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

A high-yield method for isolation of barley chromosomes in suspension, thei r analysis and sorting using flow cytometry is described. To accumulate mer istem root tip cells at metaphase, actively growing roots were subjected to subsequent treatment with 2 mmol/L hydroxyurea for 18 h, 2.5 mu mol/L amip rophos methyl for 2 h, and ice water (overnight). This treatment resulted i n metaphase indices exceeding 50%. Synchronized root tips were fixed in 2% formaldehyde for 20 min and chromosomes were released into a lysis buffer b y mechanical homogenization, producing, on average, 5 x 10(5) chromosomes f rom 50 root tips. The isolated chromosomes were morphologically intact and suitable for flow cytometric analysis and sorting. While it was possible to discriminate and sort only one chromosome from a barley cultivar with stan dard karyotype, up to three chromosomes could be sorted in translocation li nes with morphologically distinct chromosomes. The purity of chromosome fra ctions, estimated after PRINS with primers specific for GAA microsatellites , reached 97%. PCR with chromosome-specific primers confirmed the purity an d suitability of flow-sorted chromosomes for physical mapping of DNA sequen ces.