scholarly journals Internal Transcribed Spacer (ITS) as Dna Barcoding to Identify Fungal Species: a Review

Author(s):  
Nurrahmi Dewi Fajarningsih

Despite the fact that fungi are important sources of both bioactive compounds and mycotoxins, and that they are very ubiquitous in our environment, their species identification is hampered by incomplete and often unclear literature. Fungi identification is primarily based on their phenotypic and physiological characteristics. Nowadays, many molecular methods to identify fungal species have been developed. One of the methods considered as a new concept to rapidly and accurately identify unknown fungal sample is DNA Barcoding. This literature review will outline the use of DNA barcoding approach to rapidly identify fungal species and the use of ITS region that recently has been designated as primary DNA barcode for fungal kingdom. “DNA barcode” is a short, highly variable and standardized DNA region with approximately 700 nucleotides in length, which is used as a unique pattern to identify living things. Internal Transcribed Spacer (ITS) region of nuclear DNA (rDNA) has become the most sequenced region to identify fungal taxonomy at species level, and even within species. ITS region is a highly polymorphic non-coding region with enough taxonomic units. Therefore, it is able to separate sequences into species level. Even though ribosomal ITS as a universal barcode marker for fungi is still hampered by few limitations, the ITS will remain as the key choice for fungal identification. The search for alternative regions as DNA marker to improve fungal identification, especially in specific heredities, has already started. 

Botany ◽  
2019 ◽  
Vol 97 (9) ◽  
pp. 503-512 ◽  
Author(s):  
Deniz Aygoren Uluer ◽  
Rahma Alshamrani

Aesculus L. is a small genus of horticulturally important trees and shrubs, comprising 13–19 species. Frequent hybridization among species, particularly in cultivation, has contributed to taxonomic confusion and difficulties in the identification of plants. In this study, we evaluated three widely employed plant DNA barcode loci, matK, and the entire ITS region (ITS1+5.8S+ITS2) as well as subunit ITS2 for 50 individuals representing 13 species of Aesculus, excluding only A. wangii (=A. assamica). In contrast to the plastid matK region, both the ITS and ITS2 loci displayed low levels of species discrimination, especially in our “first hit” BLASTn searches. We also presented the phylogeny of Aesculus based on matK and the entire ITS region, with additional matK and ITS sequences from GenBank. Our results show that Aesculus chinensis, A. flava, A. glabra, A. pavia, and A. sylvatica are probably not monophyletic. Furthermore, with the widest taxon coverage until now, the current study highlights the importance of sampling multiple individuals, not only for DNA barcoding, but also for phylogenetic studies.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ammarah Hami ◽  
Rovidha S. Rasool ◽  
Nisar A. Khan ◽  
Sheikh Mansoor ◽  
Mudasir A. Mir ◽  
...  

AbstractChilli (Capsicum annuum L.) is one of the most significant vegetable and spice crop. Wilt caused by Fusarium Sp. has emerged as a serious problem in chilli production. Internal transcribed spacer (ITS) region is widely used as a DNA barcoding marker to characterize the diversity and composition of Fusarium communities. ITS regions are heavily used in both molecular methods and ecological studies of fungi, because of its high degree of interspecific variability, conserved primer sites and multiple copy nature in the genome. In the present study we focused on morphological and molecular characterization of pathogen causing chilli wilt. Chilli plants were collected from four districts of Kashmir valley of Himalayan region. Pathogens were isolated from infected root and stem of the plants. Isolated pathogens were subjected to DNA extraction and PCR amplification. The amplified product was sequenced and three different wilt causing fungal isolates were obtained which are reported in the current investigation. In addition to Fusarium oxysporum and Fusarium solani, a new fungal species was found in association with the chilli wilt in Kashmir valley viz., Fusarium equiseti that has never been reported before from this region. The studies were confirmed by pathogenicity test and re-confirmation by DNA barcoding.


mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Christian Milani ◽  
Giulia Alessandri ◽  
Marta Mangifesta ◽  
Leonardo Mancabelli ◽  
Gabriele Andrea Lugli ◽  
...  

