Lignosus spp. are tropical macrofungi available in regionof South China, Thailand, Malaysia, Indonesia, Philippines andPapua New Guinea (Lee et al, 2013). To date, genus Lignosusconsists of six species, including L.dimiticus, L.ekombitii,L.goetzii, L. rhinocerotis, L. sacer and L. hainanensis (Tan etal.,2010). Other than this six species, another two newspecies include L. trigis and L.cameronensis (Yap et al, 2014).

Sclerotium of Lignosus spp. is used as folk medicine forthe treatment of chronic hepatitis, gastric ulcers and livercancer in china (Lee et al., 2013). In Malaysia, the indigenouscommunities utilized this Tiger Milk Mushroom (Lignosus spp.)as traditional and complementary medicine to treat cough,asthma, fever, cancer and food poisoning (Lee et al, 2013).

Realizing the economic potential of Tiger Milk Mushroom,Nubiotek Sdn. Bhd. had initiated Research and Development(R&D) activities to cultivate and domesticate this mushroomsince 2009. In 2013, sclerotium of Tiger Milk mushroom wassuccessfully cultivated and at the present, Nubiotek has thecapacity to produce 10,000 bags of Tiger Milk mushroomsawdust.

The objective of this study is to identify and performquality assurance (QA) and quality control (QC) of Nubiotek’scultivated Tiger Milk Mushroom strain.

Figure 1: PCRamplificationof species specific primers (ITS1 and ITS 4) on DNA from Lignosus sp.strain NB08.

Figure 2: Phylogenetic position of Lignosus sp. strain NB08 withrespect to other species. Tree inferred by Neighbour-joining analyison rDNA-ITS region. The numbers against branches indicate thepercentage at which a given branch was supported in 100 bootstrapreplications.

Deoxyribonucleic acid (DNA) amplificationusing polymerase chain reaction (PCR)successfully yielded single gel band ampliconwith 500bp size. (Fig.1).Sequencing of purified amplicon was analyzedand yielded NB08 strain is closely related withL. rhinoceros strain CH2 (99%) , L.rhinocerusstrain CH31 (98%) , L.rhinocerus strain PEN94(98%) , and L.rhinocerus strain TM02 (98%).Fig. 2 shows that NB08 is closely related othercultivated L.rhinocerus with 100% bootstrapconfidence support.

MOLECULAR IDENTIFICATION OF CULTIVATED/DOMESTICATED SCLEROTIUM OF TIGER MILK MUSHROOM (Lignosus rhinocerus) STRAIN NB08

Introduction

Methodology

Result & Discussion

Present findings suggest NB08 strain is L.rhinocerotis (Cooke) Ryvarden. In addition, PCR is a rapid and accurate identification method suitable for quality assurance (QA)/ quality control (QC) activities for commercial fungi cultivation.

Conclusion

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a. DNA Extration

b. PCR Amplification

c. DNA sequencing and analysis

d. Phylogenetic Analysis

1. Extract Genomic DNA from Lignosus spp. StrainNB08 tuber sample.

2. Measure the purity and concentration of DNA .3. Store at -20℃

1. Perform using primers ITS 1 and ITS 4.2. Conduct the PCR in a reaction volume of 25L

containing 12.5 μL of MyTaqTM Red Mix–Premix, 200 ng of template DNA and 5 pmol of both forward and reverse primers..

3. Analyze by 2% agarose gel electrophoresis.4. Compare with 100bp molecular weight markers.

1. Purify using MEGA quick-spinTM Total Fragment DNA Purification kit.

2. Send purified amplicon for sequencing.

1. Analyze the phylogenetic relationship using Maximum Likelihood and Neighbor-Joining criteria.

2. Construct the phylogram using Mega 6.06.3. Compute the pairwise difference using Mega 6.06.

Reference1. Tan C. S., Ng S.T., Vikineswary S., Lo F.P. & Tee C.S., 2010. Genetic Markers for

Identification of a Malaysian Medicinal Mushroom, Lignosus Rhinocerus . 161-162.2. Lee, S. S., Enchang, F. K., Tan, N. G., Fung, S. T., & Pailoor, J.(2013) . Preclinical

toxicological evaluations of the sclerotium of Lignosus rhinocerus(Cooke), the Tiger MilkMushroom. Journal of Ethnopharmacology. Retrieved fromhttp://www.ligno.com.my/wp-content/uploads/2012/01/Preclinical-toxicological-evaluations-of-the-sclerotium-of-Lignosus-rhinocerus.pdf

3. Yap, Y.H., Fung S.Y. , Tan N.H., Ng S.T & Tan C.S., 2014. DNA barcode markers for twonew species of tiger milk mushrooms: Lignosus tigris and L. cameronensis. Int. J. Agric.Biol., 16: 841‒844

gi|210063384|gb|FJ380871.1| Lignosus rhinocerus CH2 18S ribosomal RNA gene partial sequence internal transcribed spacer 1 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequence ae

gi|228546989|gb|FJ899143.1| Lignosus rhinocerus strain CH31 18S ribosomal RNA gene partial sequence internal transcribed spacer 1 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete see

gi|384236033|gb|JQ409359.1| Lignosus rhinocerotis strain PEN94 internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequence and 28S ribose

gi|381218023|gb|JQ409479.1| Lignosus rhinocerotis strain TM02 internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene complete sequence and internal transcribed spacer 2 partial sequene

Lignosus sp. strain DFB08

gi|300115749|gb|GU580884.1| Lignosus sp. LPT-2010 tissue-type sclerotium 18S ribosomal RNA gene partial sequence internal transcribed spacer 1 5.8S ribosomal RNA gene and internal transcribed space...

gi|300115748|gb|GU580883.1| Lignosus sp. LPT-2010 tissue-type fruit body 18S ribosomal RNA gene partial sequence internal transcribed spacer 1 5.8S ribosomal RNA gene and internal transcribed space...

gi|381218028|gb|JQ409484.1| Lignosus sp. CST-2012a strain T8 internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene complete sequence and internal transcribed spacer 2 partial sequence

gi|381218027|gb|JQ409483.1| Lignosus sp. CST-2012a strain T1 internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene complete sequence and internal transcribed spacer 2 partial sequence

gi|261286809|gb|GU001675.1| Lignosus sacer isolate 15572 18S ribosomal RNA gene partial sequence internal transcribed spacer 1 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequee

gi|261286808|gb|GU001674.1| Lignosus sacer isolate 11329 18S ribosomal RNA gene partial sequence internal transcribed spacer 1 5.8S ribosomal RNA gene and internal transcribed spacer 2 complete sequee

gi|381218025|gb|JQ409481.1| Lignosus sp. CST-2012b strain K internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene complete sequence and internal transcribed spacer 2 partial sequen...

gi|381218024|gb|JQ409480.1| Lignosus sp. CST-2012b strain A internal transcribed spacer 1 partial sequence 5.8S ribosomal RNA gene complete sequence and internal transcribed spacer 2 partial sequence100

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