Development and validation of molecular markers for characterization of Boehmeria nivea var. nivea and Boehmeria nivea var. tenacissima
© Li et al; licensee BioMed Central Ltd. 2010
Received: 15 May 2010
Accepted: 29 November 2010
Published: 29 November 2010
The root of Boehmeria spp (ramie) is a hepatoprotective Chinese herbal medicine. Medicinal properties vary between Boehmeria nivea var. nivea and Boehmeria nivea var. tenacissima, which are local species found in Taiwan. As commercial preparations may use either species, there is a need for a rapid and simple assay to identify variants for quality control.
Four methods were developed and tested for their applicability in differentiating the two species. These methods were random amplified polymorphic DNA (RAPD); sequence characterized amplified regions (SCAR); single nucleotide polymorphisms (SNP) and cleaved amplified polymorphic sequences (CAPS).
Three RAPD markers were developed that produced unique bands in B. nivea var. tenacissima and B. nivea var. nivea. Based on sequenced RAPD bands, one SCAR marker was developed that produced a single DNA band in B. nivea var. nivea. Two SNP markers differentiated between B. nivea var. nivea and B. nivea var. tenacissima based on single nucleotide substitutions. A pair of CAPS oligonucleotides was developed by amplifying a 0.55-kb DNA fragment that exhibited species-specific digestion patterns with restriction enzymes Alf III and Nde I. Consistent results were obtained with all the four markers on all tested Boehmeria lines.
The present study demonstrates the use of the RAPD, SCAR, SNP and CAPS markers for rapid identification of two closely related Boehmeria species.
The root of Boehmeria species (Urticaceae), namely Boehmeria nivea var. nivea is a hepatoprotective Chinese herbal medicine  as well as an antioxidant and anti-inflammatory agent . Sancheti and colleagues have reported its glycosidase and cholinesterase inhibition properties as an anti-diabetic herb to lower blood glucose and cholesterol levels . Compared to B. nivea var. nivea, B. nivea var. tenacissima is more hepatoprotective on hepatitis B-induced liver damage . As commercial preparations may consist of one or the other variants, there is a need for rapid and simple assays to identify variants for the purpose of both commercial production and quality control. Whereas today's methods rely primarily on morphological observations, molecular genetics are a more precise tool, less susceptible to user bias.
Based on four molecular approaches, namely random amplified polymorphic DNA (RAPD), sequence characterized amplified region (SCAR), single nucleotide polymorphism (SNP) and cleaved amplified polymorphic sequence (CAPS), we developed and evaluated a set of authentication techniques for the Boehmeria species and help conserve Chinese medicinal plants in Taiwan.
RAPD is a modified polymerase chain reaction (PCR) technique involving multiple oligonucleotide primers. The resulting amplified DNA markers are random polymorphic segments with band sizes from 100 to 3000 bp depending upon the genomic DNA and the primer. SCARs are DNA fragments amplified by using specific 15-30 bp primers, designed from nucleotide sequences established in cloned RAPD fragments. By using longer PCR primers, SCARs have a higher rate of reproducibility than RAPDs. SNP analysis is more specific still but requires sequencing to identify the different nucleotides.
CAPS polymorphisms are differences in restriction fragment lengths caused by SNPs that create or abolish restriction endonuclease recognition sites in PCR amplicons. All of these markers are locus-specific with a wide range of applicability in gene mapping and marker-assisted selection [5–7]. This article describes the main results of the study.
Eight lines of B. nivea var. nivea and B. nivea var. tenacissima were collected from various locations of Taiwan and identified by one of the authors (TST), based on the criteria that B. nivea var. nivea has a white-grey color with obvious pubescence in their ventral leaf surface and B. nivea var. tenacissima has a light green-grey color . Four collections, namely CY1 (Chi-Yi-1), CY2 (Chi-Yi-2), CY3 (Chi-Yi-3) and HCn (Hsin-Chu-n) belong to B. nivea var. tenacissima and the other four, namely HCd (Hsin-Chu-d), TC (Tai-Chung), CY (Chi-Yi) and TARI (Taiwan Agricultural Research Institute) are local variants of B. nivea var. nivea.
