Phylogenetic tree of the DNA binding with one-finger (Dof) genes in Rosa chinensis. The phylogenetic tree is constructed using MEGA X (Kumar et al., 2018) by the maximum likelihood (ML) method with 1000 bootstrap replicates. Twelve major phylogenetic groups designated from group I to group XII are indicated. Each Dof subgroup is indicated by a specific color.
The motifs and the gene structure analysis of the Rosa chinensis DNA binding with one-finger (RcDof) genes. (A) The motifs of RcDof members. (B) The gene structure of RcDof members. (C) The RcDof members cis-acting element analysis of the promoter regions. The motifs, numbered 1 to 10, are displayed in boxes with different colors. The hormone-related elements including abscisic acid responsive elements (ABRE), gibberellin-responsive elements (GARE-motif, P-box and TATC-box) and auxin-responsive elements (TGA-box and TGA-element) are red, whereas cis-acting regulatory elements essential for the anaerobic induction (ARE), drought-inducibility elements (MBS), and low-temperature responsive elements (LTR) connected with stress are blue.
Phylogenetic tree of the DNA binding with one-finger (Dof) members in Rosa chinensis, Arabidopsis thaliana, Oryza sativa, and Zea mays. Phylogenetic tree is constructed using the maximum likelihood (ML) method with 1000 bootstrap replicates. The inner ring shows the gene structure of Dof members in these four species. The squares in green, yellow, and pink represent untranslated region (UTR), coding sequence (CDS), and Dof sequence. The outer ring shows 12 major phylogenetic groups designated from group I to group XII. Each of the 12 clades is indicated by a specific color.
The location and collinearity analysis of the Rosa chinensis DNA binding with one-finger (RcDof) genes. The outer ring represents the chromosomes of the location of RcDofs. The denser the red line, the greater the number of genes in this part. The inner ring denotes the collinearity analysis of RcDofs. The bright line represents collinearity, and the gray line represents all isomorphic blocks in the genome.
The collinearity analysis of the Rosa chinensis DNA binding with one-finger (RcDof) genes with Arabidopsis thaliana, Oryza sativa, Zea mays, and Glycine max. The bright line represents collinearity with RcDof, and the gray line represents all isomorphic blocks in the genomes of these five species. The yellow line represents the collinearity RcDofs with Z. mays, and the blue line represents the collinearity RcDofs with O. sativa. The green line represents the collinearity RcDofs with A. thaliana, and the blue line represents the collinearity RcDofs with G. max.
The expression analysis of 16 Rosa chinensis DNA binding with one-finger (RcDof) genes in different tissues (roots, stems, leaves, flowers, stamens, and pistils). (A) The organizational diagram of each part of R. chinensis. Each part of the tissue is painted in a different color. (B) The bar chart of 16 RcDofs expression in different tissues. Each RcDofs name is the title on each small image. Different lowercase letters in the same column indicate significant differences between different treatments using Duncan’s test (P < 0.05).
The multiple peptide sequence alignment of RcDofs using Clustal W (Thompson et al., 1994).
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The DNA binding with one finger (Dof), as an important transcription factor, plays an important role in growth and development, primary and secondary metabolism, stress resistance, and plant hormone signal transduction. However, the identification and analysis of the Dof transcription factor family in Rosa is rarely reported. In this study, 28 Rosa chinensis Dof (RcDof) members were identified, which were located on seven chromosomes. The RcDofs were divided into 12 subfamilies according to evolutionary analysis. Through motif, gene structure, and cis-acting element analyses of the 12 subfamilies, the functions of RcDofs were analyzed and predicted. Furthermore, the Dof members in R. chinensis ‘Old Blush’ and another three species (Arabidopsis thaliana, Oryza sativa, and Zea mays) were systematically analyzed. Twelve subfamilies were found in these four species and the motifs and gene structures of Dof members in each subfamily were similar, which further proves that the RcDofs analysis is accurate. Through an intra- and interspecies collinearity analysis, it was found that the collinearity between A. thaliana and R. chinensis is closer in comparison. Tissue expression analysis of RcDofs was by quantitative reverse-transcription polymerase chain reaction (PCR). Quantitative real-time PCR analysis showed expressions of the RcDofs are tissue specific. The RcDofs had higher expression in leaves, roots, and flowers than other tissues. Taken together, this study provides valuable information for future research on functional exploration of RcDof genes and molecular breeding in Rosa.
