Abstract
BackgroundIdentification 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.ResultsWe 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.ConclusionsInsertion 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 materialThe online version of this article (doi:10.1186/s13007-016-0124-8) contains supplementary material, which is available to authorized users.
Highlights
Identification of transgene insertion sites in plant genomes has practical implications for crop breeding and is a stepping stone to analyze transgene function
The insertion site of a transgene is of great importance for the transgene function [3, 4] which is influenced by its position on the chromosome, including the flanking DNA sequences [5]
The experimental workflow is shown on Fig. 1
Summary
Identification of transgene insertion sites in plant genomes has practical implications for crop breeding and is a stepping stone to analyze transgene function. Single copy sequences are not always easy to localize in large plant genomes by standard approaches. During the past 30 years, many cultivars of agricultural crops beneficial to humankind have been developed by means of genetic engineering, including plants resistant to herbicides, pests or viruses, bearing fruits with prolonged shelf life and products more suited for industrial processing [for review see 1]. The insertion site of a transgene is of great importance for the transgene function [3, 4] which is influenced by its position on the chromosome, including the flanking DNA sequences [5]. Transgene localization is not easy by routine approaches, like fluorescence in situ hybridization (FISH), or Southern blotting. In barley and common wheat, FISH enables cytological localization of cDNAs, as short as 1.5 kb, on a
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