Rapid Gene Isolation Using MutChromSeq

Steuernagel, Burkhard; Vrána, Jan; Karafiátová, Miroslava; Wulff, Brande B. H.; Doležel, Jaroslav

doi:10.1007/978-1-4939-7249-4_20

Cited by 15 publications

(16 citation statements)

References 22 publications

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“…to biotic and abiotic stresses is only possible through identifying variants at the molecular level that define their genetic control. Chromosome sorting and sequencing of wild type and multiple independent mutants, referred to as MutChromSeq, provides a lossless complexity reduction method suitable for the cloning of any gene of interest (Sánchez-Martín et al, 2016;Steuernagel et al, 2017). In this study, we used this approach to clone the leaf rust resistance gene, Rph1, from cultivated barley.…”

Section: Discussionmentioning

confidence: 99%

“…The very strength of NLR exome capture in providing a stringent complexity reduction is also its weakness by excluding, perforce (1) NLRs with significant sequence divergence to the source sequences used in bait design, (2) NLRs with exotic integrated domains (Sarris et al, 2016), and (3) R genes not conforming to the canonical structure of an NLR (Fu et al, 2009;Krattinger et al, 2009;Moore et al, 2015). This bias can be overcome by chromosome flow sorting, where only a prior knowledge of the chromosome on which the gene resides is required (Giorgi et al, 2013;Steuernagel et al, 2017). Sánchez-Martín and colleagues flowsorted and sequenced wheat chromosome 5D from six Pm2 mutants and the parental wild type.…”

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confidence: 99%

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The Coiled-Coil NLR Rph1, Confers Leaf Rust Resistance in Barley Cultivar Sudan

et al. 2018

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

The Coiled-Coil NLR Rph1, Confers Leaf Rust Resistance in Barley Cultivar Sudan

et al. 2018

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“…To avoid these complications, the MutChromSeq (Mutant Chromosome Sequencing) approach was proposed by Sanchez-Martin et al [ 22 ] for rapid gene isolation in barley and wheat. MutChromSeq is a chromosome flow sorting and sequencing based powerful, sequence-unbiased and reference-free forward genetics approach for genome complexity reduction and induced causal mutations identification without having positional fine mapping [ 163 ]. It has been effectively applied to reclone the Eceriferum-q gene (resistant to wax covered leaf sheath); Rph1 (leaf rust resistance gene) in barley [ 130 ] and clone the Pm2 gene (Powdery mildew resistance) in wheat [ 22 ].…”

Section: Ngs Based Forward Genetics For Identification and Mappingmentioning

confidence: 99%

“…It does not depend on recombination or fine-mapping for gene cloning and causal mutation identification. This approach may be performed in any crop species where, the mutagenesis is feasible; the target gene is associated with a phenotype and the chromosomal location of target gene is known [ 22 , 163 ]. Chromosomal sequence comparison of multiple independently derived mutants and their wild parents confirms the identification of causal mutations in a single candidate gene or a non-coding sequence.…”

Section: Ngs Based Forward Genetics For Identification and Mappingmentioning

confidence: 99%

Next Generation Sequencing Based Forward Genetic Approaches for Identification and Mapping of Causal Mutations in Crop Plants: A Comprehensive Review

Sahu

Sao

Mondal

et al. 2020

Plants

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The recent advancements in forward genetics have expanded the applications of mutation techniques in advanced genetics and genomics, ahead of direct use in breeding programs. The advent of next-generation sequencing (NGS) has enabled easy identification and mapping of causal mutations within a short period and at relatively low cost. Identifying the genetic mutations and genes that underlie phenotypic changes is essential for understanding a wide variety of biological functions. To accelerate the mutation mapping for crop improvement, several high-throughput and novel NGS based forward genetic approaches have been developed and applied in various crops. These techniques are highly efficient in crop plants, as it is relatively easy to grow and screen thousands of individuals. These approaches have improved the resolution in quantitative trait loci (QTL) position/point mutations and assisted in determining the functional causative variations in genes. To be successful in the interpretation of NGS data, bioinformatics computational methods are critical elements in delivering accurate assembly, alignment, and variant detection. Numerous bioinformatics tools/pipelines have been developed for such analysis. This article intends to review the recent advances in NGS based forward genetic approaches to identify and map the causal mutations in the crop genomes. The article also highlights the available bioinformatics tools/pipelines for reducing the complexity of NGS data and delivering the concluding outcomes.

