Single-feature polymorphism discovery in the barley transcriptome
© Rostoks et al.; licensee BioMed Central Ltd. 2005
Received: 8 February 2005
Accepted: 14 April 2005
Published: 11 May 2005
A probe-level model for analysis of GeneChip gene-expression data is presented which identified more than 10,000 single-feature polymorphisms (SFP) between two barley genotypes. The method has good sensitivity, as 67% of known single-nucleotide polymorphisms (SNP) were called as SFPs. This method is applicable to all oligonucleotide microarray data, accounts for SNP effects in gene-expression data and represents an efficient and versatile approach for highly parallel marker identification in large genomes.
Whole-genome sequences of Arabidopsis and rice have provided a fundamental platform for the discovery of gene content and function in dicot and monocot plants. Research on the model species has provided a wealth of knowledge on universal biochemical and genetic processes, as well as the development of analytical tools that are applicable to other plant species [1–3].
The availability of abundant, high-throughput sequence-based markers is the key for detailed genome-wide trait analysis. Single-nucleotide polymorphisms (SNP) are the most common sequence variation and a significant amount of effort has been invested in resequencing alleles to discovery SNPs. In fully sequenced small-genome model organisms SNP discovery is relatively straightforward, although high-throughput SNP discovery in natural populations remains both expensive and time-consuming .
A number of recent studies have reported the use of oligonucleotide arrays, including expression arrays, for SNP detection in a highly parallel manner . In these studies, whole genomic DNA was demonstrated to work very well for simple organisms such as yeast [6, 7], and even complex, albeit relatively small genomes, such as Arabidopsis . However, the application of oligonucleotide arrays for SNP detection in large genomes, such as human, has relied on prior complexity reduction using PCR-based enrichment [9, 10]. The use of oligonucleotide arrays for simultaneous genotyping and gene-expression analysis using RNA target has also been reported in yeast . While there is arguably little need for enhanced SNP discovery in yeast, the real power of the approach came from coupling genotyping and gene expression analysis.
For large-genome species, including crops such as wheat and barley, full-genome sequences may not be available in the near future. This has been compensated to some extent by model species that have allowed conserved biological processes to be studied. However, while Arabidopsis and rice provide insights into universal genetic, structural and developmental processes, they fail to address many topics relevant to crop-plant species, such as yield, yield stability and quality. Rice has a long history as a genetic model that has been strengthened by release of draft genome sequences [12, 13]. As a result of conservation of synteny at the genomic level it has been promoted as a model for the grasses . However, unlike the temperate cereals such as wheat and barley, rice cultivation occurs under short days and rather specific environmental conditions, its end uses are distinct and numerous exceptions to conserved synteny have now emerged [15–17]. Together, these highlight the limitations of rice as a universal genetic model for the cereal grasses.
Wheat and barley together constitute one third of world cereal production . Barley in particular is cultivated throughout the world, in environments as diverse as arctic regions of Northern Europe, subtropical regions of Africa and the highlands of the Andes and the Himalayas . Barley breeding has created varieties tailored mainly for animal feed, malt production and human food . Ultimately, environmental and agronomical variation is based on genetic (sequence) diversity of the barley genome, with expression of agronomic traits closely linked to environmental adaptability.
With genome sizes of around 5,200 megabase pairs (Mbp) for barley [21, 22] and around 16,100 Mbp for bread wheat  and genomic structure consisting of gene islands interspersed with highly repetitive retrotransposon sequences [15, 23], access to sequence-based markers is currently provided through highly developed expressed sequence tag (EST) resources .
The most important traits in crop species are generally polygenic. These have traditionally been studied using biparental mapping populations and a large pool of mapped restriction fragment length polymorphism (RFLP) and/or simple sequence repeat (SSR) markers . However, with the strong trend towards genome-wide association analyses based on linkage disequilibrium (LD) [26, 27] there is a clear need for robust high-density and high-throughput markers that can be effectively deployed, often in closely related elite germplasm. While the number and distribution of markers for LD studies in barley remains to be empirically determined, SNP markers offer both the sequence specificity and throughput necessary for the success of this approach. SNP discovery in large-genome species is currently limited to identifying SNPs in silico in EST assemblies and resequencing of EST-derived unigenes in relevant germplasm , and scaling-up such approaches requires significant investment of both time and funding [28–30]. An approach that would allow parallel screening of the whole 'gene space' for SNPs is therefore highly desirable.
