Distinguishing homozygous from hemizygous transformed plants - (Jun/20/2011 )

Dear collegues,

I have some transformed T1 barley lines that I would like to analyze for copy number and zygosity for my gene of interest. I have a question though, that I hope someone can provide me an answer to. How do you use real-time PCR to distinguish between single copy homozygous plants and double copy hemizygous plants? Or is it still necessary to do the T2 segregation analysis?

Best regards,
Dennis Eriksson

Copy number can be measured by qPCR. Design real-time primers for your gene and some single copy gene preferably on different chromosome as reference and run your plant sample with some 5-10 control samples. Dilute one control and create a standard curve for both genes. Calculate quantity for each gene and then divide your target gene by reference for sample and each control. Look at the variation of controls and see if your gene falls into it or is significantly lower.

But what I faintly remember about plant genetics, plants don't care that much about their ploidy, so maybe this method isn't much suitable for plants. But I don't know, I work with humans, they care pretty much

Trof on Tue Jun 21 11:09:55 2011 said:

Thank you for your reply. However, I still don´t understand how to distinguish between the single copy homozygous and the double copy hemizygous (for both loci), which both essentially carry two copies of my gene-of-interest in that particular generation. Wouldn´t they yield the same result in a qPCR?

The barley variety I am working with is diploid, so that is not a problem.

I maybe didn't get it. Homozygous means that in diploid organism you have two same alleles. Hemizygous means one is missing. So what exactly is "single copy homozygous" and "double copy hemizygous"? What the "copy" refers to? Copy of what, a chromosome?

Trof on Wed Jun 29 10:26:55 2011 said:

I´m sorry, I wasn´t very clear on that. I meant single or double insert, not copy. E.g. "single insert homozygous" would be where there has been transfer of only one transgene into the plant genome and that is has segregated to homozygosity (in 25% of the offspring), and "double insert hemizygous" would be where two transgenes have been transferred into the plant genome and both loci have segregated into hemizygosity (which would then also occur in 25% of that offspring in my case as I am working with self-fertilizing barley).

The thing is, when you transform plants (with Agrobacterium) it is common to get more than one insert of your gene-of-interest.

But in this very moment I think I found the answer myself. By comparing with the other T1 offspring in the same line (from the same individual T0 transformant), you would see a variation from 0 to 4 "gene copies" if the parental T0 was "double insert hemizygous", but in the case of "single insert homozygous" there are only similar T1 plants i.e. "two-gene-copy, single insert, homozygous". Sometimes one does not see the forest for all the trees.

Thanks anyway, I do appreciate your willingness to help me.

Best regards, Dennis

SweDennis on Wed Jun 29 11:12:55 2011 said:

Ay, I wrote this with my brain on stand-by. What I said goes for the T2 generation and not the T1 of course. But I can still get some clues by comparing the offspring within a single line. To get it straight; all T0 plants are hemizygous for the gene-of-interest, with one or more inserts. Those with only one insert would segregate easy, with 25% homozygous (2 gene copies), 50% heterozygous (1 gene copy), and 25% not carrying the gene-of-interest at all. A double insert hemizygous T0 would segregate in the following manner: the maximum copy number of the gene-of-interest would be 4, and the proportions of the gene copy numbers 4:3:2:1:0 would be 1:4:6:4:1 (assuming that the two loci are unlinked). So that should be enough for me to tell which parental plant was single insert or double insert. The later case would still of course have some "single loci/insert homozygous" (12,5%) and some "double loci/insert hemizygous" (25%) that I cannot tell apart, but that doesn´t matter. I will mainly be interested in the former case where the parent is single insert.

Hi,

Why not doing southern blot, it's pretty easy and it's a quality rather than quantity result. I used it many times and although it's "old fashion" it works perfect. If you think about it, I can give you more details.

Best,
Guy

One more thing,

Your assumption for unlinked insertion is not accurate; it is very common to have tandem tightly linked insertion. A southern blot will solve this issue as well

Guy

Guy on Wed Jun 29 17:56:42 2011 said:

Well, I wanted to look into the possibility of using qPCR for this, since it is faster and less laborious than Southern. Particularly the optimization of the hybridization stringency conditions for Southern may take some time in my case, since I am working with a gene (glutamine synthetase) that is found in different isoforms in barley.

Besides, I was wondering if you can really rely on the band intensity to distinguish between homozygous and hemizygous plants? Does it really give you accurate and reliable results?

Best regards, Dennis

Guy on Wed Jun 29 18:03:24 2011 said:

I suppose you are right about this. But do you have any idea how tightly linked theu may be in certain cases? If two transgenes get inserted in absolute tandem, i.e. with no sequence in between them at all, then I suppose they will appear as one single band on the blot?

Best regards, Dennis