13 replies to this topic

[SweDennis]

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

[Trof]

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

[SweDennis]

Trof, on 21 June 2011 - 03:09 AM, said:

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

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.

[Trof]

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?

[SweDennis]

Trof, on 29 June 2011 - 02:26 AM, said:

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?

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]

SweDennis, on 29 June 2011 - 03:12 AM, said:

Trof, on 29 June 2011 - 02:26 AM, said:

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?

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

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.

[Guy]

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

[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

[SweDennis]

Guy, on 29 June 2011 - 09:56 AM, said:

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

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

[SweDennis]

Guy, on 29 June 2011 - 10:03 AM, said:

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

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

[Guy]

SweDennis, on 29 June 2011 - 11:11 PM, said:

Guy, on 29 June 2011 - 10:03 AM, said:

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

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

As for the intensity of the band, It's not about that, it's about the random distribution of restriction enzyme sites in the genome. EACH insertion, even if you have tandem repeats will give you different band size and usually one should cut with a 4 cutter RE so the chance for different bans is even higher. In short the idea is to design a probe that is homologue to one of the ends of your insertion-T-DNA, after the last RE you use to cut the DNA, this way it will hybridize to a piece of DNA that its size is determined by the distance between the last restriction site in your T-DNA and the next site which is in the genome and depends on the site of insertion (usually pretty random).

Does it help??
Guy

[Guy]

Guy, on 30 June 2011 - 10:54 AM, said:

SweDennis, on 29 June 2011 - 11:11 PM, said:

Guy, on 29 June 2011 - 10:03 AM, said:

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

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

As for the intensity of the band, It's not about that, it's about the random distribution of restriction enzyme sites in the genome. EACH insertion, even if you have tandem repeats will give you different band size and usually one should cut with a 4 cutter RE so the chance for different bans is even higher. In short the idea is to design a probe that is homologue to one of the ends of your insertion-T-DNA, after the last RE you use to cut the DNA, this way it will hybridize to a piece of DNA that its size is determined by the distance between the last restriction site in your T-DNA and the next site which is in the genome and depends on the site of insertion (usually pretty random).

Does it help??
Guy

I forgot to answer about the linkage, It might be very tight, and even if it's 5 cM, which is still a few millions of bps,

you will almost never see it segregating
Guy

[SweDennis]

As for the intensity of the band, It's not about that, it's about the random distribution of restriction enzyme sites in the genome. EACH insertion, even if you have tandem repeats will give you different band size and usually one should cut with a 4 cutter RE so the chance for different bans is even higher. In short the idea is to design a probe that is homologue to one of the ends of your insertion-T-DNA, after the last RE you use to cut the DNA, this way it will hybridize to a piece of DNA that its size is determined by the distance between the last restriction site in your T-DNA and the next site which is in the genome and depends on the site of insertion (usually pretty random).

Does it help??
Guy
[/quote]

But isn´t there a high probability that the restriction sites are located in the same position on the two homologous chromosomes? Because if they are, then both chromosomes will yield bands of the same length in a homozygous plant. That is what I mean when I wondered if you can accurately estimate the band intensity to determine if a plant is homozygous or hemizygous for the inserted transgene. My idea was that a plant which is homozygous for the transgene insert would get double the amount of hybridized probes compared to a hemizygous (which is carrying the transgene only on one of the chromosomes). Let´s say that you use Southern to determine the number of transgene inserts in the T1 generation (segregating from transgene-hemizygous parental plants), and then you select those with only a single insert. My question is then still if you can use Southern to determine if those plant lines with single transgene insert are homozygous or hemizygous? If you can assume that restriction sites are quite randomly distibuted also between two homologous chromosomes (which I doubt), then yes, but what if they actually are located in the same positions?

Cheers, Dennis

[Guy]

[quote name='SweDennis' timestamp='1309526331' post='113998']
As for the intensity of the band, It's not about that, it's about the random distribution of restriction enzyme sites in the genome. EACH insertion, even if you have tandem repeats will give you different band size and usually one should cut with a 4 cutter RE so the chance for different bans is even higher. In short the idea is to design a probe that is homologue to one of the ends of your insertion-T-DNA, after the last RE you use to cut the DNA, this way it will hybridize to a piece of DNA that its size is determined by the distance between the last restriction site in your T-DNA and the next site which is in the genome and depends on the site of insertion (usually pretty random).

Does it help??
Guy

But isn´t there a high probability that the restriction sites are located in the same position on the two homologous chromosomes? Because if they are, then both chromosomes will yield bands of the same length in a homozygous plant. That is what I mean when I wondered if you can accurately estimate the band intensity to determine if a plant is homozygous or hemizygous for the inserted transgene. My idea was that a plant which is homozygous for the transgene insert would get double the amount of hybridized probes compared to a hemizygous (which is carrying the transgene only on one of the chromosomes). Let´s say that you use Southern to determine the number of transgene inserts in the T1 generation (segregating from transgene-hemizygous parental plants), and then you select those with only a single insert. My question is then still if you can use Southern to determine if those plant lines with single transgene insert are homozygous or hemizygous? If you can assume that restriction sites are quite randomly distibuted also between two homologous chromosomes (which I doubt), then yes, but what if they actually are located in the same positions?

Cheers, Dennis


Hi Dennis,

You are right, 2 homologues chromosomes have the same seq, if you need to work with single insertion, hemizygous; after determining a single insertion by SB you can do one of the following;

1) Check the offspring-whether they segregate (antibiotic or phenotype). If they do, it is/was a hemizygous
2) Cross your transgene (let's say after fixing it as homozygous) and work with F1 plants that will always be hemizygous

So after you did the SB reaction on your segregating T1s and found your precious single insertion transformants you just have to check them in the next generation, if they segregate. I don't know what is your expected phenotype (fluorescent marker, OX etc') but I guess you can check it (have to admit that I know little about barley genetics though). can you for example sow the seeds on antibiotics to see if they segregate??

Best,
Guy