I am trying to find the genomic locus (given its sequenced genome) of a morphological marker associated with a particular AFLP from an old linkage map. RAPD markers were also used, and seem just as impossible to find.
Dear Christopher,
is not easy to find the sequence corresponding to the AFLP marker. Are you looking for the AFLP markers on an assembled genome? Is yes you could try to align the primers and see if you get a sequence that is close to the expected size. After that you could design primer and screen a population segregating for the trait that the AFLP was linked to.
Uf you not even have the expected sequence length is even more difficult.
Good luck
Massimo
Thank you Massimo. Yes, I tried to blast the primers, but didn't get any results. I imagine the size is rather typical of AFLPs (they were from a standard protocol), but I'm not sure how to "manually" align them.
I have a good idea of roughly where it should be (within 1-2 Mbp). If I could narrow it down, I can compare different SNP haplotypes associated with the known phenotype differences (showing monogenic dominant/recessive segregation) linked relatively closely (3cM) to a known gene & chromosome.
Given the primers, including the selective bases, you may find quite a number of matching sequences along the genome. I mean, you cannot find where your particular AFLP polymorphism is located with just this information. You would need to sequence the particular segment of interest to locate the marker locus.
My suspicion was that it's simply impossible - that results do not translate. However, I wanted to be sure I wasn't missing a trick. I know there were several matches for each AFLP primer pair, but I know which one I want to localize (so sequence is already available). My problem is that I don't know the exact segment of interest, because I don't know which SNP the marker relates to. The full genome of one of the parents, and full SNP haplotype of the other are available, so I wondered whether there might be some way to "simulate" amplification of the polymorphic loci...
Christopher, your task is impossible for large eukaryotic genomes. Even if you have AFLP marker of certain length, it does not mean that this fragment is unique. Many if not the most of AFLP fragments have have two or more sequence variants of the same length..
Hi Christopher,
As they told you it is very difficult unless you employ a technique like the one in the attached paper "deletion analysis method".
Christopher, if you like to convert AFLP band into genome-located DNA marker you need to clone the fragment, sequence several inserts, develop SCAR/STS primers, confirm co-segregation between SCAR/STS and AFLP band, and finally, localize it on the genome. In worst case, you have no segregation for SCAR/STS and have to do chromosome walking to find polymorphic sites. Other ways - just a hope for very good luck.
OK, I have an update. I believe I've found the causal indel on a particular segment of sequence (using GWAS), and would like to confirm the placement of the associated AFLP. It's a little unclear to me how to identify how AFLP primers would localize to the sequence (it's not straight-forward complementary base-matching as in ordinary sequencing primers).
This tool (http://insilico.ehu.es/AFLP/info.html) seems to find the AFLP sites in a chromosome.
Thank you Yasir,
Unfortunately, it appear to be for Prokaryotes only.
You're right, the site says it is only for prokaryotes. I assume this is because of some size limit of chromosomes. Or is there a physiological difference in the AFLP protocol. I'd like to see what sort of alignment it actually looks for. Is it just the enzyme recognition site + selective nucleotides? If it is, then what is the purpose of the AFLP primer? For example, the MseI recognition site is supposed to be
5' TTAA
3' AATT
The cuts are
5' ---T ^ TAA--- 3'
3' ---AAT ^ T--- 5'
Now the AFLP MseI primer is given as GATGAGTCCTGAGTAA. I think only the last TTAA is part of the genome sequence that needs to align. What is the purpose of the rest of the adapter? Now suppose (one half of) the marker (with the selection nucleotides is M-CAA. I think this means that the sequence to look for M-CAA is TTAACAA (recognition site + selection) rather than GATGAGTCCTGAGTAACAA.
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