Fast Isogenic Mapping-by-Sequencing of Ethyl Methanesulfonate-Induced Mutant Bulks

Hartwig, Benjamin; James, Geo Velikkakam; Konrad, Kathryn; Schneeberger, Korbinian; Turck, Franziska

doi:10.1104/pp.112.200311

Cited by 124 publications

(140 citation statements)

References 37 publications

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“…One way to reduce the number of mutations is backcrossing to the wildtype, followed by sequencing of pooled genomes of F 2 individuals 10,15 . With this strategy we identified an unknown mutation in A. alpina.…”

Section: Discussionmentioning

confidence: 99%

“…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 . Nevertheless, all mapping-by-sequencing methods rely on resequencing, a method for whole-genome reconstruction based on aligning sequences to a reference sequence.…”

Section: A N a Ly S I Smentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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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“…1 A-C). We used fast isogenic mapping (32) in an F 2 population generated after two backcrosses to lhp1 to identify a single nucleotide polymorphism, causing conversion of a tryptophan (TGG) to a premature stop codon (TGA) in exon 1 of At3g42660 as candidate for the causative mutation (SI Appendix, Fig. S1A and Table S1).…”

Section: Resultsmentioning

confidence: 99%

Ctf4-related protein recruits LHP1-PRC2 to maintain H3K27me3 levels in dividing cells in Arabidopsis thaliana

Zhou

Tergemina

Cui

et al. 2017

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

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Polycomb Repressive Complex (PRC) 2 catalyzes the H3K27me3 modification that warrants inheritance of a repressive chromatin structure during cell division, thereby assuring stable target gene repression in differentiated cells. It is still under investigation how H3K27me3 is passed on from maternal to filial strands during DNA replication; however, cell division can reinforce H3K27me3 coverage at target regions. To identify novel factors involved in the Polycomb pathway in plants, we performed a forward genetic screen for enhancers of the like heterochromatin protein 1 (lhp1) mutant, which shows relatively mild phenotypic alterations compared with other plant PRC mutants. We mapped enhancer of lhp1 (eol) 1 to a gene related to yeast Chromosome transmission fidelity 4 (Ctf4) based on phylogenetic analysis, structural similarities, physical interaction with the CMG helicase component SLD5, and an expression pattern confined to actively dividing cells. A combination of eol1 with the curly leaf (clf) allele, carrying a mutation in the catalytic core of PRC2, strongly enhanced the clf phenotype; furthermore, H3K27me3 coverage at target genes was strongly reduced in eol1 clf double mutants compared with clf single mutants. EOL1 physically interacted with CLF, its partially redundant paralog SWINGER (SWN), and LHP1. We propose that EOL1 interacts with LHP1-PRC2 complexes during replication and thereby participates in maintaining the H3K27me3 mark at target genes.Ctf4 | epigenetic inheritance | ENHANCER OF LHP1 1 | Polycomb repressive complex 2 | replication

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“…by analyzing additional mutant lines carrying allelic series identified by TILLING. Such strategies to identify unknown mutations have recently been shown to be highly effective in the model plants Arabidopsis (Hartwig et al 2012;Schneeberger et al 2009) and rice (Abe et al 2011). …”

Section: Forward Genetics Approach Using Micro-tom Ems Mutant Collectmentioning

confidence: 99%

Micro-Tom mutants for functional analysis of target genes and discovery of new alleles in tomato

Just

Garcia

Fernández

et al. 2013

Plant Biotechnology

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Tomato is currently the model plant for fleshy fruit development and for Solanaceae species. Recent genomic approaches including transcriptome, proteome and metabolome analyses and genetic mapping have produced a wealth of candidate genes whose function needs to be assessed. The recent development in model and crop plants of TILLING (Targeting Induced Local Lesions IN Genomes), which reveals allelic series corresponding to several independent point mutations, and the current availability of deep sequencing tools further increase the interest of generating artificiallyinduced genetic diversity in tomato. We describe here the generation and use of EMS (ethyl methanesulfonate) tomato mutants in the miniature cultivar Micro-Tom and provide as example the identification of new fruit size and morphology mutants. We further propose new deep sequencing-based strategies for the discovery of mutations underlying phenotypic variations observed in mutant collections that will considerably increase the interest of exploiting Micro-Tom mutant collections for gene discovery in tomato.

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Fast Isogenic Mapping-by-Sequencing of Ethyl Methanesulfonate-Induced Mutant Bulks

Cited by 124 publications

References 37 publications

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

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

Ctf4-related protein recruits LHP1-PRC2 to maintain H3K27me3 levels in dividing cells in Arabidopsis thaliana

Micro-Tom mutants for functional analysis of target genes and discovery of new alleles in tomato

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