Lignosus spp. are tropical macrofungi available in region of South China, Thailand, Malaysia, Indonesia, Philippines and Papua New Guinea (Lee et al, 2013). To date, genus Lignosus consists of six species, including L.dimiticus, L.ekombitii, L.goetzii, L. rhinocerotis, L. sacer and L. hainanensis (Tan et al.,2010). Other than this six species, another two new species include L. trigis and L.cameronensis (Yap et al, 2014). Sclerotium of Lignosus spp. is used as folk medicine for the treatment of chronic hepatitis, gastric ulcers and liver cancer in china (Lee et al., 2013). In Malaysia, the indigenous communities 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 mushroom since 2009. In 2013, sclerotium of Tiger Milk mushroom was successfully cultivated and at the present, Nubiotek has the capacity to produce 10,000 bags of Tiger Milk mushroom sawdust. The objective of this study is to identify and perform quality assurance (QA) and quality control (QC) of Nubiotek’s cultivated Tiger Milk Mushroom strain. Figure 1: PCR amplification of species specific primers (ITS1 and ITS 4) on DNA from Lignosus sp. strain NB08. Figure 2: Phylogenetic position of Lignosus sp. strain NB08 with respect to other species. Tree inferred by Neighbour-joining analyis on rDNA-ITS region. The numbers against branches indicate the percentage at which a given branch was supported in 100 bootstrap replications. Deoxyribonucleic acid (DNA) amplification using polymerase chain reaction (PCR) successfully yielded single gel band amplicon with 500bp size. (Fig.1). Sequencing of purified amplicon was analyzed and yielded NB08 strain is closely related with L. rhinoceros strain CH2 (99%) , L.rhinocerus strain CH31 (98%) , L.rhinocerus strain PEN94 (98%) , and L.rhinocerus strain TM02 (98%). Fig. 2 shows that NB08 is closely related other cultivated L.rhinocerus with 100% bootstrap confidence 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 100 200 300 600 400 500 700 500 a. DNA Extration b. PCR Amplification c. DNA sequencing and analysis d. Phylogenetic Analysis 1. Extract Genomic DNA from Lignosus spp. Strain NB08 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 MyTaq TM 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-spin TM 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. Reference 1. 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 Milk Mushroom. Journal of Ethnopharmacology. Retrieved from http://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 two new 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 100 72 98 68