Studies On Variety Classification Of Loropetalum Chinense Var.rubrum Based On AFLP And SRAP Analysis

Loropetalum chinense var.rubrum belongs to Hamamelidaceae Rubrum,a native of Hunan unique resources.But so far,identificating the variety and quality of Loropetalum chinense var.rubrum was based on the main method for its botanical characteristics,relying on the external shape for judgement.Therefore,among the Loropetalum chinense var rubrum seedlings market often appears the same name with foreign body,or with the synonym of imagination.The DNA was directly tested by the molecular markers,in a wide range of higher organisms,the genetic material for a more comprehensive and detailed comparison.This study used AFLP and SRAP technology to research 22 Loropetalum chinense var.rubruand,one L.chinense,one L.chinense var.semper rubrum and one L.chinense var.auratum total of 25 copies of the genetic diversity,classification and relationship.Aims to provide a more scientific method for the evaluation and classification,and providing a more scientific,effective and practical method for the collection of Loropetalum chinense var.rubrum protection and rational development,as well as Loropetalum chinense var.rubrum further research and breeding Selection of work to lay a solid foundation, access to the key findings are as follows:(1) A reliable and effective system for DNA extraction detecting AFLP and SRAP was developed.The studies were made on DNA of Loropetalum chinense var.rubrum obtained from the young leaves,mature leaves,capsule.By using the improved methods of CTAB with Tris-HCl clean,CTAB,SDS with Tris-HCl clean and SDS.The results show that high molecule weight DNA and AFLP fingerprint can be obtained from these organs by using the CTAB+Tris-HCl methods.DNA extracted from young leaves of high purity but in high concentrationat,The young leaves were inconvenient to get,using CTAB method by Tris-HCl washing of DNA extraction from mature leaves purity and concentration can be guaranteed.(2) A reliable,effective and reproductive PCR reaction system for detecting SRAP was developed.It was shown that SRAP technology is a useful molecular marker system for mapping and gone tagging in Loropetalum chinense Var.rubrum.Each 50μL PCR reaction mixture consisted of 150ng of genomic DNA, 1.0mmol/L of dNTPs,1.6 mmol/L of MgCl₂,2.0μmol/L of primer and 3.6 unit of Taq polymerase.Samples were subjected to thermal profile for amplification in an oven thermocycler:4 min of denaturing at 94℃,five cycles of three steps;1 min of denaturing at 94℃,1 min of annealing at 35℃and 1 min of elongation at 72℃.In the following 30cycles the annealing temperature was increased to 55℃,with a final elongation step of 5 min at 72℃.(3) Eight primer combinations selected from 64 primer combinations produced a total of 1226,of which 1085 were polymorphic with a polymorphism percentage of 88.5%.Using Ntsys2.10 get An UPGMA dendrogram and PCA Charts based on the AFLP alleles showed that the similarity coefficients among 25 Loropetalum chinense species was between 0.6819 and 0.8975,Average is 0.7390.The distance between "Meihongchangyeqing" cultivated species and "Meihongchangyeqing" wild species was the most similar at the similarity coefficients of 0.8975,The distance between "Mizhilianghong" and "Meihongchangyeqing" cultivated was the most different at the similarity coefficients of 0.6819.all the materials were divided into2 groups with the threshold of 0.68 of similarity coefficient,5 groups with the threshold of 0.74 of similarity coefficient.8 groups with the threshold of 0.7580 of similarity coefficient.At the same time,the first group was divided into 5 subgroups with the threshold of 0.7910 of similarity coefficient.The result was shown that the diversity and complexity of the Loropetalum chinense var.rubrum.(4) Twelve primer combinations selected from 110 primer combinations produced a total of 165,of which 146 were polymorphic with a polymorphism percentage of 88.4%.Using Ntsys2.10 get An UPGMA dendrogram and PCA Charts based on the AFLP alleles showed that the similarity coefficients among 25 Loropetalum chinense species was between 0.5714 and 0.8927,Average is 0.7549.Average is 0.7549.The distance between "Mizhilianghong" and "Dayuanyeshuangmianhong" was the most similar at the similarity coefficients of 0.8927.The distance between "Dayejuanbanhong" color leaves and "Meihongxiyeqing" cultivated species was the most different at the similarity coefficients of 0.5714.all the materials were divided into 6 groups with the threshold of 0.7253 of similarity coefficient,8 subgroups with the threshold of 0.7565 of similarity coefficient.(5) Joint the original data of AFLP and SRAP together,Using Ntsys2.10 get An UPGMA dendrogram and PCA Charts based on the Joint-data.Compared the three groups of analysis,found that the CI_M index was 0.51398 between Joint-data analysis and AFLP analysis,which means that AFLP analysis gets more information than SRAP analysis.However,the variation in the identification of material on the SRAP analysis is better than AFLP analysis.Compared with morphologic markers,the genetic diversity is not entirely related to the diversity,so rely on the identification of morphological classification system is not perfect.(6) Using Joint-data to analysis the relationship among the mutation of Loropetalum chinense species,the order of evolutionary degree:L.chinense and L.chinense var.semper rubrum was nearest,than the L.chinense var.auratum,the L. chinense var.rubrum was the farthest.