Genome sequencing reveals agronomically important loci in rice using MutMap

Abe, Akira; Kosugi, Shunichi; Yoshida, Kentaro; Natsume, Satoshi; Takagi, Hiroki; Kanzaki, Hiromitsu; Matsumura, Hideo; Yoshida, Kakoto; Mitsuoka, Chikako; Tamiru, Muluneh; Innan, Hideki; Cano, Liliana; Kamoun, Sophien; Terauchi, Ryohei

doi:10.1038/nbt.2095

Cited by 1,102 publications

(930 citation statements)

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“…To compare both analyses, we carried out a functional characterization of the mutations using the reference annotations of rice 26 . For four mutants, NIKS revealed the same candidate genes reported previously 10 . Among those was the experimentally validated mutation in the sample Hit1917-pl1.…”

Section: Principles and Performance Of Nikssupporting

confidence: 74%

“…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%

“…Testing NIKS on seven previously analyzed rice mutants Ethyl methanesulfonate (EMS) mutations have been identified by resequencing isogenic bulked, or pooled, recombinants of seven rice mutants 10 . To reduce the high load of mutations introduced by EMS, researchers 10 analyzed DNA pools of backcrossed recombinants rather than single-mutant genomes.…”

Section: Principles and Performance Of Niksmentioning

confidence: 99%

“…To reduce the high load of mutations introduced by EMS, researchers 10 analyzed DNA pools of backcrossed recombinants rather than single-mutant genomes. The mutations were induced in the elite cultivar background of Hitomebore, which has no assembled reference sequence.…”

Section: Principles and Performance Of Niksmentioning

confidence: 99%

“…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.…”

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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: Principles and Performance Of Nikssupporting

confidence: 74%

Section: A N a Ly S I Smentioning

confidence: 99%

Section: Principles and Performance Of Niksmentioning

confidence: 99%

Section: Principles and Performance Of Niksmentioning

confidence: 99%

mentioning

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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Next‐Gen Sequencing‐Based Mapping and Identification of Ethyl Methanesulfonate‐Induced Mutations in Arabidopsis thaliana

Zhang

Millet²,

Ausubel

et al. 2014

CP Molecular Biology

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Forward genetic analysis using ethyl methanesulfonate (EMS) mutagenesis has proven to be a powerful tool in biological research, but identification and cloning of causal mutations by conventional genetic mapping approaches is a painstaking process. Recent advances in next-gen sequencing have greatly invigorated the process of identifying EMS-induced mutations corresponding to a specific phenotype in model genetic hosts, including the plant Arabidopsis thaliana and the nematode Caenorhabditis elegans. Next-gen sequencing of bulked F2 mutant recombinants produces a wealth of high-resolution genetic data, provides enhanced delimitation of the genomic location of mutations, and greatly reduces hands-on time while maintaining high accuracy and reproducibility. In this unit, a detailed procedure to simultaneously map and identify EMS mutations in Arabidopsis is described.

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