The universality of the cpn60 gene, its demonstrated utility to differentiate closely related species and subspecies because of the sufficient sequence difference present in cpn60 UT from closely related species (Hill et al., 2004) and its recent evaluation as a DNA barcode for bacteria (Links et al., 2012), led us to investigate whether the redesigned and novel cpn60 primers amplify cpn60 gene from a broad range of fungal taxa.
The redesigned cpn60 universal primers amplified UT sequence both from S. pombe and S. cerevisiae as expected and DNA sequence analysis evidence was available for both the templates. With novel primers, based on amplicon sizes, all appeared to have worked on both the templates. DNA sequence evidence was available for some templates. Multiple bands were observed in few cases, out of these, DNA sequence analysis evidence was obtained for S. pombe templates.
For taxa not represented in cpnDB, little reference data was available for comparison to experimental data. The experimental sequences were probably of the same taxonomic group as identified in Dr.Lèvesque`s lab unless and until there was another fungal contaminant in these samples. Low level contamination by commensal fungi or environmental spores can be a problem when using universal primers. Previously, in the Hemmingsen lab, DNA extracts prepared from spores of Arbuscular Mycorrhizal (AM) Fungi were analyzed. In some cases,
cpn60 sequences were amplified from these extracts that were consistent with a fungal source but
not consistent with an AM fungal source. In these cases the most abundant template in the extract (AM fungal genomic DNA) failed to produce an amplicon while a template representing a minor contaminant did. That means, great caution must be taken while analyzing sequence data to avoid false positive results. Some cases where reference cpn60 sequence was not available in
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databases, identification of fungal taxa will get more specific as more of fungal sequences are deposited in cpnDB or NCBI database.
The cpn60 universal target sequence was amplified from F. avenaceum (ascomycota), D. hansenii (ascomycota), P. graminis (basidiomycota), and R. littoreum
(chytridiomycota). Three other ascomycetes and a zygomycete did not amplify with UT primers. Other cpn60 gene parts were also amplified from different samples using novel cpn60 primers that were designed specifically for fungal cpn60, although some regions were not amplified in these different samples. Novel fungal primers H1787/1789 generated DNA sequences for A.
alternata (Ascomycota), C. purpurea (Ascomycota), D. hansenii (Ascomycota), and M. vinacea
(Zygomycota), except for F. avenaceum (Ascomycota). The cpn60 UT region seemed to be more often amplified (in seven out of eight samples) than other regions (three out of eight samples on 5` end and four out of eight samples on 3` end). Sequence analysis identified C. purpurea and D.
hansenii as expected when novel fungal primers H1786/H1788 were used. With same primers, P. fastigiata, an ascomycete, was identified to be 87% identical to another ascomycete in the
absence of any reference sequence.
In cases where expected sequences were obtained upon analysis of experimental sequence, it was possible to obtain the exact sequence of the degenerate primers. As an example, if expected sequence is generated by primers H1787/H1789 for DNA sample of A. alternata, the exact sequence of primer H1780 can be known from it and can be used to make more specific primers for A. alternata and this can be helpful to know another part of its cpn60 sequence. This study produced substantial evidence that redesigned cpn60 UT primers and novel primers specific for fungi have utility for detecting and identifying fungal taxa from phylogenetically diverse fungi. Therefore, the cpn60 UT can be useful for the detection of both bacterial and
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fungal taxons unlike other gene targets for detection of micro-organisms that can detect either fungi or bacteria.
The chytrid sample, LEV5712 was identified as Rhizophydium littoreum by ITS. Chytrids belong to phylum Chytridiomycota. They are characterized by the formation of
zoospores and a posterior flagellum at some stage of their lifecycle. They are mostly parasites on marine algae, other chytrids and invertebrates. The interest in chytrids was heightened in 1998, when a vertebrate parasite Batrachochytrium dendrobatidis (Bd) was discovered which was devastating populations of amphibians (Longcore et al., 1999). There were only three chytrid sequences in cpnDB (2 Piromyces and one Neocallismatix) and although there were 6
Rhizophydium sequences in Genbank, none were cpn60. Redesigned cpn60 UT primers were
able to amplify the UT part of the Rhizophydium DNA and the best nucleotide hit was
Neocallimastix patriciarum (74%) and the best peptide hit was Bd (81%) in NCBI. The sequence
on analysis showed a 20 bp insertion as compared to the cpnDB entries (Figure 6). Since the number of inserted nucleotides is not a multiple of three, this insertion should cause a frameshift mutation in the gene and render it non-functional in which case it may be a pseudogene. But if it is not making the gene non-functional, the insertion may be an intron occurring as a result of transposon insertion, a type of intron gain (Figure 7). In this type of intron creation, a transposon sequence inserts itself into sequence AGGT which is believed to be the preferential site for intron gain and the coding sequence of the gene is not altered (Yenerall and Zhou, 2012).
Whether this insertion is an intron can be demonstrated using a simple experiment of amplifying the cDNA, obtained by reverse transcription of mRNA extracted from chytrid sample, with
cpn60 primers. On further cloning and sequencing, the insertion believed to be an intron will no
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The presence of a putative intron in the Rhizophydium sequence was interesting because intron was short i.e. 20 bp. Minimum length of introns in two of the most studied fungi, S.
cerevisiae is 52 bp (Spingola et al., 1999) and in S. pombe is 35 bp. The intron size distribution
in fungi is biased towards shorter introns with 33% of the introns being shorter than 100bp (41 to 60 nt). The intron observed in the Rhizophydium sample was 20 bp long. In one of the studies on
Rhizophydium tubulin genes, 4 introns were found with lengths of 22, 23, 25 and 37 bp. From
these findings, it suggests that Rhizophydium chytrid seems to have very short introns. This is the first time that cpn60 gene has been systematically studied as a target for fungal identification; therefore, not much literature is available for the same. According to standard protocol followed for amplification of bacterial templates, amplicons obtained from PCR are run on ethidium bromide gel and bands of appropriate size are cut. In case of eukaryotic templates, the size of required PCR products may not appear to be of appropriate size on gel due to the presence of insertions in them, as a result, the product can be discarded although it is the right PCR product. Therefore, the standard protocols should be redesigned for the appropriate detection of
eukaryotic PCR products on gel. Chytrid amplicon in our lab had a 20 base internal addition that was very short and the difference between size of PCR product with and without this addition was not very visible on gel, and therefore the PCR product was extracted expecting it to be of the expected cpn60 size.