<i>Kmasker plants</i> – a tool for assessing complex sequence space in plant species

Beier, Sebastian; Ulpinnis, Chris; Schwalbe, Markus; Münch, Thomas A.; Hoffie, Robert Eric; Koeppel, Iris; Hertig, Christian; Budhagatapalli, Nagaveni; Hiekel, Stefan; Pathi, Krishna Mohan; Hensel, Goetz; Grosse, Martin; Chamas, Sindy; Gerasimova, S. V.; Kumlehn, Jochen; Scholz, Uwe; Schmutzer, Thomas

doi:10.1111/tpj.14645

Cited by 8 publications

(10 citation statements)

References 57 publications

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“…The target site-specific activity of gRNA-navigated Cas9 and the resultant mutation patterns are highly variable. The validation of gRNA activity in vivo prior to whole- plant genome modification allows one to achieve a high portion of plants with specific alterations in the target motif [17]. Here, two different test systems were used to select efficient guide RNAs for targeted mutagenesis of the NUD gene in barley.…”

Section: Discussionmentioning

confidence: 99%

Conversion of hulled into naked barley by Cas endonuclease-mediated knockout of the NUD gene

et al. 2020

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Background The naked caryopsis character in barley is a domestication-associated trait defined by loss-of-function of the NUD gene. The functional NUD gene encodes an Apetala 2/Ethylene-Response Factor (AP2/ERF) controlling the formation of a cementing layer between pericarp and both lemma and palea. The downstream genes regulated by the NUD transcription factor and molecular mechanism of a cementing layer formation are still not sufficiently described. A naturally occurring 17-kb deletion in the nud locus is associated with the emergence of naked barley. Naked barley has been traditionally used for food and nowadays is considered as a dietary component for functional nutrition. Results In the present study, we demonstrate that targeted knockout of the NUD gene using RNA-guided Cas9 endonuclease leads to the phenotype conversion from hulled to naked barley. Using in vivo pre-testing systems, highly effective guide RNAs targeting the first exon of the NUD gene were selected. Expression cassettes harboring the cas9 and guide RNA genes were used to transform barley cv. Golden Promise via Agrobacterium-mediated DNA transfer. The recessive naked grain phenotype was observed in 57% of primary transformants, which indicates a frequent occurrence of homozygous or biallelic mutations. T-DNA-free homozygous lines with independently generated mutations in the NUD gene were obtained in the T1 generation. At homozygous state, all obtained mutations including one- and two-amino acid losses with the translational reading frame being retained invariably caused the naked grain phenotype. Conclusions The hulled and naked barley isogenic lines generated are a perfect experimental model for further studies on pleiotropic consequences of nud mutations on overall plant performance under particular consideration of yield-determining traits. Due to the high β-glucan content of its grains, naked barley is considered as being of particular dietary value. The possibility to convert hulled into naked barley cultivars by targeted mutagenesis allows breeders to extend the potential utilization of barley by the provision of functional food.

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Section: Discussionmentioning

confidence: 99%

Conversion of hulled into naked barley by Cas endonuclease-mediated knockout of the NUD gene

et al. 2020

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“…To reconstruct the B chromosome origin we analyzed the WGS assembly using two independent validation methods based on (1) the sequence reads of microdissected Bs (MiDiSeq) and (2) the coverage ratio analysis approach 16 , using k-mer frequency ratios calculated by Kmasker plants 17 . For the first approach, we used quality enriched reads from microdissected Bs aligned with the constructed WGS assembly to confirm sequences from Bs.…”

Section: Resultsmentioning

confidence: 99%

Supernumerary B chromosomes of Aegilops speltoides undergo precise elimination in roots early in embryo development

Ruban

Schmutzer

et al. 2020

Nat Commun

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Not necessarily all cells of an organism contain the same genome. Some eukaryotes exhibit dramatic differences between cells of different organs, resulting from programmed elimination of chromosomes or their fragments. Here, we present a detailed analysis of programmed B chromosome elimination in plants. Using goatgrass Aegilops speltoides as a model, we demonstrate that the elimination of B chromosomes is a strictly controlled and highly efficient root-specific process. At the onset of embryo differentiation B chromosomes undergo elimination in proto-root cells. Independent of centromere activity, B chromosomes demonstrate nondisjunction of chromatids and lagging in anaphase, leading to micronucleation. Chromatin structure and DNA replication differ between micronuclei and primary nuclei and degradation of micronucleated DNA is the final step of B chromosome elimination. This process might allow root tissues to survive the detrimental expression, or overexpression of B chromosome-located root-specific genes with paralogs located on standard chromosomes.

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“…For those plants with large genomes, such as maize and wheat, potential repetitive sequences might not be well documented. When designing oligo probes in these genomes, de novo repeats detection and filtering based on shotgun sequences is another approach that can be used to improve the specificity of oligos [49][50][51]. Moreover, it should be noted that oligos might bind to unintended sites with minor mismatches (Figure 1d).…”

Section: Specificitymentioning

confidence: 99%

“…Kmasker plants is a tool designed to assess complex plant genome sequences [50]. It also offers a FISH probe analysis function based on a k-mer analysis method to enhance the specificity of the designed oligo probes.…”

Section: Development and Application Of Oligo-fish Probe Design Toolsmentioning

confidence: 99%

Single Copy Oligonucleotide Fluorescence In Situ Hybridization Probe Design Platforms: Development, Application and Evaluation

Liu

Zhang

2021

IJMS

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Oligonucleotides fluorescence in situ hybridization (Oligo-FISH) is an emerging technology and is an important tool in research areas such as detection of chromosome variation, identification of allopolyploid, and deciphering of three-dimensional (3D) genome structures. Based on the demand for highly efficient oligo probes for oligo-FISH experiments, increasing numbers of tools have been developed for probe design in recent years. Obsolete oligonucleotide design tools have been adapted for oligo-FISH probe design because of their similar considerations. With the development of DNA sequencing and large-scale synthesis, novel tools have been designed to increase the specificity of designed oligo probes and enable genome-scale oligo probe design, which has greatly improved the application of single copy oligo-FISH. Despite this, few studies have introduced the development of the oligo-FISH probe design tools and their application in FISH experiments systematically. Besides, a comprehensive comparison and evaluation is lacking for the available tools. In this review, we provide an overview of the oligo-FISH probe design process, summarize the development and application of the available tools, evaluate several state-of-art tools, and eventually provide guidance for single copy oligo-FISH probe design.

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Kmasker plants – a tool for assessing complex sequence space in plant species

Cited by 8 publications

References 57 publications

Conversion of hulled into naked barley by Cas endonuclease-mediated knockout of the NUD gene

Conversion of hulled into naked barley by Cas endonuclease-mediated knockout of the NUD gene

Supernumerary B chromosomes of Aegilops speltoides undergo precise elimination in roots early in embryo development

Single Copy Oligonucleotide Fluorescence In Situ Hybridization Probe Design Platforms: Development, Application and Evaluation

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