Frequently asked questions (FAQ)

 



  1. How can I obtain the DUPLO double mutants?
    The DUPLO mutants will eventually be distributed via NASC (the European Arabidopsis Stock Centre). The mutants are not distributed yet, because only a very limited number has been finished, and issues regarding a distribution disclaimer (MTA) still need to be sorted. Soon the lines will be available via this site for a transitional period.
  2. How were the gene pairs for double mutant construction selected?
    A couple of criteria was important for the selection: the genes in a pair have to show sequence similarity. The expression profiles according to chip data have to be similar. A third gene with similar properties must not exist in the genome. For practical reasons the duplicated genes must show enough physical distance in the genome to allow recombination after crossing of the individual DUPLO lines in a given pair. And suitable insertions need to exist for both genes.
  3. Which alleles were selected for the genes in a given pair?
    Only lines from the GABI-Kat and SALK collection were considered. If possible, null alleles for the genes were chosen. Therefore lines with the predicted insertion within coding regions of the gene were preferably selected. Only in cases in which we have started to work on a gene pair, and the addressed allele can not be confirmed, other alleles with the predicted insertion in transcribed regions (5' or 3' of the coding region or introns) are considered.
  4. What is the genetic background of the double mutants?
    The single mutants are from the GABI-Kat and from the SALK collection of T-DNA insertion lines. The genetic background in both cases is Columbia.
  5. What is the workflow for the generation of the double mutants?
    Ideally the procedure is as follows: The predicted insertions in the single mutants are confirmed in a process similar to the one used in GABI-Kat (more information). Then 12-18 independent T2 plants of each line of a given pair are genotyped to identify the homozygous single mutants. The homozygous single mutants are crossed to generate the double hemizygous double mutant, which is re-confirmed by PCR on the DNA of the plants. After selfing of these plants the double homozygous double mutants are identified by genotyping.
  6. Are GABI-Kat and SALK lines processed differently in DUPLO?
    Yes. GABI-Kat lines can be selected with sulfadiazin (more information). The T-DNA in SALK-Lines usually confers a Kanamycin resistence. Because this resistence is not stable in every case or over several generations, 18 plants of a given SALK line grown without selection are genotyped. Usually 12 plants of a GABI-Kat line selected on sulfadiazin-containing plates are genotyped. Furthermore, the T-DNA in GABI-Kat lines is larger than the T-DNA in SALK lines. Therefore there is a fair chance that an intronic insertion will result in a null allele in case of GABI-Kat lines, which is not so sure for SALK lines. This affects the alleles selected for use in DUPLO pairs.
  7. Where can I get more information about the primers that were used in the confirmation process?
    The answer is given on the Confirmation strategy page. Detailed information about the confirmation primers for a given allele will become available via SimpleSearch, or is already if the respective allele has been confirmed already (look at the show confirmation sequences link on the line/FST description page of SimpleSearch).
  8. Where can I get more information about the primers that were used in the genotyping procedure?
    Information for a given allele will become available via SimpleSearch, once DUPLO double mutants are ready for distribution.
  9. Why do I occasionally find the same gene in two different pairs and with two different partner genes?
    At the beginning of the project a preliminary prediction of DUPLO pairs has been used. The practical work on the respective pairs had been started at that point. Later an enhanced prediction for the pairs was made in which occasionally certain genes were assigned to new partner genes. In cases where the practical work on the lines only present in the older prediction already progressed to far, both combinations of DUPLO genes were finished.