The Dof transcription factors contain a DNA binding single zinc finger, and the Dof proteins are one of the plant-specific transcription factor families with a single zinc finger conserved domain (Dof domain) rich in a unique cysteine residue that belongs to the C2C2 single zinc finger protein superfamily (Diaz et al., 2002; Gupta et al., 2015). The Dof proteins usually contain only one copy of the conserved Dof domain, which consists of the nucleotide residues from 200 to 400, the N-terminal highly conserved DNA binding domain, and the C-terminal transcriptional regulatory domain (Yanagisawa, 2004). In the binding domain, it conserved the CX2CX21CX2C motif forms a single zinc finger structure, and four conserved cysteine residues in the single zinc finger structure covalently bind to one Zn2+ (Yanagisawa and Schmidt, 1999).
There are similar DNA binding properties for all Dof proteins. The C-terminus contains a transcriptional regulatory domain with multiple functions, which can interact with a variety of regulatory proteins and activate gene expression. There is great variation among different Dof members because of the poor amino acid conservation of the Dof domains (Yanagisawa and Schmidt, 1999), which leads to the functional diversity of the Dof proteins (Kisu et al., 1998).
As an important transcription factor in plants, Dofs play essential roles in growth and development, primary and secondary metabolism, stress resistance, and plant hormone signal transduction (Gupta et al., 2015). Some Dofs are involved in regulation of fruit ripening (Khaksar et al., 2019). In terms of plant growth and development, the maize ZmDof1 protein negatively regulates the expression of the pollen-specific gene Zm401 to control pollen development (Yang et al., 2011). The overexpression of ZmDof1 in Arabidopsis thaliana activated the expression of the phosphoenol/pyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) genes, which increased the amount of plant nitrogen in all of the examined organs in transgenic plants (Kurai et al., 2011). AtDof4 (a Dof in A. thaliana) directly activates the cell wall relaxation factor AtEXPA9, which regulates the development of the seed epidermis (Zou et al., 2013). ZmDof3 regulates the formation of starch and aleurone grains during Zea mays endosperm development, which affects the starch content in grains (Qi et al., 2017). AtDof2 and AtDof3 could be direct targets by the odorant-binding proteins 1 (OBP1) in transgenic A. thaliana, which could regulate the number of cells that are decreased and cause plant dwarfing (Skirycz et al., 2008). GmDof4 and GmDof11 enhanced the lipid content in the seeds of transgenic A. thaliana (Wang et al., 2007). It was predicted that the DzDof2.2 might have a role in regulating auxin biosynthesis based on its orthologue in A. thaliana (Khaksar et al., 2019). When SlCDF1 and SlCDF3, which are Dofs in Solanum lycopersicum, were overexpressed in A. thaliana, the result was direct activation of the expression of COR15, RD29A, and RD10, which increased the resistance of transgenic A. thaliana under drought and salt stress (Corrales et al., 2014). The overexpression of Dof family gene CDF3 in A. thaliana delayed flowering and increased the resistance to drought and cold stress (Corrales et al., 2017).
Dof has been identified in many plants, such as Oryza sativa (Lijavetzky et al., 2003), A. thaliana (Lijavetzky et al., 2003), Jatropha curcas (Wang et al., 2018), Vitis vinifera (Silva et al., 2016), Medicago truncatula (Shu et al., 2015), Durio zibethinus (Khaksar et al., 2019), and Chrysanthemum morifolium (Song et al., 2016), but the identification and analysis of Dof transcription factor family in Rosa is rarely reported. As one of the most important commercialized flower plants worldwide, the research on Rosa has been greatly promoted based on the Rosa genome (Raymond et al., 2018). In this study, we identified the RcDof family genes based on the Rosa chinensis genome, and their gene structures, conserved domains, and tissue-specific expression were comprehensively analyzed. Our findings provide a foundation for future functional analysis of Dof genes in Rosa.
