Sequencing of 15 622 gene-bearing BACs clarifies the gene-dense regions of the barley genome.

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley-Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant.

Development and implementation of high-throughput SNP genotyping in barley.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Coupling amplified DNA from flow-sorted chromosomes to high-density SNP mapping in barley.

BACKGROUND: Flow cytometry facilitates sorting of single chromosomes and chromosome arms which can be used for targeted genome analysis. However, the recovery of microgram amounts of DNA needed for some assays requires sorting of millions of chromosomes which is laborious and time consuming. Yet, many genomic applications such as development of genetic maps or physical mapping do not require large DNA fragments. In such cases time-consuming de novo sorting can be minimized by utilizing whole-genome amplification. RESULTS: Here we report a protocol optimized in barley including amplification of DNA from only ten thousand chromosomes, which can be isolated in less than one hour. Flow-sorted chromosomes were treated with proteinase K and amplified using Phi29 multiple displacement amplification (MDA). Overnight amplification in a 20-microlitre reaction produced 3.7 - 5.7 micrograms DNA with a majority of products between 5 and 30 kb. To determine the purity of sorted fractions and potential amplification bias we used quantitative PCR for specific genes on each chromosome. To extend the analysis to a whole genome level we performed an oligonucleotide pool assay (OPA) for interrogation of 1524 loci, of which 1153 loci had known genetic map positions. Analysis of unamplified genomic DNA of barley cv. Akcent using this OPA resulted in 1426 markers with present calls. Comparison with three replicates of amplified genomic DNA revealed >99% concordance. DNA samples from amplified chromosome 1H and a fraction containing chromosomes 2H - 7H were examined. In addition to loci with known map positions, 349 loci with unknown map positions were included. Based on this analysis 40 new loci were mapped to 1H. CONCLUSION: The results indicate a significant potential of using this approach for physical mapping. Moreover, the study showed that multiple displacement amplification of flow-sorted chromosomes is highly efficient and representative which considerably expands the potential of chromosome flow sorting in plant genomics.

Array-based genotyping and expression analysis of barley cv. Maythorpe and Golden Promise.

BACKGROUND: Golden Promise is a salt-tolerant spring barley closely related to Maythorpe. Salt tolerance in Golden Promise has been attributed to a single mutation at the Ari-e locus (on 5H) resulting from irradiation of Maythorpe. Golden Promise accumulates lower shoot Na+ compared to Maythorpe when growing under saline conditions. This study focused on elucidating the genetic basis and mechanisms involved in this difference. RESULTS: The level of polymorphism between the two genotypes was explored using the Barley1 GeneChip for single feature polymorphisms (SFPs) and an oligonucleotide pool assay for single nucleotide polymorphisms (SNPs). Polymorphism analyses revealed three haplotype blocks spanning 6.4 cM on chromosome 1H, 23.7 cM on chromosome 4H and 3.0 cM on 5H. The Barley1 GeneChip was used to examine transcript abundance in different tissues and stages during development. Several genes within the polymorphic haplotype blocks were differentially regulated. Additionally, a more global difference in the jasmonic acid pathway regulation was detected between the two genotypes. CONCLUSION: The results confirm that Golden Promise and Maythorpe are genetically very closely related but establish that they are not isogenic, as previously reported, due to three polymorphic haplotype blocks. Transcriptome analysis indicates that the response of the two genotypes to salinity stress is quite different. Additionally, the response to salinity stress in the roots and shoot tissue is strikingly different.

Large-scale expression profiling and physiological characterization of jasmonic acid-mediated adaptation of barley to salinity stress.

Barley (Hordeum vulgare L.) is a salt-tolerant member of the Triticeae. Recent transcriptome studies on salinity stress response in barley revealed regulation of jasmonic acid (JA) biosynthesis and JA-responsive genes by salt stress. From that observation and several other physiological reports, it was hypothesized that JA is involved in the adaptation of barley to salt stress. Here we tested that hypothesis by applying JA to barley plants and observing the physiological responses and transcriptome changes. Photosynthetic and sodium ion accumulation responses were compared after (1) salinity stress, (2) JA treatment and (3) JA pre-treatment followed by salinity stress. The JA-pre-treated salt-stressed plants accumulated strikingly low levels of Na(+) in the shoot tissue compared with untreated salt-stressed plants after several days of exposure to stress. In addition, pre-treatment with JA partially alleviated photosynthetic inhibition caused by salinity stress. Expression profiling after a short-term exposure to salinity stress indicated a considerable overlap between genes regulated by salinity stress and JA application. Three JA-regulated genes, arginine decarboxylase, ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) activase and apoplastic invertase are possibly involved in salinity tolerance mediated by JA. This work provides a reference data set for further study of the role of JA in salinity tolerance in barley and other plants species.