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“…172Both portions were dried to constant weight by heating in an oven at 65 °C for 3 days. 173The chromosome sequencing data was analysed using the MutChromSeq pipeline according 178toSteuernagel et al (2017). The raw data were trimmed using sickle 179 (https://github.com/najoshi/sickle) with default parameters.…”

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LYS3 encodes a prolamin-box-binding transcription factor that controls embryo growth in barley and wheat

Orman-Ligeza¹,

Borrill

Chia³

et al. 2019

Preprint

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Tel +44 (0)1223 34249. 23 24 Keywords: high lysine, PBF, large embryo, shrunken endosperm.  Wheat and barley LYS3/PBF mutants have enlarged embryos suggesting that this gene 40 suppresses embryo growth. 41  The down-stream target genes of PBF in wheat are predicted to be involved in a wide 42 range of biological processes including organ development and starch metabolism. 43 3 ABSTRACT 44 Mutations at the LYS3 locus in barley have multiple effects on grain development, including 45 an increase in embryo size and a decrease in endosperm starch content. The gene underlying 46 LYS3 was identified by genetic mapping and mutations in this gene were identified in all four 47 6 2. Materials and methods 102 2.1. Barley germplasm 103 Grains of Bomi, Morex and Risø1508 were obtained from the Germplasm Resources Unit, 104 John Innes Centre, Norwich, UK and Risø18, Risø19, M1460 and Minerva were kindly 105 supplied by Birthe Møller Jespersen, University of Copenhagen, Denmark. 106 107 2.2. Plant growth 108For mapping experiments, individual grains were germinated in Petri dishes on moist filter 109 paper. After over-night incubation at 4 C, plates were transferred to room temperature. 110When roots and shoots were established, each seedling was transplanted into a 1 L pot 111containing Levington M2 compost (Scotts Professional, Ipswich, UK) and grown in a 112 glasshouse. In winter, additional lighting was provided by sodium lamps for 16 h per day and 113 temperatures were maintained between 15 C (night) and 20 °C (day). In summer, plants 114were grown in a glasshouse under ambient conditions. 115Wheat TILLING mutants were sown directly into M2 compost, incubated at 4 C for 3 116 days and then transferred to a glasshouse with a 22-hour photoperiod and temperatures of 21 117 C (night) and 18 C (day). Supplementary lighting was provided by a mixture of high-118pressure sodium lamps and both far red and white LED lights (Conviron, Winnipeg, US). 119 120 Analysis of grain and embryo development 121Anthesis occurred whilst the ear was enveloped in the flag leaf so the exact day of anthesis 122 was difficult to determine without damaging the developing spike. Accordingly, flowering 123 time was defined as the day on which the awns of the developing ear protruded more than 1 124 cm above the leaf sheath and grain/embryo age was measured in days after flowering (DAF). 125

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Rapid Gene Isolation Using MutChromSeq

Cited by 15 publications

References 22 publications

The Coiled-Coil NLR Rph1, Confers Leaf Rust Resistance in Barley Cultivar Sudan

The Coiled-Coil NLR Rph1, Confers Leaf Rust Resistance in Barley Cultivar Sudan

Next Generation Sequencing Based Forward Genetic Approaches for Identification and Mapping of Causal Mutations in Crop Plants: A Comprehensive Review

LYS3 encodes a prolamin-box-binding transcription factor that controls embryo growth in barley and wheat

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