The protein and genome sequences of R. chinensis (PRJNA438537) were obtained from the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) database (Raymond et al., 2018), and the domain of Dof (PF02701) originated from the Pfam database (El-Gebali et al., 2019), as an HMMER structure for an HMMER search (Robert et al., 2011), which the e-value was set for e−20. After confirmation of the Dof domain of RcDofs by Interpro (Sarah et al., 2009), P3DB (Gao et al., 2012), and ExPASy (Elisabeth et al., 2003), and removing duplicates, unique names were given to each RcDof according to the positions on the reference chromosomes. The Dof protein and genome sequences for A. thaliana, O. sativa, and Z. mays were obtained from the phytozome database (Goodstein et al., 2012).
Clustal W (Thompson et al., 1994) was used to align the Dof multiple peptide sequences in R. chinensis. The MEGA X program (Kumar et al., 2018) was used to align the Dof protein sequences in R. chinensis ‘Old Blush’ and with A. thaliana, O. sativa, and Z. mays, which all contain Dof genes. The JTT+G+I model was the most optimal model predicted by MEGA X and produced bootstrap values in 1000 replicates.
The motifs of RcDofs were detected outside the domain of Dof protein sequences in RcDofs, with e-values less than 1e−20 and lengths of 10 to 50 amino acids. The order of motif was numbered according to the protein sequences and predicted by MEME software (Timothy et al., 2009). The exon/intron structure of the RcDofs was analyzed and displayed by the GSDS program (Hu et al., 2015). The location of the RcDofs was according to the position in the DNA (containing the exon and intron together) sequence by the Genewise program (Chen et al., 2016).
The length of RcDof promoters was set in 1500 base pairs (bp), and the genome sequence from the NCBI database was extracted using PlantCARE (Magali et al., 2002) and TBtools (Chen et al., 2020). The collinearity analysis of RcDofs was defaulted by the MCScanX program (Baek et al., 2016) and graphed by Circos (Krzywinski et al., 2009).
rosa hybrid ‘Month-pink’ was chosen as the plant material for analysis, which was a cultivar mainly cultivated in Heilongjiang. The stems, roots, leaves, flowers, stamens, and pistils of the R. hybrid ‘Month-pink’ were sampled during plant flowering, and ≈0.2 g of each sample was harvested. A Trelief RNAprep Pure Plant Kit (TSP411; Tsingke Biotechnology Co., Beijing, China) was used to extract the RNA from the samples, and its purity was determined using a spectrophotometer (NanoDrop 2000C; Thermo Fisher Scientific, Waltham, MA, USA), with optical density (OD) 260/280 nm values between 1.8 and 2.1 and OD 260/230 nm values ranged from 2.0 to 2.2 for all RNA samples. Complementary DNA (cDNA) was generated from the RNA samples using a Goldenstar RT6 cDNA Synthesis Kit (TSK301; Tsingke Biotechnology Co.). The primers of RcDofs for use in quantitative real-time PCR (qRT-PCR) were designed by Primer Premier 5.0 (Singh et al., 1998) software (Supplemental Table 1). A qRT-PCR detection system (CFX96 Touch; Bio-Rad, Hercules, CA, USA) was used with the 2 × TSINGKE Master qPCR Mix (SYBR Green I) (TSE201, Tsingke Biotechnology Co.), and three biological replicates were processed for each sample. RcUBR was used as the reference gene based on the preliminary studies of laboratory (Dong et al., 2021; Klie and Debener, 2011). The relative expression of RcDofs was calculated according to the formula of 2−ΔCt (Zhang et al., 2020). The gene expression data were analyzed using Duncan’s test using statistical software (IBM SPSS Statistics ver. 19.0; IBM Corp., Armonk, NY, USA).
