Genome wide SNP discovery in flax through next generation sequencing of reduced representation libraries

BACKGROUND: Flax (Linum usitatissimum L.) is a significant fibre and oilseed crop. Current flax molecular markers, including isozymes, RAPDs, AFLPs and SSRs are of limited use in the construction of high density linkage maps and for association mapping applications due to factors such as low reproducibility, intense labour requirements and/or limited numbers. We report here on the use of a reduced representation library strategy combined with next generation Illumina sequencing for rapid and large scale discovery of SNPs in eight flax genotypes. SNP discovery was performed through in silico analysis of the sequencing data against the whole genome shotgun sequence assembly of flax genotype CDC Bethune. Genotyping-by-sequencing of an F(6)-derived recombinant inbred line population provided validation of the SNPs. RESULTS: Reduced representation libraries of eight flax genotypes were sequenced on the Illumina sequencing platform resulting in sequence coverage ranging from 4.33 to 15.64X (genome equivalents). Depending on the relatedness of the genotypes and the number and length of the reads, between 78% and 93% of the reads mapped onto the CDC Bethune whole genome shotgun sequence assembly. A total of 55,465 SNPs were discovered with the largest number of SNPs belonging to the genotypes with the highest mapping coverage percentage. Approximately 84% of the SNPs discovered were identified in a single genotype, 13% were shared between any two genotypes and the remaining 3% in three or more. Nearly a quarter of the SNPs were found in genic regions. A total of 4,706 out of 4,863 SNPs discovered in Macbeth were validated using genotyping-by-sequencing of 96 F(6) individuals from a recombinant inbred line population derived from a cross between CDC Bethune and Macbeth, corresponding to a validation rate of 96.8%. CONCLUSIONS: Next generation sequencing of reduced representation libraries was successfully implemented for genome-wide SNP discovery from flax. The genotyping-by-sequencing approach proved to be efficient for validation. The SNP resources generated in this work will assist in generating high density maps of flax and facilitate QTL discovery, marker-assisted selection, phylogenetic analyses, association mapping and anchoring of the whole genome shotgun sequence

Synteny analysis in Rosids with a walnut physical map reveals slow genome evolution in long-lived woody perennials.

BackgroundMutations often accompany DNA replication. Since there may be fewer cell cycles per year in the germlines of long-lived than short-lived angiosperms, the genomes of long-lived angiosperms may be diverging more slowly than those of short-lived angiosperms. Here we test this hypothesis.ResultsWe first constructed a genetic map for walnut, a woody perennial. All linkage groups were short, and recombination rates were greatly reduced in the centromeric regions. We then used the genetic map to construct a walnut bacterial artificial chromosome (BAC) clone-based physical map, which contained 15,203 exonic BAC-end sequences, and quantified with it synteny between the walnut genome and genomes of three long-lived woody perennials, Vitis vinifera, Populus trichocarpa, and Malus domestica, and three short-lived herbs, Cucumis sativus, Medicago truncatula, and Fragaria vesca. Each measure of synteny we used showed that the genomes of woody perennials were less diverged from the walnut genome than those of herbs. We also estimated the nucleotide substitution rate at silent codon positions in the walnut lineage. It was one-fifth and one-sixth of published nucleotide substitution rates in the Medicago and Arabidopsis lineages, respectively. We uncovered a whole-genome duplication in the walnut lineage, dated it to the neighborhood of the Cretaceous-Tertiary boundary, and allocated the 16 walnut chromosomes into eight homoeologous pairs. We pointed out that during polyploidy-dysploidy cycles, the dominant tendency is to reduce the chromosome number.ConclusionSlow rates of nucleotide substitution are accompanied by slow rates of synteny erosion during genome divergence in woody perennials

