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[Arabidopsis] Misterious T-DNA line

Bernd Weisshaar via arab-gen%40net.bio.net (by bernd.weisshaar from uni-bielefeld.de)
Sat Jan 23 05:37:01 EST 2010


Hi,

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  
confirmed.

2) Have a look at this example:
http://www.gabi-kat.de/db/showseq.php?term2=009A02
The GK-line 009A02 hat several FSTs which were used to predict  
insertion locations. Three were attempted for confirmation, two were  
positive.

3) Since we also do a segregation analysis during the confirmation  
workflow, the result of the segregation for sul resistance confirms  
this.
http://www.gabi-kat.de/db/show_segre.php?plantid=009A02
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."

5) Finally:
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:

> Hello,
>
> 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  
> though
> 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  
> insertion
> 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
> Portugal
> _______________________________________________
> 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
D-33594 Bielefeld
Germany

Office: G0-102
Tel.: +49-521-106-8721
Fax: +49-521-106-6423
genomforschung from uni-bielefeld.de (Secretary)
website: http://www.genomforschung.uni-bielefeld.de/




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