After the hmmbuild analyses using the HMM profile analysis of Dof domains, and according to the retrieved protein sequences, a new hidden Markov model for the Dof protein conserved domain of Rosa was constructed. Twenty-eight protein sequences with Dof domains were identified and none of the duplicate values for the same gene number by genome-wide (Supplemental Fig. 1). They were designated as RcDof01-28 from the R. chinensis ‘Old Blush’ genome. Full-length coding sequences of RcDof01-28 ranged from 522 to 1596 bp, which encoded 28 putative proteins with 173 to 531 amino acid residues (Table 1, Supplemental Tables 2–4).
A phylogenetic tree comprising 28 Dof protein sequences from R. chinensis ‘Old Blush’ was constructed (Fig. 1). The phylogenetic tree of the RcDof sequences was generated with the maximum-likelihood method using a JTT+G+I model, which was the optimal model as determined by MEGA X software. Using the phylogenetic tree and applying the classification method to the RcDofs, 12 subfamilies were separated as I–XII. The conserved motifs of the 28 RcDofs were analyzed using MEME software. Figure 2A shows the motifs and the composition of each RcDof. The motifs of each subfamily were relatively similar, which confirmed the close associations among the same subfamilies in the evolutionary tree. The gene structures and the phylogenetic trajectories of the 28 RcDofs were examined. The Dof domain was conserved in all subfamilies, and the exon and intron numbers, ranging from 0 to 6, were similar in each subfamily (Fig. 2B). The RcDofs cis-regulatory elements in promoters in the 1500-bp upstream region were analyzed using the online software PlantCARE and TBtools. Based on the R. chinensis genome database, nine cis-regulatory elements were found based on the PlantCARE predicted (Supplemental Table 5). Six of the cis-regulatory elements, which included abscisic acid responsive elements (ABRE), gibberellin-responsive elements (GARE-motif, P-box and TATC-box), and auxin-responsive elements (TGA-box and TGA-element) were directly related to hormones, and three were related to plant stress (Fig. 2C), such as salt and heat. These elements indicated that the RcDofs may have a very important role in abiotic stress, and may respond to hormones that are produced due to stress.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05150-21
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05150-21
The Dof in A. thaliana, O. sativa, and Z. mays was retrieved by hmmsearch, and the RcDof in R. chinensis was analyzed as a whole. The phylogenetic tree still adopted the maximum-likelihood (ML) method, using the JTT+G+I model, which is the optimal model predicted by MEGA X. The gene structures of Dof in R. chinensis and others were examined for their relationship with the phylogenetic trajectories. Similarly, the gene family of Dofs was divided into 12 subfamilies in these three species (Fig. 3), and the gene structure of Dofs in each subfamily was also very similar, indicating that their function may be similar.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05150-21
The 28 RcDof members were located on the seven chromosomes (Chrs) of the R. chinensis genome. Chr 5 had the largest number of RcDofs, with seven, followed by Chr2, with six RcDofs. Chr4 had the fewest RcDofs, with only one, followed by Chr6, with two RcDofs. At least one RcDof member was mapped to each chromosome of R. chinensis ‘Old Blush’. Among the 28 RcDofs, there was collinearity for eight pairs of RcDofs, and most of them were the RcDofs between different chromosomes that produced collinearity, indicating that there may be many close relationships between their Dof genes (Fig. 4).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05150-21
The collinear analysis of Rosa Dof family genes with other species is shown in Fig. 5. RcDofs and A. thaliana genes produced more than nine pairs of collinearity, and there are only two sets of genes in O. sativa and Glycine max, respectively, and only one pair of genes in Z. mays. In comparison, there was a similarity in the relationship between Rosa and A. thaliana.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05150-21
Here, 16 RcDofs randomly distributed in all subgroups were selected for specific-tissue expression analysis by qRT-PCR in leaves, stems, roots, flowers, stamens, and pistils. RcDofs had the expression in all those tissues (leaves, stems, roots, flowers, stamens, and pistils) and the data expression in different tissues showed obvious difference, as shown in Fig. 6. The expression data of RcDofs by qRT-PCR analysis showed that RcDofs had a higher expression in the roots, flowers, or leaves compared with other tissues, which indicated that the roots, leaves, and flowers might be target tissues for future research on the function of RcDofs.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05150-21
