MINI-REVIEW

DNA barcoding to map the microbial communities:current advances and future directions

Chiranjib Chakraborty & C. George Priya Doss &Bidhan C. Patra & Sanghamitra Bandyopadhyay

Received: 12 November 2013 /Revised: 16 January 2014 /Accepted: 17 January 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract During the last two decades, the DNA barcodedevelopment towards microbial community has increaseddramatically. DNA barcode development is related to error-free and quick species identification which aid in understand-ing the microbial biodiversity, as well as the diseases related tomicrobial species. Here, we seek to evaluate the so-calledbarcoding initiatives for the microbial communities and theemerging trends in this field. In this paper, we describe thedevelopment of DNA marker-based DNA barcoding system,comparison between routine species identification and DNAbarcode, and microbial biodiversity and DNA barcode formicrobial communities. Two major topics, such as the molec-ular diversity of viruses and barcode for viruses have beendiscussed at the same time. We demonstrate the current statusand the maker of DNA barcode for bacteria, algae, fungi, andprotozoa. Furthermore, we argue about the promises, limita-tions, and present and future challenges of microbial barcodedevelopment.

Keywords Microbial community . DNA barcode . Nextgeneration sequencing . Barcodemarker gene . Biodiversity

Introduction

The last decade has proved to be an exciting era for biologicalscience which is moving very fast with the help of new rapidtechnologies. Compared to the last 10 years, technologicaladvancement has made data collection much easier, faster,and cheaper than what we can’t think before. Biologicalresearch across different disciplines and researchers areperforming collaborative research among cross subjects.Presently, most significant technologies were developed fromcomputer science. Advancement in computer science hasbecome one of the foundation components for the progressionof new biological science (Losos et al. 2013; Stein 2008).Computer science has enabled scientists to decode variousbiological problems and health-related problems, such as ge-netically inherited diseases and drug discovery and develop-ment, etc (Doss et al. 2013; Chakraborty and Agrawal 2013;Chakraborty et al. 2011; Chakraborty and Doss 2013; Georgeet al. 2013). In the ecosystem and biodiversity domain, DNAbarcoding is a recent significant effort in this direction.

More than 100 million species are existing in this planet,but only little proportion of them have been identified andcharacterized (May 1988). One of the biggest challenges is theidentification of unknown species. Very few taxonomists cansignificantly identify nearly about 0.01 % of the projected 10–15 million species (Hawksworth and Kalin-Arroyo 1995).Hebert et al. (2003a) expressed that a group of 15,000 taxon-omists or more than that will be needed to recognize livingorganisms morphologically. Again, if we look into the micro-bial world, we find astonishing numbers of microbes, which isvery thrilling for microbiologist. In this microbial world, weare not aware of the diversity of the 99 % microbes that aremainly bacteria, archaea, and single-celled eukaryotes(Editorial 2011; Woese 1996). It is an impossible task toprepare the catalogue of microbial diversity by traditionalmethods following each taxon or each specimen with the

C. Chakraborty (*)Department of Bioinformatics, School of Computer and InformationSciences, Galgotias University, Greater Noida, Indiae-mail: drchiranjib@yahoo.com

C. G. P. DossMedical Biotechnology Division, School of Biosciences andTechnology, VIT University, Vellore 632014, Tamil Nadu, India

B. C. PatraDepartment of Zoology, Vidyasagar University, Midnapore 721102, West Bengal, India

S. BandyopadhyayMachine Intelligence Unit, Indian Statistical Institute, Kolkata, India

Appl Microbiol BiotechnolDOI 10.1007/s00253-014-5550-9

morphological explanation (Lawton et al. 1988). Today’semerging technologies can support the task very easily(Godfray 2002). Considerable number of bacteria are notcultured, and few do not form colonies on agar plates(Schloss and Handelsman 2004; Rappe and Giovannoni2003). Especially, it has been observed for those bacteriawhich are living inside or on human beings (Curtin 2009;Friedrich 2008; Stone 2009; Turnbaugh et al. 2007). It is ofgreat challenge for the microbiologist to identify these mi-crobes. Recent advancement in DNA genome-based andDNA sequencing-based technologies can be an essential com-ponent in identifying these microbes. Presently, using DNAsequence data, the current “DNA barcoding” system is pro-viding a proficient platform for species-level identificationand this system ismore challenging. The scientific communityis using this process very efficiently which reflects in thepublication number. The publication in this domain is increas-ing very fast (Fig. 1).

