allelic diversity.
PMCID: PMC3349577  PMID: 22284310
6.  NGS technologies for analyzing germplasm diversity in genebanks* 
More than 70 years after the first ex situ genebanks have been established, major efforts in this field are still concerned with issues related to further completion of individual collections and securing of their storage. Attempts regarding valorization of ex situ collections for plant breeders have been hampered by the limited availability of phenotypic and genotypic information. With the advent of molecular marker technologies first efforts were made to fingerprint genebank accessions, albeit on a very small scale and mostly based on inadequate DNA marker systems. Advances in DNA sequencing technology and the development of high-throughput systems for multiparallel interrogation of thousands of single nucleotide polymorphisms (SNPs) now provide a suite of technological platforms facilitating the analysis of several hundred of Gigabases per day using state-of-the-art sequencing technology or, at the same time, of thousands of SNPs. The present review summarizes recent developments regarding the deployment of these technologies for the analysis of plant genetic resources, in order to identify patterns of genetic diversity, map quantitative traits and mine novel alleles from the vast amount of genetic resources maintained in genebanks around the world. It also refers to the various shortcomings and bottlenecks that need to be overcome to leverage the full potential of high-throughput DNA analysis for the targeted utilization of plant genetic resources.
PMCID: PMC3281264  PMID: 22257472
genetic resources; next-generation sequencing; SNP; allele mining; genetic diversity; association analysis
7.  BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.) 
BMC Genomics  2011;12:247.
Although second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place.
Five new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with <30 or >250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X.
BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.
PMCID: PMC3224359  PMID: 21595870
8.  DNA polymorphisms and haplotype patterns of transcription factors involved in barley endosperm development are associated with key agronomic traits 
BMC Plant Biology  2010;10:5.
Association mapping is receiving considerable attention in plant genetics for its potential to fine map quantitative trait loci (QTL), validate candidate genes, and identify alleles of interest. In the present study association mapping in barley (Hordeum vulgare L.) is investigated by associating DNA polymorphisms with variation in grain quality traits, plant height, and flowering time to gain further understanding of gene functions involved in the control of these traits. We focused on the four loci BLZ1, BLZ2, BPBF and HvGAMYB that play a role in the regulation of B-hordein expression, the major fraction of the barley storage protein. The association was tested in a collection of 224 spring barley accessions using a two-stage mixed model approach.
Within the sequenced fragments of four candidate genes we observed different levels of nucleotide diversity. The effect of selection on the candidate genes was tested by Tajima's D which revealed significant values for BLZ1, BLZ2, and BPBF in the subset of two-rowed barleys. Pair-wise LD estimates between the detected SNPs within each candidate gene revealed different intra-genic linkage patterns. On the basis of a more extensive examination of genomic regions surrounding the four candidate genes we found a sharp decrease of LD (r2<0.2 within 1 cM) in all but one flanking regions.
Significant marker-trait associations between SNP sites within BLZ1 and flowering time, BPBF and crude protein content and BPBF and starch content were detected. Most haplotypes occurred at frequencies <0.05 and therefore were rejected from the association analysis. Based on haplotype information, BPBF was associated to crude protein content and starch content, BLZ2 showed association to thousand-grain weight and BLZ1 was found to be associated with flowering time and plant height.
Differences in nucleotide diversity and LD pattern within the candidate genes BLZ1, BLZ2, BPBF, and HvGAMYB reflect the impact of selection on the nucleotide sequence of the four candidate loci.
Despite significant associations, the analysed candidate genes only explained a minor part of the total genetic variation although they are known to be important factors influencing the expression of seed quality traits. Therefore, we assume that grain quality as well as plant height and flowering time are influenced by many factors each contributing a small part to the expression of the phenotype. A genome-wide association analysis could provide a more comprehensive picture of loci involved in the regulation of grain quality, thousand grain weight and the other agronomic traits that were analyzed in this study. However, despite available high-throughput genotyping arrays the marker density along the barely genome is still insufficient to cover all associations in a whole genome scan. Therefore, the candidate gene-based approach will further play an important role in barley association studies.
