PCR primers that amplify fungal rRNA genes from environmental samples

Two PCR primer pairs were designed to amplify rRNA genes (rDNA) from all four major phyla of fungi: Ascomycota, Basidiomycota, Chytridomycota, and Zygomycota. PCRs performed with these primers showed that both pairs amplify DNA from organisms representing the major taxonomic groups of fungi but not from nonfungal sources. To test the ability of the primers to amplify fungal rDNA from environment samples, clone libraries from two avocado grove soils were constructed and analyzed. These soils possess different abilities to inhibit avocado root rot caused by Phythophthora cinnamomi. Analysis of the two rDNA clone libraries revealed differences in the two fungal communities. It also revealed a markedly different depiction of the soil fungal community than that generated by a culture-based analysis, confirming the value of rDNA-based approaches for identifying organisms that may not readily grow on agar media. Additional evidence of the usefulness of the primers was obtained by identifying fungi associated with avocado leaves. In both the soil and leaf analyses, no nonfungal rDNA sequences were identified, illustrating the selectivity of these PCR primers. This work demonstrates the ability of two newly developed PCR primer sets to amplify fungal rDNA from soil and plant tissue, thereby providing unique tools to examine this vast and mostly undescribed community of organisms.     

Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil

Four fungal 18S rDNA and internal transcribed spacer (ITS) polymerase chain reaction (PCR) primer pairs were tested for their specificity towards target fungal DNA in soil DNA extracts, and their ability to assess the diversity of fungal communities in a natural grassland soil was compared. Amplified PCR products were cloned, and approximately 50 clones from each library were sequenced. Phylogenetic analysis and database searches indicated that each of the sequenced cloned DNA fragments was of fungal origin for each primer pair, with the exception of the sequences generated using the 18S rDNA primers nu-SSU-0817 and nu-SSU-1196, where 35 of the 50 sequenced clones represented soil invertebrates. Although some of the primers have previously been suggested to be biased towards certain fungal taxonomic groups, the ratio of sequences representing each of the four main fungal phyla, Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, was similar for each of the primer pairs, suggesting that primer bias may be less significant than previously thought. Collector's curves were plotted to estimate the coverage obtained for each of the clone libraries after clustering the sequences into operational taxonomic units at a level of 99% sequence similarity. The curves indicated that good coverage of diversity was achieved, with the exception of the clone library constructed using primers nu-SSU-0817 and nu-SSU-1196, on account of the high number of non-fungal sequences obtained. The work demonstrates the usefulness of 18S rDNA and ITS PCR primers for assessing fungal diversity in environmental samples, and it also highlights some potential limitations of the approach with respect to PCR primer specificity and bias.     

Analysis of fungal communities on historical church window glass by denaturing gradient gel electrophoresis and phylogenetic 18S rDNA sequence analysis.

Besides lichens and bacteria, fungi play a crucial role in the biodeterioration of historical glass. In the present paper, the fungal diversity on the surface of two historical church window glasses was investigated by 18S rDNA-based denaturing gradient gel electrophoresis (DGGE) analysis. 566-bp 18S rDNA-specific clone libraries were constructed with primer set NS1/NS2+10. Positive clones were reamplified with primer sets EF4/518rGC (426-bp fragments) and NS26/518rGC (316-bp fragments), amplicons were screened by DGGE and clustered according to their position in DGGE. Results indicated that fungal 18S rDNA clone libraries should be screened with at least two different primer sets to obtain the maximum number of different clones. For phylogenetic sequence analyses, clone inserts were sequenced and compared with 18S rDNA sequences listed in the EMBL database. Similarity values ranged from 93.7% to 99.81% to known fungi. Analyses revealed complex fungal communities consisting of members and relatives of the genera Aspergillus, Aureobasidium, Coniosporum, Capnobotryella, Engyodontium, Geomyces, Kirschsteiniothelia, Leptosphaeria, Rhodotorula, Stanjemonium, Ustilago, and Verticillium. The genera Geomyces and Aureobasidium were present on both glass surfaces. Some genera had not been detected on historical glass so far.     

