Diverse cis-elements in the promoters of SiCKX genes suggest that CKX proteins are expressed in different plant tissues. For example, SiCKX1, SiCKX3, SiCKX4, SiCKX5, SiCKX8, SiCKX9, and SiCKX10 all have salt-responsive element (GT1GMSCAM4) ( Table 2) and their gene expressions were obviously up-regulated under salt condition ( Fig. 6). Conversely, other genes (SiCKX2, SiCKX6, SiCKX7, and SiCKX11)

were not responsive to salt stress compared to the control ( Fig. 6) Ku-0059436 molecular weight probably due to the lack of the salt-responsive element ( Table 2). Paralogs are genes derived from duplication events within a genome. Segmental (chromosomal segments) duplication, tandem duplication (duplications in a tandem pattern), and transposition events, can result in duplication of gene families [52]. Duplicate genes provide raw materials SB203580 solubility dmso for evolution of new gene

functions. Phylogenetic analysis has been commonly used to identify gene families and predict their functional orthologs [37], [53] and [54]. However, there is far less evolutionary information about the CKX gene family in foxtail millet. To detect the expansion of this family in S. italica in our study a phylogenetic tree was reconstructed using full-length SiCKX protein sequences ( Fig. 4). The phylogenetic tree divided the SiCKX genes into several distinct groups. Among the 11 proteins, three pairs of paralogous proteins (SiCKX1/SiCKX3, SiCKX2/SiCKX4, and SiCKX10/SiCKX11) and one tandemly duplicated protein (SiCKX5/SiCKX8) were found, suggesting that divergence in each protein pair occurred relatively late. Each of other three SiCKX proteins,

including SiCKX 6, SiCKX7, and SiCKX9, occupied a distinct branch. Furthermore, SiCKX6 was basal to SiCKX2/SiCKX4. These results suggested that SiCKX6, SiCKX7, and SiCKX9 may have diverged earlier from the other SiCKX proteins. Further investigation suggests that both segmental duplication and tandem duplication led to expansion of CKX gene family in the foxtail millet genome ( Fig. 5). Members of the CKX family in wheat, soybean, cotton, Arabidopsis, and Zea mays showed tissue-specific expression patterns. Wheat TaCKX3 was expressed in embryos, and was strongly up-regulated Miconazole by 6-BA [31]. In soybean, GmCKX12 and GmCKX16 were abundant in leaves, while GmCKX13 and GmCKX14 were highly expressed in young shoots [32]. GhCKX transcripts were found in cotton roots, hypocotyls, stems, leaves, and ovules. The highest expression level was found at − 1 DPA (day post anthesis) ovule [55]. Arabidopsis AtCKX1 had slightly higher expression in roots while AtCKX2 was better expressed in shoots [56]. In maize, ZmCKX6, ZmCKX10, and ZmCKX12 were abundant and constitutively expressed in roots, shoots, mature leaves, immature ears, and tassels, whereas ZmCKX2 and ZmCKX3 were preferentially expressed in young leaves and mature leaves, respectively [37].