PMCID: PMC2822787  PMID: 20064201
9.  Development and implementation of high-throughput SNP genotyping in barley 
BMC Genomics  2009;10:582.
High density genetic maps of plants have, nearly without exception, made use of marker datasets containing missing or questionable genotype calls derived from a variety of genic and non-genic or anonymous markers, and been presented as a single linear order of genetic loci for each linkage group. The consequences of missing or erroneous data include falsely separated markers, expansion of cM distances and incorrect marker order. These imperfections are amplified in consensus maps and problematic when fine resolution is critical including comparative genome analyses and map-based cloning. Here we provide a new paradigm, a high-density consensus genetic map of barley based only on complete and error-free datasets and genic markers, represented accurately by graphs and approximately by a best-fit linear order, and supported by a readily available SNP genotyping resource.
Approximately 22,000 SNPs were identified from barley ESTs and sequenced amplicons; 4,596 of them were tested for performance in three pilot phase Illumina GoldenGate assays. Data from three barley doubled haploid mapping populations supported the production of an initial consensus map. Over 200 germplasm selections, principally European and US breeding material, were used to estimate minor allele frequency (MAF) for each SNP. We selected 3,072 of these tested SNPs based on technical performance, map location, MAF and biological interest to fill two 1536-SNP "production" assays (BOPA1 and BOPA2), which were made available to the barley genetics community. Data were added using BOPA1 from a fourth mapping population to yield a consensus map containing 2,943 SNP loci in 975 marker bins covering a genetic distance of 1099 cM.
The unprecedented density of genic markers and marker bins enabled a high resolution comparison of the genomes of barley and rice. Low recombination in pericentric regions is evident from bins containing many more than the average number of markers, meaning that a large number of genes are recombinationally locked into the genetic centromeric regions of several barley chromosomes. Examination of US breeding germplasm illustrated the usefulness of BOPA1 and BOPA2 in that they provide excellent marker density and sensitivity for detection of minor alleles in this genetically narrow material.
PMCID: PMC2797026  PMID: 19961604
10.  De novo 454 sequencing of barcoded BAC pools for comprehensive gene survey and genome analysis in the complex genome of barley 
BMC Genomics  2009;10:547.
De novo sequencing the entire genome of a large complex plant genome like the one of barley (Hordeum vulgare L.) is a major challenge both in terms of experimental feasibility and costs. The emergence and breathtaking progress of next generation sequencing technologies has put this goal into focus and a clone based strategy combined with the 454/Roche technology is conceivable.
To test the feasibility, we sequenced 91 barcoded, pooled, gene containing barley BACs using the GS FLX platform and assembled the sequences under iterative change of parameters. The BAC assemblies were characterized by N50 of ~50 kb (N80 ~31 kb, N90 ~21 kb) and a Q40 of 94%. For ~80% of the clones, the best assemblies consisted of less than 10 contigs at 24-fold mean sequence coverage. Moreover we show that gene containing regions seem to assemble completely and uninterrupted thus making the approach suitable for detecting complete and positionally anchored genes.
By comparing the assemblies of four clones to their complete reference sequences generated by the Sanger method, we evaluated the distribution, quality and representativeness of the 454 sequences as well as the consistency and reliability of the assemblies.
The described multiplex 454 sequencing of barcoded BACs leads to sequence consensi highly representative for the clones. Assemblies are correct for the majority of contigs. Though the resolution of complex repetitive structures requires additional experimental efforts, our approach paves the way for a clone based strategy of sequencing the barley genome.
