Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.).

摘要: A high-yield method for isolation of barley chromosomes in suspension, their analysis and sorting using flow cytometry is described. To accumulate meristem root tip cells at metaphase, actively growing roots were subjected to subsequent treatment with 2 mmol/L hydroxyurea 18 h, 2.5 μmol/L amiprophos methyl ice water (overnight). This resulted metaphase indices exceeding 50%. Synchronized tips fixed 2% formaldehyde 20 min released into a lysis buffer by mechanical homogenization, producing, on average, 5 × 105 from 50 tips. The isolated morphologically intact suitable cytometric sorting. While it was possible discriminate sort only one chromosome cultivar standard karyotype, up three could be sorted translocation lines distinct chromosomes. purity fractions, estimated after PRINS primers specific GAA microsatellites, reached 97%. PCR chromosome-specific confirmed the suitability flow-sorted physical mapping DNA sequences.