Genome-wide analysis and expression profiles of PdeMYB transcription factors in colored-leaf poplar (Populus deltoids)

Background MYB transcription factors, comprising one of the largest transcription factor families in plants, play many roles in secondary metabolism, especially in anthocyanin biosynthesis. However, the functions of the PdeMYB transcription factor in colored-leaf poplar remain elusive. Results In the present study, genome-wide characterization of the PdeMYB genes in colored-leaf poplar (Populus deltoids) was conducted. A total of 302 PdeMYB transcription factors were identified, including 183 R2R3-MYB, five R1R2R3-MYB, one 4R-MYB, and 113 1R-MYB transcription factor genes. Genomic localization and paralogs of PdeMYB genes mapped 289 genes on 19 chromosomes, with collinearity relationships among genes. The conserved domain, gene structure, and evolutionary relationships of the PdeMYB genes were also established and analyzed. The expression levels of PdeMYB genes were obtained from previous data in green leaf poplar (L2025) and colored leaf poplar (QHP) as well as our own qRT-PCR analysis data in green leaf poplar (L2025) and colored leaf poplar (CHP), which provide valuable clues for further functional characterization of PdeMYB genes. Conclusions The above results provide not only comprehensive insights into the structure and functions of PdeMYB genes but also provide candidate genes for the future improvement of leaf colorization in Populus deltoids. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-021-03212-1.


Background
Transcription factors play important roles in the regulation of gene expression, which can control the rate of transcription initiation of target genes. MYB transcription factors, one of the largest and best-characterized transcription factor gene families in plants, are widely distributed in all eukaryotic organisms, including animals, plants, and fungi [1,2], which can be involved in various types of plant growth and development.
Most R2R3-MYB proteins among different species are largely conserved, and they can cluster into the same subgroups according to their sequence similarity. However, divergence between them also exists. A comparative analysis of R2R3-MYB genes among different plant species revealed that this transcription factor gene family has undergone extensive expansion during evolution. The expansion of the R2R3-MYB family in plants fits well with the observation that many R2R3-MYB members participate in various biological processes and plant-specific processes [15].
More and more R2R3-MYB transcription factors have been functionally identified since the first identification of the plant MYB gene C1 from Zea mays [16]. The R2R3-MYB transcription factors are mainly involved in stamen and pollen maturation [17], trachoma initiation [18], root formation and development [19,20], hormone signal transduction [21], embryogenesis, and stress [22][23][24]. Recently, the functions of R2R3-MYB transcription factors in the regulation of secondary metabolism (particularly anthocyanin biosynthesis and metabolism) have been reported in many species, including apple [25], grape [26], litchi [27] and strawberry [28]. Based on phylogenetic analysis, the R2R3-MYB transcription factors associated with anthocyanin biosynthesis in different species typically belong to the same subgroup. In P. trichocarpa, the anthocyanin content of male catkins is higher, and the transcript levels of R2R3-MYB transcription factors, including PtrMYB116, PtrMYB117, PtrMYB118, and PtrMYB119, in the male catkins of P. trichocarpa are also higher. Another R2R3-MYB transcription factor, PtrMYB120, could play a role in vegetative tissues by evoking anthocyanin biosynthesis to protect them from the deleterious effects of UV light [14]. Transgenic poplars overexpressing PtrMYB119, PtrMYB120, or PdeMYB118 show an elevated accumulation of anthocyanins throughout the plant [29,30]. Although some R2R3-MYB transcription factors associated with anthocyanin biosynthesis have been identified, many more MYB transcription factors associated with anthocyanin biosynthesis are required to obtain new coloredleaf tree species.
With the development of the social economy, the roles of colored-leaf trees in urban beautification are increasing. Leaf color formation is very complicated, and the distribution and concentration of anthocyanins play important roles in the formation of leaf color. Anthocyanin accumulation is controlled through the coordinated expression of genes encoding the anthocyanin biosynthetic pathway enzymes, and R2R3-MYB transcription factors are considered crucial in the regulation of anthocyanin synthesis.
In the current study, a comprehensive investigation of PdeMYB transcription factors in colored-leaf poplar (Populus deltoids) was conducted, and the analysis of phylogenetic relationships, sequence features, gene duplication, chromosome distribution, and motif recognition was performed. In addition, a comprehensive expression analysis of PdeMYB genes in green leaf poplar (L2025) and colored leaf poplar (QHP) using previous RNA-seq data was performed. The expression levels of candidate PdeMYB genes in green leaf poplar (L2025) and colored leaf poplar (CHP) were also evaluated by qRT-PCR analysis. These findings should not only provide a characterization of the PdeMYB gene superfamily but also provide valuable information for further functional elucidation of these genes in colored-leaf poplar, which is useful for generating colored-leaf tree species by genetic engineering.