The Dof genes are plant-specific transcription factors. They are components that play important roles in plant growth, plant hormone response, and environmental stress (Noguero et al., 2013). In this study, a total of 28 RcDof genes were identified from the R. chinensis reference genome. The number of Dof genes is similar to Hordeum vulgare [26 (Moreno et al., 2007)], V. vinifera [25 (Silva et al., 2016)], Sorghum bicolor [28 (Kushwaha et al., 2011)], and O. sativa [30 (Lijavetzky et al., 2003)]. Dof genes in G. max [78 (Guo et al., 2013)] are much larger than those in R. chinensis. These RcDofs were distributed in 12 subfamilies in the phylogenetic relationship analysis, and similar conclusions have been reported for Camellia sinensis (Yu et al., 2020), V. vinifera (Silva et al.,2016), Manihot esculenta (Zou et al., 2019a), Solanum melongena (Wei et al., 2018), and A. thaliana (Lijavetzky et al., 2003). After analysis of the cis-regulatory elements of RcDof members, which are related to plant hormones and plant stress tolerance, similar findings were obtained for Pyrus bretschneideri (Liu et al., 2020), Daucus carota spp. sativus (Huang et al., 2016), A. thaliana (Le and Bellini, 2013), O. sativa (Gaur et al., 2011), Brassica rapa (Ma et al., 2015), and C. sinensis (Li et al., 2016). Similar multispecies analyses have also been reported for S. bicolor with O. sativa and A. thaliana (Kushwaha et al., 2011), and Populus trichocarpa with O. sativa and A. thaliana (Yang et al., 2006).
There are eight pairs of RcDofs with collinearity among the 28 RcDofs, indicating that these RcDofs may have many close relationships. RcDofs had nine pairs of collinearity with the genes in A. thaliana, which had more collinearity pairs compared with O. sativa (2) and G. max (2). This phenomenon might be caused by the distance of the genetic relationship and the size of chromosomes of different plants. The functions of these collinear genes can be used as references when studying the functions of RcDofs. AT5G62940 (HCA2) has the function of DNA-binding transcription factor activity in phloem, xylem, and root (Guo et al., 2009; Miyashima et al., 2019). AT5G39660 (CDF2) may have an important role in the timing of the transition from the vegetative to the reproductive phase (Imaizumi et al., 2005), with a similar function for AT1G28310 (DOF1.4) (Riechmann et al., 2000), whereas AT2G46590 (DAG2) may have the function of responding to red light and water stimulus, or in seed germination (Gualberti et al., 2002; Santopolo et al., 2015). AT1G69570 (CDF5) may have the function of regulation of photoperiodism and flowering (Henriques et al., 2017). These gene functions can be studied as potential targets of RcDofs, especially the RcDofs that produce collinearity.
The RcDofs expression data showed a difference in different tissues, which indicated that RcDofs might play an important role in plant growth and development. Similarly, Dof transcription factors in some species also showed a tissue-specific expression: In V. vinifera, the expression data of Dof family members showed a significant difference in berry, flower, rachis, tendril, bud, and seed. The rachis, tendril, and bud had a higher expression (Silva et al., 2016). In S. bicolor, the Dof genes had tissue-specific expression in roots, seedings, leaves, and stems. The roots, seedings, and leaves could be used as target tissues because the Dof genes had a higher expression in these tissues (Gupta et al., 2016). In Musa nana, MaDof genes had specific expression at different development stages (Fr1, Fr2, Fr3, and Fr4) (Dong et al., 2016). In Brachypodium distachyon, BdDof members had a specific expression in four organs (leaf, root, ear, and seed) by qRT-PCR analysis (Hernando-Amado et al., 2012). All of these results showed Dof members had a specific expression in different tissues. In this study, RcDofs had a higher expression in roots, flowers, and leaves than other tissues, similar to these species, which showed a specific-tissue expression. Furthermore, some tissues could be used as target tissues because of the higher expression of Dof genes. In J. curcas, flower buds could be used as target tissue because of the high gene expression of Dof members (Zou and Zhang, 2019). In Citrullus lanatus, leaves and roots could be used as target tissues because ClDofs had a high expression (Zhou et al., 2020). It had been reported that AtDof4.2 was specifically expressed in flower, which inferred that flower could serve as the target tissue for this gene (Skirycz et al., 2007). In this study, RcDofs had a higher expression level in roots, leaves, and flowers. Our findings suggest that roots, leaves, and flowers could be used as target tissues for studying the function of RcDofs.