With the advancement of sequencing and computationaltechnologies, the scientists from different genomic centers inboth the academia as well as the industries have standardizedDNA sequence and increased their capabilities in identifyingthe microbes with the help of the short sequences (Brent 2000;Pedersen 2010). Short unique sequence may help in differen-tiating the individual species due to the existence of geneticvariation in the closely related species (Hebert et al. 2003b).Many sequences of microbes are published in the variousmicrobial databases, as well as the public databases such asNCBI. Using these sequences, many efforts have been madein identifying the different strains as well as the developmentof barcode for microbial communities (Segata et al. 2013).Here, we undertook intensive field collections to explore theconcept of biological barcoding in microbial communities.We briefly described how DNA barcode can be proficient,

with a focus on its current standing and trends for the micro-bial communities such as virus, bacteria, algae, fungi, andprotozoa. Finally, an outlook has been provided on promises,limitations, and present and future challenges of microbialbarcode growth.

Comparing routine species identification and DNAbarcode-based species identification

The process of conventional species identification has manylimitations. The first problem is the phenotypic flexibility andgenetic variability in the characters which are accounted forthe different species identification that may direct to inaccu-rate identifications. The second problem is the identificationof cryptic species complex. These complexes were wide-spread in many groups of species (Jarman and Elliott 2000)especially most frequently existing in the marine environment(Knowlton 1993). Thirdly, the taxonomists are depending onmorphological keys for the routine species identification.These keys may vary in particular in life cycle or gender togender. Therefore, many species may be wrongly identified.Highly expert manpower is needed for this type of identifica-tion system.

Conversely, when using next generation high-throughputmethods, “DNA barcoding” is proved to be faster in speciesidentification. This modern-automated method is accurate,economic, and less time-consuming when compared to thetraditional process. This automated process can classify manynumbers of species at the same time. For example, high-throughput system facility could routinely handle 1,000 sam-ples per day or more. Currently, manpower cost is rising fast,whereas the cost of automation keeps on decreasing(Tautz et al. 2003). Therefore, the “DNA barcoding” is foundto be a more cost-effective and promising method as com-pared to the traditional process.

DNA barcoding system and DNA markers

We found that the genetic diversity is varying from species tospecies. The process of the species identification throughbarcoding is generally accomplished by the retrieval of a shortDNA sequence of microbes—from a standard part of thegenome. This short sequence is defined as barcode sequence,and it is specific for a particular gene. This short, consistentsequence can help in differentiating individual species(Hajibabaei et al. 2007; Hebert et al. 2003a).

For animals, the most common short sequence or markermitochondrial cytochrome c oxidase subunit I (COI)—isprojected to serve as a provisional identifier of a specimen.COI is approximately 650 base pair long and a single mtDNA

Fig. 1 Increasing trend in the publication in the DNA barcodingresearch. Keyword searched was performed from PUBMED, NCBIdatabase. We have used the keywords “(DNA barcoding) AND("2003"[Date - Publication] : "2004"[Date - Publication])”. Searchwas performed on 12th of November 2013

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gene. The wide range applicability as random PCR primers isthe major advantage of the COI gene (Hebert et al. 2003a, b).

DNA barcoding system has proved to be a trickier task forplants where DNA barcoding is using other markers. Due tothe low rate of nucleotide substitution mitochondrial genomein plants, the researchers are not using COI as a universal plantbarcode. The plastid markers show promise for plants(Fazekas et al. 2008). Different coding and non-coding re-gions have standardized the DNA barcoding system. Fewcoding loci such as rpoB, rpoC1, rbcL, matK, and 23SrDNA and non-coding loci such as trnH-psbA, atpF-atpH,and psbK-psbI were identified and used as DNA barcodingfor plants (Fazekas et al. 2008). Hollingsworth et al. (2011)have described a brief review with combinations of sevenplastid markers by different researchers on plant DNAbarcode development. Different mixtures of these markerswere discussed in the Second International Barcode of LifeConference in Taipei. Chase et al. (2007) suggested a combi-nation of barcode marker rpoC1+rpoB+matK or rpoC1+matK+trnH-psbA, followed by Kress and Erickson (2007)another combination of barcode marker rbcL+trnH-psbA.Lahaye et al. (2008) projected that the matK marker can worksingly as a plant barcode marker. The study of Seberg andPetersen (2009) projected rpoC1, matK, and trnH-psbA.Therefore, development of DNA barcoding loci for plantsremains as a challenging task.

