The utility of flow sorting to identify chromosomes carrying a single copy transgene in wheat

Abstract

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.

Keywords: Flow cytometric sorting; Hordeum vulgare; HvSUT1; Single chromosome amplification; Transgene localization; Triticum aestivum.

Fig. 1

Experimental workflow. a Monovariate flow karyotype is dissected into small regions. From each region, 200 chromosomes are sorted. b Flow-sorted chromosomes are used as template for PCR with transgene-specific marker. c The region representing chromosome with the transgene identified on monovariate flow karyotype is dissected by sorting chromosomes from sort regions on bivariate flow karyotype. d From the sort gates on the bivariate flow karyotype, 100 chromosomes are sorted for PCR with transgene-specific marker. Moreover, 1000 chromosomes are sorted immediately afterwards onto the microscopic slides to identify flow-sorted chromosomes by FISH. e From the sort gate most enriched for the transgene-bearing chromosome (region G in this example), single chromosomes are sorted individually into PCR tubes. DNA of single-flow sorted chromosomes is amplified and resulting DNA is used as template for PCR to identify the presence of multiple transgene- and chromosome-specific sequences. This step unambiguously confirms the chromosome with integrated transgene

Fig. 2

Bivariate flow karyotypes of three transgenic HOSUT lines of wheat obtained after the analysis of chromosomes with FITC-labelled (GAA)_n microsatellites and stained by DAPI. The position of red and green regions used to sort particular chromosomes is indicated. The green sort gate was found to represent chromosomes carrying transgene. Chromosomes were flow-sorted also from the neighboring population delineated by red gate and were used as a control. Although the transgene-bearing chromosome should not be included in this region, the sorted population could potentially be contaminated with transgene-bearing chromosomes due to similarity in chromosome size and DNA content

Fig. 3

Agarose gel electrophoresis of PCR products obtained with primers for the transgene and DNA of chromosomes flow-sorted from three HOSUT lines using the green and red sort regions as shown in Fig. 1. The amplicon of HvSUT-RT (169 bp) was obtained with chromosomes sorted from the green sort region in all three HOSUT lines. When chromosomes were sorted from the red sort regions, no PCR amplification occurred for HOSUT 12/44 and HOSUT 20/6. However, a weak band was observed for HOSUT 24/31. Genomic DNA of the transgenic lines served as positive control

Fig. 4

Representative images of chromosomes flow-sorted from three HOSUT lines using the green and red sort regions on bivariate flow karyotypes as shown in Fig. 2. FISH was done using probes for Afa-family (red signals) and GAA microsatellites (green signals). Chromosomes were counterstained with DAPI (blue)

Fig. 5

Agarose gel electrophoresis of PCR products obtained using DNA produced by multiple displacement amplification of three single chromosomes flow-sorted from the sort region representing chromosome 4A in HOSUT 24/31 line. PCR with primers for the four transgenes resulted in products of expected length. The same was true for the chromosome 4A-specific STS markers. Note that none of the chromosome 7A-specific markers was detected in the samples of single chromosome DNA. PCR with genomic DNA of HOSUT 24/31 as template detected both 4A and 7A chromosome-specific markers. PCR with the positive control (represented by 1000 chromosomes sorted from green sorting region and amplified) showed slight PCR bands of chromosome 7A, which reflects a minor contamination of the sorted chromosome 4A by chromosome 7A