Genome-wide identification of the PdeMYB gene family in P. deltoids
A total of 302 PdeMYB genes were identified based on the complete genome sequences of P. deltoids. The length of amino acids for the identified PdeMYB proteins ranged from 51 bp (PdeMYB190) to 1717 bp (PdeMYB289), with an average of 342.8 bp (Additional file 1). The molecular weight of the identified PdeMYB proteins ranged from 5.6084 kDa (PdeMYB190) to 188.4509 kDa (Pde-MYB289), and the predicted isoelectric points of these ranged from 4.08 (PdeMYB223) to 10.98 (PdeMYB298). In addition, the subcellular localization of the identified PdeMYB proteins was also predicted, and 208 of 302 (approximately 69%) PdeMYB proteins were localized in the nucleus (Additional file 1).
In plants, MYB proteins are characterized by a highly conserved MYB domain at the N-terminus, which contains one to four imperfect repeats, and can be classified into four major subfamilies: 1R-like MYB, R2R3-MYB, R1R2R3-MYB, and 4R-MYB. In our study, a total of 302 PdeMYB transcription factors were identified, including 183 R2R3-MYB, five R1R2R3-MYB, one 4R-MYB, and 113 1R-MYB.

Phylogenetic analysis of the PdeMYB gene family between P. deltoids and Arabidopsis
An unrooted phylogenetic tree among the R2R3-MYB, R1R2R3-MYB, and 4R-MYB genes of P. deltoids, rice, and Arabidopsis was constructed using the neighbor-joining method with MEGA 7.0 (Fig. 1). The PdeMYB genes of P. deltoids were divided into 10 groups (I to X) according to their sequences. Each group contained a different number of PdeMYB genes. Group III contained 50 PdeMYB genes, which was the largest number in the 10 groups, while group IV contained 4 PdeMYB genes, which was the smallest number in the 10 groups. Except group IV, VII, and X, the other groups of P. deltoids possessed much more MYB gene members than these of Arabidopsis and rice in the seven out of ten groups. Each group could be further divided into several subgroups. Some subgroups just included P. deltoids MYB genes but no AtMYB and OsMYB gene, indicating that these genes may have occurred in P. deltoids during evolutionary process. While some subgroups just contained AtMYB or OsMYB genes with no P. deltoids MYB genes, suggesting that some evolutionary changes occurred in the genome -the MYB genes could have been either acquired in Arabidopsis or rice during evolution or lost in P. deltoids. The gain and loss of species-specific MYB genes could have resulted in functional divergence.  The phylogenetic tree of 1R-MYB genes in Arabidopsis, rice, and P. deltoids was constructed using the neighbor-joining method with MEGA 7.0 (Fig. 2). The PdeMYB genes in P. deltoids were divided into seven groups based on their sequences. Most groups contained the 1R-MYB genes of P. deltoids, rice and Arabidopsis, while Pr1 group just contained the 1R-MYB genes of P. deltoids and rice. Pr2 contained 20 Pde-MYB genes, which was the largest number in the seven groups, while Group Pr1 contained 9 PdeMYB genes, which was the smallest number in the seven groups. Except group Pr4 and Pr7, the other groups of P. deltoids possessed much more MYB gene members than these of Arabidopsis and rice in the five out of seven groups. In Pr1 group, one of the subgroups contained all 1R-MYB genes of P. deltoids including PdeMYB215, PdeMYB258, PdeMYB253, Pde-MYB255 and PdeMYB254. In Pr6 group, one of the    subgroups contained all 1R-MYB genes of rice, such as OsMYB173, OsMYB136, OsMYB157, OsMYB206 and so on. In Pr4 group, one of the subgroups contained all 1R-MYB genes of Arabidopsis including AtMYB156, AtMYB163, AtMYB164, AtMYB166, AtMYB169, and AtMYB170. There are much more MYB gene members of rice in Pr7 than these of P. deltoids and Arabidopsis, which indicated that these MYB genes could be either acquired in rice during evolution or lost in P. deltoids and Arabidopsis. The above results also indicated the differences during the evolutionary process of 1R-MYB genes between P. deltoids and Arabidopsis.

