Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis.

BACKGROUND: Experimental proof of gene function assignments in plants is based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades. RESULTS: We sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, resulting in about 2 T-DNA insertions per line and suggesting that this number was previously underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10 % of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days. CONCLUSIONS: Long read sequencing was demonstrated to be an effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations

SMRT sequencing only de novo assembly of the sugar beet (Beta vulgaris) chloroplast genome

Stadermann KB, Weisshaar B, Holtgräwe D. SMRT sequencing only de novo assembly of the sugar beet (Beta vulgaris) chloroplast genome. BMC Bioinformatics. 2015;16(1): 295.Background Third generation sequencing methods, like SMRT (Single Molecule, Real-Time) sequencing developed by Pacific Biosciences, offer much longer read length in comparison to Next Generation Sequencing (NGS) methods. Hence, they are well suited for de novo- or re-sequencing projects. Sequences generated for these purposes will not only contain reads originating from the nuclear genome, but also a significant amount of reads originating from the organelles of the target organism. These reads are usually discarded but they can also be used for an assembly of organellar replicons. The long read length supports resolution of repetitive regions and repeats within the organelles genome which might be problematic when just using short read data. Additionally, SMRT sequencing is less influenced by GC rich areas and by long stretches of the same base. Results We describe a workflow for a de novo assembly of the sugar beet (Beta vulgaris ssp. vulgaris) chloroplast genome sequence only based on data originating from a SMRT sequencing dataset targeted on its nuclear genome. We show that the data obtained from such an experiment are sufficient to create a high quality assembly with a higher reliability than assemblies derived from e.g. Illumina reads only. The chloroplast genome is especially challenging for de novo assembling as it contains two large inverted repeat (IR) regions. We also describe some limitations that still apply even though long reads are used for the assembly. Conclusions SMRT sequencing reads extracted from a dataset created for nuclear genome (re)sequencing can be used to obtain a high quality de novo assembly of the chloroplast of the sequenced organism. Even with a relatively small overall coverage for the nuclear genome it is possible to collect more than enough reads to generate a high quality assembly that outperforms short read based assemblies. However, even with long reads it is not always possible to clarify the order of elements of a chloroplast genome sequence reliantly which we could demonstrate with Fosmid End Sequences (FES) generated with Sanger technology. Nevertheless, this limitation also applies to short read sequencing data but is reached in this case at a much earlier stage during finishing

Horizontaler Transfer von chromosomalen bakteriellen Genen aus Agrobakterien zu Pflanzen

Huep G, Weisshaar B. Horizontaler Transfer von chromosomalen bakteriellen Genen aus Agrobakterien zu Pflanzen. GenomXPress. 2008;2008(4):17-19.Das Bodenbakterium Agrobacterium tumefaciens ist in der Lage, seine sogenannte Transfer-DNA (T-DNA) in Pflanzenzellen zu übertragen. Dieser seit etwa 27 Jahren bekannte Prozess erlaubt dem Agrobakterium, den Stoffwechsel der Pflanzenzellen so umzuprogrammieren, dass nur vom Bakterium selber verwendbare Nährstoffe produziert werden. Die T-DNA und Agrobakterien werden heute dazu verwendet, maßgeschneiderte DNA-Sequenzen in Pflanzengenome zu integrieren, um gezielt genetisch veränderte Pflanzen herzustellen. Üblicherweise sind die vom Bakterium übertragenen DNA-Bereiche auf dem Ti-Plasmid lokalisiert, welches einen vom Bakterien-Hauptgenom zu unterscheidenden, selbstreplizierenden Teil der DNA in den Bakterienzellen darstellt. Nun ist der Nachweis gelungen, dass in seltenen Fällen auch einige Gene aus dem Hauptgenom des Agrobakteriums in das Pflanzengenom übertragen werden, ohne dass diese Gene Bestandteil des Ti-Plasmids oder der T-DNA sind

GABI-Kat SimpleSearch: an Arabidopsis thaliana T-DNA mutant database with detailed information for confirmed insertions

