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DNA Barcode Markers for Two New Species of Tiger Milk Mushroom: Lignosus tigris and L. cameronensis.

Byline: Hui-Yeng Y. Yap Shin-Yee Fung Nget-Hong Tan Szu-Ting Ng and Chon-Seng Tan

Abstract

Two new species of the tiger milk mushroom namely Lignosus tigris and L. cameronensis were reported recently based on studies on morphological characteristics. Both Lignosus species are known to be medicinal fungi and have potential to be used as functional food. Since intact fungal samples of the two Lignosus species are not always available for morphology-based identification a DNA barcode marker approach based on the internal transcribed spacer (ITS) region via PCR technology has been developed. The DNA markers are highly specific and easily amplified thus allowing rapid identification and reliable authentication of these two Lignosus species. Identification and authentication of the fungi are important in view of the potential medicinal and functional food applications of the two fungi. Copyright 2014 Friends Science Publishers.

Keywords: Internal transcribed spacer (ITS); Molecular markers; Lignosus; Tiger milk mushroom.

Introduction

Lignosus Lloyd ex Torrend a genus in the family Polyporaceae; has been regarded as one of the most valued medicinal fungal taxa in Southeast Asia. Of late two new species namely L. tigris and L. cameronensis have been discovered from the tropical forest in Lata Iskandar Pahang; the central region of Peninsular Malaysia. The pore and basidiospore sizes are the foremost morphological criteria for the identification of these two Lignosus species and differentiation from the other generic members (Tan et al. 2013). However specimens obtained from the field collection are usually only the sclerotia without their cap and/or stipe intact thus making it impossible to identify whether the sclerotia were from either of the two Lignosus species. Hence there is a need to develop additional tool to enable rapid identification of the Lignosus species as well as differentiation between the two Lignosus species.

The ITS region of ribosomal DNA (rDNA) cistron has been primarily sequenced in fungi for the use of diversity documentation intra- and interspecies level classification phylogenetic analyses and environmental sampling; because of their high degree of variation than the 5S 5.8S 18S small subunits and 25S28S large subunit in rDNA (O'Brien et al. 2005; Schoch et al. 2012). In this study we report the development of a set of suitable DNA barcode markers based on the ITS region of the fungi for the identification of L. tigris and L. cameronensis.

Materials and Methods

Materials

Specimens of L. tigris and L. cameronensis were collected from tropical forest in Lata Iskandar Pahang Malaysia (4.3245N 101.3324E). L. rhinocerotis strain TM02 culture was from Ligno Biotech Sdn. Bhd. Selangor Malaysia while pure Pleurotus tuber-regium culture was purchased from Fungi Perfecti Olympia Washington USA.

DNA Extraction and Sequencing

Genomic DNA was extracted from 50 to 100 mg sclerotial internal tissues using 300 L extraction buffer containing 2% SDS 1.4 M NaCl 20 mM EDTA and 100 mM Tris- HCl (pH 8.0) with 5 L of 10 mg/mL RNAse A. The mixture was ground into fine particles and incubated at 65C for 30 min. Equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added to the mixture followed by high speed centrifugation at 13000 rpm for 5 min prior to DNA precipitation in the aqueous phase by one volume of isopropanol. Upon removal of the supernatant the DNA pellet was washed with ethanol twice air dried and suspended in 50 L TE buffer.

The fungi ITS regions (ITS-1 5.8S rRNA and ITS-2) was PCR amplified using primers ITS1 (5'- TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'- TCCTCCGCTTATTGATATGC-3') (White et al. 1990).

PCR reaction mixture of 25 mM Tris-HCl (pH 8.8) 50 mM KCl 4.0 mM MgCl2 0.2 mM dNTPs 25 pmolprimers 100 ng DNA template and 1U of Taq DNA polymerase in a total volume of 25 L was carried out as follow: 4 min initial denaturing at 94C followed by 25 cycles of amplification (94C for 45 sec 55C for 45 sec 72C for 1 min) and final extension for 5 min at 72C.

The PCR product was electrophoresed using 1% agarose gel and the amplicon band of interest was further purified using glass-milk matrix (Fermentas Maryland USA) according to manufacturer's manual. Purified PCR product was then ligated into pGEM(R)-T Easy Vector (Promega Wisconsin USA) according to manufacturer's manual followed by transformation into competent Escherichia coli JM109 strain. Purified plasmids from positive clones were subjected to commercial DNA sequencing service using M13 Forward and M13 Reverse as sequencing primers.

Data Analysis

Consequential forward and reverse sequences were compared against the ITS sequences of L .rhinocerotis

isolates CH2 (GenBank accession no. FJ380871) and TM02 (GenBank accession no. JQ409479) L. sacer isolates (GenBank accession no. GU001675 GU001674) and L. hainanensis (GenBank accession no. GU580883) via CLUSTALW multiple sequence alignment in MEGA 4.1 (Tamura et al. 2007) to design the specific primer pair; which is also the PCR-based genetic marker for L. tigris and L. cameronensis molecular identification.

