Identification And Molecular Marker Study Of Characteristic Trait Of High Aroma In Nicotiana.tabacum L.

Tobacco leaf is necessary for the tobacco industry and a kind of hobby and special merchandise. The evaluation of tobacco leaf quality ultimately depends on the consumers' taste. Therefore, aroma plays an important role in evaluating the quality of tobacco leaf and how to improve aroma of tobacco leaf is still the main direction of breeding objective in many advanced countries with tobacco research and industry. There are three propose in this study. The first porpose is to identify the characteristic trait of high aroma at the biochemical level. The second propose is to screen or mapp the genetic markers/loci which linked with gene of high aroma; The third propose is to study the application of the characteristic trait of high aroma in tobacco breeding. Therefore, A lot of tobacco plant materials include flue-cured tobacco with special aroma, flue-cured tobacco with normal aroma, oriental tobacco, sun/air-tobacco are analyzed by a series of biochemical and genetic study with many powerful technologies such as gas chromatography, molecular marker, bioinformatics computing, etc. the results are as follows:1.To genetic map the gene of characteristic trait of high aroma by SSR and TRAP, three genetic populations include F2, BC1, DH were constructed with tobacco cultivars DBJ-599 and K326 because of the obvious difference at target trait. The sensory evaluation, genetic mapping and segregation distortion analysis were adopted. The results confirmed that characteristic trait of high aroma linked to a SSR marker with genetic distance of 8.37cM and showed mendel qualitative heredity.2. The aroma components among main cultivar types of N.tabacum L. including common flue-cured tobacco, flue-cured tobacco with high aroma, oriental tobacco and sun/air-cured tobacco are analyzed by Gas Chromatography. Fifty-two aroma components are detected and 6 components are significantly correlated with type classification of tobacco. Using the 6 aroma components, flue-cured tobacco with high aroma, oriental tobacco and sun/air-cured tobacco clustered together and common flue-cured tobacco come into a single cluster. To evaluate the aroma phenotype of the materials in this study, sensory evaluation is developed and the results showed that flue-cured tobacco with high aroma, oriental tobacco and sun/air-cured tobacco all possess a characteristic aroma of oriental tobacco-like while common flue-cured tobacco didn't, which is coincident with the cluster analysis. Therefore, we analyze the aroma components comparatively between the two clusters and there are 26 aroma component detected significant difference at 0.05 or 0.01 levels, respectively. Except palmitic-acid and 2-methyltetrahydrofuran-3-one, the others 24 contents of aroma components in flue-cured tobacco are lower than other tobacco types. This study provides biology and applied chemistry research foundation for tobacco types classification and special aroma detection.3. The silver staining as a kind of DNA staining methods is important for molecular marker study. In order to evaluate the application of Bassam and Sanguinetti silver staining methods in the novel molecular markers SRAP and TRAP study. The two kinds of silver staining are compared in the detecting efficiency, expends and the images of silver staining with levels by Photoshop computer software. The results indicated that the Sanguinetti silver staining method is simpler, more economy, and easier to distinguish the bands of PCR amplication from background than the Bassam method. It also showed that the Sanguinetti silver staining method has a very good effect on SRAP and TRAP molecular markers.4. Many difficulties existed in the development of TRAP markers in former studies which influenced the application of TRAP in molecular marker analysis. Therefore, we adopt a new approach that taking the primers of EST-SSR as the fixation primers of TRAP. We find a co-dominant heredity TRAP marker which amplifies a characteristic band about 400bp. The premium results show that it linked with the gene of characteristic aroma of oriental tobacco-like through screening of Flue-cured tobacco, oriental tobacco and sun/air-cured tobacco varieties. This characteristic TRAP marker can be used in marker assisted selection (MAS) directly or transformed to SCAR marker. We also can develop novel aroma genes based on this characteristic band based on sequencing.5. We invent a new approach for ILP marker developing. According to the approach, firstly, searching primer pairs based on EST database. Secondly, develop e-PCR with the primer pairs on genomic sequences. Thirdly, compare the sequence length from ESTs and genome and search the gap between them. Finally, ILP markers come into being. In order to improve the progress as well as automation level, a software is programed by C# computer language. Based on above study, we investigate 6,859 potential ILP markers and 207 ILP markers are synthesized. Solanaceae ACGMs explonation are also studied.