Genetic Diversity Of Chimonanthus Praecox (L.) Link Based On ISSR Markers

Chimonanthus praecox(L.)Link,the special plant of China,is one of the famous and precious flower plants which have long cultural history.It has a broad distributing and many cultivars.In this study,98 cuitivars and 12 wild materials were used to analyse genetic diversity and phylogentic relationship by ISSR and morphological markers.The main results were presented as following:1.A quantitative taxonomic study was conducted on 110 different Chimonanthus praecox(L.)Link materials.Results show that the form of the middle-perianth is served as the frist classification criteria,the flower shape and the bloom color have important effects on cultivars classification.2.Different sample storing methods were employed to extract genomic DNA of Chimonanthus praecox in order to make clear how the storing methods affecting the DNA quality.The results showed that,the silica gel dehydrated desiccation preservation was a good method for sampling from open country or faraway place.3.An optimized PCR reaction system for ISSR was established,optimum ISSR reaction system of Chimonanthus praecox was every 20ul reaction system containing 1×PCR buffer,2.5mmol.l^-1MgCl₂,0.2 mmol.l^-1dNTP,0.3u mmol.l^-1primer,30ng DNA template,1U Taq DNA polymerase,the amplication was 1 cycle after initial denaturation at 94℃for 5 min,follwed by 35 cycle of 1 min at 94℃,1 min annealing at 52～58℃(depending on different primers),1 min 10 seconds at 72℃,and a final 10 min extension at 72℃,After which the samples were held at 4℃.4.Fifteen primers with good,clear bands were got,screening from one hundred ISSR primers.These fifteen primers were used to amplicated respectively the genomic DNA of 110 different Chimonanthus praecox(L.)Link materials.And a tatal of 100 bands were amplified with 60 polymorphic bands and precentage of polymorphic bands(PPB)60%.The average value of observed number of alleles, average value of effcetive number of alleles,Nei's gene diversity and Shannon's information index were 1.5900,1.3357,0.1936,0.2892 respectively.Clustring of UPGMA analysis indicated that the cultivars and wild materials and were separated into 2 categories respectively,And the cultivated species were divided into 2 categories according to the bloom color.It also shows the form of the middle-perianth has an important effect on classification.Compare dendrogram by ISSR maeker with dendrogram by morphological marker.we can find that they not only have obvious difference but also have some resemblance.