Conserved gene structure and protein motif analysis of the PdeMYB gene family in P. deltoids
To better understand the structural diversity and motif composition of PdeMYB genes, the intron-exon structure pattern was analyzed and visualized using the Gene Structure Display Server 2.0 (Fig. 3). A total of 292 PdeMYB genes possessed exons varying from 1 to 10, which accounted for 97% of the total PdeMYB genes. Sixteen PdeMYB genes lacked introns and had only one exon, including PdeMYB14, PdeMYB24, PdeMYB25, PdeMYB59, PdeMYB76, PdeMYB121, PdeMYB126, PdeMYB127, PdeMYB190, PdeMYB229, PdeMYB248, PdeMYB264, PdeMYB266, PdeMYB267, PdeMYB294, and PdeMYB295. The majority (164 of 302) of the Pde-MYBs had typical splicing, with three exons and two introns. PdeMYB256 and PdeMYB257 contained 19 exons and 18 introns, which was the greatest number of exons in the total PdeMYB genes. Moreover, the number of introns in the MYB genes appeared to be limited, as most PdeMYB genes (75%) had no more than two introns. Phylogenetic analysis of the PdeMYB gene family was performed according to the intron number and exon length. As the position(s) of the intron(s) were fully conserved, genes in the same subgroups had similar intron patterns, such as in PdeMYB238/PdeMYB246 and PdeMYB113/PdeMYB142 (Fig. 3).
As shown in Fig. 4, six conserved motifs were predicted to further reveal the diversification of PdeMYB genes in P. deltoids. In most cases, a motif is repeated only once or twice, and one gene has only one motif repeated. However, only a few special cases exist. Pde-MYB71, PdeMYB194, PdeMYB170, and PdeMYB83 contained two repeated motifs, and each motif was repeated twice. PdeMYB6 contained three repeated motifs: motif 3 repeated three times, motif 4 repeated twice, and motif 6 repeated four times. PdeMYB57 also contained three repeated motifs, and each motif was repeated twice. PdeMYB21 contained four repeated motifs: motif 2 repeated twice, motif 3 repeated twice, motif 4 repeated twice, and motif 5 repeated twice.

