**1. Introduction**

Genotyping-by-sequencing (GBS) is a powerful genomic approach for identification of genetic variation on a genome-wide scale for genetic diversity analysis of non-model plants [1–3]. This approach produces high-density, low-cost genotypic information without the requirement for a reference genome sequence [4]. The detailed GBS approach in plant diversity analysis is described in Peterson et al. [3]. In brief, the GBS analysis involves five major steps: (1) genome complexity reduction with restriction enzyme; (2) barcoding the seared genomic DNAs with indexed adaptors; (3) high-throughput sequencing of barcoded DNA fragments; (4) identification of genetic variants through a bioinformatics analysis of de-multiplexed reads; and (5) a genetic diversity analysis of sequenced samples based on sample-by-variant matrix. The GBS application, despite being a powerful approach, has certain limitations, including many missing data points, uneven genome coverage, complex bioinformatics, and issues related to polyploidy [5–8]. To overcome these limitations, a GBS-based pipeline, called Haplotag, was developed by Tinker et al. [9], which can generate tag-level haplotype and single nucleotide polymorphism (SNP) data for polyploid organisms. This approach has been successfully applied in the study of diploid and polyploid genomes in oat (*Avena sativa*) [10–12] and genetic diversity analysis of northern wheatgrass (*Elymus lanceolatus* ssp. *Lanceolatus*) [13].

Crested wheatgrass [CWG; *Agropyron cristatum* (L.) Gaertn.] is one of the perennial species of the genus *Agropyron* that comprises 10–15 species in a polyploid series of diploid (2n = 2x = 14), tetraploid (2n = 4x = 28) and hexaploid (2n = 6x = 42) forms with the P genome [14,15]. *Agropyron* species are native to temperate-frigid grassland and sandy soils of Eurasia [14,16,17], and were first introduced to Canada in 1911 [16]. CWG is the most important commercial species of the crested wheatgrass complex in Canadian grasslands [18]. It is characterized by an extensive root system, making it drought tolerant and winter hardy. CWG is considered an important pasture grass for early spring grazing, providing highly palatable and nutritious forage [19]. This species is easy to establish, has strong competitive ability, tolerates insect predation, provides high forage yield, and can be managed for multiple harvests in a season [16,19,20]. It performs well on marginal lands and semi-desert environments to moist moderately saline soils [19,20]. Due to these features, this species can be used for land reclamation of abandoned croplands, burnt and degraded areas, as well as in erosion control [21]. It has persisted as a high yielding species compared to native forage species, even in 20- to 40-year-old pastures, despite heavy grazing and trampling [19,22]. In addition, CWG is also known to possess traits of interest, including disease resistance, tolerance to abiotic stress, and high yield, which have been utilized in wheat and barley breeding [23–27]. The palatability and nutrient content of CWG declines after anthesis, and it becomes less desirable for summer grazing [19]. Thus, a goal of present CWG breeding programs is to develop later maturing cultivars that would maintain nutritive value into the summer grazing season. Development of high forage-quality, late-maturing CWG cultivars is limited by the relatively long varietal development process, few studies to assess genetic variability of the germplasm, and lack of an effective marker system for marker-assisted and/or genomic selection/breeding. Recent RNA-seq studies in CWG have identified flowering time related genes and flowering related differentially expressed genes [28,29]. This emphasizes the need for genetic diversity studies of CWG for the management and utilization of proper genetic resources in a breeding program as exogamous perennial forage species are often morphologically comparable, though they are genetically highly heterogeneous and heterozygous [30,31]. An adequate level of genetic diversity is crucial for both germplasm adaptation and the long-term sustainability of plant communities [32].

Attempts have been made to assess genetic variability within and among the genus *Agropyron* using molecular markers like amplified fragment length polymorphism (AFLP) [18] and simple sequence repeat (SSR) markers [31,33,34]. The revealed variabilities have allowed for better understanding of the extent of diversity present in the genus. However, these marker systems are unable to provide high resolution of genetic diversity and population structure information to understand the ancestry and microevolution of the populations. Research is needed to assess molecular characteristics of CWG for plant breeding. The molecular characterization is now more feasible than before with the advanced sequencing technology and reduced cost to acquire informative markers such as SNPs in non-model polyploid CWG plants. Recent GBS studies in polyploid plants [10,13] demonstrate the likelihood that GBS will unveil genetic variability on a genome-wide scale in CWG plants, and characterize CWG germplasm for breeding and genetic research.

This study was conducted with the objective to apply GBS in combination with the Universal Network Enabled Analysis Kit (UNEAK) [35] and the Haplotag pipelines to (1) identify genome-wide SNP markers; (2) assess the genetic diversity present in 12 lines of *A. cristatum*; and (3) assess whether the GBS application is useful in the genetic diversity analysis of complex polyploid plants.
