Genome-Wide Identification of the DOG1 Gene Family in Pepper (Capsicum annuum) and Its Expression Profiles During Seed Germination

Abstract

The DOG1 (Delay of Germination1) family plays key regulatory roles in seed dormancy and germination. However, a genome-wide analysis of DOG1 genes has not been performed for pepper (Capsicum annuum), one of the agriculturally important species, and no studies have been conducted to characterize their expression profiles. Based on C. annuum genome information, the identification and expression analysis of CaDOG1 gene family members through bioinformatics approaches can provide a theoretical foundation for subsequent studies on the biological functions of CaDOG1s and the improvement of seed traits in C. annuum breeding. In this study, a total of 13 CaDOG1 genes were identified in the C. annuum genome. Phylogenetic analysis showed that these CaDOG1s, along with DOG1s from thale cress (Arabidopsis thaliana), rice (Oryza sativa), and maize (Zea mays), were classified into four subgroups. All CaDOG1 genes were unevenly distributed on six C. annuum chromosomes, and they had relatively conserved exon-intron patterns, most with zero to one intron. According to the chromosomal distribution patterns and synteny analysis of the CaDOG1 genes, the CaDOG1 family expanded mainly through replication, which occurred predominantly after the divergence of dicotyledons and monocotyledons. Conserved motif analysis indicated that all encoded proteins contained Motif 2 and Motif 6, except for CaDOG1-3. Expression profile analysis using transcriptome data revealed that CaDOG1 genes were differentially expressed across various tissues and developmental stages, with notable involvement in flowers and seeds. Quantitative real-time PCR also revealed that all CaDOG1 genes were downregulated during seed germination, indicating that CaDOG1s may play negative roles in seed germination. Moreover, upon abscisic acid treatment, six CaDOG1 genes exhibited upregulation, while in response to ethylene, four CaDOG1 genes exhibited downregulation. Taken together, these findings provide an extensive description of the C. annuum DOG1 gene family and might facilitate further studies for elucidating their functions in seed germination.

Keywords: DOG1; bioinformatics; expression profiling; pepper (Capsicum annuum); seed germination.

Figure 1

Chromosome localization of CaDOG1 genes. The chromosome numbers are indicated above each vertical bar, and the scale of the chromosomes is in megabases (Mb).

Figure 2

Phylogenetic analysis of DOG1 proteins from different plant species. The DOG1 proteins from thale cress (Arabidopsis thaliana, AtDOG1s), pepper (Capsicum annuum, CaDOG1s), rice (Oryza sativa, OsDOG1s), and maize (Zea mays, ZmDOG1s), were aligned using ClustalX 2.0, and the phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with bootstrap values (>50) of 1000 replications.

Figure 3

Gene structure, motif distribution, and protein structures of CaDOG1s. (a) Exon–intron organizations of CaDOG1 genes. (b) Motif distribution of CaDOG1 proteins. (c) Predicted 3D structures of CaDOG1 proteins. The protein structure was predicted by AlphaFold3. The darker the color, the more reliable the prediction result.

Figure 4

Analysis of similarity and collinearity of CaDOG1 family genes. (a) Protein sequence identity within the CaDOG1 family. (b) Collinearity analysis of CaDOG1 family genes. The outermost circular ring stands for the gene density on the chromosome. The greater the number of red lines, the denser the genes are. The middle circle represents the GC content of the chromosome nucleotide sequence, where higher black lines signify a higher GC content. The innermost ring indicates the chromosome name as well as its length. (c) Collinearity analysis of DOG1 family genes among C. annuum, S. lycopersicum, and A. thaliana. (d) Collinearity analysis of DOG1 family genes among C. annuum, Z. mays, and O. sativa. The red lines highlight the syntenic DOG1 gene pairs.

Figure 5

cis-element analysis of CaDOG1 promoters. Blocks of different colors and quantities represent the number of different promoter elements contained within the upstream region of the CaDOG1 genes at 2000 bp. Darker colors indicate a higher number of promoter elements. The different-colored histogram represents the sum of the cis-acting elements in each category.

Figure 6

Expression patterns of CaDOG1 genes in different tissues of C. annuum. The expression levels of the CaDOG1 gene family were displayed based on RPKM-standardized values (Table S2). F1 to F9 represent flowers with lengths of 2.5, 3.5, 5.0, 7.0, 8.0, 10.0, 12.0, 14.5, and 17.0 mm. FST0 to S11 correspond to fruits collected at 3, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 days post-anthesis, from which placental tissues and seeds were dissected. FST denotes the flower, seed, and placenta complex; ST denotes the seed and placenta complex; and S denotes the seed.

Figure 7

Expression profiles of CaDOG1 genes during C. annuum seed germination. The expression levels of each CaDOG1 gene were quantified by qRT-PCR and normalized to CaActin-7. All experiments were conducted in triplicate with at least three independent biological replicates. Error bars indicate the standard error of the mean. The different lowercase letters indicate significant differences (p < 0.05), as determined using one-way ANOVA followed by the Waller–Duncan test. The sequences of CaDOGL6-1 and CaDOGL6-2 are identical; therefore, they are presented collectively in this figure.

Figure 8

Expression profiles of CaDOG1 genes in response to ACC and ABA during pepper seed germination. The expression levels of each CaDOG1 gene were quantified by qRT-PCR. The expression level for each gene in the CK plants at 0 h was normalized to 1.0. All experiments were conducted in triplicate with at least three independent biological replicates. Error bars indicate the standard error of the mean. A t-test was used to determine statistically significant differences in the expression levels under different conditions compared to CK (* p < 0.05, ** p < 0.01). The sequences of CaDOGL6-1 and CaDOGL6-2 are identical and therefore are presented collectively in this figure.