Over two decades, DNA is used as a tool to identify thebacterial strains which was described by Woese (1987). Forbacterial taxonomy, scientists rely on a combination ofcharacter-based data and genome-based data or DNA-baseddata to understand the genetic and ecological diversity (Fraseret al. 2009). DNA-DNA hybridization technique was utilizedfor the identification of bacteria. Recently, genome-orientedmethods are evolving for the description of new species ofbacteria (Stackebrandt et al. 2002; Stackebrandt and Ebers2006; Richter and Rossello-Mora 2009). Since 1990s, 16SrRNA gene, the most common housekeeping genetic markeris being used as a marker gene in bacterial identificationprocess. In this context, many scientists have used PCR-amplified 16S rRNA gene to identify microbes (Schmidt andRelman 1994; Janda and Abbott 2007). This gene is used tostudy bacterial phylogeny and taxonomy for a number ofcauses. There are some benefits to use the gene as a barcodingmarker. First, this gene is present in almost all the bacterialspecies, frequently accessible as a multigene family or op-erons. Secondly, the function of the gene eventually has notchanged. Finally, this gene is approximately 1,500 bp inlength which is sufficient for informatics methods. Due tothe above advantages, 16S rRNA gene is used as a barcodemarker frequently. (Patel 2001; Ranasinghe et al. 2012).Another gene chaperonin-60 (cpn60) was also used as abacterial barcode marker gene. This gene is approximately555 bp long (Links et al. 2012).

Other than this, many other genes have been proposed asspecies-level molecular markers such as cytb, ITS1, ITS2,18S, and 28S RNA (LaJeunesse 2001; Case et al. 2007;Hajibabaei et al. 2007). A list of the marker genes has beenprovided in Table 1 which is routinely used in barcodingstudies of microbial communities. Approximate length ofsome marker gene is noted in Fig. 2. However, the barcodemarker sequence from each unidentified species is comparedwith a library of reference barcode sequences derived fromindividuals of known identity. The final goal of the DNAbarcoding system is to build up a robust and efficient mech-anism for the species identification in a fast manner which isstandardized, simple, and scalable.

Microbial biodiversity and DNA barcode for microbialcommunities

Evolution developed biodiversity. Actually, biodiversity wascreated by the evolution during the 3.5 billion years. About600 million years ago, life of microbes such as archaea,bacteria, protozoa, and similar single-celled organisms weredeveloped and gone through the biodiversity (Wilson 1999;Snedden 2007). Therefore, the microbial world is full ofbiodiversity. In the 1990s, investigation was initiated to un-derstand the diversity of bacteria and archaea. This was per-formed using DNA sequences and DNA taxonomy. The con-cept was that microbes have highly conserved small subunitribosomal RNA (SSrRNA) gene. This gene can be isolatedfrom mass environmental samples as a form of mass “envi-ronmental DNA.” With the examination of SSrRNA genesequence from the DNA sample, we can identify how manydifferent taxa are present. Then, scientists evaluated the rela-tionship of each sequenced organism. They have found thatmicroorganisms lived in main groups which are unknown totraditional microbiology. Finally, it has been noted that thediversity of microbes is almost 100 times higher than whatwas projected earlier (Woese 1996; Pace 1997; Blaxter 2003).

Barcoding for viruses—an open question

Probably, viruses are the most abundant biological creature onearth. It has been estimated that the total number of virusparticles is more than 10 times the total number of cells(Suttle 2007). The molecular diversity of viruses is compli-cated, as virus molecular diversity of genome is also complex(Hulo et al. 2011). The “molecular entity” and virus speciesstill remain as a debate for several scientists (Casiraghi et al.2010). The identification and explanation ofmolecular entitiesof virus should be the major objective of DNA barcoding.Very few works have been incited to identify thepathogenically important virus. Therefore, researchers should

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try to develop the barcode for the detection or identification ofthe virus.

Recently, few works have been found in this direction. Weiet al. (2011) described the k-mer-based barcode image toidentify significant pathogen human enteroviruses (HEVs).In this process, Wei et al. used the condition of 1<k<7 for afixed k and a genome barcode was described in terms of the k-mer frequency distribution across the whole genome for allcombinations of k-mers. Bluetongue virus (BTV) is an animalvirus which affects the different mammals such as cattle,buffalo, sheep, deer, goats, etc (Maan et al. 2011). For thedetection of this virus, ultrasensitive technique bio-barcodeamplification assay (BCA)methodwas developed. This meth-od was used for the specific detection of the outer-core proteinVP7 of BTV. Though they have produced protein bases bio-barcode, however, signal DNA annealed to DNA strands

Table 1 Different type of markers that have routinely been included in microbial communities for barcoding studies

Microbialcommunity

Barcoding markergenes

Remark References

Virus No specific marker 1. Nanoparticle-based bio-barcode assay2. Sandwich immunoassay and fluorophore-tagged oligonucleotidesas representative barcodes

