Gardenia jasminoides Ellis and its related taxa of random amplified polymorphic DNA analysis

     【Abstract】 Objective To explore Buxus Gardenia Gardenia jasminoides and its variants, plena Gardenia Gardenia and variable between the water-related. Method from the 56 random primers screened 12 primer pairs of Gardenia jasminoides Ellis and its related taxa for RAPD analysis, application SPSS10.0 analysis software calculated the Jaccard similarity between any two coefficients of the sample, using the group clustering

    method obtained cluster tree. The results were obtained 251 valid sites. Cluster analysis showed that the first cluster Gardenia Gardenia and plena, and then with water Gardenia clustering, the final clustering with Buxus gardenia.

    Conclusion plena Gardenia Gardenia and the closest relationship, Buxus Gardenia Gardenia kinship with the farthest.

     Key words Gardenia; relatives group; random amplified polymorphic DNA

     RAPD Analysis for Gardenia jasminoides and Relatives

     Abstract: ObjectiveTo probe relative relationship among Gardenia jasminoides, its varietas (G. jasminoides Ellis. Var. Radicans and G. jasminoides Ellis. Var. Fortuniana) and its forma (G. jasminoides Ellis. F. longicarpa). MethodsRAPD analysis for samples of Gardenia jasminoides and relatives were performed by 12 random primers screened from 56. Proximity matrix was analyzed by SPSS version 10.0 and rescaled distance cluster combine was obtained by cluster analysis within groups. ResultsA total of 251 polymorphic loci

    were amplified.It showed that Gardenia jasminoides were clustered with G. jasminoides Ellis.var. fortuniana at first,

    then with G. jasminoides Ellis. f. longicarpa, and with G. jasminoides Ellis. var. radicans at last.ConclusionAmong samples, Gardenia jasminoides was the closest relative to G. jasminoides Ellis.var. fortuniana, and most distant relative to G. jasminoides Ellis. var. radicans.

     Key words: Gardenia jasminoides; Relatives; RAPD

     Gardenia jasminoides is commonly used traditional Chinese

    medicine, has Xiehuo Chufan, heat diuretic, cooling blood detoxification effect. Located in the Yangtze River and south of the region for the acid soil indicator plants. Gardenia 3 varieties and 1 form [1 ~ 7], namely, Gardenia Gardenia

    jasminoides Ellis., Buxus gardenia G. jasminoides Ellis. Var. Radicans (Thunb.) Makino, plena gardenia G. jasminoides Ellis. Var . fortuniana (Lindl.) Hara., large flower gardenia G. jasminodes var. grandiflora Nakai., water gardenia G. jasminoides Ellis. f. longicarpa ZWXie et Okada., they all have some form of structural differences, In order to explore the kinship between them, the author uses random amplified polymorphic DNA (random amplified polymorphism DNA, RAPD) technology to its analyzed.

     1 Equipment

     1.1 Instrument PTC-100TM Programmable Thermal Controller (MJ RESEARCH INC, USA), high-speed centrifuge IEC Micro-MB

    centrifuge (IEC Corporation, USA), DZKW-C-type water bath pan

    (Hebei Province Huanghua Space Instrument Factory), DYY-? -5

    Regulator steady flow electrophoresis instrument (Instrument Factory, Beijing, 61), UV gel imaging system (SYNGENE ADIVISION of SYNOPRICS LTD, USA), nucleic acid ultraviolet quantitative instrument GENEQUANTII (Pharmacia Biotech, UK).

     1.2 Reagents Taq enzyme (Huamei Bio-Engineering Company,

    200 u, 3 μl / μl), agarose (Spain, China and the United States companies to install), dNTP (China and the United States company), CTAB (Sigma Company), ethidium bromide (Fluka Company) , Tris base (Ultra Pure, China and the United States company), EDTA (China and the United States company), mercaptoethanol (Shanghai Health Industry Co., Ltd.), random primers (Shanghai Health Industry Co., Ltd.) and the remaining

reagents for the Beijing Chemical Plant production AR.

     1.3 Materials Gardenia, Buxus gardenia, plena gardenia, gardenia leaves the water collected by the author identification, rapid drying silica gel preserved samples should adopt a mixed sample, numbered in Table 1.

