Hello,
I am not knowledgeable or experienced in primer designing. Currently, I am researching my options in studying population genetics of an insect species. There are no published microsatellite primers for the species. However, there are some primers designed for another species in the same genus which cross amplifies in two other species in the genus.However, these 3 species live in a similar geographic environment (Galapagos islands), but my target species have a very different distribution.
My questions is does it worth a try to test the primers on my target species? Or is it very unlikely that they work. It is obvious that I can't know for sure until I try, however, as I said I'm not experienced with this and I thought I should get the opinions of experts.
Hi, I would definitely give it a try. I think there is a high chance for you to get amplification with some unspecific amplification (which is normal for cross-species primers). But then, all you need to do is to optimize the primers that give you amplification. I used primers designed for cultivated rice on the wild rice and most of them worked well. Of course, there are a few which did not work. This is actually no difference than testing the primers you design specifically for your targeted species. A lot of time, you will still have to optimize the primers you design.
This article might help you to understand more:
Article Design factors that influence PCR amplification success of c...
I would say that if your organism is in the same genus, there is a very good likelihood that at least some of the cross-species primers would work on your species. Sometimes cross-species primers are the only option, and there are plenty of examples of where they have served their purpose.
Having said that, if generating a genomic library and probing for novel primers in your species is an option, I'd definitely consider going that route. Cross-species primers often have problems with allele drop-out or null alleles in the non-target species due to mutations at the priming site. Also, scoring alleles in cross-species markers can be a problem due to occasional single-base indels that look like stutter. Finally, cross-species markers can often be either monomorphic OR have very low diversity in congeneric non-target species. There is much research detailing all of these issues, and I suspect they would be especially problematic in a species which has a short generation time (such as most insects). Considering only usage and not cost, novel primers in your target species are always better.
I would used the primers from the other species, they most likely work. Once you've got your first sequences and in case you don get what you expect you can design new primers based on the sequences you obtained. You can use the ncbi web page for that. You just upload the sequence and add the other of the closest relative and pick a new primer. Good luck!
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