Yin et al. 2011; Cao et al. 2009

Bacteria 16S rRNA 1. Nuclear DNA and housekeeping gene2. Coding genes are referred as 16S rDNA

Janda and Abbott 2007;Woo et al. 2008

COI Terminal enzyme in the respiratory chains in many bacteria Michel et al. 1998

cpn60 1. Chaperonin-60 (cpn60) also known as GroEL and Hsp602. A large barcode gap has been noted in cpn60

Links et al. 2012

rpoB Gene that encodes the β subunit of bacterial RNA polymerase Campbell et al. 2001

Algae COI COI was used as the barcode marker for brown and red algae Saunders and McDevit 2012

rbcL 1. Genomic location is plastid, rbcL-3P was used as the barcode markerfor diatoms and macroalgae

2. Protein coding gene

Saunders and McDevit 2012

tufA 1. tufA gene, a chloroplast gene, is a protein coding gene which encodesfor elongation factor Tu

2. It intercede the entry of an amino-acyl-tRNA into the acceptor site of aribosome during protein synthesis

Famà et al. 2002

LSU Genes coding for it are referred to as LSU rDNA such as 28S LSU rDNA Ali et al. 2001

UPA Universal plastid amplicon (UPA) used DNA barcode for red algae Sherwood et al. 2010

ITS Internal transcribed spacer (ITS) region of the ribosomal cistron Schoch et al. 2012

RuBisCO It encoded Ribulose-1,5-bisphosphate carboxylase oxygenase Meyer et al. 2012

Fungi COI COI coding region contains group I introns Cho et al. 1998

ITS Internal transcribed spacer region of the ribosomal cistron Schoch et al. 2012;Begerow et al. 2010

LSU Genes coding for LSU are referred to as LSU rDNA such as 28S LSU rDNA Begerow et al. 2010

SSU Genes coding referred to as SSU rDNA such as 16S SSU rDNA Begerow et al. 2010

RPB1, RPB2 Ribosomal polymerase B1 and B2 Seifert 2009

Protozoa COI COI as a good marker for DNA barcoding of amoebae Kosakyan et al. 2012

18S rRNA DNA encoding protozoal small subunit (SSU) ribosomal RNA Karnati et al. 2003

28S rRNA Evaluated as DNA barcode markers for piroplasma Gou et al. 2012

ITS ITS2 is used for piroplasma Gou et al. 2012

16S rRNA 16S ribosomal RNA, 28S rRNA 28S ribosomal RNA, COXI cytochrome c oxidase I, cpn60 chaperonin-60, rpoB RNA polymerase betasubunit, rbcL ribulose-bisphosphate carboxylase, tufA elongation factor Tu, UPA universal plastid amplicon, LSU large subunit, SSU small subunit, ITSinternal transcribed spacer, RuBisCO ribulose-1,5-bisphosphate carboxylase oxygenase, RPB1/ RPB2 ribosomal polymerase B1/ B2)

Fig. 2 Approximate length of some markers used in microbial commu-nities for barcoding studies

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bound with the gold nanoparticles were released by heatingand characterized by PCR and real-time fluorescence PCR(Yin et al. 2011). To detect avian influenza virus (AIV), afluorescent DNA barcode-based immunoassay was developedbased on the application of sandwich immunoassay andfluorophore-tagged oligonucleotides as representativebarcodes Cao et al. (2009).

To understand the viral biodiversity and development ofDNA barcode, no marker DNA or RNA has been developedfor viruses. However, it is the time to understand the viralbiodiversity with DNA barcoding.

DNA barcoding for bacteria

A microbial diversity picture in this planet is still not clear, asmany microbes are difficult to culture. It is very crucial tounderstand microbe association with different environmentsand their function in those environments. Gene sequences of16S rRNA from different samples especially environmentalsamples have reformed our understanding of microbial diver-sity, and it helps us in cataloging the vast diversity of micro-organisms on earth (Rappe and Giovannoni 2003; Schloss andHandelsman 2004; Lozupone and Knight 2007). Over the past20 years, more than 80,000 16S rRNA gene sequences havebeen deposited in GenBank, which can help us to understandbacterial phylogeny (Schloss and Handelsman 2004). To iden-tify different species of bacteria, 16S rRNA gene is describedas an important marker for soil and marine ammonia-oxidizing bacteria (Stephen et al. 1996), mountain lakes(Hiorns et al. 1997), rice paddy soil microcosms (Großkopfet al. 1998), and human clinical samples (Clarridge 2004). So,it is apparent that 16S rRNA gene highly conserved for eachand every species of bacteria, and it can be used as a markerfor DNA barcode for different species.

