Variant calling from 5 MB regions coming from contrasting cultivars

Hi,

I would like to compare ~5 MB genomic (QTL) regions across two groups (resistant and susceptible) and identify variants that might majorly influence resistance.
I was thinking of the following pipeline;

• use susceptible cultivar as the reference (since there is only one)
• Assemble tolerant groups (3 cultivars) using the above as a reference
• Call variants

Since I won’t be using reads here, can you suggest the most appropriate tools I can use for the purpose?
I wasn’t sure whether it is appropriate to use a small reads assembly and variant calling pipeline for this purpose (esp since those ask to give seq. platform).
I really appreciate any help you can provide.