An Affymetrix GeneChip that allows simultaneous expression analysis of 22,000 transcripts has recently become available for barley . Transcription provides a native mechanism for the enrichment of gene sequences. Polymorphisms present in DNA are transcribed into the messenger RNA and can potentially affect the hybridization to the GeneChip probes, if present in a region complementary to the probe. Polymorphisms generated during mRNA processing, such as alternative splicing and polyadenylation, could also affect hybridization of the target RNA.
Here we report the use of the Affymetrix Barley1 GeneChip to identify single-feature polymorphisms (SFP), which include not only SNPs but also the processing polymorphisms mentioned above, in barley transcript profiling data from cultivars Morex and Golden Promise. The statistical algorithm presented here allowed us to distinguish genotype-dependent hybridization differences at the probe level once overall gene-expression level was accounted for, leading to the identification of 10,504 SFPs.
Identification of SFP in Barley1 GeneChip transcription-profiling data
Gene-expression data for barley cultivars Morex and Golden Promise was generated within an international collaborative project of barley researchers (unpublished results, see Acknowledgements) and consisted of 36 GeneChip hybridizations (three replicates of six tissue types) for two genotypes. Raw microarray data are available from ArrayExpress [32, 33], BarleyBase  and . The analysis code, lists of RNA and genomic SFPs, primer sequences, and the SFP sequence confirmation table are available from our website as supplementary information . The hybridization intensities for each of the perfect match (PM) probes were extracted from the .CEL files. Background correction and quantile normalization was performed using the Bioconductor package RMA [36, 37]. The resulting data matrix of 22,801 probe sets with 11 PM probes each was analyzed using probe-level linear models that accounted for main fixed effects of genotype, tissue, and individual probe intensity, as well as tissue-specific differences across genotypes. One replicate from a single tissue sample of Golden Promise consistently clustered with the analogous Morex replicates and this sample was reclassified as Morex. The residuals from the linear model were saved into a matrix of 250,811 probes by 36 arrays and subsequently fitted for a genotype effect at the probe level to identify SFPs between the 17 Golden Promise and 19 Morex arrays. The Bioconductor package siggenes  was used to determine SFPs according to statistical analysis of microarrays (SAM) [38, 39].
SFP false discovery rate (FDR) estimates in RNA and genomic DNA hybridization data
RNA hybridization: 17 Golden Promise 19 Morex, 6 tissues; SAM analysis for the two-class unpaired case assuming unequal variances; s0 = 0.0342 (the 5% quantile of the s values); number of permutations, 500. Mean number of falsely called genes is computed.
Genomic DNA hybridization three replicates three genotypes; SAM analysis for the multi-class case with three classes; s0 = 0.0123 (the 25 % quantile of the s values); number of permutations: 100; mean number of falsely called genes is computed.
Sequence confirmation of SFP
Confirmation of SFP was done by comparison with three barley sequence datasets. Barley EST  is EST unigene assembly 21  and contained 234 contigs with 624 predicted SFP probes where both Morex and Golden Pomise sequence were available. These were examined manually to identify SNP that overlapped 25mers on the array (see SFP confirmation table in  (EST dataset)).
The second set is an experimental cDNA sequence set targeting regions with predicted SFPs. Comparative DNA sequence was generated from each genotype by targeted resequencing of reverse-transcription PCR (RT-PCR) products covering 262 probes. For each genotype we combined an equal amount of RNA from all six tissue types used for hybridization to the GeneChips and converted it to a single-stranded cDNA. PCR amplification and subsequent sequencing allowed us to obtain good-quality sequence from both genotypes (see SFP confirmation table in  (targeted dataset)).
