The utility of flow sorting to identify chromosomes carrying a single copy transgene in wheat.
BACKGROUND
Identification of transgene insertion sites in plant genomes has practical implications for crop breeding and is a stepping stone to analyze transgene function. However, single copy sequences are not always easy to localize in large plant genomes by standard approaches.


RESULTS
We employed flow cytometric chromosome sorting to determine chromosomal location of barley sucrose transporter construct in three transgenic lines of common wheat. Flow-sorted chromosomes were used as template for PCR and fluorescence in situ hybridization to identify chromosomes with transgenes. The chromosomes carrying the transgenes were then confirmed by PCR using DNA amplified from single flow-sorted chromosomes as template.


CONCLUSIONS
Insertion sites of the transgene were unambiguously localized to chromosomes 4A, 7A and 5D in three wheat transgenic lines. The procedure presented in this study is applicable for localization of any single-copy sequence not only in wheat, but in any plant species where suspension of intact mitotic chromosomes suitable for flow cytometric sorting can be prepared.
Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 78371 Olomouc, Czech Republic.
Petr
P
Cápal
Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 78371 Olomouc, Czech Republic ; Faculty of Agriculture, Ryukoku University, 1-5 Yokotani, Seta Oe-cho, Otsu, Shiga 520-2194 Japan.
Takashi R
TR
Endo
Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 78371 Olomouc, Czech Republic.
Jan
J
Vrána
Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 78371 Olomouc, Czech Republic.
Marie
M
Kubaláková
Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 78371 Olomouc, Czech Republic.
Miroslava
M
Karafiátová
Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 78371 Olomouc, Czech Republic.
Eva
E
Komínková
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstrasse 3, 06466 Stadt Seeland, Germany.
Isabel
I
Mora-Ramírez
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstrasse 3, 06466 Stadt Seeland, Germany.
Winfriede
W
Weschke
Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 78371 Olomouc, Czech Republic.
Jaroslav
J
Doležel
Flow cytometric sorting; Hordeum vulgare; HvSUT1; Single chromosome amplification; Transgene localization; Triticum aestivum