Rapid identification and determination of purity of flow-sorted plant chromosomes using C-PRINS.
BACKGROUND
Flow-sorted plant chromosomes are being increasingly used in plant genome analysis and mapping. Consequently, there is a need for a rapid method for identification of sorted chromosomes and for determination of their purity. We report on optimization of procedures for primed in situ DNA labeling (PRINS) and cycling-PRINS (C-PRINS) for fluorescent labeling of repetitive DNA sequences on sorted plant chromosomes suitable for their identification.


METHODS
Chromosomes of barley, wheat, and field bean were sorted onto microscope slides, dried, and subjected to PRINS or C-PRINS with primers for GAA microsatellites (barley and wheat) or FokI repeat (field bean). The following parameters were optimized to achieve the highest specificity and intensity of fluorescent labeling: ratio of labeled versus unlabeled nucleotides, nucleotide concentration, and the number and concentration of primers.


RESULTS
Under optimal conditions, C-PRINS resulted in strong and specific labeling of GAA microsatellites on sorted barley and wheat chromosomes and FokI repeats on sorted field bean chromosomes. The labeling patterns were characteristic for each chromosome and permitted their unequivocal identification as well as determination of purity after sorting, which ranged from 96% to 99%. A standard polymerase chain reaction (PCR) with chromosome-specific primers was not sensitive enough to detect low-frequency contamination.


CONCLUSIONS
The results indicate that a single C-PRINS assay with primers that give chromosome-specific labeling pattern is sufficient not only to determine chromosome content of peaks on flow karyotype but also to determine the purity of sorted chromosome fractions. The whole procedure can be performed in less than 3 h on the next day after sorting. Numerous applications are expected in the area of plant flow cytogenetics.
Kubaláková
M
M
Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Olomouc, Czech Republic.
Lysák
M A
MA
Vrána
J
J
Simková
H
H
Cíhalíková
J
J
Dolezel
J
J
D002875:Chromosomes / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000032:analysis; D007887:Fabaceae / Q000235:genetics; D005434:Flow Cytometry; D018745:Genome, Plant; D001467:Hordeum / Q000235:genetics; D007621:Karyotyping; D018895:Microsatellite Repeats; D008856:Microscopy, Fluorescence; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D020401:Primed In Situ Labeling / Q000379:methods*; D014908:Triticum / Q000235:genetics
D017931:DNA Primers; D018744:DNA, Plant