Genetic Diversity Of Blue Honeysuckle (Lonicera L. Subsect. Caerulea) Based On Sequence-Related Amplified Polymorphism

Lonicera Caerulea L. belongs to Caprifoliaceae family, was a perennial under shrub with strong environmental adaptability, it was widely distributed in Russia, Japan, Korea and North America. The Xingan and Changbai Mountains are rich in wildlife resources too. Fruits of Lonicera Caerulea L. are blue-purple, its processing into jams, beverages, canned food, wine and other pollution-free food both rich in nutrients such as anthocyanins, minerals, and vitamins and have medicinal value. Loved by people around the world, is one of the third generation fruit trees and can comparable with blueberries, has good market prospects.The experiment was carried out in horticultural of Northeast Agricultural University from 2009 to 2010. 58 varieties of blue honeysuckle from China's first blue honeysuckle germplasm garden was used to establish the best genomic DNA extraction method, discussed the influence of amplification factors in amplification reaction system and optimization SRAP-PCR reaction system. Study the genetic diversity in 58 blue honeysuckles from different countries or region was assessed using SRAP markers. Purpose is to understand the blue honeysuckle populations'level of genetic diversity and the protection and utilization of resources to provide the theoretical basis. The main results are as follows:1,DNA extraction is based on the analysis of genome diversity and an important basic skills in molecular biology study. Different tissues of plant material because of its metabolic products contained in this life, the type of content in different extraction methods are also different. Blue honeysuckle leaves are rich in polysaccharides and polyphenols and other secondary metabolites, these substances will affect the quality of genomic DNA extracted. In this study, the trial of conventional and modified CTAB method were compared and the results show that the modified CTAB method extraction out of the bright bands and no smearing, this method was efficient for blue honeysuckle. That is, in the process of adding liquid nitrogen grinding PVP powder, to prevent the oxidation of phenolic compounds. pre-wash the samples with SET, separated the nucleus and secondary compounds such as polysaccharides and polyphenols, DNA sample extracted by the methods had high purity, OD260/OD280 maintained at between 1.7 and 1.9, to meet the requirements of SRAP amplified.2,The reaction factors have a significant impact in PCR reaction, in this study, we design for single-factor experiment to the reaction system, Tag DNA polymerase, template DNA, dNTPs, Mg²⁺ and primer. established 20μl reaction system of SRAP for blue honeysuckle included 2.0μl 10×PCR buffer, 1.0 U Taq DNA polymerase, 30 ng template DNA, 0.2 mmol/l dNTPs, 2.0mmol/l Mg²⁺ and 0.2μmol/l primer. The reaction procedure was as follows: denaturation at 94℃for 5 min ; denaturation at 94℃for 1 min ,anneal at 35℃for 1 min ,extension at 72℃for 90 s and in total 5 cycles ; denaturation at 94℃for 1 min ,anneal at 50℃for 1 min ,extension at 72℃for 90 s and in total 35cycles ;extension at 72℃for 8min ;preservation at 4℃.3,During the primer screening, the L.caerulea subsp.altaicai×L.caerulea subsp.Kamtschatica, L.caerulea subsp.emphyllocalyx and daxinganling were used as experimental material, twenty out of 90 pairs of primer combinations were selected in order to amplify the genotypes, 172 bands were acquired by PCR, about 143 of them is diversity bands, so the rate of diversity is 83.1%, each primer pair produced 5-12 bands. The genetic similarity index among the 78 species varied from 0.53 to 0.96.4,Using selected primers and optimized system to carry out PCR amplification of the tested varieties, electrophoretic bands by 1 / 0 form of data, choose clear, reproducible and strong bands for statistical object denoted 1and no amplified bands as 0, UPGMA method based on tree building and analysis of the genetic diversity. 58 materials were clustered into 7 categories, species from the Russian regions clustered in one group; Yichun, Shangzhi, Boli species together; Altay, Daxinganling and Changbaishan wild species cluster separately, each as a group; three cultivars L.caerulea subsp.altaicai×L.caerulea subsp.Kamtschatica, Blue bird and Blue spindle cluster with E3, E6 and VIR. The 23rd unknown species together with Vladivostok, it may come from Vladivostok region or its offspring. Resources from the northeast which is divided into four groups, it can be seen, blue honeysuckle in northeast have high genetic variation, also shows that the northeast is one of the genetic diversity centers of blue honeysuckle.