The Research On Genetic Diversity By ISSR Analysis And Genetic Variation Laws Of Medicinal Melia Azedarach

Melia azedaeach L.are excellent native tree species, and widely distribute in our country. As raw material tree species of botanical pesticides, their extracts have some characteristics which are broad-spectrum, high efficient, low toxicity, no residual, target biology with a strong selective, and so on, have unique insecticidal activity, but almost no pollution on the environment. most of Melia azedarach resources in our country was very abundant, but dispersed, lacked of natural forest, to protect existing resources of Melia azedarach, to develop and use of botanical pesticides has become a hot research as raw material to Melia azedarach.In this research, separately studied genetic diversity of Melia azedarach in the south of our country and genetic variation of Melia azedarach in Fujian province from genetic diversity and genetic variation. With ISSR molecular markers, analyzed genetic diversity of Melia azedarach of different provenances in the south, effect of geographical differences to genetic differentiation of Melia azedarach, and genetic relationship of Melia azedarach of different provenances, such as, carefully surveyed and carried out elite selection to resources of Melia azedarach of Fujian province, screened plus trees of Melia azedarach with high content of medicinal components, analyzed phenotypic variation of seeds, variation of Toosendanin content, nutrient variation and effect of meteorological factors to those three sides. In this study, the conclusions were as follows:1,Set up PCR amplification program of better repeatability and stability: predegeneration 5 min at 94℃; degeneration 45s at 94℃; the time of annealing is 45s, annealing temperature corresponds to each primer; extension 90s at 72℃; it has 40 cycle from degeneration to extension in total; then extension 10min at 72℃, finally conserved at 4℃. With single factor test method and orthogonal test method, finally established the best reaction system of ISSR-PCR applicabling to Melia azedarach of 13 provenances: 0.40mmol/L dNTP, 1.00 U Taq DNA polymerase, 0.80μmol/L primer, 3.00mmol/L Mg2+, 50 ng template DNA in 20μl reaction volume.2,By repeated screening, 24 ISSR primers of clear, good stability, high polymorphism of amplified bands finally were selected to use to study genetic diversity of Melia azedarach from 100 ISSR primers. DNA of Melia azedarach of 13 provenances were amplified 382 sites in total, proportion of polymorphic bands was 98.43%, proportion of polymorphic bands of Nanning of Guangxi province(NN) and Cangshan of Fuzhou city(CS) was the highest 54.97%, proportion of polymorphic bands of Yanping of Fujian province(YP) was the lowest 43.98%, the results showed Melia azedarach of 13 provenances had rich genetic diversity.3,Variation range of Number of efficient alleles(Ne) was 1.3110-1.3887 in DNA of Melia azedarach of 13 provenances, variation range of Nei's gene diversity(H) was 0.1822 -0.2277, variation range of Shannon's Information index(I) was 0.2660-0.3324, variation range of Proportion of polymorphic bands(PPB) was 43.98-54.97, these showed that geographical factors was one of the main factors which caused rich genetic diversity among Melia azedarach of different provenances. Coefficient of gene differentiation(Gst) of Hainan provenance was the highest 0.4394, the lowest 0.2899 was Guangxi provenance; variation range of gene flow was 0.6379-1.2247, and micro-geographical environment caused by the geographical provenance variation in more than 55%, was bigger genetic variation among different provenances, showed that micro-geographical environment also have a greater impact to genetic differentiation of Melia azedarach.4,Nei's(1978) genetic distance of Melia azedarach of 13 provenances was 0.0012-0.2840, cluster analysis by UPGMA showed Melia azedarach of 13 provenances can be divided into 4 categories: The typeⅠwas Baoshan, Yunnan province(BS); the typeⅡfor Mianyang, Sichuan province (MY), Nanning, Guangxi province(NN), Changjiang, Hainan province(CJ), Yongtai, Fujian province(YT), Liuyang, Hunan province(LY), Longquan, Guangxi province(LQ), Cangshan, Fuzhou(CS); Mingxi, Fujian(MX) was the typeⅢ; The typeⅣwas Jinhua, Zhejiang province(JH), Yanping, Fujian province(YP), Jianou, Fujian province(JO), Youxi, Fujian province(YX). Generally speaking, Melia azedarach of 13 provenances can be divided into 2 categories: Baoshan, Yunnan province for a large class, other provenances for other major category.5,With uniform experimental design of U7(73) of three factors and seven levels, studing the main factors of solvent concentration, extraction time and extraction temperature, which influenced Toosendanin extraction of Melia azedarach, the results found that effect of ethanol concentration was the most significant for extraction of Toosendanin; At 7 treatment combinations, extraction consequent of the seventh treatment combination was the best, extraction amount was up to 4.259mg·g-1, the optimal treatment combination of extraction was ethanol concentration 75%, extraction temperature (water bath) 50℃, extraction time 50min.6,Determination and analysis of Toosendanin found that different parts of Toosendanin content existed greater differences of 99 candidate plus tree of Melia azedarach, in 32 areas of Fujian province. Toosendanin content of branch leaves was 0.0314-0.8105mg·kg-1, Toosendanin content of the bark was 0.1600-5.9931mg·kg-1, Toosendanin content of fruit was 0.4213-8.9917 mg·kg-1, average content of total of Toosendanin was higher than content branch leaves and bark. According to different parts of toosendanin content, through cluster analysis, 99 candidate plus tree of Melia azedarach were divided into four categories, Dehua FK4, Shaxian FK53, Shaowu FK57, Shouning FK62, Shunchang FK63, Xianyou FK74, Yongchun FK84, Yongtai FK89, Youxi FK90, were elected to plus tree of Melia azedarach of high toosendanin content. East longitude, north Latitude, altitude, annual mean temperature, average annual rainfall were the main meteorological factors which were affectted Toosendanin content, cumulative contribution rate of five meteorological factors was 86.562%, but generally speaking, the meteorological factors is not significant to impact of Toosendanin content.7,Phenotypic traits of seeds of Melia azedarach of 32 provenances had significant differences in length, width and average single weight, phenotypic traits of seeds was more abundant; the order of the mean coefficient of variation of phenotypic traits of seeds from big to small: width > length > average single weight, phenotypic traits existed some variation range in different provenances, but meteorological factors on phenotypic variation of seeds does not significantly affect; length and width of seeds were the main characters of phenotypic traits of seeds of Melia azedarach, accumulative contribution rate was up to 98.14%.8,N, P, K content of Melia azedarach of 32 provenances existed the larger differences, the order from big to small was N> K> P. N, P, K nutrient elements content among Melia azedarach of different provenances had achieved a very significant level, showed that Melia azedarach of different provenances the ability of absorbing and using nutrient elements Melia azedarach of different provenances existed significant differences. meteorological factors on N, P, K content of Melia azedarach was not obvious on the overall, only N, K content of leaves separately were significantly associated with extreme low temperature and extreme high temperature of provenance areas N content of leaves with increasing latitude downward trend. Toosendanin content of Luoyuan was the smallest, the largest for Pucheng among Melia azedarach of 32 provenances, the average content of 0.2618-2.2468mg·kg-1, and N, P, K content of nutrient elements did not significantly effect to Toosendanin content.