In the last decade, waterhemp (Amaranthus tuberculatus) has evolved resistance to 2,4-D and HPPD inhibitors in multiple states across the midwestern US. Two populations resistant to both chemistries, one from Nebraska (NEB) and one from Illinois (CHR), were studied using an RNA-seq approach to identify the genes responsible for resistance. In this study, cDNA libraries were generated and sequenced for eight replicates of herbicide-resistant (HR) and herbicide-sensitive (HS) plants from each of the two populations (32 total plants sequenced). Using both a waterhemp transcriptome assembly and a high-quality grain amaranth (A. hypochondriacus) genome as references, differential gene expression analysis was conducted to identify genes that were significantly over- or under-expressed in HR compared to HS. When these differentially expressed genes (DEGs) were mapped back to the grain amaranth genome, physical clustering of the DEGs was apparent at gene expression “hotspots” along several of the 16 grain amaranth scaffolds. Interestingly, mapping of known metabolic genes (cytochrome P450s, glutathione-S-transferases, glucosyltransferases, and ABC transporters) to the A. hypochondriacus genome also showed clustering of these genes at several of these hotspot regions. It may be that the evolutionary forces acting on weedy populations - especially the continual selection provided by herbicides - lead to the formation of genetic islands of metabolism-based resistance genes, akin to the ‘domestication islands’ of crops and ‘pathogenicity islands’ of bacteria. Furthermore, SNP calling on all 32 samples to look for statistically significant resistant-specific variants and subsequent mapping of these variants also found similar patterns of clustering, with allele-specific regions coinciding with the hotspot regions. This allele-specificity at the hotspot regions not only allows for the development of allele-specific assays to diagnose resistance problems in fields, but also provides insights into the regulation of these regions. Further work has begun to identify any potential cis-acting regulators or epigenetic variation leading to this localized difference in expression between R and S plants. These allele-specific expression hotspots are a potentially useful tool in future RNA-seq studies to narrow down the regions of true regulatory control leading to resistance, and will likely provide insights into the evolution of herbicide resistance in weeds.

Gene expression hotspots in herbicide-resistant waterhemp (Amaranthus tuberculatus)

256th National Meeting (2018)

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