The third set was an experimental random genomic DNA sequence set used as a tool for SNP discovery in barley . This dataset (SFP confirmation table in  (random dataset)) consisted of barley unigenes that had been resequenced from genomic DNA from eight barley lines, including Morex and Golden Promise, within an ongoing SNP discovery project . The selection of these genes was considered random with respect to the genes predicted to have SFP. The SNP discovery project targeted the 3' ends of unigenes, the region also selected for Affymetrix probe design. The random-sequencing dataset consisted of sequences for 300 unigene contigs and covered a total of 2,204 Affymetrix probes with high-quality sequences from both genotypes.
Single feature polymorphism (SFP) comparison with sequence-characterized SNPs
Chi-square = 2,049.2, df = 4, p-value = 0
SFP discovery in individual tissue types
Replicates (GP, MX)
False sequence polymorphism rate
% variance explained
Comparison of SFP prediction in individual tissues against the full sample
We tested the sensitivity and false SNP discovery rates of our analysis with single tissue/genotype comparisons to observe how it would perform in smaller experiments. Datasets containing three replicates per genotype for each tissue type were analyzed at the threshold that again identified 10,504 SFP. In general there was a 4-16% decrease in sensitivity of the SFP prediction, which was the expected result of reducing power. On the other hand, SFP prediction in a single tissue type decreased the false SNP discovery rate by 4-5%. This was probably due to the reduction of probe-level variation in expression across tissues. In all, more than 10,000 SFP could be reliably identified even when expression profiles of single tissues were analyzed.
Genomic DNA hybridizations
Comparison of SFP prediction in RNA and genomic DNA hybridizations
Chi-square = 107.28, df = 1, p-value = 3.863e-25
Affymetrix GeneChips designed for gene-expression analysis can be utilized for genome-wide identification of sequence polymorphisms . Whole-genome DNA has been used as a hybridization target in yeast [6, 7] and in Arabidopsis  to identify SFPs using expression arrays. While such an approach was valid in yeast and a small-genome model plant, the transfer of this approach to cereal crop plants with up to 100-fold larger genome sizes is problematic. The number of genes in barley is likely to be comparable to the estimated number of genes in Arabidopsis and rice [42, 43]. However, the amount of repetitive DNA in barley will dilute the gene-specific signal in the target labelled DNA.
Until now, PCR-based artificial enrichment for a subset of sequences has been used to tackle the complexity of large genomes [10, 9, 44]. Using RNA as a hybridization target provides a natural way of enriching for gene sequences while maintaining all the sequence diversity present in transcribed sequences. However, sequence polymorphism effects on hybridization are concealed within the overall variation in gene-expression levels and tissue-dependent and genotype-dependent differential gene expression. Additional complexity comes from posttranscriptional sequence polymorphisms, such as alternative splicing and alternative polyadenylation. New array designs that tile probes across genes and intergenic regions will help unravel this complexity as nucleotide polymorphisms may affect single features while alternative transcripts may more often affect adjacent features.
We present here a statistical approach that allows us to reliably discern the probe-level differential hybridization between two genotypes that is often caused by sequence polymorphisms once variation in overall gene-expression level is normalized. Our approach allows the use of expression array data generated from different tissue types, and thus increases its versatility and applicability to the wide range of currently available oligonucleotide microarray data.
The analysis algorithm was applied to gene-expression microarray data generated from two barley genotypes with six tissue types each for a total of 36 array hybridizations. At a stringent 0.1% FDR, 10,504 SFPs were identified. Comparison to the available sequence-verified SNP data suggested that 67% of the known SNPs were predicted, confirming a good sensitivity. Approximately 40% of the SFP probes that were sequence-verified did not reveal any polymorphisms at the sequence level; thus, the FDR was up to 13-fold higher compared to the rate for Arabidopsis genomic DNA hybridizations . The higher false-positive rates can be at least partly explained by variation in mRNA structure (for example, alternative splicing and polyadenylation) between tissues, and possibly between genotypes, which would lead to differential hybridization to probes but could not be detected by sequencing. A recent study using an EST collection concluded that at least 4% of barley genes may undergo alternative splicing ; however, more experimental data may be required to correctly model the rate of probe level variation in plant gene-expression data.
