Weed Science www.cambridge.org/wsc Research Article Cite this article: Montgomery JS, Sadeque A, Giacomini DA, Brown PJ, Tranel PJ (2019) Sex- specific markers for waterhemp (Amaranthus tuberculatus) and Palmer amaranth (Amaranthus palmeri). Weed Sci. 67: 412–418. doi: 10.1017/wsc.2019.27 Received: 12 March 2019 Revised: 2 May 2019 Accepted: 12 May 2019 Associate Editor: Ramon G. Leon, North Carolina State University Keywords: Dioecious; gender; restriction site–associated DNA sequencing; sex determination Author for correspondence: Patrick J. Tranel, University of Illinois, 1201 W. Gregory Drive, Urbana, IL, 61801. Email: tranel@illinois.edu *These authors contributed equally to this work. © Weed Science Society of America, 2019. Sex-specific markers for waterhemp (Amaranthus tuberculatus) and Palmer amaranth (Amaranthus palmeri) Jacob S. Montgomery 1, *, Ahmed Sadeque 1, *, Darci A. Giacomini 2 , Patrick J. Brown 3 and Patrick J. Tranel 4 1 Graduate Student, University of Illinois, Urbana, IL, USA; 2 Research Assistant Professor, University of Illinois, Urbana, IL, USA; 3 Assistant Professor, University of Illinois, Urbana, IL, USA; current: Associate Professor, University of California, Davis, CA, USA and 4 Professor, University of Illinois, Urbana, IL, USA Abstract Waterhemp [Amaranthus tuberculatus (Moq.) J. D. Sauer] and Palmer amaranth (Amaranthus palmeri S. Watson) are troublesome weeds of row-crop production in the United States. Their dioecious reproductive systems ensure outcrossing, facilitating rapid evolution and distribution of resistances to multiple herbicides. Little is known, however, about the genetic basis of dioecy in Amaranthus species. In this work, we use restriction site–associated DNA sequencing (RAD-Seq) to investigate the genetic basis of sex determination in A. tuberculatus and A. palmeri. For each species, approximately 200 plants of each sex were sampled and used to create RAD-Seq libraries. The resulting libraries were separately bar-coded and then pooled for sequencing with the Illumina platform, yielding millions of 64-bp reads. These reads were analyzed to identify sex-specific and sex-biased sequences. We identified 345 male-specific sequences from the A. palmeri data set and 2,754 male-specific sequences in A. tuberculatus. An unexpected 723 female-specific sequences were identified in a subset of the A. tuberculatus females; subsequent research, however, indicated female specificity of these markers was limited to the population from which they were identified. Primer sets designed to specifically amplify male-specific sequences were tested for accuracy on multiple, geographically distinct popula- tions of A. tuberculatus and A. palmeri, as well as other Amaranthus species. Two primer sets for A. palmeri and four primer sets for A. tuberculatus were each able to distinguish between male and female plants with at least 95% accuracy. In the near term, sex-specific markers will be useful to the A. tuberculatus and A. palmeri research communities (e.g., to predict sex for cross- ing experiments). In the long-term, this research will provide the foundational tools for detailed investigations into the molecular biology and evolution of dioecy in weedy Amaranthus species. Introduction The evolution of herbicide resistance in weed species presents a global threat to the sustainability of crop production (Cummins et al. 2013; Yu and Powles 2014). One particularly problematic plant family, Amaranthaceae, includes many agronomically important weeds that have evolved multiple herbicide resistances. Perhaps the most problematic weeds in this genus are Palmer amaranth (Amaranthus palmeri S. Watson) and waterhemp [Amaranthus tuberculatus (Moq.) J. D. Sauer]. Amaranthus tuberculatus and A. palmeri are herbaceous, outcrossing annual plants native to the American Midwest and Southwest, respectively (Sauer 1957). Both species have successfully infested a wide range of cropping systems across the United States, and their increasing ability to survive herbicidal treatments makes them two of the main targets for weed management pro- grams in row-crop systems. Whereas most of the species of genus Amaranthus are monoecious, A. palmeri and A. tuberculatus are dioecious (Murray 1940; Waselkov and Olsen 2014). Their dioecy ensures outcrossing to exchange genetic information and maintain high genetic diversity (Adhikary and Pratt 2015), thereby increasing the potential for the evolution and spread of her- bicide resistance. Unfortunately, the genetic components of dioecy and its evolution are still not well understood in Amaranthus (Gaines et al. 2012; Tranel et al. 2011; Trucco et al. 2009), although some recent studies have had success investigating dioecy in other plant species (Harkess et al. 2017; Henry et al. 2018). In A. palmeri and A. tuberculatus, it is thought that males are the heterogametic sex (Murray 1940; Trucco et al. 2009); however, cytological evalu- ation failed to identify heteromorphic sex chromosomes (Grant 1959). Ultimately, a deeper understanding of dioecy in A. tuberculatus and A. palmeri could provide for novel strategies to manage them. For example, a gene (or genes) controlling sex could be used in a gene-drive system to manipulate sex ratios, potentially to the point of causing local pop- ulation collapse (National Academies of Sciences, Engineering, and Medicine 2016; Neve 2018; https://www.cambridge.org/core/terms. https://doi.org/10.1017/wsc.2019.27 Downloaded from https://www.cambridge.org/core. IP address: 172.81.115.228, on 18 Jul 2019 at 15:06:18, subject to the Cambridge Core terms of use, available at