A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening

<p>Abstract</p> <p>Background</p> <p>A five-dimensional (5-D) clone pooling strategy for screening of bacterial artificial chromosome (BAC) clones with molecular markers utilizing highly-parallel Illumina GoldenGate assays and PCR facilitates high-throughput BAC clone and BAC contig anchoring on a genetic map. However, this strategy occasionally needs manual PCR to deconvolute pools and identify truly positive clones.</p> <p>Results</p> <p>A new implementation is reported here for our previously reported clone pooling strategy. Row and column pools of BAC clones are divided into sub-pools with 1~2× genome coverage. All BAC pools are screened with Illumina's GoldenGate assay and the BAC pools are deconvoluted to identify individual positive clones. Putative positive BAC clones are then further analyzed to find positive clones on the basis of them being neighbours in a contig. An exhaustive search or brute force algorithm was designed for this deconvolution and integrated into a newly developed software tool, FPCBrowser, for analyzing clone pooling data. This algorithm was used with empirical data for 55 Illumina GoldenGate SNP assays detecting SNP markers mapped on <it>Aegilops tauschii </it>chromosome 2D and <it>Ae. tauschii </it>contig maps. Clones in single contigs were successfully assigned to 48 (87%) specific SNP markers on the map with 91% precision.</p> <p>Conclusion</p> <p>A new implementation of 5-D BAC clone pooling strategy employing both GoldenGate assay screening and assembled BAC contigs is shown here to be a high-throughput, low cost, rapid, and feasible approach to screening BAC libraries and anchoring BAC clones and contigs on genetic maps. The software FPCBrowser with the integrated clone deconvolution algorithm has been developed and is downloadable at <url>http://avena.pw.usda.gov/wheatD/fpcbrowser.shtml</url>.</p

Ethanol Exposure Alters Protein Expression in a Mouse Model of Fetal Alcohol Spectrum Disorders

Alcohol exposure during development can result in variable growth retardation and facial dysmorphology known as fetal alcohol spectrum disorders. Although the mechanisms underlying the disorder are not fully understood, recent progress has been made that alcohol induces aberrant changes in gene expression and in the epigenome of embryos. To inform the gene and epigenetic changes in alcohol-induced teratology, we used whole-embryo culture to identify the alcohol-signature protein profile of neurulating C6 mice. Alcohol-treated and control cultures were homogenized, isoelectrically focused, and loaded for 2D gel electrophoresis. Stained gels were cross matched with analytical software. We identified 40 differentially expressed protein spots (P < 0.01), and 9 spots were selected for LC/MS-MS identification. Misregulated proteins include serotransferrin, triosephosphate isomerase and ubiquitin-conjugating enzyme E2 N. Misregulation of serotransferrin and triosephosphate isomerase was confirmed with immunologic analysis. Alteration of proteins with roles in cellular function, cell cycle, and the ubiquitin-proteasome pathway was induced by alcohol. Several misregulated proteins interact with effectors of the NF-κB and Myc transcription factor cascades. Using a whole-embryo culture, we have identified misregulated proteins known to be involved in nervous system development and function

X-ray Emission from the Binary Central Stars of the Planetary Nebulae HFG 1, DS 1, and LoTr 5

Close binary systems undergoing mass transfer or common envelope interactions can account for the morphological properties of some planetary nebulae. The search for close binary companions in planetary nebulae is hindered by the difficulty of detecting cool, late-type, main sequence companions in binary systems with hot pre-white dwarf primaries. However, models of binary PN progenitor systems predict that mass accretion or tidal interactions can induce rapid rotation in the companion, leading to X-ray-emitting coronae. To test such models, we have searched for, and detected, X-ray emission from three binary central stars within planetary nebulae: the post-common envelope close binaries in HFG 1 and DS 1 consisting of O-type subdwarfs with late-type, main sequence companions, and the binary system in LoTr 5 consisting of O-type subdwarf and rapidly rotating, late-type giant companion. The X-ray emission in each case is best characterized by spectral models consisting of two optically-thin thermal plasma components with characteristic temperatures of about 10 MK and 15-40 MK, and total X-ray luminosities about 10^30 erg/s. We consider the possible origin of the X-ray emission from these binary systems and conclude that the most likely origin is, in each case, a corona around the late-type companion, as predicted by models of interacting binaries.Comment: tell your friend

Annotation-based genome-wide SNP discovery in the large and complex Aegilops tauschii genome using next-generation sequencing without a reference genome sequence