For practical application the balance between the cost of replicates and the number of replicates necessary to maintain sensitivity is important. We therefore analyzed the microarray data comparing just three replicates of each tissue type from the two genotypes (Table 3). Overall sensitivity decreased, but remained above 50%. Remarkably, the false SNP discovery rate was better for single tissue comparisons, probably because variation in mRNA transcript processing among tissues was eliminated.
Certain molecular marker applications require the precise nature of sequence changes to be known. The conventional approach to SNP discovery is based on resequencing alleles, which is particularly inefficient if the polymorphism levels are low. Prescreening for polymorphisms using, for example, single-strand conformation polymorphism (SSCP)  or Eco-TILLING , allows a reduction in sequencing costs, but these approaches are time-consuming, relatively expensive and rely on PCR. SFP detection in gene-expression microarray data allows parallel screening of a large proportion of all the organisms' gene space in one experiment. The stringency of SFP calls can also be adjusted for a particular application, that is, decreasing stringency will result in additional calls at the expense of higher false-positive rates.
Gene-expression levels are currently being treated as quantitative traits and transcript abundance variation is being mapped as quantitative trait loci (QTL) [48, 49]. Incorporating SFP effects into calculations will improve accuracy of gene-expression studies and will facilitate correct assessment of allele-specific gene-expression differences. Furthermore, an SFP identified in a coding region of a gene that is differentially expressed in an allele-specific manner represents a marker linked to the regulatory regions of the gene, and as such may help distinguish between cis and trans effects in allele-specific gene expression [50–52].
Materials and methods
Affymetrix Barley1 GeneChip data
Affymetrix Barley1 GeneChip data was produced within an international collaborative project (A. Druka, G. Muehlbauer, I. Druka, R. Caldo, U. Baumann, N. Rostoks, A. Schreiber, R. Wise, T. Close, A. Kleinhofs, et al., unpublished work). Six tissue types were analyzed from two genotypes, Golden Promise (GP) and Morex (MX), with three type I replicates for a total of 36 arrays. We found that the GP genotype of one particular tissue replicate had a very high correlation with the three replicates from the comparable tissue from the MX genotype. We therefore re-assigned that replicate as genotype MX.
Genomic DNA from the wild barley Hordeum vulgare ssp. spontaneum (accession Mehola; arrays 1-3) and two morphologically diverse lines Oregon Wolfe Barley Recessive (arrays 4-6) and Oregon Wolfe Barley Dominant (arrays 7-9)  were prepared according to  and hybridized to the Affymetrix Barley1 GeneChip in triplicate according to standard methods for RNA.
SFP prediction in gene expression data
Raw .CEL files were background corrected and quantile normalized according to Bolstad et al. . Subsequently, only the 11 Perfect Match (PM) features from each of 22,801 probe sets were fit with the following linear model
log(Ytgrp) = u + tissue + genotype + genotype × tissue + probe + error,
where Y is the background corrected normalized intensity of t (tissue), g (genotype), r (replicate), and p (probe) in a probe set. u is the mean probe intensity, while tissue has six states, and genotype has two states. The genotype by tissue effect accounted for tissue specific effects dependent on genotype. The residuals (22,801 probe sets × 11 probes = 250,811) from this model were fitted for a genotype effect at the probe level to reveal SFP using the Bioconductor package siggenes [37, 36]. False discovery rates were estimated according to SAM [38, 39] by performing 500 random permutations for RNA analysis or 100 permutations for genomic DNA analysis. The expected proportion of significantly different features (p0) was set to 0.95.
SFP confirmation by SNP analysis in silico
The EST unigene assembly 21  that was used to produce the Affymetrix Barley1 GeneChip  contains 349,709 ESTs, of which 52,556 were derived from Morex (11 libraries) and 7,439 from Golden Promise (1 library). Library details are available from the HarvEST EST database . HarvEST was used to identify a total of 1,758 unigene contigs containing both Morex and Golden Promise EST.