Two bacteria phyla Firmicutes and Bacteroidetes are med-ically necessary and related to obesity. The ratio, betweenthese bacteria, phyla Firmicutes/Bacteroidetes ratio (F/B ratio)was observed to be significantly higher in obese individualsthan in lean individuals, and the bacterial ratio (F/B ratio) isrelated to weight loss over time (Ley et al. 2006). This initia-tive wasmoved to develop DNA barcode of these two bacteriaphyla Firmicutes and Bacteroidetes using 16S rRNA gene(Armougom and Raoult 2008).

COI gene is used to develop the DNA barcode for bacteria.Using COI marker, DNA barcode was developed for 22species pathogen (Jones et al. 2013). Recently, Smith et al.(2012) developed the DNA barcode forWolbachia, a commonendosymbiotic bacterium, using COI gene, and this gene isone of the five multilocus sequence typing genes which wasapplied for categorizingWolbachia. They have found very fewoverlap with the eukaryotic DNA barcode area. This studycorroborates that the COI gene can be a DNA barcode marker

for bacteria. Chaperonin-60 (cpn60), also known as GroELand Hsp60, is a molecular chaperone conserved in bacteria.Conversely, for evaluating the barcoding targets for Archaeaincluding 16S rRNA, type II chaperonin (ortholog of cpn60)was found to be another option (Chaban and Hill 2012). Linkset al (2012) suggested that cpn60 can be a common target forbacteria barcode. Some other genes are used for bacterialidentification such as rpoB gene which may be used as abarcode marker gene for bacteria (Case et al. 2007). So, wecan infer that 16S rRNA, COI gene, and cpn60 can normallybe used as markers for developing DNA barcode.

DNA barcoding for algae

Most of the area of the earth is covered with the sea. Algae arefound in freshwater and seawater through the planet. Themaximum variety of algae is found in the coasts in bothtemperate and tropical seas. It has been noted that thephylogenic diversity of the algae is very wide-ranging(Metting 1996). For the last two decades with the help of thecomputational biology, increasingly molecular tools were ap-plied to identify algal species. Several molecular markers weresuggested from time to time to identify the algal species.Examples include COI, rbcL (the rubisco operon) (Hugheyet al. 2001), the internal transcribed spacer (ITS) of the ribo-somal cistron (Tai et al. 2001; Ross et al. 2003), tufA, andlarge subunit (LSU) 28S of the ribosomal cistron, 23SUniversal Plastid Amplicon (UPA), etc. Some markers arecoming into the picture for the development of DNA barcodealgae.

Saunders and McDevit (2012) tried to develop a methodfor DNA barcode for macroalgae and diatoms such as brownalgae (Phaeophyceae), red algae (Rhodophyta), green algae(Chlorophyta), and microscopic diatoms (Bacillariophyta).They used three barcode markers such as COI-5P, rbcL-3P,and tufA. COI was used as the barcode marker for brown andred algae, tufA as the barcode marker for green algae, andrbcL-3P was used as the barcode marker for diatoms. ForCarrageenophytes, various molecular markers have been in-troduced for the molecular taxonomy such as COX1 andCOX2-3 spacer, ITS, LSU, rbcL, RuBisCO, and the 23SUPA (Conklin et al. 2009; Zuccarello et al. 2006; Fredericqet al. 1999; Zhao and He 2011). A recent study by Tan et al.(2012) suggested the efficiency of the four markers COX1,COX2, COX2-3, and rbcL in DNA barcoding on selectedKappaphycus and Eucheuma from Southeast Asia. ForDNA barcoding, development of some marine algae especial-ly the Rhodophyta, COX1 “barcode” region was used toassess the marker (Saunders 2005). Buchheim et al. (2011)assessed the internal transcribed spacer two (ITS2) from 591Chlorophycean, 741 Trebouxiophycean, and 938Ulvophycean algae, which is one of the marker development

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steps for the DNA barcode development. Using COX1marker,Robba et al. (2006) assessed DNA barcode using 48 samplesplus 31 sequences for six orders of red algae namely Bangiales,Ceramiales, Corallinales, Gigartinales, Gracilariales, andRhodymeniales. They also used COX1 with the plastidRubisco spacer for Porphyra species and discovered that itwas a more susceptible marker in revealing initial speciationand cryptic diversity. Finally, they concluded COX1 gene is apossible DNA barcode marker of red algae. From the aboveevidences, we can understand that the vivid biodiversity of thealgal community has not been neglected, and scientists aretrying to develop the barcode of the biodiversity of the algalcommunity.