Chromosomal location and duplication events of PdeMYBs in P. deltoids
To determine the genomic distribution of the PdeMYB gene family in P. deltoids, the chromosomal locations of PdeMYBs were evaluated according to their genomic sequences. There are 19 chromosomes in the P. deltoids genome, and each of the 19 P. deltoids chromosomes contained PdeMYB genes (Fig. 5). Although PdeMYB genes were distributed on all chromosomes in P. deltoids, their distribution on each chromosome seemed to be uneven. In the present results, there were 289 PdeMYB genes assigned to 19 chromosomes: 32 PdeMYBs were present on chromosome 1; 23 on chromosome 2; 20 on chromosomes 4 and 15; 19 on chromosomes 6 and 8; 18 on chromosome 17; 16 on chromosomes 3 and 10; 14 on chromosome 5; 13 on chromosomes 12, 13, and 14; 12 on chromosomes 9 and 18; 11 on chromosome 19; 9 on chromosome 9; 5 on chromosome 16; and 4 on chromosome 11. Chromosome 1 had the highest number of PdeMYB genes (32), followed by chromosome 2 (23). Chromosome 11 had the least number of PdeMYB genes (4). In addition, 13 PdeMYB genes belonged to the scaffold.
Gene duplication, especially tandem and segmental duplication events, contributes greatly to the diversity and evolution of gene families. In the present study, there were 59 duplicated PdeMYB gene pairs in the P. deltoids genome, and most of them belonged to the segmental duplication or whole genome duplication event. A total of 56 pairs of PdeMYB genes were identified as whole genome duplication or segmental duplications, including 53 duplication events between different chromosomes as well as three duplication events within the same chromosome (PdeMYB10/PdeMYB11, PdeMYB12/PdeMYB13, and PdeMYB47/PdeMYB48), while three pairs of Pde-MYBs were found as tandem repeats in P. deltoids, including PdeMYB156/PdeMYB157, PdeMYB53/PdeMYB54, and PdeMYB155/ PdeMYB156. Interestingly, genes such as PdeMYB156, PdeMYB277, PdeMYB162, and Pde-MYB47 occurred in more than two gene pairs (Additional file 2).
To evaluate the selection of the duplicated PdeMYB gene pairs, the non-synonymous to synonymous substitution ratios (Ka/Ks) were calculated according to the whole genome analysis of gene duplications. When the Ka/Ks ratio is greater than one, the identified genes are under positive selection; when the Ka/Ks ratio is one, the identified genes are under neutral selection; when the Ka/ Ks ratio is less than one, the identified genes are under negative purifying selection. In the present study, there were 57 PdeMYB gene duplicated pairs, the Ka/Ks ratios of which were less than one, indicating that these genes are under negative purifying selection and contribute largely to the maintenance of function in the PdeMYB gene family of P. deltoids. There were two PdeMYB gene duplicated pairs, the Ka/Ks ratios of which were more than one, indicating that these genes have likely  To evaluate the possible relationship between the PdeMYB genes and potential duplication events, the collinearity of the PdeMYB gene family in P. deltoids was identified using the BLASTP and MCScanX methods. A total of 56 segmental duplication events with 102 PdeMYB genes were identified in the P. deltoids genome (Fig. 6). PdeMYB genes were located within synteny blocks on all chromosomes. Intrachromosomal duplication was also observed in the P. deltoids genome.
To further evaluate the potential evolutionary mechanisms of the PdeMYB gene family in P. deltoids, two comparative syntenic maps were constructed between P. deltoids, Arabidopsis, and rice (Fig. 7). There were 60 collinear MYB gene pairs between P. deltoids and Arabidopsis and 77 orthologs between P. deltoids and rice. The details of the collinear MYB gene pairs were provided in Additional files 3 and 4. The number of orthologous

Expression profile of the PdeMYB genes in QHP and L2025 by RNA-seq
To evaluate the expression pattern of PdeMYB genes in colored-leaf poplar, the expression profiles of PdeMYB genes in the leaves and buds of QHP and L2025 were screened from the previous data. The expression levels of candidate PdeMYB genes in the leaves of QHP and L2025 were also evaluated (Fig. 8). The expression level of candidate PdeMYB genes in the leaves of QHP was more than 10 times that in L2025 or specifically expressed in the leaves of QHP or L2025, and these candidate PdeMYB genes are shown in Additional   Fig. 6 Chromosomal localization and paralogs of PdeMYB genes in P. deltoids. The chromosomal position of each PdeMYB was mapped according to the P. deltoids genome. A red link indicates that two genes belong to the PdeMYB gene family, a green link indicates that one of the genes belongs to the PdeMYB gene family, and a gray link indicates that none of the genes belong to the PdeMYB gene family file 5. The expression level of PdeMYB154 in the leaves of QHP was 13.28 times higher than that of L2025, and 33.21 times higher for PdeMYB155. Many Pde-MYB transcription factors are specifically expressed in QHP, such as PdeMYB25, PdeMYB27, PdeMYB60, Pde-MYB114, and PdeMYB160, which indicated that these genes might play important roles in the formation of colored leaves in QHP.

