Abstract
BackgroundMore 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.ResultsGABI-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.ConclusionsThe 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.
Highlights
More than 90% of the Arabidopsis thaliana genes are members of multigene families
flanking sequence tags (FSTs) and T-DNA insertion site predictions The GABI-Kat database contains insertion site predictions from about 135,000 FSTs, which were generated for the 93,504 lines in the T-DNA insertion line collection
During the generation of GABI-Kat FSTs, genomic DNA of individual T1 plants was digested with BfaI, adaptors were added, and fragments containing T-DNA borders as well as sequences of plant origin next to the T-DNA were amplified with a T-DNA- and an adaptorspecific primer [7]
Summary
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. 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. DNA and adapter ligation [7] Their sequences can be compared to the A. thaliana genome sequence using BLAST [8] to predict the insertion position(s) of the T-DNA in a given line. T2 plants of the respective line are grown and the insertion site prediction is confirmed at GABI-Kat by PCR with an insertion site-specific primer and a T-DNA border primer, followed by sequencing of the amplicon. A more detailed overview on the features of the SimpleSearch site is summarised in [9]
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