Genome-Wide Identification and Characterization of the CCT Gene Family in Foxtail Millet (Setaria italica) Response to Diurnal Rhythm and Abiotic Stress

Abstract

The CCT gene family plays important roles in diurnal rhythm and abiotic stress response, affecting crop growth and development, and thus yield. However, little information is available on the CCT family in foxtail millet (Setaria italica). In the present study, we identified 37 putative SiCCT genes from the foxtail millet genome. A phylogenetic tree was constructed from the predicted full-length SiCCT amino acid sequences, together with CCT proteins from rice and Arabidopsis as representatives of monocotyledonous and dicotyledonous plants, respectively. Based on the conserved structure and phylogenetic relationships, 13, 5, and 19 SiCCT proteins were classified in the COL, PRR, and CMF subfamilies, respectively. The gene structure and protein conserved motifs analysis exhibited highly similar compositions within the same subfamily. Whole-genome duplication analysis indicated that segmental duplication events played an important role in the expansion of the CCT gene family in foxtail millet. Analysis of transcriptome data showed that 16 SiCCT genes had significant diurnal rhythm oscillations. Under abiotic stress and exogenous hormonal treatment, the expression of many CMF subfamily genes was significantly changed. Especially after drought treatment, the expression of CMF subfamily genes except SiCCT32 was significantly up-regulated. This work provides valuable information for further study of the molecular mechanism of diurnal rhythm regulation, abiotic stress responses, and the identification of candidate genes for foxtail millet molecular breeding.

Keywords: CCT; Setaria italica; abiotic stress; diurnal rhythm regulation; hormones.

Figure 1

Chromosomal location of putative SiCCT genes. Thirty-six genes were mapped to the millet chromosomes, and one predicted gene was not mapped. The chromosome number is shown above each chromosome, and the length of each chromosome is indicated on the left in megabases (MB).

Figure 2

Phylogenetic relationships of CCT proteins. The phylogenetic tree was constructed from 100 protein sequences from millet, rice, and Arabidopsis using the maximum likelihood method with 1000 bootstrap replicates. The lines of different colors indicate different groups. Three subfamilies of CCT proteins are represented by different colored lines. Hollow circles, black stars, and red triangles represent the CCT proteins of Arabidopsis, rice, and millet, respectively.

Figure 3

Homology of CCT genes between foxtail millet and six representative plant species. Gray lines in the background represent collinear blocks in the genomes of millet and the other plant species, and red lines highlight collinear CCT gene pairs.

Figure 4

Interchromosomal relationships of millet CCT genes. Gray lines represent all common blocks in the millet genome, and red lines represent replicated CCT gene pairs.

Figure 5

Phylogenetic relationships, gene structure, and conserved motifs of SiCCT proteins. (A) Phylogenetic tree constructed based on the domains of the foxtail millet CCT proteins. In the phylogenetic tree, blue areas are members of the COL family, green areas are members of the CMF family, and yellow areas are members of the PRR family. (B) Exon–intron structure of foxtail millet CCT genes. Blue boxes indicate the 5′ and 3′ untranslated regions; a red box represents an exon, and black lines represent introns. (C) Motif composition of foxtail millet CCT proteins. The motifs, numbered 1–10, are indicated by different colored boxes.

Figure 6

Cis-acting element analysis of the CCT gene family in foxtail millet. (A) Number of different promoter elements in the CCT genes indicated by different colors and numbers. (B) Number of cis-acting elements in three functional categories.

Figure 7

Expression pattern of SiCCT proteins under long days. White and black bars represent light and dark periods, respectively. The gene expression level was the FPKM value calculated.

Figure 8

Expression profile of SiCCT genes under different treatments. Red boxes indicate up-regulation; blue boxes indicate down-regulation, and the intensity of the color indicates the intensity of expression. The color intensity of each color block is compared separately for each gene. The numbers on the graph were the expression levels of genes calculated by the 2^−ΔΔCt method.