Dear all
I know that genetically unlinked transgenic locus will segregate in the progeny and therefore need to screen many progeny population in order to find out the right combination of gene (i.e genetically linked). Thus my specific question is if I want to combine two transgenes by crossing independently transformed transgenic lines how many progeny plants do I need to screen to find out one in which both transgenes are present? What will be the population size if I want to combine three transgenes? How to calculate the population size?
Dear Mahmudul,
Assuming your transgenes are in separate chromosomes in the two lines and assuming you have homozygous lines for these two transgenes, first you have to cross the two homozygous lines to get F1 generation. Then self the F1 generation and grow F2 population..
First you have to work out the frequency of the occurrence of two independent alleles.
The probability of occurrence of a homozygous plant for one gene is 0.25 (remember AA;2Aa:aa is the segregation ratio). Probability of occurrence of two independent events together is the product of their respective probabilities. Therefore the probability of an F2 plant with both genes in homozygous state is
0.25 x 0.25 = 0.0625. This is f - frequency in Muller's formula.
Now you can use Muller's formula to calculate the number of plants that needs to be screened in F2 to get the recombined individual .
N = loge(1-P)/loge(1-f)
Where N is the minimum number of individuals you need to screen, P is the probability and f is the frequency. Again assuming you want a high probability of finding your plant we will use 0.99 probability.
N = loge(1-0.99)/loge(1-0.0625)
This works out to 72 individual F2 plants from the selfed F1 homozygous lines. If this is a breeding programme, you should try and get as many F2 homozygotes for the two genes because this is a segregating population and you have a chance to select within the F3 progenies of your selected double homozygous plants. So to get two individuals you need 72 x 2 = 144 F2 plants and so on. Once you get these F2 plants, you should grow the seeds from each plant in separate progeny lines to select.
Good luck!
Dear Mahmudul,
Assuming your transgenes are in separate chromosomes in the two lines and assuming you have homozygous lines for these two transgenes, first you have to cross the two homozygous lines to get F1 generation. Then self the F1 generation and grow F2 population..
First you have to work out the frequency of the occurrence of two independent alleles.
The probability of occurrence of a homozygous plant for one gene is 0.25 (remember AA;2Aa:aa is the segregation ratio). Probability of occurrence of two independent events together is the product of their respective probabilities. Therefore the probability of an F2 plant with both genes in homozygous state is
0.25 x 0.25 = 0.0625. This is f - frequency in Muller's formula.
Now you can use Muller's formula to calculate the number of plants that needs to be screened in F2 to get the recombined individual .
N = loge(1-P)/loge(1-f)
Where N is the minimum number of individuals you need to screen, P is the probability and f is the frequency. Again assuming you want a high probability of finding your plant we will use 0.99 probability.
N = loge(1-0.99)/loge(1-0.0625)
This works out to 72 individual F2 plants from the selfed F1 homozygous lines. If this is a breeding programme, you should try and get as many F2 homozygotes for the two genes because this is a segregating population and you have a chance to select within the F3 progenies of your selected double homozygous plants. So to get two individuals you need 72 x 2 = 144 F2 plants and so on. Once you get these F2 plants, you should grow the seeds from each plant in separate progeny lines to select.
Good luck!
And all of the above assumes you are working in a diploid organism. You never said what species of plant.
If you have transgenes and any access to a molecular laboratory, it would be very cost effective if you could use marker aided selection (especially if your gene is your marker). You could screen your seedlings and save yourself a lot of time and end up with a homozygous population of the size you want for the rest of your traits.
Yes, as Dr Leonard said, SSR markers would be ideal. If you don't have markers, sterilise the F2 seeds and grow in a medium with both selection agents. This is possible if you used two different selection agents for the two genes. This way you will also save lot of time and space. Selection will be more efficient, avoiding environmental effects in the field.
As for Dr Julia's question above, I believe that the calculations are valid even if the species is not diploid as long as you have single insertions of the gene. I am referring to allopolyploidy, not autopolyploids with distorted segregation.
Dear Dr. Ranjith, Kindly give suggestions on the above said question, if the transgenic plants are in a highly heterozygous state for other characters.
Hi, Mahmudul,
Concepts mentioned in your question......
'Combination of genes in a same genome' and 'genetically linked' are two different concepts. Combinations of genes, in your case, it means two transgenic genes are 'co-exist' in the genome (for example in two different chromosomes), they are not necessary genetically linked. They may not co-segregate together. If your 2 transgenic genes are genetically linked, they will have high co-segregation rate into next generation. For example, the gene-of-interest and the selectable marker gene inside a T-DNA cassette can be considered two genetic tightly linked genes after they are transformed into plant genome. In general, they should co-segregate into progeny together,
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