SFP confirmation by sequencing
192 primer pairs for 188 contigs were designed using Primer3 software  targeting 262 probes. Primers were supplied by Illumina. Single-stranded DNA template for PCR was synthesized from the same RNA samples that were used for hybridization to the Affymetrix GeneChips using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). For each genotype, we combined 1 μg of RNA from each of the six tissue types and converted it to a single-stranded cDNA according to the manufacturer's recommendations using oligo(dT)12-18 as a primer. Single-stranded DNA was diluted fivefold and 2 μl was used for PCR amplification using gene-specific primers and HotStart Taq polymerase (Qiagen) with the following thermocycling parameters: 15 min 95°C, followed by 40 cycles of 30 sec 95°C, 45 s 60°C and 2 min 72°C, with a 10 min final extension at 72°C. PCR products were treated with ExoSAP-IT reagent (USB Corporation) and sequenced with the same primers using BigDye Terminator v3.1 cycle sequencing kit on an ABI PRISM 3700 sequencer (Applied Biosystems). Base-calling of ABI chromatograms and assembly of each unigene were done using Mutation Surveyor software (SoftGenetics, State College, PA). Synthetic chromatograms generated for all probe and EST unigene sequences were included in assemblies for comparison. Polymorphisms were called using Mutation Surveyor software and examined manually. SNP positions were recorded symmetrically, that is, a SNP in the central nucleotide of a 25-mer was in position 13, while SNPs in either first or twenty-fifth position was assigned position 1. Probes with multiple SNPs were allocated to a single group (Figure 3). Insertions and deletions were scored as polymorphisms, but the positions of polymorphisms were not scored.
SNP discovery in a random EST contig set
An SNP discovery project is currently underway in our laboratory which is based on resequencing alleles of barley genes in a set of eight barley lines, including Morex and Golden Promise . The same EST unigene assembly that was used to design the Affymetrix Barley1 GeneChip was used in this SNP discovery study; PCR was carried out on genomic DNA templates, however. The Morex and Golden Promise sequences were reassembled separately as described for the SFP sequence set. Three hundred contigs representing essentially a random sample without any prior knowledge of polymorphisms were selected from this set on the basis that they included sequences from both genotypes; did not contain introns; sequences from both genotypes covered at least six Affymetrix Barley1 GeneChip probes for each probe set.
The gene-expression data for the barley cultivars Morex and Golden Promise was generated as part of an international collaborative project between barley researchers and is presented in a biological context in a separate manuscript (A. Druka, G. Muehlbauer, I. Druka, R. Caldo, U. Baumann, N. Rostoks, A. Schreiber, R. Wise, T. Close, A. Kleinhofs, A. Graner, A. Schulman, P. Langridge, K. Sato, P. Hayes, J. McNicol, D. Marshall, R. Waugh, personal communication). We thank those listed for pre-publication access to this dataset. Special thanks are due to Arnis Druka and Ilze Druka for assistance with microarray data and helpful discussions. We thank Yunda Huang for help and discussion with analysis. This project was funded by a BBSRC/SEERAD grant to R.W. and by start-up funds to J.O.B. from the University of Chicago.
