1) There are for sure other insertions in the T-DNA mutants. For the
GABI-Kat population there are about 2/3 of the lines for which
segregation analysis indicates a 2nd locus. For the other populations
it is realistic to expect similar numbers. Since we do the
confirmation in-house, we even have quite a number of such cases
2) Have a look at this example:
The GK-line 009A02 hat several FSTs which were used to predict
insertion locations. Three were attempted for confirmation, two were
3) Since we also do a segregation analysis during the confirmation
workflow, the result of the segregation for sul resistance confirms
The (automatic) interpretation of this result tells: Probable Number
of Loci: 2 or more
This is why one needs cosegregation analysis, backcrossing to wt, a
2nd allele, or other means to confirm the correlation of a phenotype
with a (mutated) gene.
4) Among the almost 8.000 lines that we have confirmed and donated to
NASC, there are about 100 cases in which two (and in few cases even
three) insertions have been confirmed. See here: http://www.gabi-kat.de/news-a-history.html
"In total, 7.899 T3-sets of which each represents a single GABI-Kat
line have been donated so far.
These lines cover 7.991 confirmed insertion alleles."
In the case mentioned by Joao, I would recommend to also consider the
unpleasant danger of mislabelling or mixup of seed. We do handle
really a lot of seed, and this is done by human beings. We try our
best, and I am sure all other people that distribute seed do the same,
but it is clear that errors can happen. The result will be exactly the
experimental outcome described by Joao: the expected insertion is not
found, but there is some other insertion (from the same population)
that confers resistance.
Bets regards, Bernd
On 20.01.2010, at 20:03, João Raimundo wrote:
>> I have been having some problems genotyping a T-DNA insertion line.
> I grew
> this line on kanamicin and 100% of the plants clearly resisted. I
> they were homozygous but when I did the PCR to confirm all the plants
> appeared to be wild type... Is it possible that this line as an
> elsewhere in the genome? I blasted the sequence given with this line
> and the
> score for my gene is very very high.
>> Thank you!
> João Raimundo
> BioFIG - Center for Biodiversity, Functional & Integrative Genomics
> Biology Department
> University of Minho
> 4710-057 Braga
> Arab-gen mailing list
>Arab-gen from net.bio.net>http://www.bio.net/biomail/listinfo/arab-gen
Bielefeld University, Department of Biology
Prof. Dr. Bernd Weisshaar
Chair of Genome Research / Lehrstuhl fuer Genomforschung
genomforschung from uni-bielefeld.de (Secretary)