Expression analyses of PdeMYB genes in CHP and L2025 by qRT-PCR analysis
To better explore the expression pattern of PdeMYB genes in leaf-colored poplar, the candidate PdeMYB genes were also evaluated in the leaves of CHP and L2025 (Fig. 9). The expression levels of some PdeMYB genes between CHP and L2025 were not substantially different, such as PdeMYB25, PdeMYB99, and P9de-MYB279, which may not be important in the coloration of CHP. Some PdeMYB genes such as PdeMYB60, PdeMYB70, PdeMYB96, and PdeMYB114, which are specifically expressed in the leaves of CHP, may be candidate PdeMYB genes to further explore the mechanism of leaf coloration in poplar. Other PdeMYB genes, such as PdeMYB4, PdeMYB37, PdeMYB72, and PdeMYB146, which are specifically expressed in the leaves of L2025, may also be important candidate genes for studying their functions.

Discussion
MYB transcription factors play important roles in secondary metabolism (especially in the anthocyanin pathway), development, signal transduction, and disease resistance, and comprise one of the largest transcription factor families in plants [7]. Many reports have indicated that MYB transcription factor genes can regulate the formation of leaf coloration in plants. Transgenic Arabidopsis overexpressing PAP1/AtMYB75 increases the expression level of structural genes associated with anthocyanin biosynthesis, which contributes to higher anthocyanin accumulation [7,31,32]. Tobacco overexpressing IbMYB1a can upregulate the expression levels of several structural genes, such as ANS and DFR 22, which can promote the accumulation of anthocyanin [33]. Tobacco overexpressing CsMYB6A increases the expression levels of structural genes associated with flavonoid synthesis, such as 3GT and CHS, leading to a high accumulation of anthocyanins in the leaves of transgenic tobacco [34]. Transgenic poplar overexpressing MYB6 shows a red color in the young leaves and shoots by increasing their anthocyanin accumulation [35]. PtrRML1, a repressor motif-containing poplar R3 MYB-like transcription factor, the overexpression of which in transgenic Arabidopsis can reduce their anthocyanin content in stems, petioles, and rosette leaves [36]. Overexpression of PtoMYB156, PtrMYB57, MYB182, MYB165, and MYB194 separately can also downregulate   anthocyanin biosynthesis in transgenic poplar [37][38][39][40]. Transgenic poplar overexpressing PtrMYB119 and Pde-MYB118 separately has higher anthocyanin content in leaves compared with that in wild-type poplar [29,30]. Although some MYB genes from poplar can regulate the biosynthesis of anthocyanin, many more MYB genes are needed to regulate the leaf color of poplar. To date, only PtrMYB119 and PdeMYB118 from poplar have been shown to change the leaf color of poplar under normal conditions. Therefore, genome-wide analysis of the MYB family genes in poplar was characterized, and many more MYB family genes from poplar associated with anthocyanin biosynthesis need to be identified in the future.
Many MYB gene families have been identified in different plant species. There are 198, 233, and 197 MYB genes in Arabidopsis, rice, and soybean, respectively [41,42]. In the present study, 302 PdeMYB transcription factors were identified in the P. deltoids genome. Among these, R2R3-MYBs accounted for 61% of the identified MYB transcription factors, and MYB-related proteins were the second largest subfamily of MYB proteins, accounting for 37% in our study, which is similar to the fraction of MYBrelated proteins in rice (40%) [43]. In P. trichocarpa, there are 192 R2R3-MYB and 5 R1R2R3-MYB [14], which is similar to our results in the P. deltoids genome.
