I have a double mutant of Arabidopsis which background is nramp and nramp is also single mutant which background is col-0 when mutant grow in Manganese deficiency conation in MS medium their phenotype is yellow of young leaves so I have cross with normal green plants so during f2 stage how can know that cross successful or not. And which kind of formula can used to know 3:1:1 ratio ?what is T-DNA primer? Which kind molecular marker can use for gene sequencing?
Hi Misbah,
I'm not sure to got clearly your problem. You cross the homocigous mutant with wild type Col-0? F1 should be completely heterocigous, and F2 (which should be the self fertilization of heterocigous) should exhibit phenotyping 3:1 proportion, which means 3 dominant character (that would be green leaves, I guess), and 1 of yellow leaves. I never used a "formula", only have to count the plants that have one or the other phenotype and calculate the ratio respect the total of plants.
So, if this phenotype appears, the cross was successful (you can also see the amount of seeds that F1 produced), if its normal or not.
The T-DNA primer should be some primer that match with the T-DNA sequence, which means some part of the insertion if you have an insertional mutant (a mutant that lost their function by the insertion of a big (3Kpb) segment of exogenous DNA. Depending of the seed bank where you get the seed, the sequence of the T-DNA should be different.
To sequencing, you have to choose some part of the mutant genome plant that allow you to know if you have the insertion (or mutation) or not, so you must designed primers before and after the point of insertion (or mutation). You can use the T-DNA sequence, the antibiotic or selection marker of the T-DNA or some part of the interrupted gene in the plant genome. The last option is the best choice to made some first screening in the lab, because, if you have an insertion, the primers in the plant genome will not amplify, while, if the plant does not have the insertion, will amplified the wild type gene.
There are some Arabidopsis manual, where you can find more about the segregation method (Weigel and Glazebrook, really good book).
All the best,
Mariela
PS, Please found attached the link to an Nature Review about that with a more better expanation.
https://www.ndsu.edu/pubweb/~mcclean/plsc411/arabidopsis%20screen.pdf
Hi Misbah,
I need clarification of your question: so, (A) you have 2 mutations (double-mutation) + nramp mutation? Or (B) you have 1 mutation + nramp mutation = double-mutation?
If (A), then do you know that the phenotype of "yellow young leaves" was due to nramp mutation or due to the other two mutations (double-mutant)?
If not due to nramp mutation, do you know if this phenotype is caused by one of the double-mutated genes, or caused by both mutated genes? If due to just one of the genes, and with simple complete dominant, you should see your F2 with 3:1 ratio (green vs. yellow). aa (mutant) x AA (wild) = Aa (F1), Aa x Aa = (F2) 3:1 segregation. If the trait is controlled by 2 genes ('A' and 'B'), AA and BB (diploid), then the double mutant will be aabb. You should able to use a Punnett Squre to figure out the proper F2 genetic/phenotypic ratio derived from aabb x AABB cross, if these 2 genes are not linked.
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