- Borevitz JO, Nordborg M: The impact of genomics on the study of natural variation in Arabidopsis. Plant Physiol. 2003, 132: 718-725. 10.1104/pp.103.023549.PubMedPubMed CentralView ArticleGoogle Scholar
- Borevitz JO, Chory J: Genomics tools for QTL analysis and gene discovery. Curr Opin Plant Biol. 2004, 7: 132-136. 10.1016/j.pbi.2004.01.011.PubMedView ArticleGoogle Scholar
- Rensink WA, Buell CR: Arabidopsis to rice. Applying knowledge from a weed to enhance our understanding of a crop species. Plant Physiol. 2004, 135: 622-629. 10.1104/pp.104.040170.PubMedPubMed CentralView ArticleGoogle Scholar
- Kwok PY, Chen X: Detection of single nucleotide polymorphisms. Curr Issues Mol Biol. 2003, 5: 43-60.PubMedGoogle Scholar
- Hazen SP, Kay SA: Gene arrays are not just for measuring gene expression. Trends Plant Sci. 2003, 8: 413-416. 10.1016/S1360-1385(03)00186-9.PubMedView ArticleGoogle Scholar
- Winzeler EA, Castillo-Davis CI, Oshiro G, Liang D, Richards DR, Zhou Y, Hartl DL: Genetic diversity in yeast assessed with whole-genome oligonucleotide arrays. Genetics. 2003, 163: 79-89.PubMedPubMed CentralGoogle Scholar
- Winzeler E, Richards D, Conway A, Goldstein A, Kalman S, McCullough M, McCusker JH, Stevens D, Wodicka L, Lockhart D, et al: Direct allelic variation scanning of the yeast genome. Science. 1998, 281: 1194-1197. 10.1126/science.281.5380.1194.PubMedView ArticleGoogle Scholar
- Borevitz JO, Liang D, Plouffe D, Chang HS, Zhu T, Weigel D, Berry CC, Winzeler E, Chory J: Large-scale identification of single-feature polymorphisms in complex genomes. Genome Res. 2003, 13: 513-523. 10.1101/gr.541303.PubMedPubMed CentralView ArticleGoogle Scholar
- Kennedy GC, Matsuzaki H, Dong S, Liu WM, Huang J, Liu G, Su X, Cao M, Chen W, Zhang J, et al: Large-scale genotyping of complex DNA. Nat Biotechnol. 2003, 21: 1233-1237. 10.1038/nbt869.PubMedView ArticleGoogle Scholar
- Dong S, Wang E, Hsie L, Cao Y, Chen X, Gingeras TR: Flexible use of high-density oligonucleotide arrays for single- nucleotide polymorphism discovery and validation. Genome Res. 2001, 11: 1418-1424. 10.1101/gr.171101.PubMedPubMed CentralView ArticleGoogle Scholar
- Ronald J, Akey J, Whittle J, Smith E, Yvert G, Kruglyak L: Simultaneous genotyping, gene expression measurement, and detection of allele-specific expression with oligonucleotide arrays. Genome Res. 2005, 15: 284-291. 10.1101/gr.2850605.PubMedPubMed CentralView ArticleGoogle Scholar
- Goff SA, Ricke D, Lan TH, Presting G, Wang R, Dunn M, Glazebrook J, Sessions A, Oeller P, Varma H, et al: A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science. 2002, 296: 92-100. 10.1126/science.1068275.PubMedView ArticleGoogle Scholar
- Yu J, Hu S, Wang J, Wong GK, Li S, Liu B, Deng Y, Dai L, Zhou Y, Zhang X, et al: A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science. 2002, 296: 79-92. 10.1126/science.1068037.PubMedView ArticleGoogle Scholar
- Bennetzen J, Freeling M: The unified grass genome: synergy in synteny. Genome Res. 1997, 7: 301-306.PubMedGoogle Scholar
- Caldwell KS, Langridge P, Powell W: Comparative sequence analysis of the region harboring the hardness locus in barley and its colinear region in rice. Plant Physiol. 2004, 136: 3177-3190. 10.1104/pp.104.044081.PubMedPubMed CentralView ArticleGoogle Scholar
- Brunner S, Keller B, Feuillet C: A large rearrangement involving genes and low-copy DNA interrupts the microcollinearity between rice and barley at the Rph7 locus. Genetics. 2003, 164: 673-683.PubMedPubMed CentralGoogle Scholar
