Analysis Of Genetic Diversity Of21Kinds Of Magnoliaceae Evergreen Plants

Magnoliaceae was a primitive taxa in angiosperms, most plants in it werethe tertiary relic ancient tropical flora plant and old plant.There were16genera,300species magnoliaceae plants around the world, and China had12genera,160species[1]. Magnoliaceae plant species was very rich,and had high scientific researchvalueit. It provided precious materials for the study of origin and phylogeny ofangiosperms[2].Magnoliaceae plant was not the main constitute edificators or dominant speciesof forest community, but it was important associated species to maintain ecosystemstability, therefore magnoliaceae plants plays an important role in forest ecosystembalance. Because of magnoliaceae plants unique status and the scientific researchvalue, many domestic scholars and the vast majority of garden workers have payattention to it. And did the thorough study on its growth, wood structure, classification,determination of gene sequences, the medicinal composition and breeding[4].Magnoliaceae plants was now widely used in most of the urban landscape greening.At present, the mature theory of the urban greening demands to increase theproportion of evergreen trees in urban greening tree species. But quite part ofmagnoliaceae tree species of evergreen species could satisfy this requirement.Goodability to resist pests, make the magnoliaceae trees becomes the first choice in urbangreening landscape[5].Magnoliaceae tree species, therefore, the genetic relationshipresearch, had great economic value and ecological significanceof for the protectionand utilization of this valuable ancient plant.This study was to application markers and ISSR markers for the evergreenmagnoliaceae plants about genetic diversity and genetic relationship analysis inChina.Which provided genetic information for better protection, developed moreresources of evergreen magnoliaceae plants, breeded new variety and molecularsystematics study. The main results were as follows: (1)Magnoliaceae evergreen plants genome DNA extraction:This study aimed at the characteristics of Magnoliaceae evergreen plant leaveshad rich polyphenols and polysaccharides, optimized the CTAB method of DNAextraction. Adjusted for NaCl concentration, adding chloroform/isoamyl alcoholextraction times, enhance the ability of removal of impurities such as protein,polysaccharide; Bothered by improved CTAB method of DNA through agarose gelelectrophoresis, the results showed that the improved CTAB method could beextracted from leaves of high quality, high yield rate of genomic DNA.(2)The optimizationof Magnoliaceae evergreen plant’s AFLP marker system:This study choosed MseI for high-frequency enzyme, EcoRI enzymes forlow-frequency, determined the evergreen magnoliaceae plants genome DNA20μldouble enzyme system (10×Buffer tango4μL(fermentas); EcoR I(10U/μL)0.4μL；MseI(10U/μL)0.4μL；200ng from the template DNA, increased ddH2O to20μL.)The best enzyme digestion time was3h; The connection product without dilutedirectly to pre amplification, pre amplification program for94℃pre modified3min;94℃modified45seconds;50℃renaturation45seconds,72℃for1min,26cycles;After inspection to optimize the system of the AFLP markers could be used forgenetic analysis of21species of evergreen magnoliaceae plants.(3)The genetic analysis of Magnoliaceae evergreen plant’s AFLP marker:Based on the8primers which were choosed from previous magnoliaceae plantsmarkers primers, had good repeatability, high polymorphism:E41M51C, E41M51G,E47M51A, E47M51C, E48M51C, E49M51A, E49M51G, E48M51A. Applicated this8primers in21species of evergreen magnoliaceae plants for genetic diversity andgenetic relationship analysis and amplified out1064DNA bands, they were mainlydistributed in100-1000bp, including polymorphism with1027, the percentage ofpolymorphic loci (P) was97.37%. PopGen32software was used to analyse themarkers spectrum analysis, the result showed that the number of average effectivealleles (Ne) in each site was1.5680, Nei’s genetic diversity index (H) was0.3320,Shannon information (I) was0.4976, the highest polymorphism was E41M51Gprimer combinations, as high as98.47%, E47M51C primer