Gene duplication plays an important role in gene family expansion in plants [44], either in the form of segmental duplication, tandem duplication, or transposition events [45]. When gene duplication occurs within the same chromosome, tandem duplication occurs. In different chromosomes, this is considered segmental duplication. Both segmental and tandem duplication can lead to the diversification of species, which might also be crucial for increasing the adaptability of plants to different environmental conditions [46]. In the present study, expansion of the R2R3-MYB gene family in P. deltoids resulted Fig. 9 The relative expression level of candidate PdeMYB genes in the leaves of L2025 and CHP. Bars indicate the mean ± SE, n = 3 from both segmental duplication and tandem duplication events, which are similar to those in maize and Gossypium raimondii [45,47]. There were three tandem duplications and 56 segmental duplications for PdeMYB genes in P. deltoids, which indicated that segmental duplication events were a major cause of the expansion of PdeMYB genes.
It is generally believed that PdeMYB members that fall within certain clades may have common evolutionary origins and conserved functions. Therefore, the putative functions of the P. deltoids MYB proteins can be speculated through the functional clades of Arabidopsis MYBs. Phylogenetic analyses and evolutionary relationships of the PdeMYB gene family have been systematically studied among different species, suggesting the conservation and expansion of P. deltoids MYBs. In addition, the pattern of gene structures can be used to evaluate the phylogenetic relationships in a gene family. The number of exons in the 302 PdeMYB genes ranged from one to ten, and most of the PdeMYBs had three exons and two introns, which is similar to the results in other plants [9]. Remarkably, the PdeMYB members within the same subfamily shared similar exon/intron patterns [11]. In our study, most of the PdeMYB genes were disrupted by no more than two introns, which is consistent with previous reports that most MYB-related genes in land plants contain up to two introns [48].
Phylogenetic classification of genes into subfamilies was performed according to the gene structure and domain analysis. In general, members of the same subfamily often share similar sequences, conserved motifs, and even binding partners, and are likely to share similar functions [49,50]. Therefore, when there is a lack of sufficient data, functional characterization of unknown proteins is generally carried out based on comparisons with annotated proteins [13,51]. In the present study, PdeMYB genes were clustered into the same group as those in Arabidopsis thaliana, indicating the functional conservation of R2R3-MYBs between species. In Arabidopsis thaliana, MYB75, MYB90, MYB113, and MYB114 have been reported to be involved in the regulation of anthocyanin biosynthesis [7,10,51]. PdeMYB155, PdeMYB157, PdeMYB156, PdeMYB153, and PdeMYB154 were clustered into the same group as AtMYB75, AtMYB90, AtMYB113, and AtMYB114 in Arabidopsis thaliana, indicating that these genes in poplar might be involved in the synthesis of anthocyanin. In accordance with this, the expression level of PdeMYB154 and PdeMYB155 in the leaves of QHP was much higher than these in L2025 (Fig. 8), and a similar situation occurred in another colored leaf cultivar, CHP (Fig. 9). PdeMYB60 and PdeMYB114 are specially expressed in the leaves of QHP and CHP plants, indicating that they may play important roles in leaf coloration in poplar (Figs. 8 and 9). In contrast to the expression patterns of PdeMYB60 and PdeMYB114 in QHP and CHP, PdeMYB25 was specifically expressed in the leaves of QHP, but there was no significant difference in its expression level between CHP and L2025 (Figs. 8 and 9), which indicated that PdeMYB25 might not be a critical gene during leaf coloration in poplar. Although many genes might be involved in leaf coloration in poplar, the detailed functions of these anthocyanin-related genes should be further identified by experimental assays in the future.