- Bennetzen J, Ramakrishna W: Numerous small rearrangements of gene content, order and orientation differentiate grass genomes. Plant Mol Biol. 2002, 48: 821-827. 10.1023/A:1014841515249.PubMedView ArticleGoogle Scholar
- FAO. [http://faostat.fao.org/default.jsp]
- Stanca M: Diversity in abiotic stress tolerance. Diversity in Barley. Edited by: von Bothmer R, van Hintum T, Knuepffer H, Sato K. 2003, Amsterdam: Elsevier Science, 179-199.Google Scholar
- Fischbeck G: Diversification through breeding. Diversity in Barley. Edited by: von Bothmer R, van Hintum T, Knuepffer H, Sato K. 2003, Amsterdam: Elsevier Science, 29-52.Google Scholar
- Bennett M, Leitch I: Nuclear DNA amounts in angiosperms. Annls Bot Lond. 1995, 76: 113-176.View ArticleGoogle Scholar
- Jakob SS, Meister A, Blattner FR: The considerable genome size variation of Hordeum species (poaceae) is linked to phylogeny, life form, ecology, and speciation rates. Mol Biol Evol. 2004, 21: 860-869. 10.1093/molbev/msh092.PubMedView ArticleGoogle Scholar
- Rostoks N, Park YJ, Ramakrishna W, Ma J, Druka A, Shiloff BA, SanMiguel PJ, Jiang Z, Brueggeman R, Sandhu D, et al: Genomic sequencing reveals gene content, genomic organization, and recombination relationships in barley. Funct Integr Genomics. 2002, 2: 51-59. 10.1007/s10142-002-0055-5.PubMedView ArticleGoogle Scholar
- NCBI dbEST summary. [http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html]
- Graingenes. [http://wheat.pw.usda.gov/ggpages/map_summary.html]
- Flint-Garcia SA, Thornsberry JM, Buckler ES: Structure of linkage disequilibrium in plants. Annu Rev Plant Biol. 2003, 54: 357-374. 10.1146/annurev.arplant.54.031902.134907.PubMedView ArticleGoogle Scholar
- Rafalski A: Applications of single nucleotide polymorphisms in crop genetics. Curr Opin Plant Biol. 2002, 5: 94-100. 10.1016/S1369-5266(02)00240-6.PubMedView ArticleGoogle Scholar
- Kota R, Rudd S, Facius A, Kolesov G, Thiel T, Zhang H, Stein N, Mayer K, Graner A: Snipping polymorphisms from large EST collections in barley (Hordeum vulgare L.). Mol Genet Genomics. 2003, 270: 24-33. 10.1007/s00438-003-0891-6.PubMedView ArticleGoogle Scholar
- Kota R, Varshney RK, Thiel T, Dehmer KJ, Graner A: Generation and comparison of EST-derived SSRs and SNPs in barley (Hordeum vulgare L.). Hereditas. 2001, 135: 145-151. 10.1111/j.1601-5223.2001.00145.x.PubMedView ArticleGoogle Scholar
- Rostoks N, Cardle L, Svensson J, Walia H, Rodriguez E, Wanamaker S, Hedley P, Liu H, Ramsay L, Russell J, et al: Single nucleotide polymorphism mapping of the barley genes involved in abiotic stresses. Czech J Genet Plant Breed. 2004, 40: 52-Google Scholar
- Close TJ, Wanamaker SI, Caldo RA, Turner SM, Ashlock DA, Dickerson JA, Wing RA, Muehlbauer GJ, Kleinhofs A, Wise RP: A new resource for cereal genomics: 22K barley GeneChip comes of age. Plant Physiol. 2004, 134: 960-968. 10.1104/pp.103.034462.PubMedPubMed CentralView ArticleGoogle Scholar
- ArrayExpress. [http://www.ebi.ac.uk/arrayexpress/]
- Brazma A, Parkinson H, Sarkans U, Shojatalab M, Vilo J, Abeygunawardena N, Holloway E, Kapushesky M, Kemmeren P, Lara GG, et al: ArrayExpress - a public repository for microarray gene expression data at the EBI. Nucleic Acids Res. 2003, 31: 68-71. 10.1093/nar/gkg091.PubMedPubMed CentralView ArticleGoogle Scholar
- BarleyBase. [http://www.barleybase.org/]
- Naturalvariation. [http://naturalvariation.org/barley]
- Bolstad BM, Irizarry RA, Astrand M, Speed TP: A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003, 19: 185-193. 10.1093/bioinformatics/19.2.185.PubMedView ArticleGoogle Scholar
