Technology Of DNA Molecular Markers And Its Preliminary Application In Lotus (Nelumbo)

In this study, technology of DNA molecular markers RAPD, ISSR, AFLP, and SRAP were used to study genetic relationship of lotus {Nelumbo) . The reaction conditions and procedures of the technique suiting genetics in the lotus were optimized and perfected. All the markers except RAPD were applicated for the first time in the lotus. The lotus materials used in the research include two parts: twelve accessions and thirty-eighty accessions.The twelve accessions contain Asian lotus, America lotus and their offsprings of hybridization, and Asian lotus include Chinese lotus, Japan lotus. The tested Chinese lotus are wildly planted and mainly rhizome lotus. 12 RAPD primers showed 128 fragments, 10.67 fragments on average per primer, 106 polymorphism fragments (82.81%); 7 primers of ISSR showed 91 fragments, 13 fragments on average, 75 polymorphism fragments (82.41%); 2 pairs of AFLP primers produced 72 fragments, 36 fragments on average, 64 polymorphism fragments (88.89%); 3 pairs of SRAP primers obtained 67 fragments, 22.3 fragments on average, 60 polymorphism fragments (89.55%). Establishing tree of evolution with UPGMA, the results obtained by four kinds of clustering indicated that 12 species with ISSR, AFLP and SRAP were clustered into three groups, America lotus, Asian lotus, and the hybrids of America lotus and Asian lotus. In Asian lotus, there were some differences between ISSR and AFLP, SRAP clusterings. For the result of ISSR clustering, Japan lotus - Nelumbo nucifera cv. Ohga lotus was clustered with Chinese lotus together, that's no circumscription obviously. For the results of AFLP and SRAP clusterings, Japanese lotus - Nelumbo nucifera cv.Ohga lotus and Chinese lotus has obvious circumscription. But the result of RAPD analysis showed that the hybrids of America lotus and Asian lotus are in the same group as Asian lotus, another group America lotus was independent. In the results of the SRAP analysis, some accessions could not be distinguished. Compared results among four molecular markers, RAPD, ISSR, AFLP, SRAP analyses and pedigree ofthe lotus, those of the AFLP analysis were the most consistent with their pedigree.Thirty-eight accessions belonging to two species of the Nelumbonaceae were analysed with PCR-based markers to determine their genetic relationships. RAPD analyses using 19 primers gave 212 fragments while ISSR analyses using 13 primers produced 164 fragments. Variability within the accessions was detected. Two dendrograms based on the Jaccard similarity index showed the genetic relationships in Nelumbo. Using RAPD and ISSR assay, both of the analyses showed a high Jaccard similarity coefficient between Nelumbo nucifera Gaertn. and Nelumbo lutea (Willd.) Pers., which was 0.656 and 0.785, respectively, suggesting that there is not distinct divergence between Nelumbo lutea and Nelumbo nucifera. Therefore, our results suggested that Nelumbo lutea should be considered to be a subspecies of Nelumbo nucifera. Our results also indicated that Japanese lotus might originate from Chinese lotus.