Study On Genetic Divergence Of Hainan Common Wild Rice(Oraza Rufipogon) And Shanlan Upland Rice(Oraza Sativa) Based On ITs Analysis

In this study, we sequenced Internal Transcribed Spacer (ITS) of different population of Hainan common wild rice(Oraza rufipogon) and Shanlan upland rice (Oraza sativa). By DNA sequence analysis, we attempted to construct the phylogenetic tree and establish a molecular marker to distinguish Indica and Japonica rice subspecies, so as to constitute a new research method of the oryza genus and promote the understanding of the genetic background of the primary Oraza rufipogon and shanlan upland rice and offer a molecular genetic marker for the evaluation and utilization of rice genetic resources. Main results are showed as follows:1. Sequences of the entire ITS region, including ITS1, ITS2, from the experimented materials was amplified, cloned and sequenced. The range of length of5.8S was86bp to169bp. There was no significant different of GC content in this fragment. The sequences of ITS1were from163bp to239bp. The GC contents were44.1%-77.2%. The sequences of ITS2ranged from165bp to243bp. In size and G/C content varied from44%to71%. Mutations sites of ITS2were significantly higher than that of ITS1.2. ITS sequences of the experimented materials were reconstructed by the Molecular Evolutionary Genetics Analysis version5(MEGA5) for building sequence alignments and phylogenetic trees. Of the aligned positions, there were358informative sites (124in ITS1,157in ITS2and77in5.8S). Nucleotide diversity (Pi) was0.42671in5.8S and0.6327in ITS of rDNA. It had indicated that the population genetic diversity level of the ITS region in Oraza rufipogon were higher than those between Oraza rufipogon and cultivated rice. Maximum-Likelihood phylogenetic tree based on ITS sequenceing revealed that30species of wild rice and shanlan rice varieties was clustered together by indica-japonica differentiation. The grouping of the lines was not in accordance with the available pedigree date on their geographic origin and rice regions. Phylogenetic analyses also revealed those seven common wild rice populations and one shanlan upland rice variety had a closed phylogenetic relationship with indica rice. Nineteen matericals and two materials of shanlan upland rice had a closed phylogenetic relationship with japonica rice.3. In this study, the informative sites of ITS1and ITS2account for30.7%and39.6%of the total length respectively in common wild rice, and account for29.4%and32.6%in shanlan upland rice. Seventeen subspecies differentiating sites were screened betteen common wild rice and shanlan upland rice. The feasibility of the investigation of relationships between species in the genus was confirmed by using highly and moderately repetitive DNA sequences, rDNA ITS.4. Comprehensive analysis of mutation distribution and secondary structure of RNA of ITS region between shanlan rice and the common wild rice. The results showed that constrasting with shanlan upland rice, there was a great diversity of ITS region among the common wild rice. Four ITS variation types were found in common wild rice, including two variation types in shanlan upland rice. Analyses of secondary structure of RNA of ITS showed that ITS2sequence is very conservative in the secondary structure of RNA. Specific variation types from shanlan upland rice and two conservative motifs were fond in ITS2regions. It could speculate out that they had happened gene exchange and introgression, and common wild rice appeared to be the center of genetic diversity.