Establishing and Optimizing AFLP Amplification Reaction System of Shiraia Bambusicola

DNA of Sharaia bambuiscola was extracted by the improved method of CTAB. The several key factors affecting the effect of DNA digestion and the PCR selective amplification such as the time of DNA digestion, the times of the per-amplified dilute products, the mount of the selective amplification primer, Taq concentration and dNTPs concentration were optimized and trialed with establishment of an optimized AFLP reaction system of S. bambusicola. The best time of digestion DNA with double endonucleases (EcoRIand MseI) was 3h. The optimized selection amplification system was 25μl reaction mix containing 2. 5μl the 20 times of the per-amplified dilute products, 2. 5μL 10× buffer(with Mg 2+ ), 10mmol/L primer each 1. 5μL, 2.5U·μL -1 Taq polymerase 0. 7μL, 10mmol/L dNTP 0. 5μL, and 15. 8μL ddH2O. Stable and clean DNA finger print can be obtained and 3 pairs of AFLP primers with good genetic diversity were selected according to the optimized reaction system. The results will be an effective protocol for further studying the genetic stucture and differentiation, phylogenetic trees, host specificity and artificial cultivation of Shiraia bambusicola population. Introduction Shiraia bambuiscola P. Hennings has been reported as a parasitic medicinal fungus on branches of bamboo. It is widely applied in various fields such as biological technology and medicine. But the phylogeny is unclear. Recently, the research results show that a new family Shiraiaceae (Pleosporale, Dothideomycetes, Ascomycota) was established with S. bambusicola as representative gene and species[1]. Stromata of S. bambusicola has a long history as a traditional Chinese medicine, and are the raw material for extracting of hypocrellin[2]. Hypocrellin is promising in the photodynamic therapy (PDT) for anticancer treatment[3,4]. Furthermore, it has a striking function to kill tumor cells, inhibit viruses, cure diabetic retinopathy and against human immunodeficiency virus type 1(HIV-1)[5-8]. S. bambusicola is distributed in southern China and Japan[9]. Sri Lanka is hopefully one of distribution regions[10]. But the biomass of the fungus is very limited. Driven by market demand, the wild resource of S. bambusicola has been overexploited and become scarce[11]. The fungus and its genetic diversity has not been sufficiently studied. S. bambusicola is discontinuously distributed in China and Japan[9], which may results in rich genetic diversity within the species but from different geological locations. RAPD (Random Amplified Polymorphic DNA) was used to analyze genetic differentiation of S. bambusicola populations from east China[12]. However, little has been done in the genetic diversity. International Conference on Material Science and Application (ICMSA 2015) © 2015. The authors Published by Atlantis Press 173 Materials and Methods Materials 35 test materials of Shiraia bambusicola were collected from Anhui, Zhejiang, Guizhou provinces; primers and adapter (JieLi); 10U/μL of EcoRI, 10U/μL of MseI(Thermo), 5 U/μL of T4 DNA ligase, 2.5U/μL of Taq DNA polymerase (buffer with Mg 2+ ), 10mmol/L each of dNTPs (Zomanbio) were applied.