Using Transcriptomics to Investigate the Genetic Mechanism of Glyphosate Resistance in Kochia scoparia

The confirmation of glyphosate-resistant Kochia scoparia across the US central Great Plains in 2007 has raised concerns about the long-term usefulness of the herbicide. In previous studies, gene amplification of the glyphosate targeted enzyme 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) was found in glyphosate-resistant plants collected throughout the central Great Plains States. To further investigate the genetic mechanism of glyphosate resistance, RNA was isolated from glyphosate-susceptible, and -resistant treated and untreated plants with known EPSPS gene copy numbers. Complementary DNA libraries were constructed and sequenced using the Illumina HiSeq 2000 platform. The FASTX-Toolkit and Cutadapt were used to remove primers, adapters, and low quality sequence. Oases was used for de novo transcript assembly, and transcripts were concatenated to form a reference transcriptome. Using TopHat, sequence read sets were aligned to the reference transcriptome and visualized using Tablet. Transcript expression was measured using Cufflinks, and command-line BLAST was used to annotate transcripts of interest. EPSPS splice variants were not detected in any of the samples, and the expression patterns of chorismate pathway transcripts did not appear to change with increases in EPSPS expression. Seven single nucleotide polymorphims were identified in glyphosate-susceptible reads yet none were identified in glyphosate-resistant read alignments suggesting that only one EPSPS locus was amplified in glyphosate-resistant plants.