Long-read sequencing technologies have greatly facilitated assemblies of large eukaryotic genomes. In this paper, Oxford Nanopore sequences generated on a MinION sequencer are combined with Bionano ...Genomics Direct Label and Stain (DLS) optical maps to generate a chromosome-scale de novo assembly of the repeat-rich Sorghum bicolor Tx430 genome. The final assembly consists of 29 scaffolds, encompassing in most cases entire chromosome arms. It has a scaffold N
of 33.28 Mbps and covers 90% of the expected genome length. A sequence accuracy of 99.85% is obtained after aligning the assembly against Illumina Tx430 data and 99.6% of the 34,211 public gene models align to the assembly. Comparisons of Tx430 and BTx623 DLS maps against the public BTx623 v3.0.1 genome assembly suggest substantial discrepancies whose origin remains to be determined. In summary, this study demonstrates that informative assemblies of complex plant genomes can be generated by combining nanopore sequencing with DLS optical maps.
RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using ...Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
The maize genome, with its large complement of transposons and repeats, is a paradigm for the study of epigenetic mechanisms such as paramutation and imprinting. Here, we present the genome-wide map ...of cytosine methylation for two maize inbred lines, B73 and Mo17. CG (65%) and CHG (50%) methylation (where H = A, C, or T) is highest in transposons, while CHH (5%) methylation is likely guided by 24-nt, but not 21-nt, small interfering RNAs (siRNAs). Correlations with methylation patterns suggest that CG methylation in exons (8%) may deter insertion of Mutator transposon insertion, while CHG methylation at splice acceptor sites may inhibit RNA splicing. Using the methylation map as a guide, we used low-coverage sequencing to show that parental methylation differences are inherited by recombinant inbred lines. However, frequent methylation switches, guided by siRNA, persist for up to eight generations, suggesting that epigenetic inheritance resembling paramutation is much more common than previously supposed. The methylation map will provide an invaluable resource for epigenetic studies in maize.
Genotyping-by-Sequencing in Plants Deschamps, Stéphane; Llaca, Victor; May, Gregory D
Biology,
09/2012, Letnik:
1, Številka:
3
Journal Article, Book Review
Recenzirano
Odprti dostop
The advent of next-generation DNA sequencing (NGS) technologies has led to the development of rapid genome-wide Single Nucleotide Polymorphism (SNP) detection applications in various plant species. ...Recent improvements in sequencing throughput combined with an overall decrease in costs per gigabase of sequence is allowing NGS to be applied to not only the evaluation of small subsets of parental inbred lines, but also the mapping and characterization of traits of interest in much larger populations. Such an approach, where sequences are used simultaneously to detect and score SNPs, therefore bypassing the entire marker assay development stage, is known as genotyping-by-sequencing (GBS). This review will summarize the current state of GBS in plants and the promises it holds as a genome-wide genotyping application.
Hermaphroditic (perfect) flowers were a key trait in grapevine domestication, enabling a drastic increase in yields due to the efficiency of self-pollination in the domesticated grapevine (
L. ssp.
...). In contrast, all extant wild
species are dioecious, each plant having only male or female flowers. In this study, we identified the male (M) and female (f) haplotypes of the sex-determining region (SDR) in the wild grapevine species
and confirmed the boundaries of the SDR. We also demonstrated that the SDR and its boundaries are precisely conserved across the
genus using shotgun resequencing data of 556 wild and domesticated accessions from North America, East Asia, and Europe. A high linkage disequilibrium was found at the SDR in all wild grape species, while different recombination signatures were observed along the hermaphrodite (H) haplotype of 363 cultivated accessions, revealing two distinct H haplotypes, named H1 and H2. To further examine the H2 haplotype, we sequenced the genome of two grapevine cultivars, 'Riesling' and 'Chardonnay'. By reconstructing the first two H2 haplotypes, we estimated the divergence time between H1 and H2 haplotypes at ∼6 million years ago, which predates the domestication of grapevine (∼8,000 y ago). Our findings emphasize the important role of recombination suppression in maintaining dioecy in wild grape species and lend additional support to the hypothesis that at least two independent recombination events led to the reversion to hermaphroditism in grapevine.
