Mapping of transgenic alleles in soybean using a nanopore-based sequencing strategy

Abstract

Transgenic technology was developed to introduce transgenes into various organisms to validate gene function and add genetic variations >40 years ago. However, the identification of the transgene insertion position is still challenging in organisms with complex genomes. Here, we report a nanopore-based method to map the insertion position of a Ds transposable element originating in maize in the soybean genome. In this method, an oligo probe is used to capture the DNA fragments containing the Ds element from pooled DNA samples of transgenic soybean plants. The Ds element-enriched DNAs are then sequenced using the MinION-based platform of Nanopore. This method allowed us to rapidly map the Ds insertion positions in 51 transgenic soybean lines through a single sequencing run. This strategy is high throughput, convenient, reliable, and cost-efficient. The transgenic allele mapping protocol can be easily translated to other eukaryotes with complex genomes.

Keywords: Complex genome; MinNON sequencing; cost efficient; high throughput; mapping insertion position; transgene.

Fig. 1.

MinION sequencing without Ds enrichment. (A) A schematic diagram of Ds insertion in the soybean genome. The length of the Ds insertion is 1166 bp. The positions of forward (F) and reverse (R) primers used for PCR genotyping are shown. (B) Workflow of direct genome sequencing without target enrichment. Genomic DNA was end-repaired and dA-tailed, ligated with sequencing adaptors, and sequenced on the FLO-MIN106 flow cell. (C) A schematic diagram of the Ds insertion in the Glyma.15g128600 gene. Two reads are shown. The first one covers 2347 bp in the 5'-flanking region and 3047 bp in the 3'-flanking region. The second one contains 370 bp flanking sequence in the 3' region. (D) PCR validation of the Ds insertion in Line 1. Thorne was used as control plant. The length of the DNA fragment without the Ds element in control plant is 360 bp, while the fragment length from Ds-containing Line 1 is 1526 bp.

Fig. 2.

The workflow of the enrichment of Ds-containing fragments in DNA libraries. (A) Schematic diagram of the oligo probe used to capture the Ds element. The probe is dual biotinylated at the 5' end (diamond). (B) The workflow of sequencing the enriched Ds-containing DNA fragments. Genomic DNA was sheared and ligated to PCR barcode adaptors. The Ds-containing fragments were enriched for one or two rounds. The enriched fragments were pooled and sequenced.

Fig. 3.

Sequencing results after one-round enrichment of the Ds-containing fragments. (A and B) Schematic diagram of the flanking sequences of Line 2 (A) and Line 3 (B). Partial sequences of reads are shown. (C) PCR validation of the Ds insertion in Lines 2 and 3. Thorne was used as the control plant. The lengths of the DNA fragment without the Ds element are 612 bp for Line 2 and 689 bp for Line 3. With the Ds elements, the lengths of the DNA fragments are 1778 bp for Line 2 and 1855 bp for Line 3.

Fig. 4.

Sequencing results after two-round enrichment of the Ds-containing fragments. (A and B) Efficiency of one-round (A) and two-round (B) enrichment of the Ds element-containing fragments; 2% of samples before and after probe enriching were used to perform qPCR. The amount of target fragments was normalized to that of the internal control. (C) Schematic diagram of the flanking sequences of Line 15. Partial sequences of reads are shown. (D) PCR validation of the Ds insertion in Lines 13, 14, and 15. The three individual lines were examined with three pairs of primers each. Each primer pair (labeled above the picture) recognizes a potential insertion position of the Ds element, identified by sequencing. Line 13 containing a Ds insertion in the Glyma15G128600 gene produced a 1719 bp fragment, while Lines 14 and 15 without insertions in this gene generated ~559 bp fragments (indicated with arrows). Line 14 containing a Ds insertion in the Glyma05G163800 gene produced a 1628 bp fragment, while Lines 13 and 15 without insertions in this gene generated 462 bp fragments (indicated with arrows). Line 15 containing a Ds insertion in the Glyma11G181700 gene produced a 1560 bp fragment, while Lines 13 and 14 without insertions in this gene generated 394 bp fragments (indicated with arrows).