| Objective: To explore the genetic relationship of the Valeriana officinalis L. var. latifolia Miq in Guizhou Province at molecular level, and to study the quality evaluation of Volatile oil. Methods:(1) The suitable RAPD and ISSR reaction system were establish by optimizing the concentration of Mix, template DNA and primer in this study.(2) The random primers for ISSR, RAPD and the optimal annealing temperature of ISSR were screened by optimized reaction system.(3) RAPD and ISSR molecular markers were used to analyse the genetic diversity of 20 population samples from Guizhou Province, and then the UPGMA clustering analysis was progressed by NTSYS 2.1e.(4) The correlation between the RAPD and ISSR was analyzed by SPSS 17.0.(5) Based on chloroplast DNA fragments, the trn L-F and mat K sequences of Valeriana officinalis L. var. latifolia Miq were analyzed. The genetic distance was calculated by Bioedit, Mega 5.0, and then the clustering analysis was progressed to establish the phylogenetic tree.(6) The physicochemical properties of volatile oil were determined by the Chinese Pharmacopoeia 2010 years.(7) To establish a thin layer chromatography(TLC) identification method for volatile oil.(8)To establish the determination method of borneol acetate contents in volatile oil by gas chromatography(GC).(9) The correlation between the borneol acetate contents and elevation was analyzed by SPSS 17.0. Results:(1) The optimal RAPD conditions in the experiments were as following: in 25 μL reaction system containing 2× Taq PCR Master Mix 16 μL, DNA template 100 ng, primer 0.60 μmol/L, dd H2 O completed.(2) The optimal ISSR conditions in the experiments were as following: in25 μL reaction system containing 2× Taq PCR Master Mix 14 μL, DNA template 60 ng, primer 0.70 μmol/L, dd H2 O completed.(3) With the DNA extracted from 20 samples as template, 197 bands were totally amplified from 17 random primers which were screened out from 59 RAPD primers, among which 158 were polymorphic bands, the average percentage of polymorphic bands was 86.61%. The genetic similarity coefficients was between 0.4732 and 0.8929.(4) With the DNA extractedfrom 20 samples as template, 224 bands were totally amplified from 17 random primers which were screened out from 50 ISSR primers, among which 194 were polymorphic bands, the average percentage of polymorphic bands was 80.20%. The genetic similarity coefficients was between 0.4010 and 0.9391 and the optimum annealing temperature for each primer in ISSR reaction was proposed.(5) The UPGMA cluster analysis of two methods demonstrated, that population No.4 was clustered as a class and the remained 19 samples were clustered into another group.The samples from Jianhe area were clustered into one group and the samples from Jiangkou area were clustered into another group. The samples of 2013 years were gathered into one group, and the samples of 2015 years were gathered into another group.(6) Two kinds of makers showd the significant positive correlation(0.875,P<0.01).(7) In the 23 populations of the Valeriana officinalis L. var. latifolia Miq, the lengths of trn L-F sequence ranged from 947~985 bp, of which GC% contents were between 35.92~36.55%. The sequences were aligned and final sequences 930 bp in length,which included 8 differential sites and 5 variable sites. The genetic distance ranged from 0.0000~0.0054 and the overall genetic distance was 0.0005. The branch type of phylogeny trees constructed by ML, NJ, ME and UPGMA methods and showed that samples No.15 and 18 were divided obviously with the most samples.(8)In the 23 populations of the Valeriana officinalis L. var. latifolia Miq, the lengths of mat K sequence ranged from 1080~1088 bp, of which GC% contents were between35.19~36.64%. The sequences were aligned and final sequences 1055 bp in length,which included 2 differential sites and 1 variable site. The genetic distance ranged from 0.0000~0.0009 and the overall genetic distance was 0.0003. The branch type of phylogeny trees constructed by ML, NJ, ME and UPGMA methods and showed that samples No.2, 5, 6 and 20 were clustered as a class and the remained 19 samples were clustered into another group.(9) The appearance were light yellow, transparent, and with its inherent odor and slightly bitter taste of volatile oil, its acid value was2.89~4.11 mg/g, the saponification value was 171.08~183.00 mg/g, the refractive index was 1.4728~1.4796, the specific rotation was 39.21~58.66 °.(10) TLC method has been determined of volatile oil, the developing system was cyclohexane-ethyl acetate- acetic acid(6:0.2:0.03), the silica gel G plate was saturated 15 min, the chromogenic agent was 5% vanillin sulfuric acid solution and got 8~13 rich and discernible spots.(11) Investigated by GC, borneol acetate showed good linearrelationship within the range of 1.0025~8.0200 mg/m L(r2=0.9991), with an average recovery of 98.45%. The borneol acetate contents in volatile oil was 29.64~59.65%.(12) The borneol acetate content was increased with the increase of elevation, but to a certain altitude, showed a decreased trend, and its has no significant correlation.Conclusion:(1) There is abundant genetic diversity in Valeriana officinalis L. var.latifolia Miq from Guizhou Province at species level. RAPD and ISSR markers can be used to analyze genetic diversity of Valeriana officinalis L. var. latifolia Miq and provide the base for seed selection and breeding.(2) The trn L-F and mat K sequence of Valeriana officinalis L. var. latifolia Miq in Guizhou province is highly conservative.(3) Through the quality evaluation of volatile oil, it provide the theoretical basis for reasonable development of volatile oil in future... |
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