Authentication of Bulbus Fritillariae Cirrhosae by RAPD-Derived DNA Markers

Bulbus Fritillariae is the most commonly used antitussive herb in China. Eleven species of Fritillaria are recorded as Bulbus Fritillariae in the Chinese Pharmacopoeia. Bulbus Fritillariae Cirrhosae is a group of six Fritillaria species with higher efficiency and lower toxicity derived mainly from wild sources. Because of their higher market price, five other Fritillaria species are often sold deceptively as Bulbus Fritillariae Cirrhosae in the herbal market. To ensure the efficacy and safety of medicinal herbs, the authentication of botanical resources is the first step in quality control. Here, a DNA based identification method was developed to authenticate the commercial sources of Bulbus Fritillariae Cirrhosae. A putative DNA marker (0.65 kb) specific for Bulbus Fritillariae Cirrhosae was identified using the Random Amplified Polymorphic DNA (RAPD) technique. A DNA marker representing a Sequence Characterized Amplified Region (SCAR) was developed from a RAPD amplicon. The SCAR marker was successfully applied to differentiate Bulbus Fritillariae Cirrhosae from different species of Fritillaria. Additionally, the SCAR marker was also useful in identifying the commercial samples of Bulbus Fritillariae Cirrhosae. Our results indicated that the RAPD-SCAR method was rapid, accurate and applicable in identifying Bulbus Fritillariae Cirrhosae at the DNA level.

According to the Chinese Pharmacopoeia, Bulbus Fritillariae Cirrhosae is the dried bulb of  [3]. Bulbus Fritillariae Cirrhosae is rare and expensive, because most of these Fritillaria species are only found wild and distributed at an altitude of 3000-5000 m. Studies have suggested that Bulbus Fritillariae Cirrhosae has better therapeutic effects and lower toxicity, as compared to other species of Bulbus Fritillariae [4,5]. Consequently, some producers deceptively sell other species of Fritillaria as Bulbus Fritillariae Cirrhosae in the market. Thus, the authentication of species is of primary importance for ensuring efficacy and safety of the herb, and therefore, the development of method to effectively authenticate Bulbus Fritillariae Cirrhosae would be of great value.
Authentication based on morphological, microscopic or phytochemical characteristics is not reliable enough [6][7][8] because the plant can vary due to different practices of planting, harvesting, storage and manufacturing. The use of DNA identification methods is far more reliable; because DNA sequences do not vary easily. DNA sequencing methods already have been used to identify bulb of Fritillaria species [9,10]. Based on sequence analysis of the 5S-rRNA spacer region from different species of Fritillaria, a unique sequence was found in F. cirrhosa. However, this method cannot be used to identify bulbs of other species in the Bulbus Fritillariae Cirrhosae. Li et al. developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the simultaneous identification of Bulbus Fritillariae Cirrhosae [11,12]. However, the PCR-RFLP was a two-step reaction method and required high quality DNA samples [10]. Unfortunately, it is always difficult to obtain high quality DNA from raw Chinese herbs. In this study, a SCAR marker was designed from RAPD polymorphisms for rapid and easy identification of Bulbus Fritillariae Cirrhosae. This DNA marker was further used to authenticate the commercial samples of Bulbus Fritillariae Cirrhosae. The reliability of our results was validated using the PCR-RFLP method. Overall, the RAPD-derived SCAR method proved to be a quick, simple, accurate and effective mean in identifying Bulbus Fritillariae Cirrhosae at DNA level.

Identification of RAPD Marker for Bulbus Fritillariae Cirrhosae
High molecular weight genomic DNA was isolated from dry Fritillaria bulbs, which yielded 50-300 ng of DNA per 50 mg of bulbs. An absorbance (A260/A280) ratio of 1.6-1.8 suggested an insignificant level of contaminating proteins and/or polysaccharides. Seven of the twenty-four RAPD primers, for which variability and applicability were tested in preliminary experiments, were chosen for further analysis. With these primers all specimens showed highly repeatable patterns with the finally chosen protocol and reaction conditions (data not shown). However, only the primer S158 consistently amplified a single, intense band of ~0.65 kb for the six species of Bulbus Fritillariae Cirrhosae, which was absent in other six Fritillaria species (Figure 1). This band, named as CBM08, was putatively designated as Bulbus Fritillariae Cirrhosae specific marker. Bulbs of different Fritillaria species were listed in Table 1.  (12), F. ussuriensis (13), F. thunbergii (14), F. pallidiflora (15), F. walujewii (16), and F. puqiensis (17). The ~0.65 kb CBM08 band was isolated and sequenced. The first ten nucleotides of the sequence matched with the corresponding RAPD primer used. This result clearly showed that the cloned fragment was derived from the amplified RAPD product. The length of the CBM08 marker sequence obtained was 657 bp with 47% G + C content (A: 161; C: 129; G: 182; T: 186) ( Figure 2). BLAST results revealed that the sequence showed partial homology with known plant nucleotide sequences at different similarity levels. Considerable similarity was found with the sequence of Fritillaria imperialis (Scor: 45.4 bits, and E-value: 5E-18).

