The unique genomic landscape surrounding the EPSPS gene in glyphosate resistant Amaranthus palmeri: a repetitive path to resistance

William T Molin,Christopher A Saski,Alice A Wright,Amy Lawton-Rauh

doi:10.1186/s12864-016-3336-4

William T Molin, Christopher A Saski + Show 2 more

Open Access

https://doi.org/10.1186/s12864-016-3336-4

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Abstract

BackgroundThe expanding number and global distributions of herbicide resistant weedy species threaten food, fuel, fiber and bioproduct sustainability and agroecosystem longevity. Amongst the most competitive weeds, Amaranthus palmeri S. Wats has rapidly evolved resistance to glyphosate primarily through massive amplification and insertion of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene across the genome. Increased EPSPS gene copy numbers results in higher titers of the EPSPS enzyme, the target of glyphosate, and confers resistance to glyphosate treatment. To understand the genomic unit and mechanism of EPSPS gene copy number proliferation, we developed and used a bacterial artificial chromosome (BAC) library from a highly resistant biotype to sequence the local genomic landscape flanking the EPSPS gene.ResultsBy sequencing overlapping BACs, a 297 kb sequence was generated, hereafter referred to as the “EPSPS cassette.” This region included several putative genes, dense clusters of tandem and inverted repeats, putative helitron and autonomous replication sequences, and regulatory elements. Whole genome shotgun sequencing (WGS) of two biotypes exhibiting high and no resistance to glyphosate was performed to compare genomic representation across the EPSPS cassette. Mapping of sequences for both biotypes to the reference EPSPS cassette revealed significant differences in upstream and downstream sequences relative to EPSPS with regard to both repetitive units and coding content between these biotypes. The differences in sequence may have resulted from a compounded-building mechanism such as repetitive transpositional events. The association of putative helitron sequences with the cassette suggests a possible amplification and distribution mechanism. Flow cytometry revealed that the EPSPS cassette added measurable genomic content.ConclusionsThe adoption of glyphosate resistant cropping systems in major crops such as corn, soybean, cotton and canola coupled with excessive use of glyphosate herbicide has led to evolved glyphosate resistance in several important weeds. In Amaranthus palmeri, the amplification of the EPSPS cassette, characterized by a complex array of repetitive elements and putative helitron sequences, suggests an adaptive structural genomic mechanism that drives amplification and distribution around the genome. The added genomic content not found in glyphosate sensitive plants may be driving evolution through genome expansion.

Highlights

The expanding number and global distributions of herbicide resistant weedy species threaten food, fuel, fiber and bioproduct sustainability and agroecosystem longevity
A. palmeri bacterial artificial chromosome (BAC) library construction, recruitment of enolpyruvylshikimate-3-phosphate synthase (EPSPS) BACs, tile path selection, and sequencing BAC libraries are critical tools for genomic dissection and they serve as the primary templates for targeted sequencing to facilitate accurate reconstruction of the genomic architecture around genes, especially in large repetitive plant genomes [22,23,24]
Under stringent fingerprint assembly parameters, 186 BACs clustered into a single contig, and a tile path of 4 representative BAC clones was selected for individual sequencing on the Illumina MiSeq platform and as a pool on the Pacific Biosystems (PacBio) RSII platform

Summary

Introduction

The expanding number and global distributions of herbicide resistant weedy species threaten food, fuel, fiber and bioproduct sustainability and agroecosystem longevity. Numerous management practices are available to farmers, such as tillage and cultivation, the use of herbicides remains the most effective and preferred method in minimizing weed interference. In recent years several weeds have evolved resistance to some of the most widely used herbicides, such as glyphosate, and have become a serious threat to crop production in the last decade. A. palmeri is a fast growing, highly competitive, yield-reducing weed of row crops that must be controlled throughout the crop development cycle to minimize losses. With the introduction of glyphosate resistant crops in 1996 (GR cropping systems), farmers gained an effective tool to control seedling A. palmeri. The broad species spectrum and efficacy of control with glyphosate contributed to the expansion of no- and reduced-tillage production systems [2]

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