Research Article |
Corresponding author: Karen W. Hughes ( khughes@utk.edu ) Academic editor: R. Henrik Nilsson
© 2015 Karen W. Hughes, Samuel D. Morris, Ana Reboredo Segovia.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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. https://doi.org/10.3897/mycokeys.10.5126
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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.
Basidiomycete fungi, chimera, hybrid, monokaryons, recombination, ribosomal repeat
Sequence chimeras are common when pooled DNA s are co-amplified by a PCR process (
The overall frequency of chimeras in published studies is unknown. In a study of fungal ITS amplicons derived from soil samples using a PCR/cloning process (
Most programs for checking and removing chimeric sequences were designed for 16S ribosomal sequences. DECIPHER (
With the establishment of the ribosomal ITS as the fungal barcode (
Gymnopus dichrous is a small, saprobic mushroom commonly found on oak bark and other woody debris in mid-summer in the southern Appalachian Mountains (USA). ITS sequencing identified two ITS subgroups of this mushroom called for convenience G. dichrous I and II. Gymnopus dichrous I and II differ in the ITS2 region (10% divergence) but there are no consistent or significant bp differences between G. dichrous I and II ITS1 or 5.8S regions (average divergence = 0.29% between the G. dichrous I and II ITS1 region, 0% in the 5.8s region). Homozygous collections for G. dichrous I and G. dichrous II were collected in the southern Appalachian Mountains and were designated as parental genotypes (P1). Fruitbodies that were ITS hybrids between G. dichrous I and G. dichrous II were also been collected and were designated as F1 hybrids (first filial generation as used in standard genetic crosses). For F1 hybrids, several indels in the ITS2 region obscured electropherograms and prevented recovery of the parental ITS sequences during Sanger sequencing. Cloning of the ITS1-5.8S-ITS2 PCR product was required to recover individual contributing haplotypes, however, a significant portion of recovered haplotypes were chimeric sequences.
Cloned ITS sequences were compared to P1 (parental/ homozygote) ITS sequences of Gymnopus dichrous I and II to identify chimeric and non-chimeric sequences. Below, we examine chimeras derived from cloned G. dichrous heterozygotes and show that they can be small, frequent and non-random. We also provide evidence that putative chimeras were not due to natural meiotic recombination or variation in the ribosomal repeat.
Collections. Gymnopus dichrous and G. subnudus are often collected as the same entity and are morphologically difficult to separate. Both are variable in morphology. Putative Gymnopus dichrous collections were made in the field using known morphological and environmental characteristics [e.g., small (ca. 5 cm in height) brown mushrooms, often with a darker, compressed, stem base and growing on wood, usually but not exclusively on oak bark]. Of 116 collections of putative G. dichrous, 16 were G. dichrous I-II hybrids (Table
Distribution of ITS sequences from clones of Gymnopus dichrous I-II heterozygotes.
Collection number of heterozygote | Herbarium Number |
GenBank Numbers | Number of clones |
G. dichrous I (# recovered) |
G. dichrous II (# recovered) |
G. dichrous chimeras (# recovered) |
---|---|---|---|---|---|---|
WS9223 | DEWV9223 | 7 | 2 | 2 | 3 | |
WRWV03-989 | DEWV4744 | 6 | 3 | 3 | 0 | |
WRWV04-322 | DEWV5833 | 6 | 2 | 3 | 1 | |
10454 | TENN58148 | 8 | 4 | 2 | 2 | |
10455 | TENN58149 | 2 | 2 | 0 | 0 | |
11785 | TENN60014 | JF313671-JF313672 | 4 | 3 | 1 | 0 |
11814 | TENN60027 | JF313673-JF313677 | 5 | 2 | 2 | 1 |
13361 | TENN61624 | JF313678-JF313693 | 13 | 5 | 6 | 2 |
14061 | TENN67807 | 8 | 2 | 4 | 2 | |
14094 | TENN67843 | 8 | 2 | 3 | 3 | |
14111 | TENN67859 | 8 | 3 | 4 | 1 | |
14226 | TENN68083 | 8 | 2 | 3 | 3 | |
14230 | TENN68090 | 8 | 1 | 4 | 3 | |
14237 | TENN68092 | 7 | 1 | 4 | 2 | |
14247 | TENN68105 | 16 | 4 | 9 | 3 | |
14248 | TENN68106 | 14 | 6 | 6 | 2 | |
Totals | 128 | 44 = 34.37% | 56 = 43.75% | 28 = 21.88% |
Single basidiospore isolation. Single-basidiospore isolates (SBIs) were obtained from fresh spore drops as described in
PCR and Cloning procedures. Cloning was carried out using Promega’s pGEM-T easy kit and M109 Competent cells according to manufacturer’s directions (Promega). Sanger sequencing of ITS-cloned products was performed as described in Hughes et al. (
PCR of the nuclear ribosomal ITS area was performed using primers ITS1F (
Identification of template switching regions. There are 25 regions of sequence mismatch within G. dichrous I and G. dichrous II (Fig.
