Arabidopsis T-DNA insert

[dcoman]

Dear colleagues,

I am working with a Sail T-DNA insertion line, located in an intron of a gene;
in the original background (Col3qrt1) I could select homozygous plants, based
on segregation ratio (3:1) and PCR (using genomic DNA with gene specific primers and the left
border primer). Next,I did repeted backcrosses of homozygous mutant with wild type Col 0 plants.
The following pattern occured after the 4th backcross:


-the seeds produced from the backcross segregate ,as expected, 1:1 and carry the T-DNA insert (confirmed by PCR
on genomic DNA)


-after selfing the heterozygous plants resulted from backcross, the seeds segregate as expected 3:1 BUT by PCR
all the seed from an individual silique are having only the gene specific band but NO left border
(I used positive and negative controls for primers and genomic template);

-reciprocal crosses showed no gametophytic defect;

Thus:
-in the original background(Col3qrt1) the it is possible to isolate homozygous plants;
-segregation indicates a single insert and no embryo or gametophytic defects
-backcrossing to Col 0 produces as expected heterozygous progeny
-BUT after selfing of the heterozygous plants in Col 0 background, neither homozygous nor heterozygous are identified.

I would appreciate any suggestion/ ideas to clarify this problem!

Kind regards,

Diana Coman

PhD Student
ETH Zürich