Development of two novel specific SCAR markers by cloning improved RAPD fragments from the medicinal mushroom Ganoderma lucidium (Leysser: Fr) Karst

Abstract

ABSTRACT. Development of sequence-characterized amplified region (SCAR) markers from random-amplified polymorphic DNA (RAPD) fragments is a valuable molecular approach for the genetic identification of different species. By using SCAR markers, molecular analysis is reduced to a simple polymerase chain reaction (PCR) analysis using primers designed from the amplicon sequence of RAPD. In this study, the DNA fragments from an improved RAPD amplification of Ganoderma species were cloned into a pGM-T vector; positive clones were identified by PCR amplification and enzymatic digestion, and finally, DNA fragments were sequenced using the Sanger sequencing method for developing the SCAR markers. Two SCAR markers, named LZ4-1 with 534 nucleotides, and LZ5-2 with 337 nucleotides were identified, which are specific to Ganoderma lucidium (Leysser: Fr) Karst species. BLAST of these two nucleotide sequences in the GenBank database showed no identity to other species. We deposited these sequences into the GenBank database (LZ4-1 accession No. KM391933, LZ5-2 accession No. KM391934). PCR assays confirmed them as novel molecular markers for G. lucidium (Leysser: Fr) Karst, which might be used for genetic authentication of adulterant samples. Thus, our study developed two specific SCAR markers for identifying and distinguishing the medicinal mushroom G. lucidium (Leysser: Fr) Karst from other Ganoderma species.

Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers

“…In our lab, we have successfully characterized a number of plant or fungal species by developing SCAR markers based on RAPD amplicons; namely Dimocarpus longan , Acorus species (Ryuk et al 2014), Lonicera japonica (Yang et al 2014), Trichoderma cf. harzianum (Pérez et al 2014), Cordyceps sinensis (Lam et al 2015), Litchi chinensis , Angelica sinensis , and Ganoderma lucidum (Khan et al 2016).…”

“…5'-TAATACGACTCACTATAGGG-3', SP6 primer: 5'-ATTTAGGTGACACTATAGAA-3'), then run on a 1% agarose gel electrophoresis Khan et al 2016;Fu et al 2017).…”

“…One particularly useful approach, based on initial RAPD sequencing, is Sequence Characterized Amplified Region (SCAR) analysis, which has the advantage of generating markers that are usually very stable and which therefore provides a high level of authenticity in genetic identification Khan et al 2016;Fu et al 2017). By using the SCAR markers, molecular analysis has been simplified to a PCR reaction using PCR primers designed from the sequences of RAPD amplicons (Kumla, et al 2012;Fu et al 2015).…”

“…In recent developments of biotechnology, a number of molecular techniques have been used for the genetic identification and authentication of plant, animal and bacterial species, such as Random Amplified Polymorphic DNA (RAPD), Simple Sequence Repeat (SSR), InterSimple Sequence Repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) analyses (Williams et al 1990Khan et al 2016). One particularly useful approach, based on initial RAPD sequencing, is Sequence Characterized Amplified Region (SCAR) analysis, which has the advantage of generating markers that are usually very stable and which therefore provides a high level of authenticity in genetic identification Khan et al 2016;Fu et al 2017).…”

Genetic authentication of Eclipta prostrate (Asteraceae) from Penthorum chinense (Penthoraceae) by Sequence Characterized Amplified Region (SCAR) markers

“…For example, AFLP (amplified fragment length polymorphism), ISSR (inter-simple sequence repeat), RAPD (random amplified polymorphic DNA), SSR (simple sequence repeat), SCAR (sequence characterized amplified region), STR (short tandem repeat) etc. (Williams, Kubelik, Livak, Rafalski, & Tingey, 1990;Fu, Yang, Khan, & Mei, 2013;Khan, Cheng, Mei, Wei, & Fu, 2016;Mei, Zhang, Liu, Imani, & Fu, 2017c;Fu et al, 2018). A combination of RAPD and SCAR marker analysis or SCAR marker alone provides a high level of authenticity of genetic identification (Fu et al, 2015;Fu et al, 2017).…”