     2 Methods

     2.1 DNA extraction and concentration determination of about 0.2 mg of dry leaves placed in 1.5 ml EP tube, add liquid nitrogen at about 30 s after the clamp crushed into a fine powder and immediately adding 500 μl 2 × CTAB

    extraction buffer [CTAB2%, Tris -HCl (pH 8.0) 100 mmol * L-1,

    EDTA 20 mmol * L-1, NaCl 1.4 mol * L-1, mercaptoethanol 2%]

    and 20 μl-mercaptoethanol, 60 ? water bath, after 1h heat,

    add chloroform - isoamyl alcohol (24:1) extraction, reverse the mixing of light, 10 000 r * min-1 centrifuge 10 min, the

    supernatant drawn, by adding an equal volume of CTAB precipitation buffer [CTAB 1%, Tris-HCl (pH 8.0) 50 mmol * L-

    1, EDTA 10 mmol * L-1, mercaptoethanol 1%], at room

    temperature for 0.5 h or more, 10 000 r * min-1 centrifuge 10

    min, carefully tilt to the supernatant, precipitation with 70% ethanol and ethanol each time a light wash, disposable lotion, leaving sediment Hui dry. Dissolved with 20 μl TE backup.

     With 0.8% agarose gel electrophoresis, in order to λDNA

    as control and the use of nucleic acid concentration was measured quantitatively by UV combination.

     Table 1 Experimental sources of material, acquisition time (omitted)

     2.2 DNA template concentration of the primer pairs S267 choose to use different concentrations of DNA template

    amplification results compare, and choose a good concentration of PCR amplification results.

     2.3 PCR amplification of 25 μl reaction: dNTPs10 mmol *

    L-11.5 μl, primer 10 μmol * L-10.5 μl, DNA template of

    about 42 ng, Taq enzyme 3 μl / μl 0.5μl, MgCl2 25 mmol * L-

    11.5 μl, 10 × Reaction Buffer 2.5 μl. Amplification

procedures: pre-denaturation 96 ?, 5 min; amplification cycle

    40, to 94 ? 45 s, 34 ? 1 min, 72 ? 1.5 min; after the

    extension of 72 ? 5 min. Take 8 μl PCR products in 1.8%

    agarose gel using 1 × TAE electrophoresis buffer,

    electrophoresis, gel imaging system detects ultraviolet photographs. Reposted elsewhere in the paper for free download http://

     2.4 The selection of primers in 1 and 2, two templates, with random primer amplification under the above conditions, choose the bands clear, reproducible primer as the primers used for RAPD amplification.

     2.5 Data analysis gel electrophoresis of each band (DNA fragments) are a molecular marker, and on behalf of a primer

    binding points, according to the availability of molecular markers and their migration rate statistics to be a locus of binary data, there are band counted as 1 (strong and weak band with the same account), no band counted as 0. Application SPSS10.0 analysis software calculated the Jaccard similarity between any two samples of coefficients Within group clustering method with the calculated cluster tree.

     3 Results and discussion

     3.1 from 56 primers were screened out of 12 bands clearly visible amplification products of polymorphic molecular weight of 200 ~ 2 000 bp between the primer 12 (see Table 2), by a total of 251 valid sites, of which primer S28, S180 Results of PCR amplification of samples shown in Figure 1. The SPSS10.0 analysis software have any similarity coefficient between the two samples (see Table 3) and the clustering tree (see Figure 2).

     Figure 1 S28, S180 samples amplified a product of SDS-

    PAGE (omitted)

     Figure 2 clustering tree (omitted)

     3.2 Figure 2 shows that the sample 4 and 5 of the first cluster on its close relatives, the greatest similarity coefficient (0.575) also illustrates this point, they are different for water Gardenia populations, but the results of the sample 4 falls far short of sample 5, sample 4 was born in

    Jiangxi College of Physic Garden, surrounded by a lack of water, habitat is relatively dry, and surrounded by high trees block some of the sun, while the samples of five grew up in the ditch on both sides, surrounded by paddy fields, sun and

    adequate moisture; samples 4 and 5, the difference may be caused by differences in the ecological environment of the.

     Table 2 Primers used in this experiment sequence (abbreviated)

     Table 3 similarity coefficient between the sample matrix

    (abbreviated)

     3.3 Table 3 shows that samples 1 and 3 similarity coefficient of 0.574, the sample 1 and sample 2 similarity coefficient of 0.421, the sample 1 and sample 4 similarity coefficient of 0.477, genetic distance calculation formula [8]

    as: DS = 1-SI , DS for the genetic distance, SI for the similarity coefficient, the greater the similarity coefficient, the smaller the genetic distance, genetic relationship is closer. Similarity coefficient and sample a gardenia comparison, the sample 3 plena Gardenia (0.574) "Sample 4 Water Gardenia (0.477)" Sample 2 Buxus Gardenia (0.421), samples were taken from 1 to 4 of Traditional Chinese Medicine, Jiangxi Medical Botanical Garden, and their growing environment the same, indicating that their differences come from within the species. Experiments show that plena Gardenia Gardenia and the closest relationship, Buxus Gardenia Gardenia kinship with the farthest. From the morphological point of view, for the short Buxus gardenia shrubs, Ye, flowers small

    and the plena, and other kinds of these characteristics vary greatly, Figure 2 cluster results also show that some relations with another Buxus Gardenia distant .

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