Creating gapless telomere-to-telomere assemblies of complex genomes is one of the ultimate challenges in genomics. We use two independent assemblies and an optical map-based merging pipeline to ...produce a maize genome (B73-Ab10) composed of 63 contigs and a contig N50 of 162 Mb. This genome includes gapless assemblies of chromosome 3 (236 Mb) and chromosome 9 (162 Mb), and 53 Mb of the Ab10 meiotic drive haplotype. The data also reveal the internal structure of seven centromeres and five heterochromatic knobs, showing that the major tandem repeat arrays (CentC, knob180, and TR-1) are discontinuous and frequently interspersed with retroelements.
CRISPR-Cas is a powerful double-strand-break technology with wide-ranging applications from gene discovery to commercial product development. Thus far, this tool has been almost exclusively used for ...gene knockouts and deletions, with a few examples of gene edits and targeted gene insertions. Here, we demonstrate the application of CRISPR-Cas9 technology to mediate targeted 75.5-Mb pericentric inversion in chromosome 2 in one of the elite maize inbred lines from Corteva Agriscience. This inversion unlocks a large chromosomal region containing substantial genetic variance for recombination, thus providing opportunities for the development of new maize varieties with improved phenotypes.
Phytophthora root and stem rot caused by P. sojae is a destructive soybean soil-borne disease found worldwide. Discovery of genes conferring broad-spectrum resistance to the pathogen is a need to ...prevent the outbreak of the disease. Here, we show that soybean Rps11 is a 27.7-kb nucleotide-binding site-leucine-rich repeat (NBS-LRR or NLR) gene conferring broad-spectrum resistance to the pathogen. Rps11 is located in a genomic region harboring a cluster of large NLR genes of a single origin in soybean, and is derived from rounds of unequal recombination. Such events result in promoter fusion and LRR expansion that may contribute to the broad resistance spectrum. The NLR gene cluster exhibits drastic structural diversification among phylogenetically representative varieties, including gene copy number variation ranging from five to 23 copies, and absence of allelic copies of Rps11 in any of the non-Rps11-donor varieties examined, exemplifying innovative evolution of NLR genes and NLR gene clusters.
Understanding and exploiting genetic diversity is a key factor for the productive and stable production of rice. Here, we utilize 73 high-quality genomes that encompass the subpopulation structure of ...Asian rice (Oryza sativa), plus the genomes of two wild relatives (O. rufipogon and O. punctata), to build a pan-genome inversion index of 1769 non-redundant inversions that span an average of ~29% of the O. sativa cv. Nipponbare reference genome sequence. Using this index, we estimate an inversion rate of ~700 inversions per million years in Asian rice, which is 16 to 50 times higher than previously estimated for plants. Detailed analyses of these inversions show evidence of their effects on gene expression, recombination rate, and linkage disequilibrium. Our study uncovers the prevalence and scale of large inversions (≥100 bp) across the pan-genome of Asian rice and hints at their largely unexplored role in functional biology and crop performance.
Phyllotaxy describes the geometric arrangement of leaves and is important for plant productivity. Auxin is well known to regulate phyllotactic patterns via PIN1-dependent auxin polar transport, and ...studies of maize (Zea mays) aberrant phyllotaxy1 (abph1) mutants suggest the importance of auxin and cytokinin signaling for control of phyllotaxy. However, whether additional regulators control these patterns is poorly understood. Here, we report a new dominant maize mutant, Aberrant phyllotaxy2 (Abph2), in which the shoot meristems are enlarged and the phyllotactic pattern switches from alternate to decussate. Map-based cloning revealed that the Abph2 mutation was caused by transposition of a glutaredoxin gene, MALE STERILE CONVERTED ANTHER1 (MSCA1), which gained an altered expression pattern in Abph2 mutant embryos. msca1 loss-of-function mutants have reduced meristem size and revealed a novel function of glutaredoxins in meristem growth. In addition, MSCA1 interacts with a TGA transcription factor, FASCIATED EAR4, suggesting a novel regulatory module for regulating shoot meristem size.
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