| The medicinal material honeysuckle comes from the dried buds or primary flowers of Lonicera japonica Thunb. which belong to the Caprifoliaceae family. Honeysuckle can clear away heat and toxic material, expel wind and heat. At the same time, it’s a major raw material which can be used in health drinks, food and chemical industry. L. confus DC. come from the dried buds or primary flowers of L. macranthoides Hand.-Mazz.、L. hypoglauca Miq. or L. confusa DC. L. macranthoides Hand.-Mazz. is the main merchant variety of honeysuckle in Hunan, the planting area reaches more than 20 million mu. Its output takes more than 60 percent market share. Longhui is the biggest producing area in China. Since a long-term natural selection and artifical selection, it has formed a rich extremely germplasm resources, especially in recent years, the majority of scientific and technology personnel in the practice has been a series of carefully selection of quality, evaluation of germplasm resources, selection of superior varieties and promoting are a long-term task to scientific and technological personnel. ISSR molecular marker is suitable for analyzing genetic diversity of L. macranthoides Hand.-Mazz. through the reaction system optimization, PCR has a clear electrophoretogram. It provides another powerful tool for the majority of technology personnel for its varieties of identification and cultivation. The main findings are as follows:1、In the experiment, five essential factors which may affect the result of ISSR were optimized by five single factor experiments and orthogonal experiment, and one single factor experiments of annealing temperature. The result showed that the optimal conditions of ISSR-PCR system and reaction parameters were 2.0 mmol·L-1 Mg2+,0.20 mmol-L’1 dNTP,0.3μmol·L-1 primer,1.0 U Taq polymerase,20 ng DNA in 25μL system. The reaction procedure was 94℃in 5 min, then 35 cycle times with 94℃in 30 s,54℃in 45 s,72℃in 90 s, lastly 72℃in 5 min, and stop reaction at 4℃.2、There were 100 primers used for the PCR amplification of genomic DNA fragment.15 ISSR primers amplified a total of 177 DNA bands, of which 161 were polymorphic, accounting for 91.0%. Every primer could amplify 4 to 16 polymorphic bands, with an average of 11.8 bands. Shannon’s information index was 0.4314. Effective number of alleles was 1.4707. Genetic similarity index varied from 0.39 to 0.91, with an average of 0.70, standard deviation was 0.10.3、After analyzing electrophoretograms, fingerprint spectrums of graph about DNA of L. macrathoides Hands.-Mazz. were built by ultraviolet gelatin imaging system.4、Dendrogram of cluster analysis of L. macrathoides Hands.-Mazz. based on ISSR maker technology was completed on the basis of genetic similarity index more than 0.72.18 populations were divided into six groups. The first group is L. japonica Thunb. from Liuyang; The second group is general from Yanbei; The third group is Taihu once and twice from Xiao Shajiang; The forth group includes Xianglei sixth from Longtan and Xianglei twice from Yanbei; The fifth group is general from Longtan; Others are the sixth group, includes Xianglei once、thrid、forth and fifth from Longtan, Xianglei once、thrid、forth、Baiyun and once from Yanbei, general from Xiao Shajiang, as well as Huawang from Hu Xingshan. |
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