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

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.

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

Sequence-characterized amplified region (SCAR) is a valuable molecular marker for the genetic identification of any species. This marker is mainly derived from molecular cloning of random amplified polymorphic DNA (RAPD). We have previously reported the use of an improved RAPD technique for the genetic characterization of different samples of Canarium album (Lour.) Raeusch (C. album). In this study, DNA fragments were amplified using improved RAPD amplified from different samples of C. album. The amplified DNA fragment was excised, purified from an agarose gel and cloned into a pGM-T vector; subsequently, a positive clone, called QG12-5 was identified by PCR amplification and enzymatic digestion and sequenced by Sanger di-deoxy sequencing method. This clone was revealed consisting of 510 nucleotides of C. album. The SCAR marker QG12-5 was developed using specifically designed PCR primers and optimized PCR conditions. This SCAR marker expressed seven continuous "TATG" [(TATG)n] tandem repeats, which was found to characterize C. album. Subsequently, this novel SCAR marker was deposited in GenBank with accession No. KT359568. Therefore, we successfully developed a C. album-specific SCAR marker for the identification and authentication of different C. album species in this study.

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

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, and KM391938, respectively. Thus, in this study we developed specific SCAR markers for the identification and authentication of different Ganoderma species.

Genetic characterization and authentication of Dimocarpus longan Lour. using an improved RAPD technique.

Dimocarpus longan Lour. is an edible and traditional herb in China, commonly referred to as longon. An improved randomly amplified polymorphic DNA (RAPD) protocol was here developed in order to determine the geographical origins of D. longan samples collected from 5 provinces in the southern and southwestern areas of China, including Sichuan, Hainan, Fujian, Guangdong, and Guangxi. Generally, the improved RAPD method generated good fingerprinting of the 5 samples using the selected 17 primers. In particular, primers SBS-A5, SBS-A13, SBS-I9, SBS-I20, SBS-M1, and SBS-Q12 produced distinguishable bands that clearly separated all 5 cultivars, suggesting that there are variations in RAPD genetic sites among the samples. The similarity index ranged from 0.69 to 0.76. The Sichuan and Hainan clades clustered together with a 0.73 similarity index. The Guangxi and Fujian clades clustered together with a 0.76 similarity index, and they formed the sister clade to the Sichuan/Hainan clade with a 0.71 similarity index. The Guangdong clade was in a basal polytomy with a 0.70 similarity index. Based on the abundant DNA polymorphisms, these longan accessions are distinguishable using our improved RAPD technique. Therefore, RAPD analysis is an effective technique in distinguishing the geographical origins of D. longan. Moreover, the improved method could also be employed for a variety of applications including genetic diversity and fingerprinting analyses.