While detecting the Mi-9 gene that is responsible for providing resistance to nematodes in tomato, we used C8B primer sets to amplify the genes. after crossing several parents that carry the gene in heterozygous state, we were not able to get the 25% dominant or recessive individuals in the F2.
We always get only Mi-9\mi-9, but not Mi-9\Mi-9 OR mi-9\mi-9.
Any suggestions or ideas why?
Thank you in advance.
Hi Mustafa,
I meet this problem long time ago with a deletion in a gene that I never found homozygous, even if heterozygous state frequency was very high. I concluded that this gene was particularly important for cell life and that the deletion was deleterious. is it your case, I guess yes.
fred
I like Frederic Lepretre answer but another possibility is reagent contamination with pcr product from a heterozygote taking over the reaction and just out amplifying the homozygotes. What does your negative pcr control look like?
Hi Paul,
of course it could be an answer. to get a real idea it could therefore be a good point to design a multiplex PCR with the target amplicon and a control amplicon, just to be sure the PCR turns well.
fred
Not being able to recover homozygous wildtype individuals is a classic sign of a chromosomal translocation mutation. First, make sure your PCR is working properly. It's easy to have contamination so use a full set of controls. But if you are really seeing an over-abundance of heterozygous individuals, very few homozygous mutants AND very few homozygous wildtype, then you almost certainly have a chromosomal translocation mutation near your gene of interest. This is very well documented in Arabidopsis plants.
Article Chromosomal translocations are a common phenomenon in Arabid...
Here's a good paper with a detailed discussion of the underlying genetics for how chromosomes behave during meiosis in plants with translocations.
Article Reciprocal chromosome translocation associated with TDNA-ins...
Thanks Paul Rutland
I will check with my technician if he included controls.
Katie A Burnette I really appreciate your answer and sharing the paper
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