Establishment Of Sequence Characteristic Amplified Region Markers Of Tobacco And Application Research

In this study, for the materials with cured leaves,RAPD marker was converted intoSCAR marker,established tobacco SCAR marker technology transformation method, andapplied the tobacco SCAR technology constructed tobacco SCAR fingerprint library, get thefollowing main conclusions:(1) The optimization of genomic DNA extraction method. The single factor optimizationresults showed that the volume of CTAB precipitation buffer was1.5volume of DNA solution,cell lysates time was40min, the Tris-Phenol extracted once, the RNase A（10mg/mL，1μL）hydrolysed10min in37℃,the cured leaves genomic DNA electrophoretic band wassingle,bright and clear, the ratio of OD260/OD280was1.7～1.9,the concentration reached to382.500ng/μL,and was suitable for PCR analysis.(2) RAPD primers screening.RAPD analysis showed4～14bands obtained from everyprimer,for example,primer L112amplified14bands and8bands were polymorphicfragments.15primers can obtain steady fingerprints for identification Tobacco cultivars andthe polymorphic loci can converted into SCAR marker.(3) The genetic relationship analysis. Variation range of genetic distance among12cultivars was between0.1875and0.8421with an average of0.5721, Among the15tobaccospecies, genetic distance between Red-flower Dajinyuan and NC102was minimum of0.1875,maximal of0.8923between Yunyan87and Longjiang911. The cluster analysis showed that12tobacco species were divided into3clusters based on genetic distance coefficient of0.57.ClusterⅠcontained8species and clusterⅡcontained3species, KRK26clustered one clusterlonely.(4) RAPD marker converted into SCAR marker. Taking a specific and repeatable RAPDmarker L34313bpas an example, the specific DNA fragments were cloned and sequenced.Based on the sequencing, primer pairs were designed and the SCAR markers for RAPDmarker L34313bpwere obtained, and established tobacco SCAR marker technologytransformation method.(5) Application of tobacco SCAR technology. Based on detected difference in SCARphenotypes, the molecular ID and distinguishing flow of tobacco cultivars were obtained, Theresults indicated that the6SCAR markers not only can differentiate of tobacco cultivars with lesser genetic distance or the same resistance or susceptible character accurately and quickly,but also can differentiates every cultivar from others.