How to design primers for PCR amplification and sequencing of a specific locus on a chromosomes differentiating from its homeologous sequences that are more than 90% identical. Sequence of one of the homeolgous chromosome is unknown.
First I would make a multiple alignment of the homeologous regions, so you can see the positions that vary. MAFFT is probably good for this.
Once you can see which positions vary, and which are constant, between homeologues, you can choose the variable regions to make primers that would differentiate between the copies, and you can choose the constant regions to make universal primers that amplify all copies.
I would first amplify all the regions, using the universal primers, so then you might be able to estimate the sequence of the missing homeologue, then you can design primers that differentiate between the copies.
Since the sequence information of one of the homeologous regions is not know, primers can be designed on the conserved region using the known sequences for the same region. Then, mutilple clones can be sequenced to capture the sequence of the unknown homeologous region.
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