DNA barcoding for fungi

Identification of fungi to species level is the primary prob-lem for fungal research. Proper treatment for fungal dis-eases in humans is depending on appropriate identificationof the disease-causing agents (Bialek et al. 2005; Rickertset al. 2006). Moreover, species level identification plays afundamental role in biology conservation. With the help ofmolecular biology and system biology, DNA-based taxon-omy and DNA barcoding are the most interesting researchareas for the rapid level identification of all species. Theabsence of a general DNA barcode marker for fungi is aserious limitation for multitaxon ecological and biodiversi-ty studies (Schoch et al. 2012). It is interesting for molec-ular mycological community that fungal sequences areincreasing day by day in NCBI. Therefore, many studieshave been facilitated in DNA-based taxonomy and DNAbarcoding for fungi (Begerow et al. 2010). Stockinger et al.(2010) described a practical approach to choose DNAbarcode marker for arbuscular mycorrhizal fungi. Theyconsidered different criteria like universality, feasibility,and species resolution. For fungal species, the commonlyused DNA barcoding marker are—COI (Seifert et al. 2007),SSU rRNA gene (Poll et al. 2009), ITS sequences (Hugheset al. 2009), LSU, rRNA gene (Stockinger et al. 2010),ribosomal polymerase B1 and B2 (RPB1, RPB2) (Seifert2009), etc. One recent study has evaluated the potentialityof a number of fungal genes as barcode marker for fungi. Inthis study, from the barcoding database of 2,920 samples,the researchers have chosen a subset of 742 strains withsequences for four markers namely ITS, LSU, SSU, andRPB1. This subset was separated into four taxonomicallydelimited datasets which are 416 strains of Pezizomycotina,81 strains of Saccharomycotina, 202 strains ofBasidiomycota,and 43 strains from the collective lineages, and they haveconcluded that the ITS gene, located between the area of theribosomal cistron, can be used to identify a broad range offungi species; also, the distinct barcode gap has been

produced among interspecific and intraspecific variants(Schoch et al. 2012).

For more than two decades, the rRNA cistron is widelyused for fungal diagnostics and scientists are also using thisas a marker in phylogenetics (Begerow et al. 2010). 28Snuclear ribosomal LSU rRNA gene data are not availablefor some species, although the gene is usually more con-served than the ITS. However, the LSU or a combinedLSU+ITS will increase the degree of promise for barcode(Eberhardt 2010). However, for various yeasts, the D1/D2region of LSU was adopted for characterizing species longbefore the concept of DNA barcoding was promoted(Fell et al. 2000; Scorzetti et al. 2002). To understand themolecular phylogeny, evolution, and biodiversity ofarbuscular mycorrhizal fungi (AMF), two overlapping nu-clear DNA regions, totaling c. 3 kb, were analyzed: thesmall subunit (SSU) rRNA gene (up to 1,800 bp) and afragment spanning c. 250 bp of the SSU rDNA, the ITSregion (c. 475–520 bp), and c. 800 bp of the LSU rRNAgene were analyzed by Krüger et al (2012) which may be areference data for DNA barcoding. Chinese caterpillar fun-gus (Ophiocordyceps sinensis) has a medicinal value. ITSsequence as a DNA barcode was analyzed for this fungus.The result indicated that DNA barcoding is one of the fastand accurate identification methods to identify this fungus(O. sinensis) and closely related species (Xiang et al. 2013).In another study by Li et al. (2013), ITS sequence was usedto identify this fungus. Quaedvlieg et al. (2012) developedDNA barcoding ofMycosphaerella species from Europe. Inthis study, seven nuclear genomic loci were assessed foridentification of Mycosphaerella species as well as associ-ated anamorphs. Here, researchers considered seven nucle-ar genomic loci such as ITS regions of the nrDNA operon,28S nrDNA (LSU), β-tubulin (Btub), calmodulin (Cal),actin (Act), translation elongation factor 1-alpha (EF1α),and RNA polymerase II second largest subunit (RPB2).They concluded that the combination of ITS with eitherEF1α or Btub can be considered as the barcoding loci forEPPO A1/A2-listed Mycosphaerella species. Establishedbarcode loci are important for Cladonia spp. Pino-Bodaset al (2013) studied the loci IGS rDNA, EF1α, RPB2, andCOX1, where they have evaluated a total number of 782sequences from 36 species. In this study, three markersshowed the least intraspecific genetic distance range(COX1, ITS rDNA, and EF1α). Wickerhamomycesanomalus plays a important role in the food industry. β-tubulin gene was evaluated to understand the DNAbarcode marker of Wickerhamomyces strains. Species-specific PCR assay was developed for β-tubulin gene tounderstand the phylogenetic relationships between strainsW. anomalus (Huang et al. 2012).

