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Citation: Hughes KW, Morris SD, Reboredo-Segovia A (2015) Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences -a test involving heterozygotes between Gymnopus dichrous taxa I and II. MycoKeys 10: 45-56. AbstractGymnopus dichrous exists in the southern Appalachians (USA) as two distinct entities with essentially identical nuclear ribosomal ITS1 sequences but differing ITS2 and LSU sequences (for convenience, called G. dichrous I and II). F 1 ITS heterozygotes between the two are routinely collected from nature. Cloning of ITS PCR products from F 1 heterozygotes produced sequences of both parental haplotypes but also numerous chimeric sequences (21.9%). The location of template switching was non-random leading to recovery of the same chimera several times and the chimeric region varied from 45bp to 300bp. By comparison, single-basidiospore isolates from heterozygote F 1 fruitbodies showed no recombinant haplotypes within the ITS + LSU span and clones derived from P 1 homozygotes were identical to the P 1 parent. Thus, chimeric sequences are likely an artifact of the PCR-cloning process and not a consequence of natural recombination events found in nature, nor are they due to hidden existing variation within the ribosomal repeat. Chimeras and PCR-induced mutations are common in cloned PCR products and may result in incorrect sequence information in public databases. A peer-reviewed open-access journal MycoKeysLaunched to accelerate biodiversity research RESEARCH ARTICLE Karen W. Hughes et al. / MycoKeys 10: 45-56 (2015)

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