PMCID: PMC2784808  PMID: 19930547
11.  High level of conservation between genes coding for the GAMYB transcription factor in barley (Hordeum vulgare L.) and bread wheat (Triticum aestivum L.) collections 
The transcription factor GAMYB is involved in gibberellin signalling in cereal aleurone cells and in plant developmental processes. Nucleotide diversity of HvGAMYB and TaGAMYB was investigated in 155 barley (Hordeum vulgare) and 42 wheat (Triticum aestivum) accessions, respectively. Polymorphisms defined 18 haplotypes in the barley collection and 1, 7 and 3 haplotypes for the A, B, and D genomes of wheat, respectively. We found that (1) Hv- and TaGAMYB genes have identical structures. (2) Both genes show a high level of nucleotide identity (>95%) in the coding sequences and the distribution of polymorphisms is similar in both collections. At the protein level the functional domain is identical in both species. (3) GAMYB genes map to a syntenic position on chromosome 3. GAMYB genes are different in both collections with respect to the Tajima D statistic and linkage disequilibrium (LD). A moderate level of LD was observed in the barley collection. In wheat, LD is absolute between polymorphic sites, mostly located in the first intron, while it decays within the gene. Differences in Tajima D values might be due to a lower selection pressure on HvGAMYB, compared to its wheat orthologue. Altogether our results provide evidence that there have been only few evolutionary changes in Hv- and TaGAMYB. This confirms the close relationship between these species and also highlights the functional importance of this transcription factor.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-008-0777-4) contains supplementary material, which is available to authorized users.
PMCID: PMC2755743  PMID: 18488187
12.  Evidence and evolutionary analysis of ancient whole-genome duplication in barley predating the divergence from rice 
Well preserved genomic colinearity among agronomically important grass species such as rice, maize, Sorghum, wheat and barley provides access to whole-genome structure information even in species lacking a reference genome sequence. We investigated footprints of whole-genome duplication (WGD) in barley that shaped the cereal ancestor genome by analyzing shared synteny with rice using a ~2000 gene-based barley genetic map and the rice genome reference sequence.
Based on a recent annotation of the rice genome, we reviewed the WGD in rice and identified 24 pairs of duplicated genomic segments involving 70% of the rice genome. Using 968 putative orthologous gene pairs, synteny covered 89% of the barley genetic map and 63% of the rice genome. We found strong evidence for seven shared segmental genome duplications, corresponding to more than 50% of the segmental genome duplications previously determined in rice. Analysis of synonymous substitution rates (Ks) suggested that shared duplications originated before the divergence of these two species. While major genome rearrangements affected the ancestral genome of both species, small paracentric inversions were found to be species specific.
We provide a thorough analysis of comparative genome evolution between barley and rice. A barley genetic map of approximately 2000 non-redundant EST sequences provided sufficient density to allow a detailed view of shared synteny with the rice genome. Using an indirect approach that included the localization of WGD-derived duplicated genome segments in the rice genome, we determined the current extent of shared WGD-derived genome duplications that occurred prior to species divergence.
PMCID: PMC2746218  PMID: 19698139
13.  Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage 
Journal of Experimental Botany  2009;60(12):3531-3544.
Drought tolerance is a key trait for increasing and stabilizing barley productivity in dry areas worldwide. Identification of the genes responsible for drought tolerance in barley (Hordeum vulgare L.) will facilitate understanding of the molecular mechanisms of drought tolerance, and also facilitate the genetic improvement of barley through marker-assisted selection or gene transformation. To monitor the changes in gene expression at the transcriptional level in barley leaves during the reproductive stage under drought conditions, the 22K Affymetrix Barley 1 microarray was used to screen two drought-tolerant barley genotypes, Martin and Hordeum spontaneum 41-1 (HS41-1), and one drought-sensitive genotype Moroc9-75. Seventeen genes were expressed exclusively in the two drought-tolerant genotypes under drought stress, and their encoded proteins may play significant roles in enhancing drought tolerance through controlling stomatal closure via carbon metabolism (NADP malic enzyme, NADP-ME, and pyruvate dehydrogenase, PDH), synthesizing the osmoprotectant glycine-betaine (C-4 sterol methyl oxidase, CSMO), generating protectants against reactive-oxygen-species scavenging (aldehyde dehydrogenase,ALDH, ascorbate-dependent oxidoreductase, ADOR), and stabilizing membranes and proteins (heat-shock protein 17.8, HSP17.8, and dehydrin 3, DHN3). Moreover, 17 genes were abundantly expressed in Martin and HS41-1 compared with Moroc9-75 under both drought and control conditions. These genes were possibly constitutively expressed in drought-tolerant genotypes. Among them, seven known annotated genes might enhance drought tolerance through signalling [such as calcium-dependent protein kinase (CDPK) and membrane steroid binding protein (MSBP)], anti-senescence (G2 pea dark accumulated protein, GDA2), and detoxification (glutathione S-transferase, GST) pathways. In addition, 18 genes, including those encoding Δl-pyrroline-5-carboxylate synthetase (P5CS), protein phosphatase 2C-like protein (PP2C), and several chaperones, were differentially expressed in all genotypes under drought; thus they were more likely to be general drought-responsive genes in barley. These results could provide new insights into further understanding of drought-tolerance mechanisms in barley.