DNA extraction was performed according to the method described by Arasl et al.. Briefly, 100 mg fresh leaves were ground in liquid nitrogen and transferred to tubes containing 5 mL CTAB/PVPP extraction buffer which consisted of 0.1 M Tris HCl, 1 M NaCl, 20 mM EDTA, 1% hexadecyl trimethylammonium bromide (CTAB; w/vol) and 1% polyvinylpolypyrrolidone (PVPP; w/vol). The mixture was incubated at 65°C for 20 minutes and extracted with an equal volume of chloroform/isoamylalcohol (24:1). After centrifugation (8,000×g, Sigma 3-18 K, Germany) for 5 minutes, the supernatant was transferred to a clean tube and precipitated with two volumes of precipitation buffer (50 mM Tris HCl, 4 mM NaCl, 10 mM EDTA and 1% CATB) at 10, 000×g for 20 minutes. The pellet was re-suspended in 350 μL 1.2 M NaCl and incubated with 10 mg/mL RNase at 37°C for 30 minutes. After extraction with an equal volume of chloroform/isoamylalcohol (24:1), the DNA pellet was re-precipitated with ice-cold isopropanol, washed with 70% ethanol, vacuum dried and dissolved in 200 μL TE buffer.
Primers used for marker analysis
Annealing temperature (°C)
Name of the primer
Number of polymorphic bands
0.25, 0.3 (Afl III)
0.2, 0.35 (Drd I)
Three pairs of oligonucleotides were used in the SCAR assays , namely forward oligonucleotide SR-S343-F1 and the three reverse oligonucleotides SR-S343-R1, SR-S343-R2 and SR-S343-R3 (Table 1). The SCAR reaction was performed with an initial denaturation step at 95°C for five minutes, followed by 35 cycles of 94°C for two minutes, 60°C for one minute, 72°C for one minute and a 10-minute final extension at 72°C.
PCR fragments were cloned with TA cloning technology using pGEM-T-Easy vectors (Promega, USA) and used to transform the Escherichia coli strain XL-2 Blue (Stratagene, USA). DNA sequence analysis was carried out with the BLAST sequence analysis programs at the National Center for Biotechnology Information (NCBI) . Alignments were edited with the online ClustalW program from DNA Data Bank of Japan .
A sequence from a RAPD DNA fragment, namely RP-S62-0.6, was chosen for SNP detection. The procedures were performed according to the manufacturer's instructions  with one modification, i.e. the mixture solution was diluted 1:8 with magnesium buffer (400 mM Tris pH 9 and 10 mM MgCl2). Each reaction contained 0.5 μL of the SNaPshot™ Multiplex Ready Reaction Mix (Applied Biosystems, USA), 2.0 μL of PCR product, 1.0 μL of extension primers and water up to 10 μL. Thermal cycling and post-extension were run on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, USA).
CAPS analyses were performed according to published methods . The RP-S62-0.6 DNA fragment was amplified from the eight Boehmeria lines with primers CP-S62-f and CP-S62-r. DNA fragments were digested with restriction enzymes (i.e. Afl III, Bsr FI, Msp I, Drd I and Nde I) and separated on a 1.5% agarose gel for polymorphism detection.
A mixture of DNA was used to identify the basis of all the markers for quality control. Samples contained DNA from both B. nivea var. nivea and B. nivea var. tenacissima in the ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1:9 respectively.
Results and Discussion
RAPD markers for quick screening
RAPD analysis is fast and economical  as long as suitable primers are available. In the present study, only two primers out of 100 species-specific patterns were easily visualized in electrophoresis.
Conversion of RAPD into SCAR and SNP
Only a fraction of the recommended SNaPshot mixtures was used in this study because the quality of SNP detection can be maintained using only one eighth of the recommended amounts of the reagents.
Conversion of SNP sites into CAPS
Potential applications in quality control
Based on known SNPs, CAPS is a suitable strategy for the analysis of mixed samples. The present study identified the Boehmeria species in a sample and characterized the relative amounts of one species vs. another in a single sample. While CAPS may be used as a rapid analysis kit for Boehmeria-based preparation compared with the time-consuming RFLP analysis, however it is rather laborious and requires the use of restriction enzymes.
The present study demonstrates the usefulness of the RAPD, SCAR, SNP and CAPS markers for rapid identification of variants between two closely related Boehmeria species. In particular, CAPS would be a suitable strategy for the analysis of mixed samples.
random amplified polymorphic DNA
sequence characterized amplified regions
single nucleotide polymorphism
cleaved amplified polymorphic sequences
Taiwan Agricultural Research Institute
hexadecyl trimethylammonium bromide
polymerase chain reaction
The authors thank Dr. Pelle Stolt for editorial comments on the manuscript. This work was financially support by the Council of Agriculture, the Executive Yuan (93N705-921-G) through NSTP.AB and the Industrial Technology Research Institute (93-EC-17-A-20-R7-0318), Taiwan.
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