- Bioconductor. [http://bioconductor.org]
- Schwender H, Krause A, Ickstadt K: Comparison of the Empirical Bayes and the Significance Analysis of Microarrays. 2003, Technical Report. SFB 475: Dortmund, Germany: University of DortmundGoogle Scholar
- Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA. 2001, 98: 5116-5121. 10.1073/pnas.091062498.PubMedPubMed CentralView ArticleGoogle Scholar
- HarvEST. [http://harvest.ucr.edu]
- Costa JM, Corey A, Hayes PM, Jobet C, Kleinhofs A, Kopisch Obusch A, Kramer SF, Kudrna D, Li M, Riera Lizarazu O, et al: Molecular mapping of the Oregon Wolfe Barleys: A phenotypically polymorphic doubled-haploid population. Theor Appl Genet. 2001, 103: 415-424. 10.1007/s001220100622.View ArticleGoogle Scholar
- Bancroft I: Insights into cereal genomes from two draft genome sequences of rice. Genome Biol. 2002, 3: reviews1015.1-1015.3. 10.1186/gb-2002-3-6-reviews1015.View ArticleGoogle Scholar
- Bennetzen JL, Coleman C, Liu R, Ma J, Ramakrishna W: Consistent over-estimation of gene number in complex plant genomes. Curr Opin Plant Biol. 2004, 7: 732-736. 10.1016/j.pbi.2004.09.003.PubMedView ArticleGoogle Scholar
- Vos P, Hogers R, Bleeker M, Reijans M, van de LT, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, et al: AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 1995, 23: 4407-4414.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang H, Sreenivasulu N, Weschke W, Stein N, Rudd S, Radchuk V, Potokina E, Scholz U, Schweizer P, Zierold U, et al: Large-scale analysis of the barley transcriptome based on expressed sequence tags. Plant J. 2004, 40: 276-290. 10.1111/j.1365-313X.2004.02209.x.PubMedView ArticleGoogle Scholar
- Andersen PS, Jespersgaard C, Vuust J, Christiansen M, Larsen LA: Capillary electrophoresis-based single strand DNA conformation analysis in high-throughput mutation screening. Hum Mutat. 2003, 21: 455-465. 10.1002/humu.10169.PubMedView ArticleGoogle Scholar
- Comai L, Young K, Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, et al: Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. Plant J. 2004, 37: 778-786. 10.1111/j.0960-7412.2003.01999.x.PubMedView ArticleGoogle Scholar
- Brem RB, Yvert G, Clinton R, Kruglyak L: Genetic dissection of transcriptional regulation in budding yeast. Science. 2002, 296: 752-755. 10.1126/science.1069516.PubMedView ArticleGoogle Scholar
- Schadt EE, Monks SA, Drake TA, Lusis AJ, Che N, Colinayo V, Ruff TG, Milligan SB, Lamb JR, Cavet G, et al: Genetics of gene expression surveyed in maize, mouse and man. Nature. 2003, 422: 297-302. 10.1038/nature01434.PubMedView ArticleGoogle Scholar
- Cowles CR, Hirschhorn JN, Altshuler D, Lander ES: Detection of regulatory variation in mouse genes. Nat Genet. 2002, 32: 432-437. 10.1038/ng992.PubMedView ArticleGoogle Scholar
- Morley M, Molony CM, Weber TM, Devlin JL, Ewens KG, Spielman RS, Cheung VG: Genetic analysis of genome-wide variation in human gene expression. Nature. 2004, 430: 743-747. 10.1038/nature02797.PubMedPubMed CentralView ArticleGoogle Scholar
- Wittkopp PJ, Haerum BK, Clark AG: Evolutionary changes in cis and trans gene regulation. Nature. 2004, 430: 85-88. 10.1038/nature02698.PubMedView ArticleGoogle Scholar
- Arabidopsis methods. [http://naturalvariation.org/methods]
- Rozen S, Skaletsky H: Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000, 132: 365-386.PubMedGoogle Scholar
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