Insertional mutagenesis approaches, especially by T-DNA, play important roles in gene function studies of the model plant Arabidopsis thaliana. GABI-Kat SimpleSearch () is a Flanking Sequence Tag (FST)-based database for T-DNA insertion mutants generated by the GABI-Kat project. Currently, the database contains >108 000 mapped FSTs from ∼64 000 lines which cover 64% of all annotated A.thaliana protein-coding genes. The web interface allows searching for relevant insertions by gene code, keyword, line identifier, GenBank accession number of the FST, and also by BLAST. A graphic display of the genome region around the gene or the FST assists users to select insertion lines of their interests. About 3500 insertions were confirmed in the offspring of the plant from which the original FST was generated, and the seeds of these lines are available from the Nottingham Arabidopsis Stock Centre. The database now also contains additional information such as segregation data, gene-specific primers and confirmation sequences. This information not only helps users to evaluate the usefulness of the mutant lines, but also covers a big part of the molecular characterization of the insertion alleles

Chloroplast genome sequence of Arabidopsis thaliana accession Landsberg erecta assembled from Single-Molecule, Real-Time sequencing data

Stadermann KB, Holtgräwe D, Weisshaar B. Chloroplast genome sequence of Arabidopsis thaliana accession Landsberg erecta assembled from Single-Molecule, Real-Time sequencing data. Genome Announcements. 2016;4(5): e00975-16.A publicly available data-set from Pacific Biosciences was used to create an assembly of the chloroplast genome sequence of the Arabidopsis thaliana Landsberg erecta genotype. The assembly is solely based on SMRT sequencing data and hence provides high resolution of the two inverted repeat regions typically contained in chloroplast genomes

Expression analysis of flavonoid biosynthesis genes during Arabidopsis thaliana silique and seed development with a primary focus on the proanthocyanidin biosynthetic pathway

Kleindt CK, Stracke R, Mehrtens F, Weisshaar B. Expression analysis of flavonoid biosynthesis genes during Arabidopsis thaliana silique and seed development with a primary focus on the proanthocyanidin biosynthetic pathway. BMC Research Notes. 2010;3(1): 255.Background: The coordinated activity of different flavonoid biosynthesis genes in Arabidopsis thaliana results in tissue-specific accumulation of flavonols, anthocyanins and proanthocyanidins (PAs). These compounds possess diverse functions in plants including light-attenuation and oxidative stress protection. Flavonoids accumulate in a stimulus- and/or development-dependent manner in specific parts of the plant. PAs accumulate in the seed coat (testa). Findings: We describe the biological material and the preparation of total RNA for the AtGenExpress developmental silique and seed series. AtGenExpress ATH1 GeneChip expression data from the different stages were reanalyzed and verified using quantitative real time PCR (qPCR). We observed organ-specific transcript accumulation of specific flavonoid biosynthetic genes consistent with previously published data and our PA compound accumulation data. In addition, we investigated the regulation of PA accumulation in developing A. thaliana seeds by correlating gene expression patterns of specific flavonoid biosynthesis genes with different seed embryonic developmental stages and organs and present two useful marker genes for isolated valve and replum organs, as well as one seed-specific marker. Conclusions: Potential caveats of array-based expression data are discussed based on comparisons with qPCR data. Results from ATH1 microarray and qPCR experiments revealed a shift in gene activity from general flavonoid biosynthesis at early stages of seed development to PA synthesis at late (mature) stages of embryogenesis. The examined PA accumulation-associated genes, including biosynthetic and regulatory genes, were found to be exclusively expressed in immature seeds. Accumulation of PAs initiates at the early heart stage of silique and seed development. Our findings provide new insights for further studies targeting the PA pathway in seeds

Enhancing the GABI-Kat Arabidopsis thaliana T-DNA insertion mutant database by incorporating Araport11 annotation

