Analysis Of Phylogenetic Relationship Among Several Varieties In Narcissus Tazetta L. Using Molecular Biological Approaches

Narcissus is a member of Narcissuses in Amaryllidaceaes, which has very high ornamental characters and worth. Narcissus tazetta L. is the native species of FuJian. The major cultivar is China narcissus (N. tazetta var. Chinensis Roem.), which has white corolla and light yellow vice-corolla. The white-flower species and yellow-flower species are also cultured. The studies on morphology characteristics and chromosome karyotypes analysis had been done to find the phylogenetic process and relationship among them, but these results lacked convincingness for the shortage of molecular evidence. Seven narcissus species were used in this experiment. The results of peroxide isozyme and RAPD showed that the relationship among them, which support the major viewpoint of traditional classification. The origination of triploid narcissus was studied through in situ hybridization technology. In addition, microdissected and microcloned systems of narcissus were set up; the single chromosome was amplified and generated chromosome-special libraries. Main results and conclusions are the following:1 The analysis of peroxide isozymeSeven species of narcissus tried to gather into three kinds of cluster pictures by the analysis of peroxide isozyme. III-1(bicolor flower, 2n=30)and III-2(bicolor flower, 2n=30) were grouped together; II-1 (yellow flower, 2n=20) and II-2 (yellow flower, 2n=30)were grouped together;I-1 (white flower, 2n=22), I-2(white flower, 2n=22) and III-3(bicolor flower, 2n=32) were grouped together. The analysis of peroxide isozyme could better deduce the distances of relationships among these types than the analysis of morphology.2 The analysis of RAPDBased on the polymorphic hands amplified by RAPD, seven cultured species of narcissus were divided into four groups. III-land III-2 were grouped together; II-1 and II-2 were grouped together;I-1 and I-2 were grouped together. The last group consisted of III-3. It could objectively reflect the relationships and evolution among 7 variety types than the analysis of peroxide isozyme and morphology.3 The analysis of in situ hybridization1) The digoxigenin-dUTP labeled probes of 45 S rDNA and 5S rDNA were probed to the metaphase chromosomes which were prepared from the root tips harvested from I-1, II-1and III-3. The relationship of the three species wasdeduced.2) With II-1 and 1-1 genomic DNA as probes, they proved that III - 3 of the chromosome groups were included one genome of x =10 and two genomes of x =11.This results illustrated that III-3 is autoalloploid composed of one genome of x = 10 and two genomes of x=11.3) With II-2 ( 2 n =30) genomic DNA as probes, there were 10 pieces of chromosome have hybridization signals in the narcissus of III - 3 too. It had offered evidence that II-2 of narcissus was the homology triploid of II-1.4 Microisolation and microcloning of single chromosome in narcissus1) Using the roots of narcissus 1-1 as material, many well-scattered metaphase chromosomes were acquired. The single chromosome of middle centromere in 1-1 of narcissus was successfully microisolated by glass needles with the help of micromanipulation equipment.2) The isolated single narcissus chromosomes were amplified by LA-PCR. After two rounds of PCR amplification, single chromosome DNA pools with fragments ranging from 500bp and 2500bp was acquired.3) The second-round PCR products of single chromosome were cloned into plasmid vectors to generate chromosome-special libraries. Clones randomly selected for size evaluation showed that the size of the inserts in the library varied from 600-2500bp.