An easy-to-use primer design tool to address paralogous loci and T-DNA insertion sites in the genome of Arabidopsis thaliana

Huep G, Kleinbölting N, Weisshaar B (2014)
Plant Methods 10: 28.

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Journal Article | Published | English
Abstract
BACKGROUND: More than 90% of the Arabidopsis thaliana genes are members of multigene families. DNA sequence similarities present in such related genes can cause trouble, e.g. when molecularly analysing mutant alleles of these genes. Also, flanking-sequence-tag (FST) based predictions of T-DNA insertion positions are often located within paralogous regions of the genome. In such cases, the prediction of the correct insertion site must include careful sequence analyses on the one hand and a paralog specific primer design for experimental confirmation of the prediction on the other hand. RESULTS: GABI-Kat is a large A. thaliana insertion line resource, which uses in-house confirmation to provide highly reliable access to T-DNA insertion alleles. To offer trustworthy mutant alleles of paralogous loci, we considered multiple insertion site predictions for single FSTs and implemented this 1-to-N relation in our database. The resulting paralogous predictions were addressed experimentally and the correct insertion locus was identified in most cases, including cases in which there were multiple predictions with identical prediction scores. A newly developed primer design tool that takes paralogous regions into account was developed to streamline the confirmation process for paralogs. The tool is suitable for all parts of the genome and is freely available at the GABI-Kat website. Although the tool was initially designed for the analysis of T-DNA insertion mutants, it can be used for any experiment that requires locus-specific primers for the A. thaliana genome. It is easy to use and also able to design amplimers with two genome-specific primers as required for genotyping segregating families of insertion mutants when looking for homozygous offspring. CONCLUSIONS: The paralog-aware confirmation process significantly improved the reliability of the insertion site assignment when paralogous regions of the genome were affected. An automatic online primer design tool that incorporates experience from the in-house confirmation of T-DNA insertion lines has been made available. It provides easy access to primers for the analysis of T-DNA insertion alleles, but it is also beneficial for other applications as well.
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Article Processing Charge funded by the Deutsche Forschungsgemeinschaft and the Open Access Publication Fund of Bielefeld University.
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Huep G, Kleinbölting N, Weisshaar B. An easy-to-use primer design tool to address paralogous loci and T-DNA insertion sites in the genome of Arabidopsis thaliana. Plant Methods. 2014;10: 28.
Huep, G., Kleinbölting, N., & Weisshaar, B. (2014). An easy-to-use primer design tool to address paralogous loci and T-DNA insertion sites in the genome of Arabidopsis thaliana. Plant Methods, 10: 28.
Huep, G., Kleinbölting, N., and Weisshaar, B. (2014). An easy-to-use primer design tool to address paralogous loci and T-DNA insertion sites in the genome of Arabidopsis thaliana. Plant Methods 10:28.
Huep, G., Kleinbölting, N., & Weisshaar, B., 2014. An easy-to-use primer design tool to address paralogous loci and T-DNA insertion sites in the genome of Arabidopsis thaliana. Plant Methods, 10: 28.
G. Huep, N. Kleinbölting, and B. Weisshaar, “An easy-to-use primer design tool to address paralogous loci and T-DNA insertion sites in the genome of Arabidopsis thaliana”, Plant Methods, vol. 10, 2014, : 28.
Huep, G., Kleinbölting, N., Weisshaar, B.: An easy-to-use primer design tool to address paralogous loci and T-DNA insertion sites in the genome of Arabidopsis thaliana. Plant Methods. 10, : 28 (2014).
Huep, Gunnar, Kleinbölting, Nils, and Weisshaar, Bernd. “An easy-to-use primer design tool to address paralogous loci and T-DNA insertion sites in the genome of Arabidopsis thaliana”. Plant Methods 10 (2014): 28.
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25 References

Data provided by Europe PubMed Central.

Analysis of the genome sequence of the flowering plant Arabidopsis thaliana.
Arabidopsis Genome Initiative., Nature 408(6814), 2000
PMID: 11130711
An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics.
Rosso MG, Li Y, Strizhov N, Reiss B, Dekker K, Weisshaar B., Plant Mol. Biol. 53(1-2), 2003
PMID: 14756321
Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome.
Szabados L, Kovacs I, Oberschall A, Abraham E, Kerekes I, Zsigmond L, Nagy R, Alvarado M, Krasovskaja I, Gal M, Berente A, Redei GP, Haim AB, Koncz C., Plant J. 32(2), 2002
PMID: 12383088
High-throughput generation of sequence indexes from T-DNA mutagenized Arabidopsis thaliana lines.
Strizhov N, Li Y, Rosso MG, Viehoever P, Dekker KA, Weisshaar B., BioTechniques 35(6), 2003
PMID: 14682050
Basic local alignment search tool.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol. 215(3), 1990
PMID: 2231712
GABI-Kat SimpleSearch: new features of the Arabidopsis thaliana T-DNA mutant database.
Kleinboelting N, Huep G, Kloetgen A, Viehoever P, Weisshaar B., Nucleic Acids Res. 40(Database issue), 2012
PMID: 22080561
GABI-Kat Website
AUTHOR UNKNOWN, 0
Seed and molecular resources for Arabidopsis.
Scholl RL, May ST, Ware DH., Plant Physiol. 124(4), 2000
PMID: 11115863
SimpleSearch
AUTHOR UNKNOWN, 0
Unique genes in plants: specificities and conserved features throughout evolution.
Armisen D, Lecharny A, Aubourg S., BMC Evol. Biol. 8(), 2008
PMID: 18847470
GABI-DUPLO: a collection of double mutants to overcome genetic redundancy in Arabidopsis thaliana.
Bolle C, Huep G, Kleinbolting N, Haberer G, Mayer K, Leister D, Weisshaar B., Plant J. 75(1), 2013
PMID: 23573814
SEQATOMS: a web tool for identifying missing regions in PDB in sequence context.
Brandt BW, Heringa J, Leunissen JA., Nucleic Acids Res. 36(Web Server issue), 2008
PMID: 18463137
Birth, death and subfunctionalization in the Arabidopsis genome.
Rutter MT, Cross KV, Van Woert PA., Trends Plant Sci. 17(4), 2012
PMID: 22326563
Linking genotype to phenotype using the Arabidopsis unimutant collection.
O'Malley RC, Ecker JR., Plant J. 61(6), 2010
PMID: 20409268
Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction
AUTHOR UNKNOWN, 2012
GABI-Kat primer design
AUTHOR UNKNOWN, 0
GABI-Kat SimpleSearch: an Arabidopsis thaliana T-DNA mutant database with detailed information for confirmed insertions.
Li Y, Rosso MG, Viehoever P, Weisshaar B., Nucleic Acids Res. 35(Database issue), 2007
PMID: 17062622
The Arabidopsis Information Resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant.
Huala E, Dickerman AW, Garcia-Hernandez M, Weems D, Reiser L, LaFond F, Hanley D, Kiphart D, Zhuang M, Huang W, Mueller LA, Bhattacharyya D, Bhaya D, Sobral BW, Beavis W, Meinke DW, Town CD, Somerville C, Rhee SY., Nucleic Acids Res. 29(1), 2001
PMID: 11125061
Primer3--new capabilities and interfaces.
Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG., Nucleic Acids Res. 40(15), 2012
PMID: 22730293
Clustal W and Clustal X version 2.0.
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG., Bioinformatics 23(21), 2007
PMID: 17846036

AUTHOR UNKNOWN, 1989

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