Genome-wide transcriptional analysis of salinity stressed japonica and indica rice genotypes during panicle initiation stage.

Rice yield is most sensitive to salinity stress imposed during the panicle initiation (PI) stage. In this study, we have focused on physiological and transcriptional responses of four rice genotypes exposed to salinity stress during PI. The genotypes selected included a pair of indicas (IR63731 and IR29) and a pair of japonica (Agami and M103) rice subspecies with contrasting salt tolerance. Physiological characterization showed that tolerant genotypes maintained a much lower shoot Na+ concentration relative to sensitive genotypes under salinity stress. Global gene expression analysis revealed a strikingly large number of genes which are induced by salinity stress in sensitive genotypes, IR29 and M103 relative to tolerant lines. We found 19 probe sets to be commonly induced in all four genotypes. We found several salinity modulated, ion homeostasis related genes from our analysis. We also studied the expression of SKC1, a cation transporter reported by others as a major source of variation in salt tolerance in rice. The transcript abundance of SKC1 did not change in response to salinity stress at PI stage in the shoot tissue of all four genotypes. However, we found the transcript abundance of SKC1 to be significantly higher in tolerant japonica Agami relative to sensitive japonica M103 under control and stressed conditions during PI stage.

An improved method to identify BAC clones using pooled overgos.

Hybridization using overgo probes is an established approach for screening arrayed bacterial artificial chromosome (BAC) libraries. We have improved the use of overgos by increasing the yield of positive clones using reduced levels of radioisotopes and enzyme. The strategy involves labeling with all four radiolabeled nucleotides in a hot pulse followed by a cold nucleotide chase and then extending the exposure time to compensate for reduced specific activity of the probes. The resulting cost savings and reduced human exposure to radiation make the use of highly pooled overgo probes a more attractive approach for screening of BAC libraries from organisms with large genomes.

Association of specific expansins with growth in maize leaves is maintained under environmental, genetic, and developmental sources of variation.

We aimed to evaluate whether changes in maize (Zea mays) leaf expansion rate in response to environmental stimuli or developmental gradients are mediated by common or specific expansins, a class of proteins known to enhance cell wall extensibility. Among the 33 maize expansin or putative expansin genes analyzed, 19 were preferentially expressed at some point of the leaf elongation zone and these expansins could be organized into three clusters related to cell division, maximal leaf expansion, and cell wall differentiation. Further analysis of the spatial distribution of expression was carried out for three expansins in leaves displaying a large range of expansion rates due to water deficit, genotype, and leaf developmental stage. With most sources of variation, the three genes showed similar changes in expression and consistent association with changes in leaf expansion. Moreover, our analysis also suggested preferential association of each expansin with elongation, widening, or both of these processes. Finally, using in situ hybridization, expression of two of these genes was increased in load-bearing tissues such as the epidermis and differentiating xylem. Together, these results suggest that some expansins may be preferentially related to elongation and widening after integrating several spatial, environmental, genetic, and developmental cues.

Expression analysis of barley (Hordeum vulgare L.) during salinity stress.

Barley (Hordeum vulgare L.) is a salt-tolerant crop species with considerable economic importance in salinity-affected arid and semiarid regions of the world. In this work, barley cultivar Morex was used for transcriptional profiling during salinity stress using a microarray containing approximately 22,750 probe sets. The experiment was designed to target the early responses of genes to a salinity stress at seedling stage. We found a comparable number of probe sets up-regulated and down-regulated in response to salinity. The differentially expressed genes were broadly characterized using gene ontology and through expression-based hierarchical clustering to identify interesting features in the data. A prominent feature of the response to salinity was the induction of genes involved in jasmonic acid biosynthesis and genes known to respond to jasmonic acid treatment. A large number of abiotic stress (heat, drought, and low temperature) related genes were also found to be responsive to salinity stress. Our results also indicate osmoprotection to be an early response of barley under salinity stress. Additionally, we compared the results of our studies with two other reports characterizing gene expression of barley under salinity stress and found very few genes in common.

