| Ophiopogon japonicus(L.f.)Ker-Gawl. is a perennial herb which is belongs to Ophiopogon of Liliaceae, and recorded in different versions of Chinese Pharmacopoeia. The underground block root is used as Nourishing Yin herbs, and it is sweet, a little bitter, slightly cold. Dwarf lilyturf mainly distributed in Sichuan, Zhejiang and other places in China. This paper studies the genetic diversity of 22 wild and 2 cultivated O. japonicus resources in Sichuan and Chongqing by morphological markers, RAMP molecular markers and ITS sequence. The main aim of this research is to reveal the abundance of genetic basis to discuss the relationship between wild and cultivated speices, and to provide theoretical basis for breeding excellent varieties and protecting the O. japonicus resources. The main results were showed as follows:1.8 main biological characters of 24 O.japonicus materials were studied, the results showed that there were different degrees of variation between the morphological characteristics of different materials, the varieties were significant in root number, the root length and root diameter was the least (0.20). Principal component analysis showed that root diameter and leaf width were the characteristics trait for O. japonicus. The result of Q cluster analysis showed that we can separate O. japonicus of different region by morphological characteristics, and there was no correlation between clustering and its geographic sources. The material (No.1) which was from Chongqing Fuling was clustered into a separate category because of its differences with other materials, and the rest materials were clustered into another category. The morphological characteristics of two cultivated species were very similar, they were always clusted together as a class. O. japonicus resources of Sichuan and Chongqing have abundant genetic variation, gene resources and excellent genetic diversity, it can provide basic materials for breeding excellent varieties and improving the genetic of germplasm resources for O. japonicus.2.19 RAMP primers combinations selected from 50 primers combinations were used to amplify the 24 O. japonicus materials and amplified 235 bands at all, of which 223 were polymorphic and the polymorphic rate was 94.9%. The genetic similarity coefficient of 24 O. japonicus materials was 0.699. So the O. japonicus resources has high polymorphism and abundant genetic diversity (H=0.373). The O. japonicus materials can be divided two categories and four small classies by useing UPGMA, and the cluster of 24 O. japonicus materials does not contact with their geographical origin closely, but there was a higher connection with their morphological characteristics.14 populations, divided by the source (municipal unit) of 24 O. japonicus materials, we can know Ht (total gene diversity) was 0.369 and Hs (gene diversity within populations) was 0.623 through the analysis, which most of the mutations occur between the populations. Gst (genetic diversity inter populations) was 0.623, which the genetic differentiation within populations was smaller than between populations. Gene flow among populations (Nm) was 0.302. There were strong differentiation in populations and it could be the causes of high degree of genetic differentiation. Cultivated O. japonicus was in low genetic diversity and have a small genetic variation, there were genetic differences between with wild O. japonicus resources.3. ITS analysis in the 24 O. japonicus materials of different regions showed that the length of ITS sequence was 692-707 bp, ITS1 and ITS2 sequence length were 257-276 bp and 261-273 bp, and 5.8S rDNA sequences district length was 163～164 bp. The order of the proportion of each base size was G(32.6%)>C(31.6%)>A(19.8%)>T(16%), and the G+C percentage of ITS sequence was ranging from 56.2 percent to 68.5 percent. There were 714 bp in tota through sequence alignment, including 223 variable,187 parsimony informative and 36 singleton sites. The genetic differentiation distance of O. japonicus materials were ranged from 0.001 to 0.245, with a mean of 0.086. Phylogenetic tree based on ITS sequence, the results showed that the length of the phylogenetic tree branches was 0.903. All of O. japonicus samples can be effectively separated and they were divided into two categories, each containing 12 materials. Clustering result showed that two cultivated species and No.20 O. japonicus were clustered into one clade, this illustrate that there was close relationship between the two cultivated species than with other wild O. japonicus resources except No.20. The classification result of phylogenetic tree based on ITS sequence has relationship with the morphological characteristics, and has no obvious relation with the geographical distribution. |
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