Figure 2.
Nucleotide sequence of the RAPD amplicon CBM08; the RAPD primers (a pair of S158) and SCAR primers (CBM-S and CBM-AS) were indicated.

Amplification using SCAR Primers
The designed SCAR primer pair (Table 2), CBM-S and CBM-AS, was used to amplify genomic DNA from the twelve Fritillaria species (Figure 2 and Figure 3A). A single and distinct band of 231 bp was obtained in DNA isolated from all the six Fritillaria species of Bulbus Fritillariae Cirrhosae, while no specific amplification was observed for any of the other six Fritillaria species ( Figure 3B). The SCAR primers were further used to amplify DNA from nine commercial samples of Fritillaria bulbs. A sharp and reproducible band (231 bp) was observed for five samples ( Figure 4A), each purchased from different drug shops in Hong Kong, which verified these samples were authentic Bulbus Fritillariae Cirrhosae.   In contrast, no specific band was observed for the other four Fritillaria bulbs, which indicated that these samples were not Bulbus Fritillariae Cirrhosae ( Figure 4A).

Validation of Results
In order to validate the reliability of our RAPD-SCAR method, the authentication of commercial samples was validated using the previously reported PCR-RFLP method. Two fragments (~190 and ~118 bp) were obtained following the digestion of the PCR products from five samples (i.e., samples 1-5) digested by SmaI ( Figure 4B), which indicated that these samples were authentic Bulbus Fritillariae Cirrhosae. The fragments from other four commercial samples (samples 6-9) could not be digested by SmaI ( Figure 4B), which indicated that they were not Bulbus Fritillariae Cirrhosae. The corroborative results of these two methods were totally coincidental. The basic features of each method are compared in Table 3.

Discussion
This work aimed to improve the authentication process of Bulbus Fritillariae Cirrhosae by using a RAPD-derived molecular method. RAPD analysis can reveal a high degree of polymorphism, and which does not require prior DNA sequence information of the species. More important, this method is simple and straightforward, which makes it suitable for application in the Traditional Chinese Medicine industries. Many researchers have explored the use of RAPD markers for authentication of traditional Chinese medicines like Panax [13], Cordyceps sinensis [14] and Atractylodes [15]. In our RAPD analysis, significant genetic polymorphism was observed among the Fritillaria species surveyed. As recorded in the 2010 Chinese Pharmacopoeia [3], there are six Fritillaria species used as source material of Bulbus Fritillariae Cirrhosae. In our study, the random primer S158 yielded a ~0.65 kb amplicon specific for Bulbus Fritillariae Cirrhosae. We selected the monomorphic band (CBM08) for SCAR marker development. In SCAR, pairs of oligonucleotide primers specific to the sequence of polymorphic bands can be used to amplify the characterized regions from genomic DNA. Based on the sequence of CBM08, SCAR primers (CBM-S, CBM-AS) were designed to amplify a small region of 231 bp.
Utility of CBM-S and CBM-AS was tested for the identification of Bulbus Fritillariae Cirrhosae from the commercial samples of dried bulbs. The SCAR primers amplified an expected 231 bp DNA fragment in five of tested samples, but not in other four samples. The reliability of the above method was demonstrated by the previously developed PCR-RFLP method. Consistent results demonstrated that the novel SCAR marker was able to distinguish Bulbus Fritillariae Cirrhosae from other Fritillaria species.
The SCAR marker has advantages for large scale analyses because of low cost, high reproducibility, and ease of use without elaborate electrophoresis. We expect that our SCAR marker will complement other molecular methods used to identify Bulbus Fritillariae Cirrhosae.

Materials
Bulbs of different Fritillaria species were collected in China. Plant specimens were identified by the authors and voucher specimens were deposited in the Center for Chinese Medicine of Hong Kong University of Science and Technology. In addition, nine commercial samples of Fritillaria bulbs (1)(2)(3)(4)(5)(6)(7)(8)(9) were collected in Hong Kong herbal markets.

DNA Extraction
For DNA extraction, the dried Bulbs were ground into a fine powder. The extraction was performed with a commercial kit (DNeasy ® Plant Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer's protocol. The DNA quantification was done with a NanoDrop ® ND-1000 spectrophotometer (DiaMed China Limited, Hong Kong, China).

RAPD Analysis
PCR for amplification conditions were optimized to increase the reproducibility of the banding pattern. With the optimized reaction conditions and PCR protocol, the patterns were highly reproducible in multiple experiments. The RAPD was carried out with 5 μL of genomic DNA (~50 ng) in a 50 μL reaction containing 10× PCR buffer (with Mg 2+ at a 1X conc. of 1.5 mM and contains loading dye), 0.5 mM dNTP, 400 nM primer and 1.5 U of Taq Polymerase (KAPA Taq DNA Polymerase with dye, KAPA Biosystems, Woburn, MA, USA). The PCR was carried out with a GeneAmp ® PCR System 9700 (Applied Biosystems; Foster City, CA, USA). The cycling conditions consisted of an initial 5 min at 94 °C followed by 1 min denaturing for at 94 °C, 1 min annealing at 36 °C, and 1 min elongation at 72 °C repeated 40 cycles and with 10 min of final extension at 72 °C. An aliquot (5 μL) of the amplification product was separated on a 1.2% agarose gel and detected under UV-light after staining in ethidium bromide.

