Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis
Abstract 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 decad...
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| Format: | Article |
| Language: | English |
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BMC
2021-08-01
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| Series: | BMC Genomics |
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| Online Access: | https://doi.org/10.1186/s12864-021-07877-8 |
| _version_ | 1818607221080064000 |
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| author | Boas Pucker Nils Kleinbölting Bernd Weisshaar |
| author_facet | Boas Pucker Nils Kleinbölting Bernd Weisshaar |
| author_sort | Boas Pucker |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-12-16T14:23:18Z |
| format | Article |
| id | doaj.art-4956c9e56d1f45f6a9ba113d733bcfaf |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-16T14:23:18Z |
| publishDate | 2021-08-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-4956c9e56d1f45f6a9ba113d733bcfaf2022-12-21T22:28:26ZengBMCBMC Genomics1471-21642021-08-0122112110.1186/s12864-021-07877-8Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesisBoas Pucker0Nils Kleinbölting1Bernd Weisshaar2Genetics and Genomics of Plants, Center for Biotechnology (CeBiTec), Bielefeld UniversityBioinformatics Resource Facility, Center for Biotechnology (CeBiTec, Bielefeld UniversityGenetics and Genomics of Plants, Center for Biotechnology (CeBiTec), Bielefeld UniversityAbstract 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.https://doi.org/10.1186/s12864-021-07877-8Long read sequencingGenome assemblyStructural variantsTranslocationsChromosome fusionsReverse genetics |
| spellingShingle | Boas Pucker Nils Kleinbölting Bernd Weisshaar Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis BMC Genomics Long read sequencing Genome assembly Structural variants Translocations Chromosome fusions Reverse genetics |
| title | Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis |
| title_full | Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis |
| title_fullStr | Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis |
| title_full_unstemmed | Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis |
| title_short | Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis |
| title_sort | large scale genomic rearrangements in selected arabidopsis thaliana t dna lines are caused by t dna insertion mutagenesis |
| topic | Long read sequencing Genome assembly Structural variants Translocations Chromosome fusions Reverse genetics |
| url | https://doi.org/10.1186/s12864-021-07877-8 |
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