Kleinbölting N, Huep G, Weisshaar B. Enhancing the GABI-Kat Arabidopsis thaliana T-DNA insertion mutant database by incorporating Araport11 annotation. Plant and Cell Physiology. 2017;58(1): e7.SimpleSearch provides access to a database containing information about T-DNA insertion lines of the GABI-Kat collection of Arabidopsis thaliana mutants. These mutants are an important tool for reverse genetics, and GABI-Kat is the second largest collection of such T-DNA insertion mutants. Insertion sites were deduced from flanking sequence tags (FSTs), and the database contains information about mutant plant lines as well as insertion alleles. Here, we describe improvements within the interface (available at http://www.gabi-kat.de/db/genehits.php) and with regard to the database content that have been realized in the last five years. These improvements include the integration of the Araport11 genome sequence annotation data containing the recently updated A. thaliana structural gene descriptions, an updated visualization component that displays groups of insertions with very similar insertion positions, mapped confirmation sequences, and primers. The visualization component provides a quick way to identify insertions of interest, and access to improved data about the exact structure of confirmed insertion alleles. In addition, the database content has been extended by incorporating additional insertion alleles that were detected during the confirmation process, as well as by adding new FSTs that have been produced during continued efforts to complement gaps in FST availability. Finally, the current database content regarding predicted and confirmed insertion alleles as well as primer sequences has been made available as downloadable flat files

Reference-based QUantification Of gene Dispensability (QUOD).

Funder: Projekt DEALBACKGROUND: Dispensability of genes in a phylogenetic lineage, e.g. a species, genus, or higher-level clade, is gaining relevance as most genome sequencing projects move to a pangenome level. Most analyses classify genes as core genes, which are present in all investigated individual genomes, and dispensable genes, which only occur in a single or a few investigated genomes. The binary classification as 'core' or 'dispensable' is often based on arbitrary cutoffs of presence/absence in the analysed genomes. Even when extended to 'conditionally dispensable', this concept still requires the assignment of genes to distinct groups. RESULTS: Here, we present a new method which overcomes this distinct classification by quantifying gene dispensability and present a dedicated tool for reference-based QUantification Of gene Dispensability (QUOD). As a proof of concept, sequence data of 966 Arabidopsis thaliana accessions (Ath-966) were processed to calculate a gene-specific dispensability score for each gene based on normalised coverage in read mappings. We validated this score by comparison of highly conserved Benchmarking Universal Single Copy Orthologs (BUSCOs) to all other genes. The average scores of BUSCOs were significantly lower than the scores of non-BUSCOs. Analysis of variation demonstrated lower variation values between replicates of a single accession than between iteratively, randomly selected accessions from the whole dataset Ath-966. Functional investigations revealed defense and antimicrobial response genes among the genes with high-dispensability scores. CONCLUSIONS: Instead of classifying a gene as core or dispensable, QUOD assigns a dispensability score to each gene. Hence, QUOD facilitates the identification of candidate dispensable genes, associated with high dispensability scores, which often underlie lineage-specific adaptation to varying environmental conditions

High Contiguity De Novo Genome Sequence Assembly of Trifoliate Yam (Dioscorea dumetorum) Using Long Read Sequencing

Siadjeu C, Pucker B, Viehöver P, Albach DC, Weisshaar B. High Contiguity De Novo Genome Sequence Assembly of Trifoliate Yam (Dioscorea dumetorum) Using Long Read Sequencing. Genes. 2020;11(3): 274.Trifoliate yam (Dioscorea dumetorum) is one example of an orphan crop, not traded internationally. Post-harvest hardening of the tubers of this species starts within 24 h after harvesting and renders the tubers inedible. Genomic resources are required for D. dumetorum to improve breeding for non-hardening varieties as well as for other traits. We sequenced the D. dumetorum genome and generated the corresponding annotation. The two haplophases of this highly heterozygous genome were separated to a large extent. The assembly represents 485 Mbp of the genome with an N50 of over 3.2 Mbp. A total of 35,269 protein-encoding gene models as well as 9941 non-coding RNA genes were predicted, and functional annotations were assigned