Karafiátová, M., Bartoš, J., Kopecký, D., Ma, L., Sato, K., Houben, A., Stein, N., Doležel, J.
CHROMOSOME RESEARCH
21:
739-751,
2013
Klíčová slova:
cDNA, multicolor FISH, low-copy FISH, nonrecombining regions, physical mapping, genetic mapping
Abstrakt:
Fluorescence in situ hybridization (FISH) is
a widely used method to localize DNA sequences on
chromosomes. Out of the many uses, FISH facilitates
construction of physical maps by ordering contigs of
large-insert DNA clones, typically bacterial artificial
chromosome (BAC) and establishing their orientation.
This is important in genomic regions with low recombination
frequency where genetic maps suffer from
poor resolution. While BAC clones can be mapped
directly by FISH in plants with small genomes, excess
of repetitive DNA hampers this application in species
with large genomes. Mapping single-copy sequences
such as complementary DNA (cDNA) is an attractive
alternative. Unfortunately, localization of single-copy
sequences shorter than 10 kb remains a challenging
task in plants. Here, we present a highly efficient FISH
technique that enables unambiguous localization of
single copy genes.We demonstrated its utility by mapping
13 out of 15 full-length cDNAs of variable length
(2,127–3,400 bp), which were genetically defined to
centromeric and pericentromeric regions of barley
chromosome 7H. We showed that a region of 1.2 cM
(0.7 %) on genetic map represented more than 40 % of
the physical length of the chromosome. Surprisingly,
all cDNA probes occasionally revealed hybridization
signals on other chromosomes, indicating the presence
of partially homologous sequences. We confirmed the
order of 10 cDNA clones and suggested a different
position for three cDNAs as compared to published
genetic order. These results underline the need for
alternative approaches such as FISH, which can
resolve the order of markers in genomic regions where
genetic mapping fails.
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Autoři z ÚEB: Jan Bartoš,
Jaroslav Doležel,
Miroslava Karafiá...,
David Kopecky
Čeština
English