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

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

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

Development and implementation of high-throughput SNP genotyping in barley

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

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. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s11103-006-9112-0 and is accessible for authorized users

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

[EN] 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 waslimited. To gain access to the gene-containing portion of the barley genome at high resolution, we identif ied 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 mostof thegene-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 barleyAe. 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 relevantSIThis work was supported by the USDA Initiative for Future Agriculture and Food Systems 01-52100-11346, North American Barley Genome Project (USDA-CSREES 2001-34213-10511), USDA-CSREES National Research Initiative (NRI) 2002-35300-12548, NSF Plant Genome Research Program DBI-0321756, BarleyCAP (USDA-CSREES-NRI 2006-55606-16722 and USDA-AFRI-NIFA 2009-85606-05701), USDA-AFRI-NIFA 2009-65300-05645, TriticeaeCAP (USDA-NIFA 2010-15718-10), NSF-ABI DBI-1062301, and UC Riverside Agricultural Experiment Station Hatch Project CA-R-BPS-5306-H. The work conducted by the US Department of Energy Joint Genome Institute was supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231. H.Š and J.D. have been supported by grant award LO1204 from the National Program of Sustainability

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.Keywords: Aegilops tauschii, Barley, centromere BACs, HarvEST:Barley, gene distribution, synteny, recombination frequency, Hordeum vulgare L., BAC sequencingThis is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by John Wiley & Sons Ltd. on behalf of the Society for Experimental Biology. The published article can be found at: http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291365-313X. Supporting information is available online at: http://onlinelibrary.wiley.com/doi/10.1111/tpj.12959/abstrac

The spread of a replicating-competant MuLV retroviral vector can be efficiently blocked by deletion variants

International audienceA retroviral vector in which the gag and pol genes have been replaced by the NLS-lacZ reporter gene was derived from a cloned AKV-like virus. A complementing cell line expressing the gag and pol retroviral genes was constructed. The retroviral vector was demonstrated to replicate in the complementing cells. Since transfection is known to generate deletion variants of the introduced plasmid, we have examined whether it can give rise to viral forms with a replicating advantage over the initial vector. After transfection in complementing cells the spread of the vector was followed by X-gal staining. The fraction of stained cells increased for the first 10 days following transfection and was then stabilized to about 20% stained cells, thus defining two cell types; one with LacZ+ phenotype and one with LacZ- phenotype. Molecular analysis showed that the latter contains a deleted form of the virus preventing cell infection by the vector presumably through a mechanism of interference involving the viral env gene. Thus, interference results in the efficient block of vector expansion

Properties of a MuLV replicating vector in mouse cells

International audienc