Comparative transcriptional profiling of two contrasting rice genotypes under salinity stress during the vegetative growth stage.

Rice (Oryza sativa), a salt-sensitive species, has considerable genetic variation for salt tolerance within the cultivated gene pool. Two indica rice genotypes, FL478, a recombinant inbred line derived from a population developed for salinity tolerance studies, and IR29, the sensitive parent of the population, were selected for this study. We used the Affymetrix rice genome array containing 55,515 probe sets to explore the transcriptome of the salt-tolerant and salt-sensitive genotypes under control and salinity-stressed conditions during vegetative growth. Response of the sensitive genotype IR29 is characterized by induction of a relatively large number of probe sets compared to tolerant FL478. Salinity stress induced a number of genes involved in the flavonoid biosynthesis pathway in IR29 but not in FL478. Cell wall-related genes were responsive in both genotypes, suggesting cell wall restructuring is a general adaptive mechanism during salinity stress, although the two genotypes also had some differences. Additionally, the expression of genes mapping to the Saltol region of chromosome 1 were examined in both genotypes. Single-feature polymorphism analysis of expression data revealed that IR29 was the source of the Saltol region in FL478, contrary to expectation. This study provides a genome-wide transcriptional analysis of two well-characterized, genetically related rice genotypes differing in salinity tolerance during a gradually imposed salinity stress under greenhouse conditions.

Detecting single-feature polymorphisms using oligonucleotide arrays and robustified projection pursuit.

MOTIVATION: Genomic DNA was hybridized to oligonucleotide microarrays to identify single-feature polymorphisms (SFP) for Arabidopsis, which has a genome size of approximately 130 Mb. However, that method does not work well for organisms such as barley, with a much larger 5200 Mb genome. In the present study, we demonstrate SFP detection using a small number of replicate datasets and complex RNA as a surrogate for barley DNA. To identify single probes defining SFPs in the data, we developed a method using robustified projection pursuit (RPP). This method first evaluates, for each probe set, the overall differentiation of signal intensities between two genotypes and then measures the contribution of the individual probes within the probe set to the overall differentiation. RESULTS: RNA from whole seedlings with and without dehydration stress provided 'present' calls for approximately 75% of probe sets. Using triplicated data, among the 5% of 'present' probe sets identified as most likely to contain at least one SFP probe, at least 80% are correctly predicted. This was determined by direct sequencing of PCR amplicons derived from barley genomic DNA. Using a 5 percentile cutoff, we defined 2007 SFP probes contained in 1684 probe sets by combining three parental genotype comparisons: Steptoe versus Morex, Morex versus Barke and Oregon Wolfe Barley Dominant versus Recessive. AVAILABILITY: The algorithm is available upon request from the corresponding author. CONTACT: xinping.cui@ucr.edu SUPPLEMENTARY INFORMATION: http://faculty.ucr.edu/~xpcui.

Maize cystatins respond to developmental cues, cold stress and drought.

Comprehensive searches of maize EST data allowed us to identify 8 novel Corn Cystatin (CC) genes in addition to the previously known genes CCI and CCII. The deduced amino acid sequences of all 10 genes contain the typical cystatin family signature. In addition, they show an extended overall similarity with cystatins from other species that belong to several different phyto-cystatin subfamilies. To gain further insight into their respective roles in the maize plant, gene-specific expression profiles were established by semi-quantitative RT-PCR. While 7 CC genes were expressed in two or more tissues varying from gene to gene, CCI was preferentially expressed in immature tassels and CC8 and CC10 in developing kernels. As shown by in situ hybridisation of maize kernels, CC8 was specifically expressed in the basal region of the endosperm and CC10 both in the starchy endosperm and the scutellum of the embryo. The remaining, not kernel-specific genes, all had distinct expression kinetics during kernel development, generally with peaks during the early stages. In addition to developmental regulation, the effect of cold stress and water starvation were tested on cystatin expression. Two genes (CC8 and CC9) were induced by cold stress and 5 genes (CCII, CC3, CC4, CC5 and CC9) were down-regulated in response to water starvation. Taken together our data suggest distinct functions for CC genes in the maize plant.