Genomic variation and DNA repair associated with soybean transgenesis: a comparison to cultivars and mutagenized plants

Abstract

Background: The safety of mutagenized and genetically transformed plants remains a subject of scrutiny. Data gathered and communicated on the phenotypic and molecular variation induced by gene transfer technologies will provide a scientific-based means to rationally address such concerns. In this study, genomic structural variation (e.g. large deletions and duplications) and single nucleotide polymorphism rates were assessed among a sample of soybean cultivars, fast neutron-derived mutants, and five genetically transformed plants developed through Agrobacterium based transformation methods.

Results: On average, the number of genes affected by structural variations in transgenic plants was one order of magnitude less than that of fast neutron mutants and two orders of magnitude less than the rates observed between cultivars. Structural variants in transgenic plants, while rare, occurred adjacent to the transgenes, and at unlinked loci on different chromosomes. DNA repair junctions at both transgenic and unlinked sites were consistent with sequence microhomology across breakpoints. The single nucleotide substitution rates were modest in both fast neutron and transformed plants, exhibiting fewer than 100 substitutions genome-wide, while inter-cultivar comparisons identified over one-million single nucleotide polymorphisms.

Conclusions: Overall, these patterns provide a fresh perspective on the genomic variation associated with high-energy induced mutagenesis and genetically transformed plants. The genetic transformation process infrequently results in novel genetic variation and these rare events are analogous to genetic variants occurring spontaneously, already present in the existing germplasm, or induced through other types of mutagenesis. It remains unclear how broadly these results can be applied to other crops or transformation methods.

Keywords: Biotechnology; Genetic engineering; Somaclonal variation; Soybean; Structural variation; Transgenic crops.

Fig. 1

Visual comparison of CGH data for individuals from the three germplasm classes and control. Each black dot represents a single probe and its log₂ ratio score. Data are shown from chromosome 11 on the left and chromosome 18 on the right for all samples. a The standing inter-cultivar variation between lines LD02-9050 and ‘Williams 82’ is shown. b Fast neutron (No-phenotype) plant 1R19C96Cfr293aMN11 is compared to the FN parent line ‘M92-220’. c Transgenic plant WPT_389-2-2 is compared to the parent line ‘Bert’; it shows relatively little noise and one true SV on chromosome 11. d The control CGH compared ‘Bert-MN-01’ to itself

Fig. 2

Distribution of genic SV from the three germplasm classes. Genic SV are found in individuals as standing variation in diverse cultivars (41 SoyNAM parents), induced by fast neutron mutagenesis (10 FN plants with a mutant phenotype and 35 FN plants with no obvious mutant phenotypes), or induced by the transformation process (five plants with unique constructs). Each column in the graph is a single genotype. Light gray bars represent “Duplicated Genes,” those overlapping putatively duplicated regions. Dark gray bars represent “Deleted Genes,” those overlapping putatively deleted regions

Fig. 3

A novel deletion on chromosome 11 in transgenic plant WPT_389-2-2. a A plot of CGH data for the transgenic plant versus ‘Bert’ is shown, zoomed in on the chromosome 11 deletion seen in Fig. 1c. Probes are plotted as dots corresponding to the log₂ ratio from the CGH array. Dark gray dots represent probes within significant SV segments that exceed the empirical threshold. Even with the extremely low detection threshold, part of this deletion could not be verified via CGH alone, necessitating visual inspection and sequencing of the deletion breakpoint. b Graphical interpretation of the hemizigous deletion found in WPT_389-2-2 is shown. c Sequence data from the breakpoint junction shows moderate homology on either end of the breakpoint

Fig. 4

A novel duplication on chromosome 13 in transgenic plant WPT_301-3-13. a A plot of CGH data for the transgenic plant versus ‘Williams 82’ is shown, zoomed in on the chromosome 13 duplication. Probes are plotted as dots corresponding to the log₂ ratio from the CGH array. Dark gray dots represent probes within significant SV segments that exceed the empirical threshold. b A graphical interpretation of the heterozygous duplication found in WPT_301-3-13, which includes a portion of Glyma13g17730 and a portion of Glyma13g17740, is shown. c The sequence data from the breakpoint junction shows five base pairs of homology on either end of the breakpoint

Fig. 5

Transgene insertion locus and induced homozygous deletions in transgenic plant WPT_389-2-2. a A graphical interpretation of the transgene orientation and induced deletions at this locus is shown. The transgene insertion on chromosome 13 contains four primary elements between the left and right borders: Pong, mPing, Tpase, and BAR. Colored lines correspond to the breakpoint sequence results. b Results of breakpoint sequencing show a 1,533 bp deletion adjacent to the T-DNA right border (dark blue). The deletion results in a unique junction connecting two genomic segments (red and green) immediately adjacent to a 6 bp track of filler sequence (light blue), and then the T-DNA right border (dark blue). c A 37 bp deletion is found at the left border-genome junction (orange and purple, respectively). Microhomology occurs across the large deletion and between the left border and the genome

Fig. 6

Genome wide view of induced variation detected through CGH and resequencing. The genomic locations of nucleotide substitutions (black bars), large duplications (blue bars), and large deletions (bright red bars) are shown for ten fast neutron plants (a) and two transgenic plants (b). Regions were filtered for background line heterogeneity such that only variants unique to one individual are shown. a Fast neutron plants, including the parent ‘M92-220’ (outer ring) and FN02-FN11 (inner rings) are shown. Background is shaded according to fast neutron irradiation dosage: gray is the non-irradiated parent ‘M92-220’, light red is 32 Gy (FN 09, 05 and 10), and green is 16 Gy (FN 02, 03, 04, 06, 07, 08, and 11). b Unique genetic variation in two different sequenced ‘Bert’ parent individuals (gray background), and transgenic plants WPT_391-1-6 and WPT_389-2-2 (yellow backgrounds) is shown. Transgene insertion sites are noted by green arrows and bars