<p>Abstract</p> <p>Background</p> <p>Many plants have large and complex genomes with an abundance of repeated sequences. Many plants are also polyploid. Both of these attributes typify the genome architecture in the tribe Triticeae, whose members include economically important wheat, rye and barley. Large genome sizes, an abundance of repeated sequences, and polyploidy present challenges to genome-wide SNP discovery using next-generation sequencing (NGS) of total genomic DNA by making alignment and clustering of short reads generated by the NGS platforms difficult, particularly in the absence of a reference genome sequence.</p> <p>Results</p> <p>An annotation-based, genome-wide SNP discovery pipeline is reported using NGS data for large and complex genomes without a reference genome sequence. Roche 454 shotgun reads with low genome coverage of one genotype are annotated in order to distinguish single-copy sequences and repeat junctions from repetitive sequences and sequences shared by paralogous genes. Multiple genome equivalents of shotgun reads of another genotype generated with SOLiD or Solexa are then mapped to the annotated Roche 454 reads to identify putative SNPs. A pipeline program package, AGSNP, was developed and used for genome-wide SNP discovery in <it>Aegilops tauschii-</it>the diploid source of the wheat D genome, and with a genome size of 4.02 Gb, of which 90% is repetitive sequences. Genomic DNA of <it>Ae. tauschii </it>accession AL8/78 was sequenced with the Roche 454 NGS platform. Genomic DNA and cDNA of <it>Ae. tauschii </it>accession AS75 was sequenced primarily with SOLiD, although some Solexa and Roche 454 genomic sequences were also generated. A total of 195,631 putative SNPs were discovered in gene sequences, 155,580 putative SNPs were discovered in uncharacterized single-copy regions, and another 145,907 putative SNPs were discovered in repeat junctions. These SNPs were dispersed across the entire <it>Ae. tauschii </it>genome. To assess the false positive SNP discovery rate, DNA containing putative SNPs was amplified by PCR from AL8/78 and AS75 and resequenced with the ABI 3730 xl. In a sample of 302 randomly selected putative SNPs, 84.0% in gene regions, 88.0% in repeat junctions, and 81.3% in uncharacterized regions were validated.</p> <p>Conclusion</p> <p>An annotation-based genome-wide SNP discovery pipeline for NGS platforms was developed. The pipeline is suitable for SNP discovery in genomic libraries of complex genomes and does not require a reference genome sequence. The pipeline is applicable to all current NGS platforms, provided that at least one such platform generates relatively long reads. The pipeline package, AGSNP, and the discovered 497,118 <it>Ae. tauschii </it>SNPs can be accessed at (<url>http://avena.pw.usda.gov/wheatD/agsnp.shtml</url>).</p

Genome-Wide Association Studies for Pasmo Resistance in Flax (Linum usitatissimum L.)

Pasmo is one of the most widespread diseases threatening flax production. To identify genetic regions associated with pasmo resistance (PR), a genome-wide association study was performed on 370 accessions from the flax core collection. Evaluation of pasmo severity was performed in the field from 2012 to 2016 in Morden, MB, Canada. Genotyping-by-sequencing has identified 258,873 single nucleotide polymorphisms (SNPs) distributed on all 15 flax chromosomes. Marker-trait associations were identified using ten different statistical models. A total of 692 unique quantitative trait nucleotides (QTNs) associated with 500 putative quantitative trait loci (QTL) were detected from six phenotypic PR datasets (five individual years and average across years). Different QTNs were identified with various statistical models and from individual PR datasets, indicative of the complementation between analytical methods and/or genotype × environment interactions of the QTL effects. The single-locus models tended to identify large-effect QTNs while the multi-loci models were able to detect QTNs with smaller effects. Among the putative QTL, 67 had large effects (3–23%), were stable across all datasets and explained 32–64% of the total variation for PR in the various datasets. Forty-five of these QTL spanned 85 resistance gene analogs including a large toll interleukin receptor, nucleotide-binding site, leucine-rich repeat (TNL) type gene cluster on chromosome 8. The number of QTL with positive-effect or favorite alleles (NPQTL) in accessions was significantly correlated with PR (R2 = 0.55), suggesting that these QTL effects are mainly additive. NPQTL was also significantly associated with morphotype (R2 = 0.52) and major QTL with positive effect alleles were present in the fiber type accessions. The 67 large effect QTL are suited for marker-assisted selection and the 500 QTL for effective genomic prediction in PR molecular breeding