Dear Evoldir Members, Thank you for your response to my question about the intragenomic variation (multi-copy nuclear marker): how to deal with that when we want to to evaluate the among-individual or population variation. See the answers below. Best regards, Justyna Wolinska >From Richard Nichols: You might wish to consider the pyrosequencing approach that we implemented to look at the frequency of rDNA variants within individuals, between individuals and between populations. It is published in Keller, Veltsos & Nichols 2008. THE FREQUENCY OF rDNA VARIANTS WITHIN INDIVIDUALS PROVIDES EVIDENCE OF POPULATION HISTORY AND GENE FLOW ACROSS A GRASSHOPPER HYBRID ZONE. Evolution, 62:833-844 http://www3.interscience.wiley.com/journal/120084325/abstract?CRETRY=1&SRETRY=0 >From Magdalena Zarowiecki: Your first problem is to find out how much intra-genomic variation there really is. If you are cloning, it is well-known that you will introduce a much higher mutation rate, so the intra-genomic variation you see is most likely just cloning-error. When I sequence ITS2 from PCR products, I sometimes find variation within individuals, but in this case there are clear double-peaks in the sequence chromatograms, and there is only a small number of polymorphic sites. If you do cloning you shouldn't find any variation in cloned products that you don't find when you sequence PCR product from the same individual. If you do, it is cloning-error. If you can map your SNPs by sequencing a subset, you can always just do PCRs followed by restriction enzyme digestion to score variation within individuals for your bulk sample. To evaluate among-individual and population variation you should do an hierarchical AMOVA, as in Arlequin for example. >From Gert Woerheide: a few years ago we have done similar things with sponges, which have been published in MPE in 2004. Wörheide, G., Nichols, S., & Goldberg, J. (2004). Intragenomic variation of the rDNA internal transcribed spacers in sponges (Phylum Porifera): implications for phylogenetic studies. Molecular Phylogenetics and Evolution, 33(3), 816-830. >From Nathaniel Jue: We have found some significant error rates in data when using a general Taq instead of one with a proofreader. Errors came from both PCR reactions and the processing of clones, if you haven't checked that out, it might help you out with repeatability issues. -- Justyna Wolinska Ludwig-Maximilians-Universität, München Department Biologie II Evolutionsökologie Grosshaderner Str. 2 82152 Planegg-Martinsried, Germany Phone: +49 (0)89 2180 74201 Fax: +49 (0)89 2180 74204 email: wolinska@bio.lmu.de http://www.biologie.uni-muenchen.de/ou/ecology/evol_e/people_wolinska_e.html Justyna Wolinska