To understand the fungal biodiversity, the ecological roles,and geographical distribution in pathogenic important fungi,

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DNA barcoding was proved to be worthful with enormouspotential. In the near future, DNA barcoding will definitelyplay a crucial role towards mycological research.

DNA barcoding for protozoa

Protozoan species identification and classification are difficulttasks because of the less number of morphological characters(Smirnov and Goodkov 1999), and it is true for protozoa withsimple morphology such as amoebae. The light microscopicfeatures in most of the protozoan species didn’t provide reli-able species differences (Smirnov 2002). Therefore, it is im-portant to develop a novel rapid tool in precise identificationof pathogenic protozoan species. With the development of themolecular system, the protozoa species identification is foundto be an interesting subject with the aid of DNA barcoding.

Amoebae are the most extensive group of protists residingin most of all the environments such as marine, freshwater,and terrestrial biosphere. Recently, attempts have been madeto identify this widespread protozoan species with molecularmarkers. Three genes, namely, SSU, ITS, and COI genes havebeen cloned to develop the molecular marker. This studyconcluded stating COI as a good marker for DNA barcodingof amoebae. However, further studies are needed to confirmthe accuracy of the COI gene as a barcode marker in othergroups of amoebae such as Gymnamoebae species. At thesame time, we have to understand the taxonomic significanceof COI variations in the different amoebaean species(Nassonova et al. 2010). Recently, Hoef-Emden (2012) triedto establish DNA barcoding systems in protists using speciesidentification technique—the blast in search with COI-5P and5′-partial LSU rDNA (domains A to D of the nuclear LSUrRNA gene). In this study, two different 5′-partial LSU rDNAdata sets have been applied to observe the evolutionarymodels. Nature of the frequency distribution of possiblebarcode gaps and genetic distances can be correlated withthe taxon sample shape. For some protozoa in genusTetrahymena such as Tetrahymena hegewischi, Tetrahymenacanadensis, Tetrahymena rostrata, Tetrahymena pyriformis,Tetrahymena thermophila, and Tetrahymena tropicalis,COX1 marker has been used for the development of barcode(Kher et al. 2011). Using molecular tools, species distinctionis a priority for piroplasma. Therefore, 18S rRNA, 28S rRNA,ITS regions, and COI genes have been evaluated as DNAbarcode markers for Piroplasma. ITS2 is defined as the mostideal DNA barcode based on the current database forPiroplasma (Gou et al. 2012). Animal parasites have beenstudied extensively in this direction. Diplostomoidmetacercariae is a freshwater fish parasite distributed world-wide. To study their diversity, barcode marker COI was usedto discriminate species range of 1,088 diplostomoids, which

was isolated from different fish from the St. Lawrence River,Canada (Locke et al. 2010).

In 1993, Plasmodium falciparum, the medically importantparasite, gene was examined through PCR and tried to devel-op “barcodes” to identify parasite stocks and lineages (Arnotet al. 1993). This study reflected the phenomenon that scien-tists are putting their efforts to barcode development for thespeedy identification of medically important parasites.

Present challenges, promises, and limitations of microbialbarcode development

In the last few years, DNA barcoding is moving very fast andmoved from dream to reality. However, list of some of themajor unsolved problems are existing for barcode develop-ment. The “cryptic species” identification is very problematicbecause morphologically these species are very similar.Taxonomies based on morphological analysis of such speciescan be challenging (Hawksworth 2006; Kemler et al. 2006,2009; Paulus et al. 2007). Many studies have shown that theoccurrence is due to convergence evolution (Lorenz et al.2005). However, DNA barcoding can solve this problemand can identify “cryptic species” quickly with support ofthe advanced barcode marker. This work is under progressto identify unique barcode marker considering genetic dis-tance boundary conditions. It is possible to identify geneticdistance boundary conditions within two individuals betweenthe related species. Even though the boundary seems to betaxon related, it was noted that the value of genetic distancebetween two DNA barcode sequences equivalent to or morethan 3 % (D≥0.03) identifies distinct species.

However, using genetic distance approaches, the develop-ment of DNA barcodes have several limitations mainly indefining the species boundaries (Witt et al. 2006). One reasonis that the DNA which we are selecting as a barcode markerhas the rate of evolution that varies substantially between andwithin species and between different groups of species, con-sequently resulting in large overlaps of intraspecific and inter-specific distances (Kipling and Rubinoff 2004; Rubinoff2006; Rubinoff et al. 2006).

