Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II

By

Hughes, Karen
Morris, Samuel
Reboredo Segovia, Ana

Type

Article

Date of Publication

2015

Original Publication

BioRisk : biodiversity & ecosystem risk assessment

Volume

10

Pages

45-56

Subjects

Basidiomycete fungi , chimera , hybrid , monokaryons , recombination , ribosomal repeat

Contributed by

Pensoft Publishers

Language

English

Published by

Sofia, Bulgaria Pensoft

Identifiers

DOI: https://doi.org/10.3897/mycokeys.10.5126

 

Abstract

Gymnopus 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). F1 ITS heterozygotes between the two are routinely collected from nature. Cloning of ITS PCR products from F1 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 F1 fruitbodies showed no recombinant haplotypes within the ITS + LSU span and clones derived from P1 homozygotes were identical to the P1 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.