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      Twenty-Five Years of Propagation in Suspension Cell Culture Results in Substantial Alterations of the Arabidopsis Thaliana Genome

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          Abstract

          Arabidopsis thaliana is one of the best studied plant model organisms. Besides cultivation in greenhouses, cells of this plant can also be propagated in suspension cell culture. At7 is one such cell line that was established about 25 years ago. Here, we report the sequencing and the analysis of the At7 genome. Large scale duplications and deletions compared to the Columbia-0 (Col-0) reference sequence were detected. The number of deletions exceeds the number of insertions, thus indicating that a haploid genome size reduction is ongoing. Patterns of small sequence variants differ from the ones observed between A. thaliana accessions, e.g., the number of single nucleotide variants matches the number of insertions/deletions. RNA-Seq analysis reveals that disrupted alleles are less frequent in the transcriptome than the native ones.

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          The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana.

          To take complete advantage of information on within-species polymorphism and divergence from close relatives, one needs to know the rate and the molecular spectrum of spontaneous mutations. To this end, we have searched for de novo spontaneous mutations in the complete nuclear genomes of five Arabidopsis thaliana mutation accumulation lines that had been maintained by single-seed descent for 30 generations. We identified and validated 99 base substitutions and 17 small and large insertions and deletions. Our results imply a spontaneous mutation rate of 7 x 10(-9) base substitutions per site per generation, the majority of which are G:C-->A:T transitions. We explain this very biased spectrum of base substitution mutations as a result of two main processes: deamination of methylated cytosines and ultraviolet light-induced mutagenesis.
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            Whole-genome sequencing of multiple Arabidopsis thaliana populations.

            The plant Arabidopsis thaliana occurs naturally in many different habitats throughout Eurasia. As a foundation for identifying genetic variation contributing to adaptation to diverse environments, a 1001 Genomes Project to sequence geographically diverse A. thaliana strains has been initiated. Here we present the first phase of this project, based on population-scale sequencing of 80 strains drawn from eight regions throughout the species' native range. We describe the majority of common small-scale polymorphisms as well as many larger insertions and deletions in the A. thaliana pan-genome, their effects on gene function, and the patterns of local and global linkage among these variants. The action of processes other than spontaneous mutation is identified by comparing the spectrum of mutations that have accumulated since A. thaliana diverged from its closest relative 10 million years ago with the spectrum observed in the laboratory. Recent species-wide selective sweeps are rare, and potentially deleterious mutations are more common in marginal populations.
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              TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana.

              Genetic analyses have demonstrated that together with TTG1, a WD-repeat (WDR) protein, TT2 (MYB), and TT8 (bHLH) are necessary for the correct expression of BANYULS (BAN). This gene codes for the core enzyme of proanthocyanidin biosynthesis in Arabidopsis thaliana seed coat. The interplays of TT2, TT8, and their closest MYB/bHLH relatives, with TTG1 and the BAN promoter have been investigated using a combination of genetic and molecular approaches, both in yeast and in planta. The results obtained using glucocorticoid receptor fusion proteins in planta strongly suggest that TT2, TT8, and TTG1 can directly activate BAN expression. Experiments using yeast two- and three-hybrid clearly demonstrated that TT2, TT8, and TTG1 can form a stable ternary complex. Furthermore, although TT2 and TT8 were able to bind to the BAN promoter when simultaneously expressed in yeast, the activity of the complex correlated with the level of TTG1 expression in A. thaliana protoplasts. In addition, transient expression experiments revealed that TTG1 acts mainly through the bHLH partner (i.e. TT8 or related proteins) and that TT2 cannot be replaced by any other related A. thaliana MYB proteins to activate BAN. Finally and consistent with these results, the ectopic expression of TT2 was sufficient to trigger BAN activation in vegetative parts, but only where TTG1 was expressed. Taken together, these results indicate that TT2, TT8, and TTG1 can form a ternary complex directly regulating BAN expression in planta.
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                Author and article information

                Journal
                Genes (Basel)
                Genes (Basel)
                genes
                Genes
                MDPI
                2073-4425
                02 September 2019
                September 2019
                : 10
                : 9
                : 671
                Affiliations
                [1 ]Genetics and Genomics of Plants, Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, Sequenz 1, 33615 Bielefeld, NRW, Germany (R.S.) (P.V.) (B.W.)
                [2 ]Microbial Genomics and Biotechnology, Center for Biotechnology (CeBiTec), Bielefeld University, Sequenz 1, 33615 Bielefeld, NRW, Germany (C.R.) (J.K.)
                Author notes
                Author information
                https://orcid.org/0000-0002-3321-7471
                https://orcid.org/0000-0002-9722-4435
                https://orcid.org/0000-0002-9261-2279
                https://orcid.org/0000-0003-3286-4121
                https://orcid.org/0000-0002-9052-1998
                https://orcid.org/0000-0002-7635-3473
                Article
                genes-10-00671
                10.3390/genes10090671
                6770967
                31480756
                9260a7ae-3f9b-475c-aa1f-dcef87a36513
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 26 July 2019
                : 29 August 2019
                Categories
                Article

                copy number variations,variant calling,next generation sequencing,long read sequencing

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