PMCID: PMC2724701  PMID: 19561048
Barley; drought stress; drought tolerance; microarray; reproductive stage
14.  Low-pass shotgun sequencing of the barley genome facilitates rapid identification of genes, conserved non-coding sequences and novel repeats 
BMC Genomics  2008;9:518.
Barley has one of the largest and most complex genomes of all economically important food crops. The rise of new short read sequencing technologies such as Illumina/Solexa permits such large genomes to be effectively sampled at relatively low cost. Based on the corresponding sequence reads a Mathematically Defined Repeat (MDR) index can be generated to map repetitive regions in genomic sequences.
We have generated 574 Mbp of Illumina/Solexa sequences from barley total genomic DNA, representing about 10% of a genome equivalent. From these sequences we generated an MDR index which was then used to identify and mark repetitive regions in the barley genome. Comparison of the MDR plots with expert repeat annotation drawing on the information already available for known repetitive elements revealed a significant correspondence between the two methods. MDR-based annotation allowed for the identification of dozens of novel repeat sequences, though, which were not recognised by hand-annotation. The MDR data was also used to identify gene-containing regions by masking of repetitive sequences in eight de-novo sequenced bacterial artificial chromosome (BAC) clones. For half of the identified candidate gene islands indeed gene sequences could be identified. MDR data were only of limited use, when mapped on genomic sequences from the closely related species Triticum monococcum as only a fraction of the repetitive sequences was recognised.
An MDR index for barley, which was obtained by whole-genome Illumina/Solexa sequencing, proved as efficient in repeat identification as manual expert annotation. Circumventing the labour-intensive step of producing a specific repeat library for expert annotation, an MDR index provides an elegant and efficient resource for the identification of repetitive and low-copy (i.e. potentially gene-containing sequences) regions in uncharacterised genomic sequences. The restriction that a particular MDR index can not be used across species is outweighed by the low costs of Illumina/Solexa sequencing which makes any chosen genome accessible for whole-genome sequence sampling.
PMCID: PMC2584661  PMID: 18976483
15.  Transferability and polymorphism of barley EST-SSR markers used for phylogenetic analysis in Hordeum chilense 
BMC Plant Biology  2008;8:97.
Hordeum chilense, a native South American diploid wild barley, is a potential source of useful genes for cereal breeding. The use of this wild species to increase genetic variation in cereals will be greatly facilitated by marker-assisted selection. Different economically feasible approaches have been undertaken for this wild species with limited direct agricultural use in a search for suitable and cost-effective markers. The availability of Expressed Sequence Tags (EST) derived microsatellites or simple sequence repeat (SSR) markers, commonly called as EST-SSRs, for barley (Hordeum vulgare) represents a promising source to increase the number of genetic markers available for the H. chilense genome.
All of the 82 barley EST-derived SSR primer pairs tested for transferability to H. chilense amplified products of correct size from this species. Of these 82 barley EST-SSRs, 21 (26%) showed polymorphism among H. chilense lines. Identified polymorphic markers were used to test the transferability and polymorphism in other Poaceae family species with the aim of establishing H. chilense phylogenetic relationships. Triticum aestivum-H. chilense addition lines allowed us to determine the chromosomal localizations of EST-SSR markers and confirm conservation of the linkage group.
From the present study a set of 21 polymorphic EST-SSR markers have been identified to be useful for diversity analysis of H. chilense, related wild barleys like H. murinum, and for wheat marker-assisted introgression breeding. Across-genera transferability of the barley EST-SSR markers has allowed phylogenetic inference within the Triticeae complex.
