Cellular Level Research On Classification Standard Of Three AA Rice Genome Species And Subspecies Based On FISH

In this paper, three cultivated rice species were used as research materials, including O.indica cv guangluai No.4, O.japonica cv Nipponbare and O.glaberrima Steud.. Two kinds of repeats Cot-1 DNA and RCS2 were applied to perform cytogenetic positioning so as to explore the distribution of repetitive sequences in the genome as well as the role of highly and moderately repetitive DNA in species differentiation. This work is also helpful for the feasibility of Cot-1 DNA FISH in subspecific classification. The main results are as following:1. In order to reveal the difference of the highly and moderately repetitive DNA sequences between three AA genome subspecies of O. sativa L. Indica, Japonica and O.glaberrima, as well as the role of highly and moderately repetitive DNA in species differentiation, fluoreseence in situ hybridization (FISH) procedure was adopted to analyse the genomes of indica and japonica using Cot-1 DNA and gDNA from indica as a probe.Karyotype analysis between indica and japonica was also made tomparatively based on their similar band patterns of the hybridization signals. FISH showed that the distribution and coverage percentage of Cot-1 DNA and gDNA in the three different species were very similar. The coverage percentage (%) of Cot-1 DNA in Indica, Japonica and O.glaberrima were 47.10±0.16, 38.61±0.13, 44.38±0.13, there was a high homology and close relationship between Indica, Japonica and O. glaberrima. But through the analysis of hybridization signals, the distribution of highly and moderately repetitive DNA sequences in the three different species has its own characteristics, indicating that the highly and moderately repetitive DNA sequences of these three rice genome have different directions in the evolution. Further integration of the results of GISH, the chromosomes which contain lower highly and moderately repetitive DNA sequences is more active in the rice genome composition. This study shows that by adjusting the post-hybridization washing stringency, Cot-1 DNA FISH is very effective in the reach of relationship between different species which have the same Genome type. The feasibility of Cot-1 DNA FISH in subspecific classification are discussed.2. Fluoreseence in situ hybridization (FISH) procedure was adopted to analyse the genomes of indica, japonica and O.glaberrima using centromeric repetitive sequence RCS2 as a probe.Karyotype analysis of indica, japonica and O.glaberrima was also made tomparatively based on their similar band patterns of the hybridization signals. FISH showed that RCS2 sequence is distributed in the centromere of chromosome. These regions were important for preserving the stability of chromosome .But the Distribution of RCS2 sequence in the Centromere of three different species has its own characteristics, indicating that RCS2 DNA sequences of these three rice genome have different directions in the evolution. Comparative analysis of Cot-1 DNA FISH showed that differention of other regions a obviously.It may be one of the most important reasons why the specific chromosomeformed.The advantage of FISH with RCS2 as probe in Karyotype are discussed.