Comparative Illumina RNA-Seq and Tonoplast Proteomic Approach to Study Glyphosate Resistant Mechanisms in Conyza canadensis biotypes

Weeds are responsible for an estimated $36 billion loss annually in the USA alone and represent the most difficult pest for farmers to control.  Glyphosate is the most widely used herbicide in the world. However, the widespread use of glyphosate has exerted selection pressure on various species of weeds. Horseweed (Conyza canadensis L.) was the first broadleaf weed to evolve glyphosate resistance in agriculture, and also is the most widely distributed glyphosate-resistant weed in the United States and the world. Horseweed is a true diploid (2n=18), and has the smallest genome (~335 Mb) of any agricultural weeds, and was suggested to be among the best candidates of model weedy plants. We have recently participated in a phylogeographic study that gives evidence that horseweed has evolved glyphosate resistance independently in many locations in the USA. Is there a shared mechanism of glyphosate resistance for biotypes that evolved independently? If not, how many mechanisms exist for glyphosate resistance, at least for the existing resistant biotypes? What genes and pathways are involved in the glyphosate resistance? To address these issues, we performed comparative transcriptome analysis of leaves samples of 8 biotypes from different locations (CA, DE, IN and TN) plus glyphosate treated and untreated controls using Illumina RNA-seq technology. Illumina runs of 16 bar-coded samples generated ~500 million reads and 51 Gb data. De novo genome and transcriptome assembly resulted in 25,439 unique genes with average length of 2,226 bp and over 72% of them could be annotated using known protein database. Gene duplication of interested families has been studied. Comparative global gene expression analyses have been performed between susceptible and resistant, treated and un-treated samples. Quantitative protein profiling of tonoplast samples comparing between TN-R and TN-S biotypes have been performed by using LC-MS/MS peptide sequencing platform. By searching public and custom protein database, specific expressing gene/proteins in R samples were isolated and suggested involved in the resistance via alternative glyphosate translocation pattern.