PMCID: PMC2569940  PMID: 18822176
16.  Barley Genomics: An Overview 
Barley (Hordeum vulgare), first domesticated in the Near East, is a well-studied crop in terms of genetics, genomics, and breeding and qualifies as a model plant for Triticeae research. Recent advances made in barley genomics mainly include the following: (i) rapid accumulation of EST sequence data, (ii) growing number of studies on transcriptome, proteome, and metabolome, (iii) new modeling techniques, (iv) availability of genome-wide knockout collections as well as efficient transformation techniques, and (v) the recently started genome sequencing effort. These developments pave the way for a comprehensive functional analysis and understanding of gene expression networks linked to agronomically important traits. Here, we selectively review important technological developments in barley genomics and related fields and discuss the relevance for understanding genotype-phenotype relationships by using approaches such as genetical genomics and association studies. High-throughput genotyping platforms that have recently become available will allow the construction of high-density genetic maps that will further promote marker-assisted selection as well as physical map construction. Systems biology approaches will further enhance our knowledge and largely increase our abilities to design refined breeding strategies on the basis of detailed molecular physiological knowledge.
PMCID: PMC2276607  PMID: 18382615
17.  454 sequencing put to the test using the complex genome of barley 
BMC Genomics  2006;7:275.
During the past decade, Sanger sequencing has been used to completely sequence hundreds of microbial and a few higher eukaryote genomes. In recent years, a number of alternative technologies became available, among them adaptations of the pyrosequencing procedure (i.e. "454 sequencing"), promising a ~100-fold increase in throughput over Sanger technology – an advancement which is needed to make large and complex genomes more amenable to full genome sequencing at affordable costs. Although several studies have demonstrated its potential usefulness for sequencing small and compact microbial genomes, it was unclear how the new technology would perform in large and highly repetitive genomes such as those of wheat or barley.
To study its performance in complex genomes, we used 454 technology to sequence four barley Bacterial Artificial Chromosome (BAC) clones and compared the results to those from ABI-Sanger sequencing. All gene containing regions were covered efficiently and at high quality with 454 sequencing whereas repetitive sequences were more problematic with 454 sequencing than with ABI-Sanger sequencing. 454 sequencing provided a much more even coverage of the BAC clones than ABI-Sanger sequencing, resulting in almost complete assembly of all genic sequences even at only 9 to 10-fold coverage. To obtain highly advanced working draft sequences for the BACs, we developed a strategy to assemble large parts of the BAC sequences by combining comparative genomics, detailed repeat analysis and use of low-quality reads from 454 sequencing. Additionally, we describe an approach of including small numbers of ABI-Sanger sequences to produce hybrid assemblies to partly compensate the short read length of 454 sequences.
Our data indicate that 454 pyrosequencing allows rapid and cost-effective sequencing of the gene-containing portions of large and complex genomes and that its combination with ABI-Sanger sequencing and targeted sequence analysis can result in large regions of high-quality finished genomic sequences.
PMCID: PMC1633745  PMID: 17067373
18.  SNP2CAPS: a SNP and INDEL analysis tool for CAPS marker development 
Nucleic Acids Research  2004;32(1):e5.
With the influx of various SNP genotyping assays in recent years, there has been a need for an assay that is robust, yet cost effective, and could be performed using standard gel-based procedures. In this context, CAPS markers have been shown to meet these criteria. However, converting SNPs to CAPS markers can be a difficult process if done manually. In order to address this problem, we describe a computer program, SNP2CAPS, that facilitates the computational conversion of SNP markers into CAPS markers. 413 multiple aligned sequences derived from barley ESTs were analysed for the presence of polymorphisms in 235 distinct restriction sites. 282 (90%) of 314 alignments that contain sequence variation due to SNPs and InDels revealed at least one polymorphic restriction site. After reducing the number of restriction enzymes from 235 to 10, 31% of the polymorphic sites could still be detected. In order to demonstrate the usefulness of this tool for marker development, we experimentally validated some of the results predicted by SNP2CAPS.
PMCID: PMC373308  PMID: 14704362

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