Good discussion Lawrence.

May I know, which crop You are working on?

In general, for gene mapping, we need the mapping populations first.

You can follow the procedure:

1. phenotype the germplasm under waterlogging conditions

2. The select two contrasting genotypes possessing high and low tolerance to waterlog stress

3. Do the crossing between selected genotypes to get F1 progeny

4. Validate the F1 progeny for heterozygosity using molecular markers

5. Do selfing of F1 progeny to generate F2 populations

6 Do marker analysis of F2 population along with tolerant, susceptible, and F1 progeny to know the segregation behavior of trait

7. Select the F2 plants showing the alleles corresponding to tolerant genotypes

8. Try to establish linkage between trait and linked molecular markers

9. Do the map-based cloning (if genome sequence is available)

10. Sequence validation of cloned gene

11. Functional validation through transgenic approach

Regards

Chet Ram

Thanks for Reply Chet Ram

I am working on Ixora, a shrub. The procedure you highlighted above is annual plants or crops.

I have done a preliminary study on it, I found out that Ixora develops some adaptive features to survive in the waterlogging environment. So, I want to see how I can Isolate the gene from the plant and see which one is responsible for the production of the feature in the plant and where it is inducive or inherent in the plant.

Dear Lawrence,

The protocol mostly pertains to annual crops.

Ok, so you are working on the perennial shrub. In such cases, you can isolate the genes of interest by analyzing the differential gene expression (transcriptome analysis) between tolerant and susceptible plants.

Take some highly up-regulated genes and further validate them with expression analysis using RT-PCR and validate their functions in heterologous plants (model plants) using a transgenic approach.

All the best

Regards

Chet Ram