Genomic DNA Extraction and RAPD Analysis for Gastrodia elata Bl. (Orchidaceae) Using an Improved Method
One improved randomly amplified polymorphic DNA (RAPD) technique has been used in animal phylogenetic relationships and fingerprints analysis by prolonging the ramp time. However, little was known whether the PCR ramp time and G/C content of primers effect on RAPD analysis of medicinal Gastrodia elata Bl. plants. The present study was conducted with the objectives to extract genomic DNA of G. elata, execute RAPD analysis with different PCR ramp time and discuss the relationship between the amplification efficiency and the G/C content of RAPD primers subsequently. The Cetyltrimethylammonium Bromide (CTAB) protocol was used in the genomic DNA extraction. Ten RAPD primers were randomly selected in PCR amplifications. The ramp time parameters from annealing to extension were used 0.3 and 3°C sec-1, respectively. The concentration of G. elata genomic DNA were about 50 ng μL-1 with a 1.93 purity. Obviously, the amplified band numbers and resolution were improved when using a 0.3°C sec-1 ramp time in the RAPD analysis. The band number increase is closely related to the G/(G+C) ratio of RAPD primers. Therefore, the extracted G. elata genomic DNA is suitable for PCR amplifications and the prolonged ramp time is helpful to improve the RAPD resolution and production. These are valuable references for molecular identification and biodiversity analysis of G. elata populations.
February 08, 2012; Accepted: March 29, 2012;
Published: June 12, 2012
Gastrodia elata Bl. in genus Gastrodia R. Br. is a saprophytic
perennial herb of the Orchidaceae family. The species are widely distributed
in China living in forest and shrubbery edges (Chen, 1999;
Guan et al., 2007). As a rare traditional Chinese
medicine, the rhizomatous herb of G. elata is useful in remedying
headache, dizziness and numbness. Also, the herb has curative effects in reducing
blood pressure, relieving pain and fortifying brain (Li
et al., 2005). Due to the rarity of wild G. elata,
the locality identification and biodiversity analysis of G. elata
populations are needed and in which the Randomly Amplified Polymorphic DNA (RAPD)
technique is used widely with rapid and simple advantages. RAPD technique has
been shown to be useful in authentication and genetic diversity analysis on
molecular level (Zou et al., 2006; Zhao
et al., 2006; Shaptadvipa and Sarma, 2009;
Duan et al., 2011; Thangaraj
et al., 2011).
However, the application of RAPD technique is limited in its stability and
reproducibility (Huang et al., 2008). An improved
method for increasing the efficiency via altering ramp time has been reported
in animal RAPD analysis (Fu et al., 2000) but
we still know little about its effects on the RAPD analysis of G.
elata. Also, whether the increased amplification efficiency is related to
the G/C content of RAPD primers in the improved method are obscure too.
To discuss the effects of PCR ramp time and G/C content of primers on RAPD analysis of G. elata, we will extract the G. elata genomic DNA and execute RAPD analysis with different ramp parameters and G/C content of primers in the present study.
MATERIALS AND METHODS
The study was conducted from February 2011 to December 2011 at Luzhou city, Research Center for Preclinical Medicine of Luzhou Medical College.
Plant materials: The fresh G. elata rhizoma used in this study are collected from Yanyuan county of Xichang city in Sichuan province. The rhizomas were germinated and grown in the perennial nursery of Medicinal Botanical Garden, Luzhou Medical College. The mature G. elata were carefully identified by Dr. Haiqing Yu. The voucher specimens (accession number GETY110201) have been deposited at the Medicinal Botanical Association of Zhongshan Mountain (MBAZM), Luzhou Medical College.
DNA extraction and purification: About 2 g rhizomas were ground in mortar
by adding quartz sands and the slurry was transferred to a 5 mL microcentrifuge
tube. Genomic DNA was extracted and purified with the Cetyltrimethylammonium
Bromide (CTAB) procedure introduced by Doyle and Doyle (1990).