The ITS2 region of two haplotypes of Gymnopus dichrous: Haplotypes DI (represented by TENN68084) and DII (represented by TENN68078). The TC pair at position 15 and the indel at position 25 were used to determine which the haplotype was represented by the 5’ end of a cloned sequence. Bases in red are points where DI and DII haplotypes differ in sequence and were used to determine if template switching had occurred in a cloned PCR product. Eight base pairs at which template switching can be detected are indicated by numbers 1-8. The possible area in which template switching (ts) could have occurred is indicated by vertical arrows and the number of observed template switching events is given above the vertical arrow. Bases that may be involved in intra-strand base pairing as determined by MFOLD are outlined with black boxes. Ambiguity codes indicate intraspecific variation.
DNA folding. Potential DNA folding of the ITS2 region for G. dichrous I and II exemplars was estimated at 72 °C (extension phase of PCR) using the MFOLD web server (http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form) with a MgCl setting of 3 mM (
Chi square analysis. The ITS 2 region was divided into 25 segments of varying length between bases 25 and 332. Each segment was flanked by a sequence difference between G. dichrous I and II that was informative for diagnosing template switching. For each segment, the number of template switching events was recorded, ranging from zero to six. These constituted observed values. Expected values were based on a null hypothesis of random template switching (each base has an equal probability of template switching).
The proportion of ITS chimeras obtained from PCR amplification of the ITS regions of G. dichrous I-II heterozygotes is given in Table
The distribution of template switch points resulting in chimeras was not random along the ITS2 region (Chi square = 35.72, P<0.05). Of 25 possible diagnostic base pair sites, template switching was observed between only 8 points (Fig.
The non-random nature of template switching would suggest that some stable mechanism influences template switching. 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. Ribosomal ITS2 RNA is known to have secondary structure at normal cellular temperatures and conditions (
The most frequent template switching occurred in a region between bases 122 and 138. This region overlaps and follows a small area of folding (a 4bp neck and 4bp loop) in G. dichrous I templates. The region between 140 and 200 is involved in complex folding patterns that are not consistent from model to model but present in all models. Five template switching events occur in this region. Between bases 200 and the end of the template at base 332, there is no consistently predicted secondary structure and fewer template switching events. Thus there is a loose agreement between secondary structure and regions of template switching but we cannot conclude cause and effect.
The size of detectable chimeras varied from chimeras occurring at the 5’ end of the ITS2 sequence (approximately 300bp) to those occurring near the 3’ end (45bp). Chimeras occurring between bp15 and bp25 would not have been recorded as such by our procedure but may have occurred (4 discontinuities may be due to template switching or to PCR generated mutation-see methods). We note that there is an area of secondary structure which overlaps bp15 and could be involved in template switching.
Areas where G. dichrous DI and DII differ extensively including indels (bases 147–151, 198–205) do not seem to be involved in template switching. This has been noted in other studies as well (
To evaluate whether cloning simply uncovered existing variation in the ribosomal repeat region (
Collection number of heterozygote | Herbarium Number | Number of SBIs | G. dichrous I | G. dichrous II | Number of meiotic recombinants In ITS+LSU |
---|---|---|---|---|---|
F1 G. dichrous I-II heterozygotes | |||||
14111 | TENN67859 | 11 | 5 | 6 | 0 |
14226 | TENN68083 | 8 | 5 | 3 | 0 |
14237 | TENN68092 | 5 | 1 | 4 | 0 |
Totals | 11 (44%) | 13 (52%) | 0 | ||
P1 G. dichrous I homozygotes | |||||
13716 | TENN65070 | 10 | 10 | 0 | 0 |
14232 | TENN68091 | 10 | 10 | 0 | 0 |
P1 G. dichrous II homozygotes | |||||
Braaton | No specimen | 4 | 0 | 4 | 0 |
Chimeras are common in cloned PCR products and tend to obscure contributing parental haplotypes, thus potentially creating errors in DNA sequence repositories. In this study, we show:
Template switching is non-random. Of 25 possible markers where ITS2 sequences of G. dichrous I and II differ, only 8 show template switching and template switching is higher in specific regions of the ITS2 sequences. The non-random nature of chimeras could lead to the misinterpretation of chimeras as parental haplotypes when the same chimera is recovered multiple times.
There is a loose correlation between areas predicted to form secondary structure and regions where template switching is high. We conclude that formation of secondary structure may affect template switching but speculate that secondary structure formation could either enhance or repress template switching, depending on location and the size of the stem-loop structure.
Chimeras occurring near the end of a template may be short and thus not easily detected.
Chimeras are not due to recovery of underlying variability in the ribosomal repeat in this system. The origins of chimeras remain obscure and may be due to multiple factors.
Chimera control should be exercised in environmental sampling studies and taxonomic studies wherever possible in order to minimize problems with persistent errors in sequence data repositories.
This study was supported by NSF DEB1144974 to R. H. Petersen, KWH, and Brian O’Meara. We thank reviewers Henrik Nilsson, Martin Ryberg Leho Tedersoo, Shawn Brown and an anonymous reviewer for their careful reviews, comments and suggestions.