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

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