Development of RAPD-SCAR markers for different Ganoderma species authentication by improved RAPD amplification and molecular cloning

Abstract

ABSTRACT. The sequence-characterized amplified region (SCAR) is a valuable molecular technique for the genetic identification of any species. This method is mainly derived from the molecular cloning of the amplified DNA fragments achieved from the random amplified polymorphic DNA (RAPD). In this study, we collected DNA from 10 species of Ganoderma mushroom and amplified the DNA using an improved RAPD technique. The amplified fragments were then cloned into a T-vector, and positive clones were screened, indentified, and sequenced for the development of SCAR markers. After designing PCR primers and optimizing PCR conditions, 4 SCAR markers, named LZ1-4, LZ2-2, LZ8-2, and LZ9-15, were developed, which were specific to Ganoderma gibbosum (LZ1-4 and LZ8-2), Ganoderma sinense (LZ2-2 and LZ8-2), Ganoderma tropicum (LZ8-2), and Ganoderma lucidum HG (LZ9-15). These 4 novel SCAR markers were deposited into GenBank with the accession Nos. KM391935, KM391936, KM391937, 2 J.J. Fu et al. ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (2): 5667-5676 (2015) and KM391938, respectively. Thus, in this study we developed specific SCAR markers for the identification and authentication of different Ganoderma species.

Development and significance of SCAR marker QG12-5 for Canarium album (Lour.) Raeusch by molecular cloning from improved RAPD amplification

“…DNA was extracted from different samples of C. album (Mei et al, 2014) and other medicinal plant samples using a standard protocol (Fu, 2012;Fu et al, 2000Fu et al, , 2013Fu et al, , 2015Khan et al, 2015). DNA samples were then diluted to a final concentration of 10 ng/mL and stored at -20°C until further use, as described in a previous study (Fu et al, 2015).…”

“…DNA samples were then diluted to a final concentration of 10 ng/mL and stored at -20°C until further use, as described in a previous study (Fu et al, 2015).…”

“…The protocol used for amplification and recovery of improved RAPD fragments was as described previously (Fu et al, 2015). Briefly, the 15-mL PCR mixture, comprising 7.5 mL 2X Taq PCR MasterMix (Tiangen Biotech, Beijing, China), 1.5 mL 2.5 mM primer pairs, 1.5 mL genomic DNA, and ddH 2 O, was amplified on a Veriti ® 96-Well thermal cycler (Applied Biosystems, Foster City, CA, USA), using the following reaction protocol: initial denaturation at 95°C for 90 s; 40 cycles of denaturation at 94°C for 40 s, annealing at 36°C for 60 s, and extension at 72°C for 90 s, with the annealing to extension temperature increasing at a rate of 0.125°C/s (5% ramp rate); and a final extension at 72°C for 5 min.…”

“…The sequence characterized amplified region (SCAR) marker is another molecular marker that is more stable and is generally derived from RAPD, ISSR, or other markers (Dnyaneshwar et al, 2006;Su et al, 2007;Kumla et al, 2012;Fu et al, 2013;Rajesh et al, 2013;Mei et al, 2015;Zhang et al, 2015). Combining SCAR with RAPD simplifies the molecular analysis procedure, as the PCR primers used to develop SCAR markers are designed from the sequence of the RAPD amplicon (Kumla et al, 2012;Rajesh et al, 2013;Dutta et al, 2014;Fu et al, 2015).…”

“…Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), simple sequence repeat (SSR), and amplified fragment length polymorphism or restriction fragment length polymorphism analyses are the major molecular marker technologies that are currently being used for the genetic characterization and identification of an organism (Williams et al, 1990;Agarwal et al, 2008;Noormohammadi et al, 2013;Mei et al, 2014;Fu et al, 2000Fu et al, , 2013Fu et al, , 2015Long et al, 2015;Zhan et al, 2015). The sequence characterized amplified region (SCAR) marker is another molecular marker that is more stable and is generally derived from RAPD, ISSR, or other markers (Dnyaneshwar et al, 2006;Su et al, 2007;Kumla et al, 2012;Fu et al, 2013;Rajesh et al, 2013;Mei et al, 2015;Zhang et al, 2015).…”