Comparative Analysis Of Allopolyploid Oryza Genomes With Moderately Repetitive Sequences

In this paper, three allopolyploid wild rice species were used as research materials, including O. latifolia, O. alta and O. grandiglumis. Two kinds of repeats C0t-1 DNA and ITS were applied to perform cytogenetic positioning and sequence analysis so as to explore the distribution of repetitive sequences in the genome as well as the relationship among allopolyploid wild rice species. This work is also helpful for providing certain evidence for the underlying genetic events. The main results are as following:1. Comparative analysis of allopolyploid wild rice with C0t-1 DNAThe O.officinalis C0t-1 DNA (CC genome) was used as probe to make comparative analyses of O. latifolia,O. alta and O. grandiglumis genome. Under the same experimental condition, we found that the relationship between C,D genome of O. alta and O. grandiglumis was closer than that of O. latifolia primitively. Based on it, we used the rice C0t-1 DNA and O. officinalis C0t-1 DNA as probes to analysis the distribution of moderately repetitive sequnces in the O. latifolia by changing the wash strigency. The results showed that the location of moderately repetitive sequnces featured specificity, which was also demonstrated by the similar research of O. grandiglumis. It means that the conservative distribution of moderately repetitive sequnces could provide evidence for the relationship among allopolyploid wild rices, which further confirm that C0t-1 DNA FISH could be an effective way to analysis the relationship among species.2. ITS sequence analysisIn order to conduct phylogenetic analyses of the allopolyploid species, we amplified and sequenced the complete ITS sequence of 5 wild species by polymerase chain reaction (PCR) technique. The 5 species included O. punctata,O. officinalis,O. latifolia,O. alta and O. grandiglumis. Sequence results showed that the length of ITS1 and ITS2 sequences were conservative, but the content of G/C for ITS sequence were different between allopolyploid species and diploid species, the content of allopolyploid species was lower than that of diploid species. Further more, phylogenetic relationship of these species was estimated according to the dendrogram constructed from the ITS sequencing data. O. latifolia and O. grandiglumis was clustered together, showing close sequence homology, and they showed far sequence homology with O. alta. However, these three allopolyploid species may all originate from O. officinalis. By combining the ITS sequence analysis with C0t-1 DNA FISH, it could create a general view toward allopolyploid so that the results would be more accurate and reliable.3. Exploration of BAC-FISHEnlightened by C0t-1 DNA FISH, we applied FISH to locate resistance gene pi-36 with the BAC clone B-16 as probe, under the experimental condition which not relied on C0t-1 DNA as block. Resistance gene pi-36 was proved to locate on the short arm of 8 chromosome of rice. By changing the wash strigency, we located this gene on 6 chromosome of O. officinalis. The improvement of BAC-FISH facilitated the complicated experimental method of BAC-FISH, which should be a beneficial exploration.