Ploidy Determination In Lagerstroemia L.using Flow Cytometry And Its Polymorphism Of CpDNA

Lagerstroemia indica is one of the significant flowering trees in Summer. It has the superiors of longer florescence ,beautiful flowers and resistance to pollution.But,it is short of varieties in actual application. While polyploidy have the advantages of bigger flower, more colourful and stronger resistibility. Polyploidy breeding of ornamental plants has been widely applied in breeding research. Polyploidy breeding research of Lagerstroemia indica is still at the start stage. The ploidy level analyses of Lagerstroemia has never reported in China. This paper determined 16 species of Lagerstroemia's ploidy by FCM(flow cytometry) , which will provide the theory guidance for the variation ,inheritance and evolution of Lagerstroemia. We also have studied the phylogenetic relationships within Lagerstroemia based on cpDNA trnT intron sequences.The results are as follows:1.In flow cytometric analysis Nuclear lysis buffers influence the integrity of nucleus which release from cytoplasmthe, different plant tissue structure and chemical composition, appropriate lysis buffers are different also. Five lysis buffers was used to extract nucleus, the best lysis buffer was obtained via experiment comparison, which is Otto buffers, LB01 is in the second place. DNA content resolution is in relation to lysis buffer, and the material.The DNA content resolution of fresh tissues are high than dry tissues's. The mechanism of the effects are for the sake of further studies.2.It's the first time to analysis ploidy by FCM with silica gel dried leaves as materials witch have never been reported because of the difficulty of extracting nucleus from dry cells. The study optimized the sample preparation methods by digesting the cell wall with cellulase and pectic enzymes . And at the same time designed the gradient experiment to ensure the enzymatic hydrolysis time ,the conclusion shows that 1h is more appropriate.3. It's the first time to determine Lagerstroemia's ploidy level by FCM .The result shows that FCM (flow cytometry) has the advantages of high speed, more accuracy and easy operation. The FCM (flow cytometry) should be used widely in the future. As a standard with diploid crape myrtle, and use Summit v4.0 diploid software (DakoCotomation company) to analysis the ploidy level of Lagerstroemia, the result shows that L.glabra, L.excelsa, L. micrantha, L.fordii , L.subcostata, L.villosa , L.venusta, L.limii , L.caudata, L.suprareticulata, L.stenopetala, are diploids .L.guilingensi is tetraploid, L.intermedia L.speciosa are hexaploid, L.balansae is aneuploidy.4. By analyzing the sequences of 16 species chloroplast trnT gene intron, found that there are 18 mutation loci, 14 information site (excluding foreign groups), GC content (27.5%), which is low. And found that the species with the same ploidy level are of very close relationship, such as L.intermedia and L.speciosa, L.glabra and L.stenopetala etc. The double Kimura value between L.venusta and L.guilingensi is 0.024.5.Using Cuphea hyssopifolia as outgroup,in the most parsimonious(MP) tree (length=82; CI=0.9534,RI=0.9755),there are four clades.The first clade include eight L.indica varieties, L.guilingensis,L.limii and L.subcostata. The second clade included L.siamica, L.villosa and L.balansae. The third clade was consiste of L.speciosa and L.intermedia. The fourth clade include only L.venusta. The genetic relationship of L.venusta with other species is the farthest, and it may be early differentiate from the other species of this genus.