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.

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.

Genetic characterization of the polycotyledon locus in tomato.

Developmental mutants serve as a useful material to unravel the mechanisms necessary for organ development. The polycotyledon (poc) mutant of tomato, with multiple cotyledons in the seedling and varied phenotypic effects in the adult plant is one such mutant. Studies using physiological and anatomical methods in our lab suggest that POC is involved in the negative regulation of polar auxin transport, which is likely the reason for the pleiotropic phenotype in the mutant. Because of the physiological significance of the polycotyledon mutant described in this paper and also being first of its kind in tomato and also other plant species, we are using a map-based cloning approach to map the polycotyledon gene. Molecular mapping of this locus using segregating interspecific F2 mapping population localized polycotyledon gene close to TG424 marker on the long arm of chromosome 9. The closest marker mapped was a PCR marker identified in this study, E8A2 at a distance of 7.4 cM from the poc locus. The absence of tightly linked RAPD markers and the non-availability of more mapped markers in this region led us to initiate chromosome walk to polycotyledon gene. Both the flanking markers TG248 and E8A2 were used to screen the BAC library and a contig was developed for TG248 marker. The BAC-end sequences were analyzed for their use as RFLP markers to enrich this region for markers. Analysis of the BAC-end sequences revealed that poc is localized in the region surrounded by copia-like retrotransposon elements explaining the absence of markers in the euchromatin region on long arm of chromosome 9. Further studies identified two BAC-end sequences which mapped around the poc locus and also indicated very low physical versus genetic distance ratio in this region. The double mutant analyses of poc with the other two known polycotyledon mutants of tomato, pct and dem revealed allelism with pct; therefore, the poc mutant was named as pct1-2, and also the original pct mutant was renamed as pct1-1.

OligoSpawn: a software tool for the design of overgo probes from large unigene datasets.

BACKGROUND: Expressed sequence tag (EST) datasets represent perhaps the largest collection of genetic information. ESTs can be exploited in a variety of biological experiments and analysis. Here we are interested in the design of overlapping oligonucleotide (overgo) probes from large unigene (EST-contigs) datasets. RESULTS: OLIGOSPAWN is a suite of software tools that offers two complementary services, namely (1) the selection of "unique" oligos each of which appears in one unigene but does not occur (exactly or approximately) in any other and (2) the selection of "popular" oligos each of which occurs (exactly or approximately) in as many unigenes as possible. In this paper, we describe the functionalities of OLIGOSPAWN and the computational methods it employs, and we report on experimental results for the overgo probes designed with it. CONCLUSION: The algorithms we designed are highly efficient and capable of processing unigene datasets of sizes on the order of several tens of Mb in a few hours on a regular PC. The software has been used to design overgo probes employed to screen a barley BAC library (Hordeum vulgare). OLIGOSPAWN is freely available at http://oligospawn.ucr.edu/.