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Pokroky v průtokové cytogenetice ječmene
Suchánková, Pavla ; Kubaláková, Marie ; Kovářová, Pavlína ; Bartoš, Jan ; Čihalíková, Jarmila ; Endo, T. R. ; Doležel, Jaroslav
Flow cytogenetics is an attractive tool that facilitates genome mapping. Flow cytometric analysis permits classification of isolated chromosomes according to their DNA content As thousands of chromosomes are analysed in each sample, quantitative detection of structural and numerical chromosome changes is possible. Any chromosome that can be discriminated can also be sorted in large quantities and high purities for subsequent molecular analysis. Unfortunately, only the smallest chromosome 5(1H) can be discriminated and sorted in barley. This is due to similarity in relative DNA content among the chromosomes 1 - 4,6 and 7 (2H - 7H). Cytogenetic stocks facilitate sorting of those translocation chromosomes that differ in DNA content Even if the sorted translocation chromosomes were found useful for mapping DNA sequences to subchromosomal regions, their use remained limited. Here we describe a novel approach that facilitates sorting of any of the 14 barley chromosome arms. The strategy is based on using wheat-barley addition lines carrying barley chromosome arms (telocentric chromosomes). Furthermore, we will be able to sort subarm chromosomal segments that are generated in the wheat-barley chromosome addition lines by the gametocidal system. These advances make flow cytogenetics an attractive tool that can simplify barley genome mapping. A possibility to purify large quantities of individual chromosome arms and their segments opens avenues for targeted isolation of low-copy "genie" sequences, preparation of specific probes for screening EST arrays, HAPPY mapping, and construction of chromosome arm-specific BAG libraries.
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Rozdělení velkých genomů čeledi Triticeae tříděných chromozomů
Doležel, Jaroslav ; Kubaláková, Marie ; Suchánková, Pavla ; Šafář, Jan ; Janda, Jaroslav ; Kovářová, Pavlína ; Bartoš, Jan ; Čihalíková, Jarmila ; Šimková, Hana
Nuclear genomes of some Triticeae species are characterized by large size and prevalence of repetitive DNA sequences. These features hamper their physical mapping and gene cloning. Purification of individual chromosomes by flow cytometry can simplify these tasks by providing small and defined genome fractions. This lecture reviews the development of the methodology and its potential for genome mapping in barley, rye and wheat Due to small differences in relative DNA content, only one chromosome type can be discriminated and sorted in each of the three species. Cytogenetic stocks facilitate separation of other parts of the genomes as individual chromosomes, translocation chromosomes and chromosome arms. Chromosome analysis by flow cytometry permits quantitative detection of structural and numerical chromosome changes. Chromosomes sorted onto microscopic slides have been used for discovery of rare structural changes and for high-resolution cytogenetic mapping using FISH. The use of sorted chromosomes for HAPPY mapping, targeted isolation of low copy "genie" sequences, and high-throughput physical mapping of ESTs on microarrays are attractive options. As millions of chromosomes with intact DNA may be sorted, construction of BAC libraries is possible. Subgenomic, chromosome-specific and chromosome arm-specific BAC libraries have already been produced in wheat and represent unique resources for genomics of cereals.
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