PCR Primers That Amplify Fungal rRNA Genes from Environmental Samples

Two PCR primer pairs were designed to amplify rRNA genes (rDNA) from all four major phyla of fungi: Ascomycota, Basidiomycota, Chytridomycota, and Zygomycota. PCRs performed with these primers showed that both pairs amplify DNA from organisms representing the major taxonomic groups of fungi but not from nonfungal sources. To test the ability of the primers to amplify fungal rDNA from environment samples, clone libraries from two avocado grove soils were constructed and analyzed. These soils possess different abilities to inhibit avocado root rot caused by Phythophthora cinnamomi. Analysis of the two rDNA clone libraries revealed differences in the two fungal communities. It also revealed a markedly different depiction of the soil fungal community than that generated by a culture-based analysis, confirming the value of rDNA-based approaches for identifying organisms that may not readily grow on agar media. Additional evidence of the usefulness of the primers was obtained by identifying fungi associated with avocado leaves. In both the soil and leaf analyses, no nonfungal rDNA sequences were identified, illustrating the selectivity of these PCR primers. This work demonstrates the ability of two newly developed PCR primer sets to amplify fungal rDNA from soil and plant tissue, thereby providing unique tools to examine this vast and mostly undescribed community of organisms.     

ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts

We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a 'universal' primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the 'universal' ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.     

Occurrence of ammonia-oxidizing Archaea in activated sludges of a laboratory scale reactor and two wastewater treatment plants

Aims:  Characterization of the ammonia-oxidizing archaea (AOA) community in activated sludge from a nitrogen removal bioreactor and wastewater treatment plants (WWTPs).
Methods and Results:  Three primer sets specific for ammonia mono-oxygenase α -subunit ( amoA ) were used to construct clone libraries for activated sludge sample from a nitrogen removal bioreactor. One primer set resulted in strong nonspecific PCR products. The other two clone libraries retrieved both shared and unique AOA amoA sequences. One primer set was chosen to study the AOA communities of activated sludge samples from Shatin and Stanley WWTPs. In total, 18 AOA amoA sequences were recovered and compared to the previous reported sequences. A phylogenetic analysis indicated that sequences found in this study fell into three clusters.
Conclusions:  Different primers resulted in varied AOA communities from the same sample. The AOA found from Hong Kong WWTPs were closely similar to those from sediment and soil, but distinct from those from activated sludge in other places. A comparison of clone libraries between Shatin WWTP and bioreactor indicated the AOA community significantly shifted only after 30-day enrichment.
Significance and Impact of the Study:  This study confirmed the occurrence of AOA in a laboratory scale nitrogen removal bioreactor and Hong Kong WWTPs treating saline or freshwater wastewater. AOA communities found in this study were significantly different from those found in other places. To retrieve diverse AOA communities from environmental samples, a combination of different primers for the amoA gene is needed.     

Detection and Identification of Decay Fungi in Spruce Wood by Restriction Fragment Length Polymorphism Analysis of Amplified Genes Encoding rRNA

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.     

Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes.

We constructed nine sets of oligonucleotide primers on the basis of the results of DNA hybridization of cloned genes from Neurospora crassa and Aspergillus nidulans to the genomes of select filamentous ascomycetes and deuteromycetes (with filamentous ascomycete affiliations). Nine sets of primers were designed to amplify segments of DNA that span one or more introns in conserved genes. PCR DNA amplification with the nine primer sets with genomic DNA from ascomycetes, deuteromycetes, basidiomycetes, and plants revealed that five of the primer sets amplified a product only from DNA of the filamentous ascomycetes and deuteromycetes. The five primer sets were constructed from the N. crassa genes for histone 3, histone 4, beta-tubulin, and the plasma membrane ATPase. With these five primer sets, polymorphisms were observed in both the size of and restriction enzyme sites in the amplified products from the filamentous ascomycetes. The primer sets described here may provide useful tools for phylogenetic studies and genome analyses in filamentous ascomycetes and deuteromycetes (with ascomycete affiliations), as well as for the rapid differentiation of fungal species by PCR.     

Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.     

Advantage of using inosine at the 3' termini of 16S rRNA gene universal primers for the study of microbial diversity

To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3' termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54 degrees C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50 degrees C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3' termini in studying the microbial diversity of environmental samples.     

Metabarcoding of fungal communities associated with bark beetles

Many species of fungi are closely allied with bark beetles, including many tree pathogens, but their species richness and patterns of distribution remain largely unknown. We established a protocol for metabarcoding of fungal communities directly from total genomic DNA extracted from individual beetles, showing that the ITS3/4 primer pair selectively amplifies the fungal ITS. Using three specimens of bark beetle from different species, we assess the fungal diversity associated with these specimens and the repeatability of these estimates in PCRs conducted with different primer tags. The combined replicates produced 727 fungal Operational Taxonomic Units (OTUs) for the specimen of Hylastes ater, 435 OTUs for Tomicus piniperda, and 294 OTUs for Trypodendron lineatum, while individual PCR reactions produced on average only 229, 54, and 31 OTUs for the three specimens, respectively. Yet, communities from PCR replicates were very similar in pairwise comparisons, in particular when considering species abundance, but differed greatly among the three beetle specimens. Different primer tags or the inclusion of amplicons in separate libraries did not impact the species composition. The ITS2 sequences were identified with the Lowest Common Ancestor approach and correspond to diverse lineages of fungi, including Ophiostomaceae and Leotiomycetes widely found to be tree pathogens. We conclude that Illumina MiSeq metabarcoding reliably captures fungal diversity associated with bark beetles, although numerous PCR replicates are recommended for an exhaustive sample. Direct PCR from beetle DNA extractions provides a rapid method for future surveys of fungal species diversity and their associations with bark beetles and environmental variables.     

Microbial communities in the surface mucopolysaccharide layer and the black band microbial mat of black band-diseased Siderastrea siderea

Microbial community profiles and species composition associated with two black band-diseased colonies of the coral Siderastrea siderea were studied by 16S rRNA-targeted gene cloning, sequencing, and amplicon-length heterogeneity PCR (LH-PCR). Bacterial communities associated with the surface mucopolysaccharide layer (SML) of apparently healthy tissues of the infected colonies, together with samples of the black band disease (BBD) infections, were analyzed using the same techniques for comparison. Gene sequences, ranging from 424 to 1,537 bp, were retrieved from all positive clones (n = 43 to 48) in each of the four clone libraries generated and used for comparative sequence analysis. In addition to LH-PCR community profiling, all of the clone sequences were aligned with LH-PCR primer sequences, and the theoretical lengths of the amplicons were determined. Results revealed that the community profiles were significantly different between BBD and SML samples. The SML samples were dominated by gamma-proteobacteria (53 to 64%), followed by beta-proteobacteria (18 to 21%) and alpha-proteobacteria (5 to 11%). In contrast, both BBD clone libraries were dominated by alpha-proteobacteria (58 to 87%), followed by verrucomicrobia (2 to 10%) and 0 to 6% each of delta-proteobacteria, bacteroidetes, firmicutes, and cyanobacteria. Alphaproteobacterial sequence types related to the bacteria associated with toxin-producing dinoflagellates were observed in BBD clone libraries but were not found in the SML libraries. Similarly, sequences affiliated with the family Desulfobacteraceae and toxin-producing cyanobacteria, both believed to be involved in BBD pathogenesis, were found only in BBD libraries. These data provide evidence for an association of numerous toxin-producing heterotrophic microorganisms with BBD of corals.     

Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts

Background  

The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmental samples provide varying degrees of success at discriminating against plant DNA while maintaining a broad range of compatibility. Typically, it has been necessary to use multiple primer sets to accommodate the range of fungi under study, potentially creating artificial distinctions for fungal sequences that amplify with more than one primer set.     

