Identification of functional centromeric sequences in barley through analysis of structurally modified chromosomes.

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In order to identify functional centromeric sequences on barley chromosomes, we analyzed the structurally-modified barley chromosomes in wheat. First, we identified two structurally-modified barley chromosomes (7HS^* and 7HS^<**>)that were originated from an isochromosome of short arm of barley chromosome 7H. Our cytological analyses indicated that both 7HS^* and 7HS^** chromosomes did not possess centromeric C-bands as well as centromere-specific repetitive sequences (cereba and (GAAAGA)n satellite sequences). These truncated chromosome normally transmit to next generation, indicating the normal formation of kinetochores in both mitotic and meiotic division. Formation of functional centromeres in mitosis was confirmed by immunostaining of centromere-specific proteins (Nasuda et al. 2005a). Although we tried to isolated centromeric sequences from 7HS^* and 7HS^<**> chromosomes with various method, our attempt was not successful. One reason was difficulty of ChIP analysis in hexaploid. And also the contamination of wheat chromosomes in flow-sorting was problematic. However, mapping the breakpoints in 7HS^* and 7HS^<**> is ongoing (Nasuda et al. 2005b). So far, we obtained a marker at very proximal region of the short arm that is eventually missing in the modified chromosomes. I am planning to use genomic information of barley and rice to further clarify the breakpoints.Identification of functional centromeric sequences in normal barley was successful (Houben et al. 2007). By ChIP (chromatin immunoprecipitation) analyses of nuclei isolated from leaf tissue, we could clearly indicate that the cereba retrotransposons and (AGGGAG)n satellite sequences have interaction with the centromere specific histone H3 (CENH3), which localize to functional centromeres in all eukaryotic species so far analyzed. The levels of interaction were not significantly different between cereba and (AGGGAG)n sequences, indicating that both form the centromeric chromatin in barley.