The grass genus Setaria Beauv., a member of the tribe Paniceae, in the subfamily Panicoideae of the Poaceae, has approximately 125 species worldwide in tropical, sub-tropical and temperate regions, including crop and weed species with different life cycles and ploidy levels [1, 2]. However, the actual number of species in this genus is confused by the presence of multiple names for some species and multiple species under the same name, as well as overlapping morphological characters both within and between species [3, 4]. There are approximately 74 species native to Africa, 25 species in America and the remainder in Eurasia . Setaria italica (foxtail millet), a crop that was domesticated more than 10 thousand years ago , is still cultivated in China, India, Japan and other countries in more arid and semi-arid regions as a stable food grain, and is used as a forage crop in North America, Africa and Australia . Setaria glauca (Weigel) Hubb. (yellow foxtail) is also domesticated and cultivated in India as to complement other food sources [3, 4]. Most other species of the genus are problematic weeds for agricultural crop production , including S. verticillata (L.) Beauv. (bristly foxtail) and S. faberi Herrm. (giant foxtail). S. viridis (L.) Beauv. (green foxtail) is a wide spread species in Eurasia and is known for its repeated evolution of herbicide resistance in North America farms . In the latest phylogenetic analysis, Setaria was found to be polyphyletic, with separate groups correlated by geography rather than the existing sub-generic classification .
Evolutionary relationships within Setaria remain unclear, even after several molecular phylogenetic studies [8, 9]. However, several groups within Setaria were shown to be monophyletic, including the close relationship between S. viridis and S. italica. Numerous studies have shown that the domesticated S. italica has been shown to be most likely derived from the wild S. viridis, including cytological genetical studies , RAPDs , RFLPs , ISSRs  and molecular phylogenetic studies [8, 9, 14]. Detailed studies of the phylogenetic relationships of Setaria, using more than 50 species from all over the world and the knotted1 and ndhF gene markers, found that Setaria is polyphyletic, with some species of the New World classified into other genera [8, 9].
The basic chromosome number of the genus and its close relatives is x = 9 [15, 16], but the genome constitution of the group is so far poorly studied. The diploid genome of S. italica (2n = 2x = 18) was designated as genome A by Li et al . Diploid S. viridis shares the same A genome as S. italica, verified by hybrid fertility and cytogenomic, enzymatic and molecular marker studies [12, 14, 17–19]. S. adhaerans (Forssk.) Link ex Chiov. (2n = 2x = 18) was identified as carrying a distinct genome from genome A (labeled B) by genomic in situ hybridization (GISH) . The genome constitution of the tetraploid S. faberi (giant foxtail) and S. verticillata (bristle foxtail) was identified as being AABB, with 2n = 4x = 36 . GISH studies also indicated that S. glauca bears an unknown genome type that is not related to either the A or B genome . The diploid genome of S. grisebachii Fourn. ex Hemsl (2n = 2x = 18) was identified as genome C due to their poor hybridization signals with both A of S. viridis and B of S. adhaerans by GISH . S. queenslandica (Domin) was detected as being the first autotetraploid in the genus, with a genome constitution of AAAA, with 2n = 4x = 36 . The genome constitutions of most other species of the Setaria genus remain unknown.
The most recent phylogenetic analysis of the genus using the chloroplast marker ndhF shows accessions of S. faberi and S. verticillata grouping with S. viridis and S. italica. However, other accessions of S. verticillata are placed elsewhere, and the authors suggest that this is caused either by the multiple origins of the polyploid and/or homoplasy in the distinguishing characteristic of the retrorse barbs on the sterile bristles in the inflorescence. An earlier study that used the knotted1 nuclear marker found both multiple placements of separate accessions of S. verticillata as well as multiple placements of copies from single accessions . Benalbdelmouna  showed that the genome constitution of both S. faberi and S. verticillata was AABB, which supports the placement of one gene copy of S. verticillata with the A genome species S. italica and S. viridis, and the other copy elsewhere. In the ndhF phylogeny two accessions of S. verticillata are placed with S. adhaerens, shown by Benalbdelmouna to possess genome B. The relationships of S. faberi are less clear, primarily because of insufficient sampling, as the ndhF phylogeny only contains a single accession of S. faberi, and the knotted1 phylogeny does not contain that species.
Due to its small genome size, diploid nature and self-fertilization, S. italica is becoming a new model for functional and evolutionary studies in the grasses, while S. viridis is a model for C4 photosynthesis [21–23]. The release of the genomic sequences of foxtail millet has accelerated the establishment of these model systems [23, 24]. Understanding the genetic relationships of Setaria genome types will be helpful in managing Setaria germplasm, and contribute to our understanding of the evolution history of this group of species. Genomic in situ hybridization (GISH) provides a visual and direct method for investigating genomic composition among species, and is especially useful in elucidating the complex origins of polyploid plants. This technique has been already applied in many groups such as Triticeae, Brassica, Nicotiana, Andropogon, and Setaria species [19, 20]. In this report, GISH was applied to chromosome preparations of Setaria species of Eurasian origin with unknown genome constitution. The known genome types of A, B and C were used as probes to detect the genome composition of these species and to identify new genomes in the Setaria group. To further confirm the results obtained by GISH, 5S rDNA and knotted1 gene sequences were analyzed using Bayesian methods to elucidate their phylogenetic relationships. Sequences of 5S rDNA and knotted1 genes have already useful in the phylogenetic study of many plant species and of Setaria relationship in particular [9, 14, 27].