Cytology Analysis And ISSR Molecular Identification Of Germplasm Resources Of Chongming Narcissus

Chongming narcissus belonged to perennial herbaceous plant of genus Narcissus Linn, and family Amaryllidaceae, with Zhangzhou narcissus, Putuo narcissus, Pingtan narcissus in Fujian and Zhoushan narcissus in Zhejiang referred to as one cultivation type of Chinese narcissus(Narcissus tazella Linn.var. chinensis Roem.), which had cultivation history of 400 years in Chongming county. Chongming narcissus had characteristics of elegant floral and long period of water cultivation and florescence, especially multiple-valve flower Chongming narcissus was famous for its full-bodied floral at domestic and overseas flower market as British rose. But now, the effects of rarely cultivated varieties, intermixed with species, species qualification to degenerate,quality decline and viral disease occurrence made Chongming narcissus on the edge of extinction. In 2007, Shanghai city already listed Chongming narcissus in Shanghai special rescue plan. In this paper, we researched the cytology background, reaction' system of ISSR-PCR and the molecular identification of germplasm resources of Chongming narcissus, so as to determine high quality provenance, improve corm and pattern, regulate florescence, formulate feasible cultivation and breeds measures, and to lay the foundation for popularizing and applying of Chongming narcissus. The results were as following:1. The cytology background of Chongming narcissus was studied through the karyotype analysis method. Found that could get more metaphase cells using of p-dichlorobenzene saturated solution to pretreat the roots of Chongming narcissus in the process of production, and appropriate time of pretreatment is 12h. Chongming narcissus-. Zhangzhou narcissus and variety Kwame brown narcissus of Zhangzhou narcissus are triploid, the somatic chromosome number were 30(2n=3x=30), all with one pair satellite chromosome on the 7th group chromosome; The karyotype formula of multiple-valve flower Chongming narcissus is 2n=3x=30=15st+15sm(3SAT), the karyotype formula of univalve flower Chongming narcissus is 2n=3x=30=15st(3SAT)+15sm, The karyotype formula of multiple-valve flower Zhangzhou narcissus is 2n=3x=30=15st+15sm(3SAT), the karyotype formula of univalve flower Zhangzhou narcissus is 2n=3x=30=15st(3SAT)+15sm, The karyotype formula of Kwame brown narcissus is 2n=3x=30::=15st+15sm(3SAT), which belonged to 3B type. Compared the karyotype of Chongming narcissus and Zhangzhou narcissus, result shows that they are segment heterologous triploid.2. Through analysis of molecular markers of Chongming narcissus ISSR, display that Chongming narcissus genomes exist very rich (CT) microsatellite, to provide reference basis for the design and synthesis of primer on the following molecular level of Chongming narcissus; The ISSR-PCR reaction system optimized by orthogonal design is more stable than the one optimized by single factor, according to orthogonal design optimization, the ISSR-PCR reaction system of Chongming narcissus is:30 ng formwork DNA,1U Taq DNA polymerases,0.5μM primer,0.3 mMdNTPs and 2.5μ100 x PCR buffer in 20μl reaction system, and the best annealing temperature is 56.8℃; PCR product map shows that obtains 115 ISSR makers loci through augmentated, including 36 polymorphisms loci which is 31.3% of total loci, the average Nei's similarity of coefficient of Chongming narcissus and Zhangzhou narcissus is 0.965, so they have relatively closer relationship.Based on the research results, proposal to use colchicine to double the organizations and organs of Chongming narcissus, induced to produce mutations, using transgenic means to transfer other floral-color genes into chongming narcissus, and expected to get Chongming narcissus new varieties which made bulb increase, side feet and flower branch increase, rich floral-color and floral organ enlarge.