Identification of a Spontaneous Frame Shift Mutation in a Nonreference Arabidopsis Accession Using Whole Genome Sequencing

Laitinen, Roosa A. E.; Schneeberger, Korbinian; Jelly, Noémie S.; Ossowski, Stephan; Weigel, Detlef

doi:10.1104/pp.110.156448

Cited by 39 publications

(30 citation statements)

References 9 publications

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“…This approach is particularly attractive when the phenotypic characterization necessary for conventional positional cloning is made difficult by high individual variation among genotypically identical individuals, requiring confirmation in F3 lines, as is the case for circadian phenotypes. In Arabidopsis, high-throughput short-read sequencing has identified spontaneous and chemically induced mutations in the reference (34,35) and nonreference genomes (36). Accordingly, we first resequenced the Col-3 parent used for the mutagenesis to an average base coverage of 25-fold (122 million reads of 35 bp were mapped) and identified ∼6,000 heterozygous and homozygous SNPs relative to the Col-0 sequence ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana

Hong

Song

Lutz

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

119

118

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Posttranslational modification is an important element in circadian clock function from cyanobacteria through plants and mammals. For example, a number of key clock components are phosphorylated and thereby marked for subsequent ubiquitination and degradation. Through forward genetic analysis we demonstrate that protein arginine methyltransferase 5 (PRMT5; At4g31120) is a critical determinant of circadian period in Arabidopsis. PRMT5 is coregulated with a set of 1,253 genes that shows alterations in phase of expression in response to entrainment to thermocycles versus photocycles in constant temperature. PRMT5 encodes a type II protein arginine methyltransferase that catalyzes the symmetric dimethylation of arginine residues (Rsme2). Rsme2 modification has been observed in many taxa, and targets include histones, components of the transcription complex, and components of the spliceosome. Neither arginine methylation nor PRMT5 has been implicated previously in circadian clock function, but the period lengthening associated with mutational disruption of prmt5 indicates that Rsme2 is a decoration important for the Arabidopsis clock and possibly for clocks in general.circadian rhythms | circadian clock | luciferase

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Section: Resultsmentioning

confidence: 99%

Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana

Hong

Song

Lutz

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

119

118

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“…We used paired-end reads of 36-80 bp generated on the Illumina Genome Analyzer platform, with average library insert lengths from 177 to 4,700 bp (Table S1). Some of the reads had been produced previously (24,25). Filtering and alignment of the short reads against the A. thaliana reference sequence were performed using GenomeMapper (19).…”

Section: Resultsmentioning

confidence: 99%

Reference-guided assembly of four diverse Arabidopsis thaliana genomes

Schneeberger

Ossowski

Ott

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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233

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We present whole-genome assemblies of four divergent Arabidopsis thaliana strains that complement the 125-Mb reference genome sequence released a decade ago. Using a newly developed reference-guided approach, we assembled large contigs from 9 to 42 Gb of Illumina short-read data from the Landsberg erecta (Ler-1), C24, Bur-0, and Kro-0 strains, which have been sequenced as part of the 1,001 Genomes Project for this species. Using alignments against the reference sequence, we first reduced the complexity of the de novo assembly and later integrated reads without similarity to the reference sequence. As an example, half of the noncentromeric C24 genome was covered by scaffolds that are longer than 260 kb, with a maximum of 2.2 Mb. Moreover, over 96% of the reference genome was covered by the reference-guided assembly, compared with only 87% with a complete de novo assembly. Comparisons with 2 Mb of dideoxy sequence reveal that the per-base error rate of the reference-guided assemblies was below 1 in 10,000. Our assemblies provide a detailed, genomewide picture of large-scale differences between A. thaliana individuals, most of which are difficult to access with alignment-consensus methods only. We demonstrate their practical relevance in studying the expression differences of polymorphic genes and show how the analysis of sRNA sequencing data can lead to erroneous conclusions if aligned against the reference genome alone. Genome assemblies, raw reads, and further information are accessible through http://1001genomes.org/projects/assemblies.html.

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“…Advances in DNA sequencing technologies have tremendously accelerated genetic mapping by combining bulk segregant analysis, that is, pooling recombinant genomes, with whole-genome sequencing, usually referred to as mapping by sequencing 2,3 . This approach is now becoming standard for mutation mapping and identification in many model species [3][4][5][6][7][8][9][10][11][12] and has even been applied to decipher quantitative traits with complex genetic architectures 13,14 . Recently, mutagen-induced changes have been used as novel markers, allowing mapping of mutations using isogenic mapping populations 10,15 .…”

Section: A N a Ly S I Smentioning

confidence: 99%

Mutation identification by direct comparison of whole-genome sequencing data from mutant and wild-type individuals using k-mers

et al. 2013

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Genes underlying mutant phenotypes can be isolated by combining marker discovery, genetic mapping and resequencing, but a more straightforward strategy for mapping mutations would be the direct comparison of mutant and wild-type genomes. Applying such an approach, however, is hampered by the need for reference sequences and by mutational loads that confound the unambiguous identification of causal mutations. Here we introduce NIKS (needle in the k-stack), a reference-free algorithm based on comparing k-mers in whole-genome sequencing data for precise discovery of homozygous mutations. We applied NIKS to eight mutants induced in nonreference rice cultivars and to two mutants of the nonmodel species Arabis alpina. In both species, comparing pooled F 2 individuals selected for mutant phenotypes revealed small sets of mutations including the causal changes. Moreover, comparing M 3 seedlings of two allelic mutants unambiguously identified the causal gene. Thus, for any species amenable to mutagenesis, NIKS enables forward genetics without requiring segregating populations, genetic maps and reference sequences.Forward genetic screens have been of fundamental importance in elucidating biological mechanisms in model species 1 . Their success, however, has relied on the feasibility of mutant gene isolation. Identification of causal mutations typically begins with genetic mapping, followed by candidate gene sequencing and complementation studies using transformation. Advances in DNA sequencing technologies have tremendously accelerated genetic mapping by combining bulk segregant analysis, that is, pooling recombinant genomes, with whole-genome sequencing, usually referred to as mapping by sequencing 2,3 . This approach is now becoming standard for mutation mapping and identification in many model species 3-12 and has even been applied to decipher quantitative traits with complex genetic architectures 13,14 . Recently, mutagen-induced changes have been used as novel markers, allowing mapping of mutations using isogenic mapping populations 10,15 . Nevertheless, all mapping-by-sequencing methods rely on resequencing, a method for whole-genome reconstruction based on aligning sequences to a reference sequence. Therefore, this requirement restricts the application of the technique to species for which such a reference genome sequence is available.Many reference-sequence assembly projects are currently in progress, including ones for most of the major crop species and breeding animals. However, even with an existing reference sequence, extending mapping-by-sequencing methods beyond the sequenced reference accessions has proved technically challenging. Mutant alleles of genes that are not present in the reference sequence cannot be identified within resequencing data alone. In particular, fast-evolving genes, such as those involved in disease resistance, might not always be represented in the reference sequence 16,17 .Alternative solutions for mapping-by-sequencing in species without reference sequences have been proposed, such ...

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Identification of a Spontaneous Frame Shift Mutation in a Nonreference Arabidopsis Accession Using Whole Genome Sequencing

Cited by 39 publications

References 9 publications

Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana

Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana

Reference-guided assembly of four diverse Arabidopsis thaliana genomes

Mutation identification by direct comparison of whole-genome sequencing data from mutant and wild-type individuals using k-mers

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