ITS Sequences Of RDNA In Wild Rices And Cultivated Rices And Their Phylogenetic Implications

ITS (internal transcribed spacer) sequences of two ordinary wild rices (Oryza rufipogori) i.e. distributed in Chaling and Anren of Hunan Province, and six cultivated varieties i.e. three indica subspecies (9311, Nanjing11, Guangluai4) and three japonica subspecies (Qiuguang, Ribenqing, Zhaowadao) were studied. By ITS sequences, we attempt to construct their phylogenetic tree and establish a molecular marker distinguishing between indica subspecies and japonica subspecies so as to constitute a new research method of the genus Oryza and promote the understanding of the genetic background of the primary cultivated rices at present, and offer a molecular genetic marker for the crossbreeding and the parent selection in heterosis application. Main results are showed as follows:1. Characters of ITS sequencesThe internal transcribed spacer (including ITS1, ITS2, and 5.8S rDNA gene) of nuclear ribosomal DNA from the experimented materials was amplified using PCR technology and sequenced with automated fluorecent sequencing. The complete concordance of 5.8S rDNA indicates that the ribosomal gene is highly conservative. The sequences of ITS1 ranged from 193bp to 196bp in size and G/C content varied from 72.31% to 74.38%. The sequences of ITS2 ranged from 224bp to 233bp in size and G/C content varied from 74.12% to 76.86%. The sequences of ITS of Chaling wild rice is the longest, while Zhaowadao's is the shortest. The size and G/C content of ITS 1 varied slightly, especially between Qiuguang and Ribenqing, 9311 and Nanjing 11 respectively. The size and G/C content of ITS2 varied more greatly than that of ITS 1, but the differences between Qiuguangand Ribenqing, Nanjing 11 and 9311 are still the least. It is evident that Qiuguang and Ribenqing, Nanjing 11 and 9311 have a close predestined relationship respectively. The similarity in size and G/C content between Zhaowadao and two japonica varieties is quite high. Though there are some discrepancies between early indica variety-Guangluai 4 and two medium indica varieties-Nanjing 11 and 9311, their similarity reached 96.5%. Thus it can be seen that the varying regularity about the size and G/C content of DNA sequences from ITS nuclear ribosomal DNA in the experimented materials has a high concordance with its traditional classification.2. Alignment and phylogenetic analysis based on the sequences of ITSITS sequences of the experimented materials were arranged by the CLUSTRAL W software. The result shows that there are 431 characters in all in the alignment and 28 characters are variable, 6.49% the total, and 8 characters, 29.7% of variable sites, can be used for phylogenetic analysis.Among eight informative sites, three japonica varieties share the same type of nucleotide respectively in the 70th, 191st, 259th and 342ed position of the alignment, which are A, T, T and A. Meanwhile, in the four corresponding sites, the type of nucleotide of three indica varieties are A, C, C and A. Therefore, these four sites can be taken as the molecular marker distinguishing between indica and japonica subspecies.There are 17 variable sites in the ITS sequences of three indica varieties, but only 4 discrepant sites between Nanjing 11 and 9311. However, there are 6 variable sites in the ITS sequences of three japonica varieties (including Zhaowadao used to be another subspecies), but only 3 different sites between Qiuguang and Ribenqing. It indicates that there is a wider genetic diversity in indica varieties than in Japonica varieties.A three-Branch unrooted tree from PAUP (version 4.0) analysis based on DNA sequences from ITS nuclear ribosomal DNA of the investigated materials was formed With Chaling wild rice as an outgroup. The results Showed: (1) Nanjing 11, 9311, Anren wild rice (might have been influenced by indica rice already) and Guanglu ai 4 belong to a monophyietic group, and Qiuguang, Ribenqing and Zhaowadao belong to another; (2) with ITS sequences, the different ecological types of the same indica subspecies can be distinguished, such as Guanglu ai 4 and N...