Screening Strategy and Identification of Specific RAPD
Twenty-four random primers were screened for their ability to produce a marker specific to Bulbus Fritillariae Cirrhosae. Amplification of DNA from the twelve Fritillaria species including six Fritillaria species of Bulbus Fritillariae Cirrhosae was carried out using a single primer. The amplicon was monomorphic in all the Fritillaria species of Bulbus Fritillariae Cirrhosae but absent in other six Fritillaria species.

Cloning of the RAPD Amplicon
The putative marker amplified by the random primer S158 was excised from 1.2% agarose gel with sterile gel slicer and purified using QIAquick Genei Gel Extraction kit (Qiagen). It was then used as a template for its re-amplification using the same primer. TA cloning strategy was employed using the Invitrogen TA Cloning system (Invitrogen, Carlsbad, CA, USA). The standard reaction consisted of 10× ligation buffer (1 μL), PCR Vector (2 μL, 50 ng), PCR product (6 μL), and T4 DNA ligase (1 μL). The reaction mixture was mixed by pipetting, and incubated at 4 °C overnight. Transformation was carried out using high efficiency competent cells (DH-5α strain of Escherichia coli) following protocol for transformation by calcium chloride as described by Sambrook et al. [16]. Ten distinct white colonies were picked up from the LB-ampicillin plate. Then, the recombinant plasmid DNA from E. coli was isolated using Qiagen's QIAprep ® Mini prep kit following the manufacturer's directions. Confirmation of the clones was done by amplifying the DNA using M13 and T7 universal primers.

Sequencing, SCAR Primer Designing
After the purified RAPD amplicon was cloned, both ends of the recombinant plasmid were sequenced on an ABI 3700 automated sequencer (Applied Biosystems). Homology searches were performed within Genebank's non-redundant database using the BLAST algorithm [17]. Based on the sequenced RAPD amplicon, a pair of SCAR oligonucleotide primer (CBM-S and CBM-AS), which could amplify approximately 231 bp fragment in Bulbus Fritillariae Cirrhosae was designed. CBM-S was designed as the forward primer and CBM-AS as the reverse primer. The sequences were custom synthesized from Life Technologies Limited, Hong Kong.

Amplification of the Genomic Region
The SCAR primers pair was used for PCR amplifications of genomic DNA from the twelve Fritillaria species (including six Fritillaria species of Bulbus Fritillariae Cirrhosae). Thermal cycling conditions for amplification using SCAR primers were optimized as: 94 °C for 5 min; 40 cycles at 94 °C for 1 min, 59 °C for 1 min., and 72 °C for 1 min; and a final extension at 72 °C for 10 min. PCR amplification for authentication of commercial samples was done by using the SCAR primer pair with the above-mentioned thermal cycling conditions.

Validation of the Authentication Result
The result of authentication of commercial samples was validated by the PCR-RFLP method. The identification of Bulbus Fritillariae Cirrhosae by PCR-RFLP was carried out using the protocol described by Li et al. [11]. Briefly, the genomic DNA of nine commercial samples was first amplified with previously reported primers ITS-P1 (5'-CGTAACAAGGTTTCCGTAGGTGAA-3') and ITS-P3 (5'-GCTACGTTCTTCATCGAT-3'). Thermal cycling conditions for amplification were: 95 °C for 4 min; 30 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 5 min. Then, the PCR products were digested using the incision enzyme SmaI. As reported [11], there was a SmaI digestion site in Bulbus Fritillariae Cirrhosae DNA sequence (CCCGGG), but not in other Bulbus Fritillariae (CTCGGG). Digestions with SmaI (10 U/μL, Roche, Basel, Switzerland) were performed in a total volume of 20 μL containing 6 μL PCR products, 5U of SmaI enzyme, 2 μL 10× digestion buffer as recommended by the manufacturer, and 11.5 μL of PCR H 2 O; The incubation was at 37 °C for 1.5 h.

Conclusions
The developed RAPD-SCAR method effectively distinguished the Bulbus Fritillariae Cirrhosae from other species of Fritillaria. It was also successfully applied to identifying commercial samples of dried bulbs, which was then analyzed by PCR-RFLP method for verification. The results demonstrated that the RAPD-SCAR method was simple, accurate and reliable in identifying Bulbus Fritillariae Cirrhosae at the DNA level. Our study suggests that the use of RAPD-derived DNA markers to authenticate Bulbus Fritillariae Cirrhosae could be helpful to ensuring the quality and clinical efficacy of Fritillaria bulbs. Additionally, this method could facilitate the testing and certification of other Chinese herbal materials in the plant pharmaceutical industry.