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

Karen W Hughes,Samuel D Morris,Ana Reboredo Segovia

doi: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.

Highlights

There has been speculation that chimeras may be the result of incomplete extension of PCR products which subsequently act as primers for the amplification cycle and, there seems to be a reduction in chimeric PCR products when extension times are increased (Meyerhans et al 1990; Qiu et al 2001a; Smyth et al 2010; Thompson et al 2002) or by optomizing the PCR protocol (Qiu et al 2001b; Wang and Wang 1997)
The possibility of identical chimeras occurring in GenBank and being interpreted as valid taxa was noted by Nilsson et al (2012)
We investigated the possibility that secondary structure formation during the PCR process might lead to non-random chimera formation, perhaps by briefly stalling taq polymerase transcription at the point of secondary folding and allowing template switching

Summary

Introduction

Sequence chimeras are common when pooled DNA s are co-amplified by a PCR process (Edgar et al 2011; Judo et al 1998; Jumpponen 2007; Meyerhans et al 1990; Odelberg et al 1995; Qiu et al 2001a; Smyth et al 2010; Tedersoo et al 2014; Wang and Wang 1997). Odelberg et al (1995) demonstrated that chimeras can be generated in a single round of PCR amplification in the absence of heat denaturation and re-annealing which suggests that some polymerase template switching may occur. Fonseca et al (2012) demonstrated that nuclear SSU chimeras were produced at high levels during the PCR-process when mixed templates were present. Their results with a nematode population demonstrated that chimera formation is higher in species-diverse PCR pools than in pools that are genetically less diverse but that the breakpoints were in regions of sequence similarity

Methods

Results

Conclusion