combination ratio ofpolymorphic loci, the lowest was only96.27%. Article E49M51G generated, themaximum number of bands in145; Most Numbers of polymorphic sites, there were140reasons. E48M51A primer combinations produced with minimum number of only119, but the resulting bands polymorphic loci rate is as high as98.32%. Showed that genetic variation between21species of Magnoliaceae evergreen plants, there isabundant genetic diversity. UPGMA method to construct kinship relationship tree,8of21kinds of primers could be completely separated, with genetic similaritycoefficient (Sg)0.462for threshold,21kinds of them could be divided into threegroups, and showed the interspecific genetic relationship at the molecular level.(4) The optimization of Magnoliaceae evergreen plant’s ISSR-PCR reaction systemThis study for the first time used orthogonal design, discussed the templateDNA、Mg2+dNTPs、primers and TaqDNA polymerase five factors influence onevergreen ISSR Magnoliaceae evergreen plants-PCR, and used the method ofweighted average and centesimal system to rate the result of the ISSR-PCRamplification reaction system, using the DPS data processing system to analysis theexperimental data, discuss the factors and the different level influence the result of thereaction of various factors inherent regularity. Results showed that the size of thevarious factors affecting the PCR reaction, in order: Mg2+> dNTPs> TaqDNApolymerase> template DNA primers, the differences between the five factors ofdifferent levels were reached very significant level; Set up the optimum reactionsystem of pomegranate ISSR-PCR was（20μl）Mg2+mmol/L、dNTPs mmol/L、TaqDNA polymerase mmol/L、 template DNA ng. The gradient experiment wascarried out for he best annealing temperature of evergreen magnoliaceae plantsISSR-PCR reaction system, and determined the optimal annealing temperature of eachprimer.(5) The ISSR genetic analysis of evergreen magnoliaceae plants:This experiment choosed10good repeatability and high polymorphism primersfrom100ISSR primers: UBC825, UBC827, R5, UBC880, UBC840, UBC812, UBC816, UBC834, UBC835, R11,and then analysed the DNA polymorphism of21evergreen magnoliaceae plants used those10primers:8primers detected out108loci,and107of them were had polymorphic, so the ratio of polymorphic loci was99.04%and the selected21species of evergreen magnoliaceae plants had rich geneticdiversity. POPGEN1.32software were used to calculate the Shannon informationindex (I) with a mean of0.5600, the genetic diversity (H) with a mean of0.3800, theobserved number of alleles (Na) with a mean of1.9900. That’s all showed that those21species of evergreen magnoliaceae plants had higher genetic variation in theprocess of cultivation, and constituted a rich resources gene pool. Using NTSYSpc 2.10UPGMA method constructed genetic relationship tree,21were completelyseparated, and divided into four groups.（6）Guiding significance of scientific research and productionMagnoliaceae plants in China throughout south China, southwest region,andand few planted in north areas of the Yellow River coast, in order to enrich largebroadleaf evergreen plant species in north China, the experiment respectivelycollected Magnolia grandiflora L、Magnolia grandiflora L.var.Lanceolata Ait.、Michelia martinii(HLevl.)Finet、Michelia figo(Lour.) Spreng、Michelia skinnerianaDunn、Michelia fallax Dandy、Michelia foveolata Merr.ex Dandy var.CinerascensLaw et Y.F.Wu、Michelia cavaleriei Finet et Gagnep、Michelia fulgens Dandy、Michelia szechuanica Dandy、Michelia crassipes Law、Michelia wilsonii Finet etGagnep、 Michelia macclurei Dandy var.sublanea Dandy、 Michelia platypetalaHand.-Mazz、Michelia foveolata Merr.ex Dandy、Michelia maudiae、Micheliachapensis Dandy、Manglietia szechuanica Hu、Manglietia chingii Dandy、Manglietiapatungensis Hu、Manglietia duclouxii Finet et Gagnep，a total of21species ofmagnoliaceae evergreen plants in genetic diversity analysis and research in state-runnursery of NaZhao county, henan provence.And revealed the genetic basis ofmagnoliaceae evergreen plants through the experiment.Those provided certaintheoretical basis for protect, introduction and breeding of Magnoliaceae evergreenplants in north China region.