Specificity Test

Specificity of the designated primer pair was evaluated using BLAST search (http://www.ncbi.nlm.nih.gov/tools/ primer-blast/) against nr database for primer string test targeting on short and nearly exact matches. DNA extracted from L. rhinocerotis strain TM02 and P. tuber-regium was used for PCR comparison analysis to validate the primers' specificity strength. PCR reaction mixture of 25 mM Tris- HCl (pH 8.8) 5 0 mM KCl 4.0 mM MgCl2 0.2 mM dNTPs 25 pmol primers 100 ng DNA template and 1 U of Taq DNA polymerase in a total volume of 25 L was carried out as follow: 5 min initial denaturing at 94C followed by 25 cycles of amplification (94C for 45 sec 50C for 45 sec 72C for 1 min) and final extension for 5 min at 72C. PCR products were electrophoresed using 1% agarose gel at 95 V.

ResultsThe PCR-amplified ITS regional (ITS-1 5.8S rRNA andITS-2) sequences shared similar nucleotide identity to itsrespective strain as reported earlier; L. tigris strain K and T and L. cameronensis strain T1 and T8 with GenBank accession numbers JQ409481 JQ409482 JQ409483 and JQ409484 respectively (Tan et al. 2013). Sequences from each strain of L. tigris shared 100% identity while strains of L. cameronensis shared 99% identity with nine bases substitution.Unique variable nucleotide sequences of L. tigris and L. cameronensis were determined by aligning to the ITS sequences of L. hainanensis L. rhinocerotis and L. sacer (Fig. 1). From the multiple sequence alignment we managed to identify two highly variable regions in L. cameronensis located at ITS-1 (5'- TTgreater than GGCCCTTgreater than CCCTTgreater than CTGGCGG-3') and ITS-2 (5'- GAGGCGCAGCCCTTCACTT-3') while there is only a single region in L. tigris at ITS-1 (5'- GTTATGGGCAGCGTCGCCT-3') with significant degree of variation. The sequence-variable regions were then targeted for specific primers design. Designations and sequences of the primers are shown in Table 1.Amplification of the corresponding ITS region with FwLT-ITS4 and FwLC-RvLC primer-pair combinations yielded PCR product of 526 and 572 bp in size for L. tigris and L. cameronensis isolates respectively (Fig. 2). The primer pairs' specificity was supported with the absence of significant homology in the submitted primer string and their specificity strength was further validated by amplification of other closely related species; L. rhinocerotis strain TM02 and P. tuber-regium which showed negative amplification (Fig. 2) signifying high degree of specificity and reliability in identifying the respective Lignosus species based on PCR approaches.

Discussion

Development of rapid and precise molecular-based marker for species identification is imperative to complement the primitive yet sometimes perplexing morphology based taxonomy taxonomy (Alexopoulos et al. 1996; O'Brien et al. 2005). To make matters worse specimens obtained from the field collection are often without their cap and/or stipe intact and left with only the sclerotia (the medicinally useful part) thus making it impossible to make identification based on the pore and basidiospore sizes.

In this study two highly variable regions in L. cameronensis located at ITS-1 and ITS-2 while only a single region in L. tigris at ITS-1 with significant degree of

Table 1: Specific primer sequence designated as molecular marker for L. tigris and L. cameronensis

Species###Primer Primer sequence###Length GC %

###(bp)

L. tigris###FwLT 5'-GTTATGGGCAGCGTCGCCT- 19###63.16

###3'

L.###FwLC 5'-TGGCCCTCCCTCTGGCGG-3' 18###77.78

cameronensis RvLC 5'-AAGTGAAGGGCTGCGCCTC-3' 19###63.16

variation were identified suggesting L. tigris is more evolutionarily conserved than L. cameronensis (Tan et al.2013). Although three bases substitution were detected in the hypervariable ITS-1 region of L. cameronensis strains; it does not affect the primer specificity as confirmed by the specificity test where non-specific amplification of the designated primers to other species such as L.rhinocerotis strain TM02 and P. tuber-regium pure culturesis negative.In conclusion the easily amplified short standard genetic markers targeting the ITS region are evidently a superior tool for identification of the two Lignosus species than the taxonomy method based morphological characteristics. As such it can be used for rapid identification and reliable authentication of the two Lignosus species.

Acknowledgments

This research was supported by UMRG No. RG075/12BIO and PV024/2012A from University of Malaya Malaysia.

References

Alexopoulos C.J. C.W. Mims and M.M. Blackwell 1996. Introductory Mycology 4th edition. John Wiley and Sons Inc. New York USACorpet F. 1988. Multiple sequence alignment with hierarchical clustering.Nucl. Acids Res. 16: 1088110890O'Brien H.E. J.L. Parrent J.A. Jackson J.M. Moncalvo and R.Vilgalys 2005. Fungal community analysis by large-scale sequencing of environmental samples. Appl. Environ. Microbiol.71: 55445550Schoch C.L. K.A. Seifert S. Huhndorf V. Robert J.L. Spouge C.A.Levesque W. Chen and Fungal Barcoding Consortium 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc. Nat. Acad. Sci. USA 109: 62416246Tamura K. J. Dudley M. Nei and S. Kumar 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 15961599Tan C.S. S.T. Ng and J. Tan 2013. Two new species of Lignosus (Polyporaceae) from Malaysia - L. tigris and L. cameronensis. Mycotaxon 123: 193204White T.J. T.D. Bruns S.B. Lee and J.W. Taylor 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications pp: 315322. M.A. Innis D.H. Gelfand J.J. Sninsky and T.J. White (eds). Academic Press New York USA
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Publication:International Journal of Agriculture and Biology
Article Type:Report
Date:Aug 31, 2014
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