The utility of flow sorting to identify chromosomes carrying a single copy transgene in wheat
| Autor: | Eva Komínková, Takashi R. Endo, Marie Kubaláková, Petr Cápal, Winfriede Weschke, Jaroslav Doležel, Isabel Mora-Ramírez, Jan Vrána, Miroslava Karafiátová |
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| Jazyk: | angličtina |
| Předmět: |
0106 biological sciences
0301 basic medicine Flow cytometric sorting Transgene Triticum aestivum Transgene localization Plant Science Biology 01 natural sciences law.invention 03 medical and health sciences chemistry.chemical_compound law medicine Genetics Common wheat Mitosis Polymerase chain reaction Hordeum vulgare medicine.diagnostic_test Methodology Chromosome food and beverages HvSUT1 030104 developmental biology chemistry Single chromosome amplification DNA 010606 plant biology & botany Fluorescence in situ hybridization Biotechnology |
| Zdroj: | Plant Methods |
| ISSN: | 1746-4811 |
| DOI: | 10.1186/s13007-016-0124-8 |
| Popis: | 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. Electronic supplementary material The online version of this article (doi:10.1186/s13007-016-0124-8) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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