ABSTRACT 16S small-subunit (SSU) rRNA gene-based bacterial profiling is the gold standard for cost-effective taxonomic reconstruction of complex bacterial populations down to the genus level. However, it has been proven ineffective in clinical and research settings requiring higher taxonomic resolution. We therefore developed a bacterial profiling method based on the internal transcribed spacer (ITS) region employing optimized primers and a comprehensive ITS database for accurate cataloguing of bacterial communities at (sub)species resolution. Performance of the microbial ITS profiling pipeline was tested through analysis of host-associated, food, and environmental matrices, while its efficacy in clinical settings was assessed through analysis of mucosal biopsy specimens of colorectal cancer, leading to the identification of putative novel biomarkers. The data collected indicate that the proposed pipeline represents a major step forward in cost-effective identification and screening of microbial biomarkers at (sub)species level, with relevant impact in research, industrial, and clinical settings. IMPORTANCE We developed a novel method for accurate cataloguing of bacterial communities at (sub)species level involving amplification of the internal transcribed spacer (ITS) region through optimized primers, followed by next-generation sequencing and taxonomic classification of amplicons by means of a comprehensive database of bacterial ITS sequences. Host-associated, food, and environmental matrices were employed to test the performance of the microbial ITS profiling pipeline. Moreover, mucosal biopsy samples from colorectal cancer patients were analyzed to demonstrate the scientific relevance of this profiling approach in a clinical setting through identification of putative novel biomarkers. The results indicate that the ITS-based profiling pipeline proposed here represents a key metagenomic tool with major relevance for research, industrial, and clinical settings.


2012 ◽  
Vol 109 (16) ◽  
pp. 6241-6246 ◽  
Author(s):  
C. L. Schoch ◽  
K. A. Seifert ◽  
S. Huhndorf ◽  
V. Robert ◽  
J. L. Spouge ◽  
...  

2000 ◽  
Vol 38 (4) ◽  
pp. 1510-1515 ◽  
Author(s):  
Travis Henry ◽  
Peter C. Iwen ◽  
Steven H. Hinrichs

Aspergillus species are the most frequent cause of invasive mold infections in immunocompromised patients. Although over 180 species are found within the genus, 3 species, Aspergillus flavus, A. fumigatus, and A. terreus, account for most cases of invasive aspergillosis (IA), with A. nidulans, A. niger, and A. ustus being rare causes of IA. The ability to distinguish between the various clinically relevant Aspergillus species may have diagnostic value, as certain species are associated with higher mortality and increased virulence and vary in their resistance to antifungal therapy. A method to identify Aspergillus at the species level and differentiate it from other true pathogenic and opportunistic molds was developed using the 18S and 28S rRNA genes for primer binding sites. The contiguous internal transcribed spacer (ITS) region, ITS 1–5.8S–ITS 2, from referenced strains and clinical isolates of aspergilli and other fungi were amplified, sequenced, and compared with non-reference strain sequences in GenBank. ITS amplicons fromAspergillus species ranged in size from 565 to 613 bp. Comparison of reference strains and GenBank sequences demonstrated that both ITS 1 and ITS 2 regions were needed for accurate identification ofAspergillus at the species level. Intraspecies variation among clinical isolates and reference strains was minimal. Sixteen other pathogenic molds demonstrated less than 89% similarity withAspergillus ITS 1 and 2 sequences. A blind study of 11 clinical isolates was performed, and each was correctly identified. Clinical application of this approach may allow for earlier diagnosis and selection of effective antifungal agents for patients with IA.


2021 ◽  
Vol 9 ◽  
Author(s):  
Parvin Aghayeva ◽  
Salvatore Cozzolino ◽  
Donata Cafasso ◽  
Valida Ali-zade ◽  
Silvia Fineschi ◽  
...  

DNA barcoding has rapidly become a useful complementary tool in floristic investigations particularly for identifying specimens that lack diagnostic characters. Here, we assess the capability of three DNA barcode markers (chloroplast rpoB, accD and nuclear ITS) for correct species assignment in a floristic survey on the Caucasus. We focused on two herbal groups with potential for ornamental applications, namely orchids and asterids. On these two plant groups, we tested whether our selection of barcode markers allows identification of the “barcoding gap” in sequence identity and to distinguish between monophyletic species when employing distance-based methods. All markers successfully amplified most specimens, but we found that the rate of species-level resolution amongst selected markers largely varied in the two plant groups. Overall, for both lineages, plastid markers had a species-level assignment success rate lower than the nuclear ITS marker. The latter confirmed, in orchids, both the existence of a barcoding gap and that all accessions of the same species clustered together in monophyletic groups. Further, it also allowed the detection of a phylogeographic signal.The ITS marker resulted in its being the best performing barcode for asterids; however, none of the three tested markers showed high discriminatory ability. Even if ITS were revealed as the most promising plant barcode marker, we argue that the ability of this barcode for species assignment is strongly dependent on the evolutionary history of the investigated plant lineage.


