ISSR And RAPD-based Genetic Diversity Detection, Fad Gene Cloning And Agrobacterium-mediated Genetic Transformation In Three Economic Plants (Rehmannia Glutinosa Libosch, Dioscorea Opposita Thunb And Glycine Max Merr)

The objectives of this dissertation are: Ⅰ .to establish an efficient ISSR and RAPD-based genetic diversity detection system of three economic plants (Rehmannia glutinosa Libosch, Dioscorea opposita Thunb and Glycine max Merr); Ⅱ .to establish an appropriate transformation and regeneration protocol for Rehmannia glutinosa Libosch .f. hueichingensis(Chao et schih) Hsiao; Ⅲ.to clone the Oleoyl- △12-desaturase gene from Glycine max Merr and to construct its antisense expression vector in order to lay the theoretical and technological foundation for the DNA marker-based cultivar identification and improvement in three plants as well as the genetic breeding via Agrobacterium-mediated transformation in Glycine max Merr and Rehmannia glutinosa Libosch .f. hueichingensis(Chao et schih) Hsiao.Ⅰ .RAPD and ISSR markers were used to investigate the genetic diversity of three economical plants, ISSR marker of which was used for the first time.The main progresses obtained are as follows: (1).By means of CTAB method, the DNAs were extracted from 10 cultivars (lines) in Rehmannia glutinosa Libosch, 28 cultivars in Dioscorea opposita Thunb, 10 cultivars in Glycine max Merr and 16 individuals in Rehmannia glutinosa Libosch .f. hueichingensis(Chao et schih) Hsiao, respectively. CTAB method for the DNA extraction from Dioscorea opposita Thunb was improved. (2).The genomic DNA, from a young regenerated plant of Rehmannia glutinosa Libosch .f. hueichingensis (Chao et schih) Hsiao, was used as a template to optimize ISSR-PCR amplification conditions for Rehmannia glutinosa Libosch as follows: proper annealing temperatures from 53℃ to 55 ℃ and a PCR reaction volume of 25μL, including 1.0-1.5 U Taq DNA polymerase, 3.0 mmol/L MgCl2, 1 x Taq DNA polymerase buffer (10mmol/L Tris-HCl, 50mmol/L KCl, 0.1% Trion X-100, pH9.0 ), 60ng template DNA, 0.4μm mol/L primer, 0.4 m mol/L each of dATP, dGTP, dCTP and dTTP. (3). Based on the optimized conditions, among 44 ISSR primers and 80 RAPD primers tested, the 10,7 and 8 ISSR primers were selected for the ISSR analyses in Rehmannia glutinosa Libosch, Dioscorea opposita Thunb and Glycine max Merr, respectively; 17 and 2 RAPD primers were done for the RAPD analyses in Rehmanniaglutinosa Libosch and Dioscorea opposita Thunb. (4).Among the 10 cultivars in Rehmannia glutinosa Libosch, the 10 ISSR primers amplified 110 bands with a percentage of polymorphic bands (PPB) of 71.82% and 17 RAPD primers amplified 177 bands with one (PPB) of 61.58%. The genetic diversity, estimated by Shannon's index, was 0.3577 by ISSR markers and 0.3135 by RAPD markers. Genetic similarity (GS) value was from 0.557 to 0.979, whose mean value was 0.665 by ISSR markers. GS value range was from 0.63 to 0.93, whose mean value was 0.7545 by RAPD markers. Furthermore, the correlation coefficient of 0.649 between RAPD and ISSR markers indicated that both markers were significantly correlated. Among the 16 individuals, the 2 ISSR primers amplified 17 bands with one (PPB) of 64.71% and 3 RAPD primers amplified 7 bands with one (PPB) of 57.14%. (5). Among the 28 cultivars, the 7 ISSR ISSR primers amplified 65 bands with one (PPB) of 83.01% and 2 RAPD primers amplified 23 bands with one (PPB) of 82. 6%. The genetic diversity, estimated by Shannon's index, was 0.4379 by ISSR markers. GS value range was from 0.33 to 0.96, whose mean value was 0.6246 by ISSR markers. (6). Among the 10 cultivars in Glycine max Merr, the 8 ISSR primers amplified 89 bands with one (PPB) of 62.5%. The genetic diversity, estimated by Shannon's index, was 0.2865. GS value range was from 0.60 to 0.75, whose mean value was 0.6912 by ISSR markers. (7). 10 Rehmannia cultivars (lines) could be divided into two similar groups by the dendrograms performed by both markers; 28 yam cultivars 10 soybean cultivars could be divided into four groups and 10 soybean cultivars could be done into two groups by the dendrograms performed by ISSR markers. (8). Primary content analysis was employed to evaluated the resolving power of the markers to differentiate between the cultivars.,whose results were very consistent to that of the above dendrograms.The results revealed that the quality of the extracted DNAs reached to the standads of both markers .the optimized reaction system was appropriate for ISSR analysis; Both markers could not only detect a quite high PPB and Shannon'index (I) among different cultivars in each of these three plants but among different individuals in Rehmannia glutinosa Libosch .f. hueichingensis (Chao et schih) Hsiao. However, different plants had different PPBs, Is,GS value ranges and mean GS values.The genetic diversity(PPB and I) among different cultivars in Rehmannia glutinosa Libosch was higher that among different individuals in Rehmannia glutinosa Libosch .f. hueichingensis (Chao et schih) Hsiao. Among the three plants' PPBs and Is by ISSR markers, Yam's PPB and I were the biggest,and Rehmannia's PPB and I were gigger than soybean's; Yam's PPB and I were higher than Rehmannia's by RAPD markers. While the result "yam GS value range > Rehmannia GS one>soybean GS one" was revealed , the result "yam mean GS value range < Rehmannia mean GS one