Dear all scientists,

I want to do RNA-seq study on a medicinal herb which grows in wild nature. I am new in this field and I don’t how to do sampling for my experiment. I should mention each individual plant of the herb has different genotype and they are not the same genotype as a variety. I should also say that I want to use an abiotic stress to study the plant major biochemical compounds and corresponding pathways and moreover I can just afford to pay two samples to do RNA-seq. My question is: I) Should I use some genotypes for normal condition (for example the genotype A, B, C, D, E and F) and some other genotypes for treated condition (for example the genotypes  G, H, I, J, K and L). Then collect one sample from each genotypes. After forward pool samples “A” to “F”  together and also pool samples “G” to “L” together to have one pooled sample for normal condition and one pooled sample for treated condition? OR II) Should I use just ONE plant (for example, a plant from genotype “A”) for normal condition and ONE plant for treated condition (for example, a plant from genotype “G”)? OR III) Should I make some clones (vegetative propagation) from genotype “A” (for example, 6 clones from genotype “A”) and genotype “G” (for example 6 clones from genotype G), then collect samples from each plant. After forward pool 6 clones from genotype “A” together and also pool 6 clones from genotype “G” together to have one pooled sample for normal condition and one pooled sample for treated condition?   I wonder if I take solution number II or III. Since, I just use two genotypes (one genotype for normal and one genotype for stress condition), is there possibility that I miss some genes? With taking to account that it is an herb which grows in mountain and each individual plant of the herb has different genotype. Therefore the nature of each genotype is not known.

As a result, we can just choose genotypes by random. Just imagine that a sensitive genotype to the studied stress is chosen by chance. So, I will miss expression of some resistance genes in my dataset. The solution number I, is not better? Thanks in advance