In this study, 28 RcDofs were identified using the published genome of R. chinensis, and a comprehensive analysis of RcDofs was performed for their detailed information. The evolutionary tree, gene structure motifs, cis-elements, and collinearity had been analyzed. The RcDofs were divided into 12 subfamilies and the gene structure motifs and cis-elements of RcDofs in each subfamily were similar. In collinear analysis, RcDofs had nine pair collinear genes with A. thaliana, which these nine RcDofs might have had a similar function with these A. thaliana genes. Also, the RcDofs showed the tissue-specific expression in qRT-PCR analysis. The expression of RcDofs was higher in roots, leaves, and flowers compared with other tissues, which showed roots, leaves, and flowers could be used as target tissues for studying the function of RcDofs. These results provide a theoretical basis for the follow-up exploration of the functions of RcDofs in Rosa.
Phylogenetic tree of the DNA binding with one-finger (Dof) genes in Rosa chinensis. The phylogenetic tree is constructed using MEGA X (Kumar et al., 2018) by the maximum likelihood (ML) method with 1000 bootstrap replicates. Twelve major phylogenetic groups designated from group I to group XII are indicated. Each Dof subgroup is indicated by a specific color.
The motifs and the gene structure analysis of the Rosa chinensis DNA binding with one-finger (RcDof) genes. (A) The motifs of RcDof members. (B) The gene structure of RcDof members. (C) The RcDof members cis-acting element analysis of the promoter regions. The motifs, numbered 1 to 10, are displayed in boxes with different colors. The hormone-related elements including abscisic acid responsive elements (ABRE), gibberellin-responsive elements (GARE-motif, P-box and TATC-box) and auxin-responsive elements (TGA-box and TGA-element) are red, whereas cis-acting regulatory elements essential for the anaerobic induction (ARE), drought-inducibility elements (MBS), and low-temperature responsive elements (LTR) connected with stress are blue.
Phylogenetic tree of the DNA binding with one-finger (Dof) members in Rosa chinensis, Arabidopsis thaliana, Oryza sativa, and Zea mays. Phylogenetic tree is constructed using the maximum likelihood (ML) method with 1000 bootstrap replicates. The inner ring shows the gene structure of Dof members in these four species. The squares in green, yellow, and pink represent untranslated region (UTR), coding sequence (CDS), and Dof sequence. The outer ring shows 12 major phylogenetic groups designated from group I to group XII. Each of the 12 clades is indicated by a specific color.
The location and collinearity analysis of the Rosa chinensis DNA binding with one-finger (RcDof) genes. The outer ring represents the chromosomes of the location of RcDofs. The denser the red line, the greater the number of genes in this part. The inner ring denotes the collinearity analysis of RcDofs. The bright line represents collinearity, and the gray line represents all isomorphic blocks in the genome.
The collinearity analysis of the Rosa chinensis DNA binding with one-finger (RcDof) genes with Arabidopsis thaliana, Oryza sativa, Zea mays, and Glycine max. The bright line represents collinearity with RcDof, and the gray line represents all isomorphic blocks in the genomes of these five species. The yellow line represents the collinearity RcDofs with Z. mays, and the blue line represents the collinearity RcDofs with O. sativa. The green line represents the collinearity RcDofs with A. thaliana, and the blue line represents the collinearity RcDofs with G. max.