Author(s):  
Takeru Nakazato

DNA barcoding technology has become employed widely for biodiversity and molecular biology researchers to identify species and analyze their phylogeny. Recently, DNA metabarcoding and environmental DNA (eDNA) technology have developed by expanding the concept of DNA barcoding. These techniques analyze the diversity and quantity of organisms within an environment by detecting biogenic DNA in water and soil. It is particularly popular for monitoring fish species living in rivers and lakes (Takahara et al. 2012). BOLD Systems (Barcode of Life Database systems, Ratnasingham and Hebert 2007) is a database for DNA barcoding, archiving 8.5 million of barcodes (as of August 2020) along with the voucher specimen, from which the DNA barcode sequence is derived, including taxonomy, collected country, and museum vouchered as metadata (e.g. https://www.boldsystems.org/index.php/Public_RecordView?processid=TRIBS054-16). Also, many barcoding data are submitted to GenBank (Sayers et al. 2020), which is a database for DNA sequences managed by NCBI (National Center for Biotechnology Information, US). The number of the records of DNA barcodes, i.e. COI (cytochrome c oxidase I) gene for animal, has grown significantly (Porter and Hajibabaei 2018). BOLD imports DNA barcoding data from GenBank, and lots of DNA barcoding data in GenBank are also assigned BOLD IDs. However, we have to refer to both BOLD and GenBank data when performing DNA barcoding. I have previously investigated the registration of DNA barcoding data in GenBank, especially the association with BOLD, using insects and flowering plants as examples (Nakazato 2019). Here, I surveyed the number of species covered by BOLD and GenBank. I used fish data as an example because eDNA research is particularly focused on fish. I downloaded all GenBank files for vertebrates from NCBI FTP (File Transfer Protocol) sites (as of November 2019). Of the GenBank fish entries, 86,958 (7.3%) were assigned BOLD identifiers (IDs). The NCBI taxonomy database has registrations for 39,127 species of fish, and 20,987 scientific names at the species level (i.e., excluding names that included sp., cf. or aff.). GenBank entries with BOLD IDs covered 11,784 species (30.1%) and 8,665 species-level names (41.3%). I also obtained whole "specimens and sequences combined data" for fish from BOLD systems (as of November 2019). In the BOLD, there are 273,426 entries that are registered as fish. Of these entries, 211,589 BOLD entries were assigned GenBank IDs, i.e. with values in “genbank_accession” column, and 121,748 entries were imported from GenBank, i.e. with "Mined from GenBank, NCBI" description in "institution_storing" column. The BOLD data covered 18,952 fish species and 15,063 species-level names, but 35,500 entries were assigned no species-level names and 22,123 entries were not even filled with family-level names. At the species level, 8,067 names co-occurred in GenBank and BOLD, with 6,997 BOLD-specific names and 599 GenBank-specific names. GenBank has 425,732 fish entries with voucher IDs, of which 340,386 were not assigned a BOLD ID. Of these 340,386 entries, 43,872 entries are registrations for COI genes, which could be candidates for DNA barcodes. These candidates include 4,201 species that are not included in BOLD, thus adding these data will enable us to identify 19,863 fish to the species level. For researchers, it would be very useful if both BOLD and GenBank DNA barcoding data could be searched in one place. For this purpose, it is necessary to integrate data from the two databases. A lot of biodiversity data are recorded based on the Darwin Core standard while DNA sequencing data are sometimes integrated or cross-linked by RDF (Resource Description Framework). It may not be technically difficult to integrate these data, but the species data referenced differ from the EoL (The Encyclopedia of Life) for BOLD and the NCBI taxonomy for GenBank, and the differences in taxonomic systems make it difficult to match by scientific name description. GenBank has fields for the latitude and longitude of the specimens sampled, and Porter and Hajibabaei 2018 argue that this information should be enhanced. However, this information may be better described in the specimen and occurrence databases. The integration of barcoding data with the specimen and occurrence data will solve these problems. Most importantly, it will save the researcher from having to register the same information in multiple databases. In the field of biodiversity, only DNA barcode sequences may have been focused on and used as gene sequences. The museomics community regards museum-preserved specimens as rich resources for DNA studies because their biodiversity information can accompany the extraction and analysis of their DNA (Nakazato 2018). GenBank is useful for biodiversity studies due to its low rate of mislabelling (Leray et al. 2019). In the future, we will be working with a variety of DNA, including genomes from museum specimens as well as DNA barcoding. This will require more integrated use of biodiversity information and DNA sequence data. This integration is also of interest to molecular biologists and bioinformaticians.


