Analysis On Phylogenetic Relationship Of Ornamental Fritillaria Based On ISSR Markers

Fritillaria L. belongs to Liliaceae and is important officinal resource in China. It has great ornamental appreciation to be grown in courtyards and used to decorating environments, sometimes to making miniscape. Because the distribution of Fritillaria L.is abord, heteromorphosis mong species is very complex and character cross is very common, its heredity background is complex. In this study, we analyzed the phylogentic relationship of ornamental fritillaria by ISSR and morphological markers. The main results were presented as follows:1. Analysed 13 cultivars, phylogenetic relationship of ornamental fritillaria by morphological marker.We choosed 12 quantitative multistate characters and 7 qualitative multistate characters to observe biology characters and phenological period. Analysis indicated that the 13 cultivars were separated into 2 categories.2. Different sample storing methods and extaction methods were employed to extract genomic DNA of ornamental fritillaria in order to make clear how the methods affecting the DNA quality.The results showed that the silica gel dehydrated desiccation preservation was a good method for sampling from faraway place.3.An optimized PCR reaction system for ISSR was established, optimum ISSR reaction system of ornamental fritillaria was every 20ul reaction system containing 1×PCR burrer, 10ngDNA template, 0.5μmol·L-1primer, 2.5mmol·L-1 Mg2+, 0.20 mmol·L-1dNTP, 2UTaq DNA polymerase, the amplication was 1 cycle after initial denaturation at 94℃for 5min, follwed by 38 cycle of 40 seconds at 94℃,45 seconds annealing at 52-59℃(depending on different primers), 1.5min at 72℃, and a final 8 min extension at 72℃, after which the samples were held at 4℃.4. Analysed 22 cultivars, phylogenetic relationship of ornamental fritillaria by ISSR marker.13 primers were screened from 100 arbitrary ISSR primers. A total of 178 DNA fragments ranging from 200-2000bp were amplified.The average number of DNA band produced by each primer was 12.3. According to the result of PCR, the genetic similarity analysis for 22 cultivars was calculated by the software of NTSYSpc-2.1e. The result of genetic analysis showed that the Jaccard coefficient ranged from 0.2338 to 0.8788. When the similarity coefficient value was 0.50, the 22 cultivars of ornamental fritillaria were divided into 2 groups. The species in one sect had closer phylogenetic relationship. Compare dendrogram by ISSR marker with dendrogram by morphological marker, we can find that they not only have obvious difference but also have some resemblance.