The expression analysis of 16 Rosa chinensis DNA binding with one-finger (RcDof) genes in different tissues (roots, stems, leaves, flowers, stamens, and pistils). (A) The organizational diagram of each part of R. chinensis. Each part of the tissue is painted in a different color. (B) The bar chart of 16 RcDofs expression in different tissues. Each RcDofs name is the title on each small image. Different lowercase letters in the same column indicate significant differences between different treatments using Duncan’s test (P < 0.05).
The multiple peptide sequence alignment of RcDofs using Clustal W (Thompson et al., 1994).
Contributor Notes
This study was supported by the Joint Guiding Project of the Natural Science Foundation of Heilongjiang Province (No. LH2020C014), the China Postdoctoral Science Foundation (2016M591506), and the “Academic Backbone” Project of Northeast Agricultural University of China (19XG04). The authors declare no conflicts.
Q.C. is the corresponding author. E-mail: qshchen@126.com.
Phylogenetic tree of the DNA binding with one-finger (Dof) genes in Rosa chinensis. The phylogenetic tree is constructed using MEGA X (Kumar et al., 2018) by the maximum likelihood (ML) method with 1000 bootstrap replicates. Twelve major phylogenetic groups designated from group I to group XII are indicated. Each Dof subgroup is indicated by a specific color.
The motifs and the gene structure analysis of the Rosa chinensis DNA binding with one-finger (RcDof) genes. (A) The motifs of RcDof members. (B) The gene structure of RcDof members. (C) The RcDof members cis-acting element analysis of the promoter regions. The motifs, numbered 1 to 10, are displayed in boxes with different colors. The hormone-related elements including abscisic acid responsive elements (ABRE), gibberellin-responsive elements (GARE-motif, P-box and TATC-box) and auxin-responsive elements (TGA-box and TGA-element) are red, whereas cis-acting regulatory elements essential for the anaerobic induction (ARE), drought-inducibility elements (MBS), and low-temperature responsive elements (LTR) connected with stress are blue.
Phylogenetic tree of the DNA binding with one-finger (Dof) members in Rosa chinensis, Arabidopsis thaliana, Oryza sativa, and Zea mays. Phylogenetic tree is constructed using the maximum likelihood (ML) method with 1000 bootstrap replicates. The inner ring shows the gene structure of Dof members in these four species. The squares in green, yellow, and pink represent untranslated region (UTR), coding sequence (CDS), and Dof sequence. The outer ring shows 12 major phylogenetic groups designated from group I to group XII. Each of the 12 clades is indicated by a specific color.
The location and collinearity analysis of the Rosa chinensis DNA binding with one-finger (RcDof) genes. The outer ring represents the chromosomes of the location of RcDofs. The denser the red line, the greater the number of genes in this part. The inner ring denotes the collinearity analysis of RcDofs. The bright line represents collinearity, and the gray line represents all isomorphic blocks in the genome.
The collinearity analysis of the Rosa chinensis DNA binding with one-finger (RcDof) genes with Arabidopsis thaliana, Oryza sativa, Zea mays, and Glycine max. The bright line represents collinearity with RcDof, and the gray line represents all isomorphic blocks in the genomes of these five species. The yellow line represents the collinearity RcDofs with Z. mays, and the blue line represents the collinearity RcDofs with O. sativa. The green line represents the collinearity RcDofs with A. thaliana, and the blue line represents the collinearity RcDofs with G. max.
The expression analysis of 16 Rosa chinensis DNA binding with one-finger (RcDof) genes in different tissues (roots, stems, leaves, flowers, stamens, and pistils). (A) The organizational diagram of each part of R. chinensis. Each part of the tissue is painted in a different color. (B) The bar chart of 16 RcDofs expression in different tissues. Each RcDofs name is the title on each small image. Different lowercase letters in the same column indicate significant differences between different treatments using Duncan’s test (P < 0.05).
The multiple peptide sequence alignment of RcDofs using Clustal W (Thompson et al., 1994).