2022 ◽  
Vol 2022 ◽  
pp. 1-19
Author(s):  
Sarfraz Ahmed ◽  
Muhammad Ibrahim ◽  
Chanin Nantasenamat ◽  
Muhammad Farrukh Nisar ◽  
Aijaz Ahmad Malik ◽  
...  

DNA barcodes are regarded as hereditary succession codes that serve as a recognition marker to address several queries relating to the identification, classification, community ecology, and evolution of certain functional traits in organisms. The mitochondrial cytochrome c oxidase 1 (CO1) gene as a DNA barcode is highly efficient for discriminating vertebrate and invertebrate animal species. Similarly, different specific markers are used for other organisms, including ribulose bisphosphate carboxylase (rbcL), maturase kinase (matK), transfer RNA-H and photosystem II D1-ApbsArabidopsis thaliana (trnH-psbA), and internal transcribed spacer (ITS) for plant species; 16S ribosomal RNA (16S rRNA), elongation factor Tu gene (Tuf gene), and chaperonin for bacterial strains; and nuclear ITS for fungal strains. Nevertheless, the taxon coverage of reference sequences is far from complete for genus or species-level identification. Applying the next-generation sequencing approach to the parallel acquisition of DNA barcode sequences could greatly expand the potential for library preparation or accurate identification in biodiversity research. Overall, this review articulates on the DNA barcoding technology as applied to different organisms, its universality, applicability, and innovative approach to handling DNA-based species identification.


Toxins ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 467
Author(s):  
Richard Dooso Oloo ◽  
Sheila Okoth ◽  
Peter Wachira ◽  
Samuel Mutiga ◽  
Phillis Ochieng ◽  
...  

Highly toxigenic strains of Aspergillus flavus have been reported to frequently contaminate maize, causing fatal aflatoxin poisoning in Kenya. To gain insights into the environmental and genetic factors that influence toxigenicity, fungi (n = 218) that were culturally identified as A. flavus were isolated from maize grains samples (n = 120) from three regions of Kenya. The fungi were further characterized to confirm their identities using a PCR-sequence analysis of the internal transcribed spacer (ITS) region of rDNA which also revealed all of them to be A. flavus. A subset of 72 isolates representing ITS sequence-based phylogeny cluster and the agroecological origin of maize samples was constituted for subsequent analysis. The analysis of partial calmodulin gene sequences showed that the subset consisted of A. flavus (87%) and Aspergillus minisclerotigenes (13%). No obvious association was detected between the presence of seven aflatoxin biosynthesis genes and fungal species or region. However, the presence of the aflD and aflS genes showed some association with aflatoxin production. The assessment of toxigenicity showed higher aflatoxin production potential in A. minisclerotigenes isolates. Given that A. minisclerotigenes were mainly observed in maize samples from Eastern Kenya, a known aflatoxin hotspot, we speculate that production of copious aflatoxin is an adaptative trait of this recently discovered species in the region.


Phytotaxa ◽  
2019 ◽  
Vol 413 (1) ◽  
pp. 11-26 ◽  
Author(s):  
FATEMEH HOJJATI ◽  
ROBERT P. ADAMS ◽  
RANDALL G. TERRY

Previous studies of nrDNA (nuclear DNA) of Juniperus seravschanica indicated its nuclear DNA (ITS) was from an ancestor of J. polycarpos. However, analysis of cpDNA (chloroplast DNA) suggested the taxon had derived its chloroplast from an ancestor of J. foetidissima. That study has been viewed as putative, because the ITS region is sometimes unreliable for the detection of ancestral hybrids due to concerted evolution and lineage sorting. The recent availability of several single copy nuclear genes (SCNGs) with primers specifically designed for Juniperus presented an opportunity to fully investigate this case of putative chloroplast capture. Three phylogenetic analyses using five SCNGs (LHCA4, maldehy, myb, CnAIP3 and 4CL), ITS region, and four cpDNAs (petN- psbM, trnD-trnT, trnL-trnF and trnS-trnG) were performed on J. seravschanica, as well as other members of the J. excelsa complex: J. excelsa, J. polycarpos, and J. p. var. turcomanica. Analyses revealed incongruence between SCNGs, ITS region and cpDNA showing that J. seravschanica contains an ancestral J. foetidissima/ J. thruifera cp genome. In addition, the phylogenies indicate that the J. excelsa complex is composed of three distinct clades at the species level: J. excelsa, J. polycarpos and J. seravschanica and two varieties of J. polycarpos: J. p. var. polycarpos and J. p. var. turcomanica.


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