A new promising phylogenetic marker to study the diversity of fungal communities: The Glycoside Hydrolase 63 gene

In molecular ecology, the development of efficient molecular markers for fungi remains an important research domain. Nuclear ribosomal internal transcribed spacer (ITS) region was proposed as universal DNA barcode marker for fungi, but this marker was criticized for Indel‐induced alignment problems and its potential lack of phylogenetic resolution. Our main aim was to develop a new phylogenetic gene and a putative functional marker, from single‐copy gene, to describe fungal diversity. Thus, we developed a series of primers to amplify a polymorphic region of the Glycoside Hydrolase GH63 gene, encoding exo‐acting α‐glucosidases, in basidiomycetes. These primers were validated on 125 different fungal genomic DNAs, and GH63 amplification yield was compared with that of already published functional markers targeting genes coding for laccases, N‐acetylhexosaminidases, cellobiohydrolases and class II peroxidases. Specific amplicons were recovered for 95% of the fungal species tested, and GH63 amplification success was strikingly higher than rates obtained with other functional genes. We downloaded the GH63 sequences from 483 fungal genomes publicly available at the JGI mycocosm database. GH63 was present in 461 fungal genomes belonging to all phyla, except Microsporidia and Neocallimastigomycota divisions. Moreover, the phylogenetic trees built with both GH63 and Rpb1 protein sequences revealed that GH63 is also a promising phylogenetic marker. Finally, a very high proportion of GH63 proteins was predicted to be secreted. This molecular tool could be a new phylogenetic marker of fungal species as well as potential indicator of functional diversity of basidiomycetes fungal communities in term of secretory capacities.     

Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis.

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1. 4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.     

Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity

Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5′ domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values.     

Microbial Communities in the Surface Mucopolysaccharide Layer and the Black Band Microbial Mat of Black Band-Diseased Siderastrea siderea

Microbial community profiles and species composition associated with two black band-diseased colonies of the coral Siderastrea siderea were studied by 16S rRNA-targeted gene cloning, sequencing, and amplicon-length heterogeneity PCR (LH-PCR). Bacterial communities associated with the surface mucopolysaccharide layer (SML) of apparently healthy tissues of the infected colonies, together with samples of the black band disease (BBD) infections, were analyzed using the same techniques for comparison. Gene sequences, ranging from 424 to 1,537 bp, were retrieved from all positive clones (n = 43 to 48) in each of the four clone libraries generated and used for comparative sequence analysis. In addition to LH-PCR community profiling, all of the clone sequences were aligned with LH-PCR primer sequences, and the theoretical lengths of the amplicons were determined. Results revealed that the community profiles were significantly different between BBD and SML samples. The SML samples were dominated by γ-proteobacteria (53 to 64%), followed by β-proteobacteria (18 to 21%) and α-proteobacteria (5 to 11%). In contrast, both BBD clone libraries were dominated by α-proteobacteria (58 to 87%), followed by verrucomicrobia (2 to 10%) and 0 to 6% each of δ-proteobacteria, bacteroidetes, firmicutes, and cyanobacteria. Alphaproteobacterial sequence types related to the bacteria associated with toxin-producing dinoflagellates were observed in BBD clone libraries but were not found in the SML libraries. Similarly, sequences affiliated with the family Desulfobacteraceae and toxin-producing cyanobacteria, both believed to be involved in BBD pathogenesis, were found only in BBD libraries. These data provide evidence for an association of numerous toxin-producing heterotrophic microorganisms with BBD of corals.     

