I working with a highly heterozygous species. I want to find a region of the genome with no polymorphism.
I am looking to design PCR primers as a positive control for a presence/absence marker. The PCR primers should amplify a non-polymorphic area of genome. The amplicon size should be 50-75 bp.
I thought about using the BUSCO gene set, and placing the primers in the exon of a BUSCO gene. The exon of a BUSCO region is likely to be non-polymorphic, but I am worried about contamination of the reaction with DNA from another species. Since BUSCO genes are highly conserved across all species, the primers might amplify the DNA of another contaminating species.
Does anyone have any tips on finding a non-polymorphic area of the genome which can be used as a template for PCR primer design?
PS - I don't have sequencing data for all the individuals which will be subject to this marker and it's positive control.