The concentration and purity of genomic DNA were detected using ND-1000 (NanoDrop
RAPD PCR amplification: The PCR reaction was carried out using SBS primer
sets (Beijing SBS Genetech Co., Ltd, China). Ten random selected SBS primers
were listed in Table 1. RAPD was performed in a total volume
of 20 μL containing 30 ng DNA, 1xreaction buffer, 2 mM MgCl2,
0.25 μM of each primer, 200 μM of each dNTP (TakaRa Biotechnology
(Dalian) Co., Ltd), one unit of rTaq DNA polymerase (TakaRa) and sterile
water to the final volume. One drop of mineral oil was added in each reaction
tube. The thermocycling profile consisted of an initial denaturation at 94°C
for 4 min, followed by 40 cycles of 1 min at 94°C 1 min at 36°C 2 min
at 72°C and final extension of 10 min at 72°C. PCR reactions were carried
out in a Mastercycler 5331 (Eppendorf, Germany). The amplified PCR products
were resolved by electrophoresis on 1.5% agarose gel in 1xTAE buffer. Gels were
visualized by 0.5 μg mL-1 ethidium bromide staining and the
images were documented using the ChemiDoc XRS (Bio-Rad, USA). The ramp time
parameters from annealing to extension were used 3 and 0.3°C sec-1,
respectively as suggested (Fu et al., 2000).
The concentration and purity of extracted G. elata genomic DNA were about 50 ng μL-1 and 1.93, respectively. The PCR reactions were repeated five times and the band patterns were highly reproducible (data not shown). To obtain the great accuracy of UV illumination, exact band numbers were observed directly by using the clear acrylic UV protection screen.
Results of RAPD analysis were shown in Fig. 1 (a-b).
The amplified band numbers and G/(G+C) ratios are listed in Table
|| Primers used in RAPD analysis
||RAPD profiles of G. elata generated by ten primers,
(a) The ramp is 0.3°C sec-1. (b) The ramp is 3°C sec-1.
RAPD primers are showed above the ten lanes , respectively. M: DL2000 DNA
ladder (bp), low ramp RAPD analysis demonstrated the increasing of the resolution
and production with the exception of primers SBS-A1 and SBS-Q19
As, Fig. 1 displayed, the PCR bands were more clearly and
the resolution in RAPD analysis was obviously improved by prolonging the ramp
time from annealing to extension. Generally, eight of ten primers generated
weak or strong PCR bands while primers SBS-A1 and SBS-Q19 produced no products
in two different ramps (Fig. 1a-b).
Among ten RAPD primers, different band number increases were found after prolonging
(Table 1). No increased bands were detected when using primers
SBS-A1, SBS-M3 and SBS-Q19 and their G/(G+C) ratios are 0.3, 1 and 0, respectively.
Interestingly, primers SBS-A4, SBS-A12, SBS-I13 and SBS-N14 all increased four
bands which are the most augment in ten primers, with the same 0.7 G/(G+C) ratio.
Primers SBS-A11, SBS-I19, SBS-N2 gained two, one and two bands with a 0.3, 0.8
and 0.6 G/(G+C) ratio, respectively.
In the current study, high quality genomic DNA of G. elata was extracted which indicated that fresh rhizoma was easily obtained by adding quartz sands in mortar via CTAB method. Also, the reproducible PCR bands suggested that the extracted genomic DNA is suitable for RAPD amplifications.
After prolonging the ramp time, the RAPD resolution and PCR products were increased
obviously. This showed that the improved RAPD method is also useful in G.
elata RAPD analysis. Fu et al. ( 2000) considered
that the properly prolonged ramp time is perhaps helpful in binding stability
between primer and DNA template. Thus, the RAPD resolution and band numbers
are increased. Since the G/C content is one of most important factor in primer
specificity of PCR amplification, it probably plays effective in the improved
RAPD technique. We found that the most increase displayed in RAPD primers with
a 0.7 G/(G+C) ratio which suggested that these primers are likely more suitable
in the improved RAPD analysis.
In recent years, an increasing quality of G. elata is needed
owing to their well therapeutic effects. There are a lot of G. elata
cultivars in the market with different localities and sale prices and the wild
G. elata resources are rare due to excessive exploitation (Zou
et al., 2006). Commonly, the genuine regional drug which has the
specific growing locality, has better curative effects than those from other
localities (Yu et al., 2011a, b).
Therefore, it is necessary to develop an effective RAPD strategy in locality
identification and biodiversity analysis of G. elata populations.
In this study, the prolonged ramp time is helpful to improve the PCR resolution and production in RAPD analysis of G. elata. To obtain the most band number increase, the G/(G+C) ratio of RAPD primers needs to be considered. These are valuable references for RAPD identification and biodiversity analysis of different herbs.
The authors contribute equally to the present work. We are thankful to the Youth Foundation, the National Natural Science Pre-Research Foundation and the Scientific Research Starting Foundation for Doctors of Luzhou Medical College (No. 409, 676 and 606). These authors particularly thank persons for providing fresh G. elata rhizoma.
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