Fungal Community Analysis in the Deep-Sea Sediments of the Central Indian Basin by Culture-Independent Approach

Few studies have addressed the occurrence of fungi in deep-sea sediments, characterized by elevated hydrostatic pressure, low temperature, and fluctuating nutrient conditions. We evaluated the diversity of fungi at three locations of the Central Indian Basin (CIB) at a depth of ~5,000 m using culture-independent approach. Community DNA isolated from these sediments was amplified using universal and fungal-specific internal transcribed spacers and universal 18S rDNA primer pairs. A total of 39 fungal operational taxonomic units, with 32 distinct fungal taxa were recovered from 768 clones generated from 16 environmental clone libraries. The application of multiple primers enabled the recovery of eight sequences that appeared to be new. The majority of the recovered sequences belonged to diverse phylotypes of Ascomycota and Basidiomycota. Our results suggested the existence of cosmopolitan marine fungi in the sediments of CIB. This study further demonstrated that diversity of fungi varied spatially in the CIB. Individual primer set appeared to amplify different fungal taxa occasionally. This is the first report on culture-independent diversity of fungi from the Indian Ocean.     

Evaluation of PCR primers for denaturing gradient gel electrophoresis analysis of fungal communities in compost

AIMS: Three previously published fungal specific PCR primer sets, referred to as the NS, EF and NL primer sets, were evaluated for use in compost microbial community analysis by PCR and denaturing gradient gel electrophoresis (DGGE). METHODS AND RESULTS: Primers were first evaluated based on their tolerance to PCR inhibitors. Due to its sensitivity to inhibitors, the NS primer set was determined to require a 10-fold smaller volume addition of compost DNA to PCR than the EF and NL primer sets, based on a logistic regression model for a 75% PCR success rate. Further evaluation of the EF and NL primer sets involved testing the resolution of PCR products from pure fungal cultures on DGGE. The NL primer set, which targets the more variable 28S rDNA, resulted in multiple bands for each pure culture. Thus, the EF primer set was used to monitor the microbial community during compost colonization studies, where three fungi were inoculated onto autoclaved grape pomace and rice straw compost. CONCLUSIONS: Of the three primer sets evaluated, the EF primer set was determined to be the best for PCR-DGGE of compost fungal populations; however, concerns with the EF primer set included the lack of sequence divergence in the targeted region of 18S rDNA and PCR artifacts which interfered with detection of inoculated fungi in the colonization studies. SIGNIFICANCE AND IMPACT OF THE STUDY: There are many factors related to PCR primers that need to be assessed prior to applying PCR-DGGE to fungal communities in complex environments such as compost.     

16S/18S/ITS扩增子高通量测序引物的生物信息学评估和改进

【背景】在过去的十几年里,基于核糖体RNA基因的扩增子测序技术被广泛用于各种生态系统中微生物群落的多样性检测。扩增子测序的使用极大地促进了土壤、水体、空气等环境中微生物生态的相关研究。【目的】随着高通量测序技术的不断发展和参考数据库的不断更新,针对不同的环境样本的引物选择和改进仍然需要更深入的校验。【方法】本文收集了目前在微生物群落研究中被广泛采用的标记基因扩增通用引物,包括16S rRNA基因扩增常用的8对通用引物和2对古菌引物、9对真菌转录间隔区(internal transcribed spacer,ITS)基因扩增引物,以及18S rRNA基因扩增的4对真核微生物通用引物和1对真菌特异性引物。这些引物中包括了地球微生物组计划(Earth Microbiome Project,EMP)推荐的2对16S rRNA基因扩增引物、1对ITS1基因扩增引物和1对18S rRNA基因扩增引物。采用最近更新的标准数据库对这些引物进行了覆盖度和特异性评价。【结果】EMP推荐的引物依然具有较高的覆盖度,而其他引物在覆盖度及对特定环境或类群的特异性上也各有特点。此外,最近有研究对这些通用引物进行了一些改进,而我们也发现,一个碱基的变化都可能会导致评价结果或扩增产物发生明显变化,简并碱基的引入既可以覆盖更多的物种,但同时也会在一定程度上降低关注物种的特异性。【结论】研究结果为微生态研究中标记基因的引物选择提供了一个广泛的指导,但在关注具体科学问题时,引物的选择仍需数据指导与实验尝试。