Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.).
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 for 18 h, 2.5 micromol/L amiprophos methyl for 2 h, and ice water (overnight). This treatment resulted in metaphase indices exceeding 50%. Synchronized root tips were fixed in 2% formaldehyde for 20 min and chromosomes were released into a lysis buffer by mechanical homogenization, producing, on average, 5 x 10(5) chromosomes from 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 standard karyotype, up to three chromosomes could be sorted in translocation lines with morphologically distinct chromosomes. The purity of chromosome fractions, estimated after PRINS with primers specific for GAA microsatellites, reached 97%. PCR with chromosome-specific primers confirmed the purity and suitability of flow-sorted chromosomes for physical mapping of DNA sequences.
Lysák
M A
MA
Institute of Experimental Botany, Olomouc, Czech Republic.
Cíhalíková
J
J
Kubaláková
M
M
Simková
H
H
Künzel
G
G
Dolezel
J
J
D002875:Chromosomes / Q000235:genetics*; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry; D001467:Hordeum / Q000235:genetics*; D006918:Hydroxyurea / Q000494:pharmacology; D017404:In Situ Hybridization, Fluorescence; D007306:Insecticides / Q000494:pharmacology; D007621:Karyotyping; D008677:Metaphase; D018895:Microsatellite Repeats / Q000235:genetics; D008938:Mitosis / Q000235:genetics; D009578:Nitrobenzenes; D009946:Organothiophosphorus Compounds / Q000494:pharmacology; D020161:Physical Chromosome Mapping; D018517:Plant Roots / Q000235:genetics; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D007306:Insecticides; D009578:Nitrobenzenes; D009946:Organothiophosphorus Compounds; C011402:amiprophos; D006918:Hydroxyurea