Table 2 Numbers of dif-ferent marker genes listedfrom GenBank, NCBI(11 November 2013)

Marker gene Number of sequences

16S rRNA 40,546

cpn60 6,258

rpoB 3,375

COI 2,967

rbcL 1,026

tufA 549

18S RNA SSU 100

28S RNA LSU 87

Appl Microbiol Biotechnol

The second challenge remains as a major challenge inidentifying the appropriate barcode marker development fordifferent microbial communities. Different kinds of DNAmarkers have been described for the microbial species identi-fication. It has been noted that the sequence of differentmarker gene from microbial species is growing which canbe noted from NCBI (Table 2). It is a challenge to select theappropriate sequence also. For example, many barcodemarkers have been described for fungi, such as nuclear largeribosomal subunit (LSU rDNA) (Kurtzman and Robnett1998), nuclear small ribosomal subunit (SSU rDNA)(Baayen et al. 2001), internal transcribed spacer (ITS)(Druzhinina et al. 2005), β-tubulin (BenA) (Geiser et al.2007) , elongation factor 1-α (EF1α) (O'Donnell et al.2008), and second largest subunit of RNA polymerase II(RPB2) (O'Donnell et al. 2008; Ertz et al. 2009). Severalinitiatives were taken in this direction to choose the appropri-ate marker (Santamaria et al. 2009). The software packagenamed DNA-BAR has been developed which can chooseDNA probes exploitable for molecular categorization of mi-croorganisms. This interactive package is produced byDasGupta et al. (2005) and written the script in degenbarwhich finds the near-minimum number of distinguishers andalso allow the users to choose the sets of these distinguishers.

The third challenge is that the morphological identificationof species is sometimes difficult, especially for the microbialcommunity. It has been noted that the morphological charac-teristics varied in most of the life history stages and gender togender. Therefore, it is very much difficult to distinguish thespecies from the mixture of biological species. The DNAbarcoding will make this task much easier. With the help ofthe barcode marker, microbial diseases sample can be identi-fied by the physicians or veterinarians without difficulty. Also,it will be easier to identify the assortment of microbial speciescollected from the environment (Besansky et al. 2003; Wongand Hanner 2008; Valentini et al. 2008). Pons et al. (2006)projected a collective population model with a Yule model ofspeciation (Yule 1924) that permits us to define “species” as acluster of specimens in a particular time frame.

The purpose and objective of the barcoding should provideelectropherograms along with the raw sequences as well assequence quality information (Consortium for the barcode oflife 2009). At present, the sequences are not following most ofthe aim and objectives which are in the public sequencedatabases.

Future challenges and concluding remarks

In summary, DNA barcoding will provide balanced taxonom-ic research, population genetics, phylogenetics, and finally,computational biology for hologram-based barcode develop-ment. It is still debatable how a scientist can identify, discover,

and illustrate the feature of the unidentified species with thehelp of the DNA barcode. It can be noted from the paper ofHebert et al. (2003a, b) that they promised us that “taxonomicexpertise is collapsing”. Conversely, Packer et al. (2009) gavean opinion that systematists should not “panic”. However, thestarting phase of the barcode development is very interestingon the basis of recent developments. It is to be noted that thebarcode databases are growing very fast to more than100,000specimens per year. Therefore, we have also found an opti-mism that synergy is present between barcode developers andpracticing systematists where in many publications their skillsets are coexistent.

International Barcode of Life describes that “DNA se-quence can be used to identify different species, in the sameway a supermarket scanner can use a familiar black strip of theUPC barcode to identify your purchases”. It means that wehave to develop a digital barcode hologram eventually. Withthe digital barcode hologram, we can use the barcode reader todetect the species. Very little work has been initiated so far(Merker et al. 2013). However, the development of digitalbarcode hologram for life is still in its infancy stage. The firststage of such work is to develop the barcode with digitalinformation, i.e., DNA-based digital information. DNA hasmany potential advantages for unchallengeable, high informa-tion storage which can carry digital information also. Churchet al. (2012) published a work related to next-generationdigital information storage in DNA that will help us to readthe barcode sequences. But, more works are needed to under-stand about barcode density, redundant encodings, paritychecks, and error correction to improve density, error rate,and safety for DNA-based barcoding. We are very hopeful tonotice condensed, digital barcode hologram and error-free andappropriate barcode for microbial communities in the nearfuture which will enable us to identify microbial species veryfast.

Acknowledgments The authors take this opportunity to thank themanagement of Galgotias University and VIT University for providingthe facilities and encouragement to carry out this work. The authors havedeclared that no conflict of interest exists.

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