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Improvement of fluorescent chromosome in situ PCR and its application in the phylogeny of the genus Fagopyrum Mill. using nuclear genes of chloroplast origin (cpDNA)

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Abstract

Fluorescent chromosome in situ PCR method plays an important role in many fields of biology and can be used for determining physical maps, chromosomal structures and phylogeny. In the present study, improvements are made to fluorescent chromosome in situ PCR protocol by incorporating the use of SYBR Green I. All the complex procedures in this method have been removed, including the fixing of PCR products, the linkage step of antigen and antibody and the necessary detection the fluorescence signal. This new method is useful for the types of studies mentioned above. As an example, this improved technique was performed using primers for the 16S rDNA, 4.5S rDNA and psbA chloroplast DNA (cpDNA) genes to investigate the phylogeny of buckwheat, the introgression of cpDNA genes into nuclear genome and the chromosomal location of these genes for the construction of a physical map. The results showed that the 16S rDNA, 4.5S rDNA and psbA cpDNA genetic markers were found with different abundances and physical distributions in the nuclear genomes of the seven buckwheat species (10 accessions in total) under investigation. These data were used to confirm the phylogeny of these buckwheat species by constructing a phylogenetic tree.

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Acknowledgments

We are grateful to the Natural Science Foundation of China (31060207, 31171609, 30471116), the Earmarked Fund for China Agriculture Research System (CARS-08-A4), the Project of the Scientific and Technological Innovation Teams in Guizhou (QianKeHe RenCai TuanDui [2011]4007), Guizhou Key Agricultural Project (QianKeHe NY Zi [2010]3094) and Guizhou Animal and Plant Breeding Project (QianNongYuZhuanZi [2010] 023) for providing funds, and to Shanghai Office ELIXIGEN CO. for proofing the English.

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Authors and Affiliations

  1. Research Center of Buckwheat Industry Technology, Institute of Plant Genetics and Breeding, School of Life Sciences, Guizhou Normal University, Baoshan Beilu 116, Guiyang, 550001, People’s Republic of China

    Fen-Long Li, Kai-Feng Huang, Tao-Xiong Shi & Qing-Fu Chen

  2. Bavarian State Research Centre for Agriculture (LfL), Institute of Crop Science and Plant Breeding, 5354, Freising-Weihenstephan, Germany

    Friedrich J. Zeller

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  1. Fen-Long Li
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  2. Friedrich J. Zeller
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  4. Tao-Xiong Shi
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  5. Qing-Fu Chen
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Correspondence to Qing-Fu Chen.

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Li, FL., Zeller, F.J., Huang, KF. et al. Improvement of fluorescent chromosome in situ PCR and its application in the phylogeny of the genus Fagopyrum Mill. using nuclear genes of chloroplast origin (cpDNA). Plant Syst Evol 299, 1679–1691 (2013). https://doi.org/10.1007/s00606-013-0825-7

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