Identification of the PdeMYB gene family in P. deltoids
The hidden Markov Model profile of MYB binding domain with accession number was obtained from the Pfam database (http:// pfam. xfam. org/) [52], which was used to search candidate PdeMYB genes from P. deltoids genome with HMMER 3.1, with a cutoff value of 0.01. The P. deltoids genome was downloaded from the Joint Genome Institute's Plant Genomics Portal (https:// phyto zome-next. jgi. doe. gov/), and HMMER 3.1 was downloaded from http:// hmmer. org/ downl oad. html. In addition, MYB genes in Arabidopsis thaliana and rice were used to further identify candidate Pde-MYB genes in P. deltoids genome, which were downloaded from the Arabidopsis Information Resource (TAIR; http:// www. arabi dopsis. org/) and RGAP release 7 (http:// rice. plant biolo gy. msu. edu/). The SMART and NCBI-CDD were used to confirm the acquired Pde-MYB proteins in P. deltoids, the website of which were http:// smart. embl-heide lberg. de and http:// www. ncbi. nlm. nih. gov/ Struc ture/ cdd/wrpsb.cgi.

Sequence analysis and structural characterization of PdeMYB genes in P. deltoids
Based on the conserved domains and genome sequence of the PdeMYB genes, the exon-intron organization of the PdeMYB genes, including intron distribution patterns, phases, and intron-exon boundaries, was graphically displayed by the Gene Structure Display Server GSDS2.0 (http:// gsds. cbi. pku. edu. cn/). The conserved motifs of the PdeMYB transcription factors were predicted using a MEME Suite analysis [53], and the software was downloaded from http:// meme-suite. org/ tools/ meme. The maximum number of motifs was set to identify 20 motifs, and the optimum width of motifs was set from 6 to 100 amino acids.

Chromosome distribution, gene duplication, and synteny of PdeMYB genes in P. deltoids
The chromosome distribution of PdeMYB genes was obtained from the database of the P. deltoids genome (https:// phyto zome-next. jgi. doe. gov/), and the chromosomal locations of the PdeMYB genes were visualized using MapChart software [54]. Segmental duplication, tandem duplication, and synteny blocks of the orthologous PdeMYB genes between P. deltoids and Arabidopsis as well as P. deltoids and rice were evaluated using MCscanX (Multiple Collinearity Scan toolkit; http:// chibba. pgml. uga. edu/ mcsca n2/) [55]. The synonymous (Ks) and nonsynonymous (Ka) substitutions of PdeMYB genes were calculated using KaKs_Calculator 2.0 to estimate their duplication events [56], and circos v0.69 was used to graphically present the synteny blocks of the orthologous PdeMYB genes between P. deltoids and Arabidopsis, P. deltoids, and rice [57].

Phylogenetic analysis and classification of PdeMYB proteins in P. deltoids
Phylogenetic analysis was performed using the fulllength amino acid sequences of PdeMYB, OsMYB, and AtMYB proteins. An unrooted neighbor-joining phylogenetic tree was constructed through multiple sequence alignments of these MYB proteins using MEGA 7.0, and these MYB proteins were classified into different groups based on the topology of the phylogenetic tree. The parameters were as follows: pairwise deletion, Poisson model, and 1000 bootstrap replications [58]. The data matrices and resulting trees were deposited in TreeBase (http:// purl. org/ phylo/ treeb ase/ phylo ws/ study/ TB2: S28602 and http:// purl. org/ phylo/ treeb ase/ phylo ws/ study/ TB2: S28603), and the accession numbers are 28602 and 28603, respectively.

Expression analyses of PdeMYB genes in Quanhong poplar (QHP) and Populus sp. Linn. 2025 (L2025) by RNA-seq
To explore the expression pattern of PdeMYB genes in colored-leaf polar QHP and green leaf poplar L2025, the expression profiles of the putative PdeMYB genes in the leaves and buds of the QHP and L2025 were retrieved from previous research with PdeMYB gene IDs as the queries [59]. The transcript abundance of PdeMYB genes was calculated as fragments per kilobase of exon model per million mapped reads (FPKM). The log 2 (FPKM +1) from the RNA-seq data were subjected to hierarchical clustering with Cluster 3.0, and the results were graphically displayed using Java TreeView [53].

Expression analyses of PdeMYB genes between CHP and L2025 by qRT-PCR analysis
Colored leaf poplar with bright red leaves, CHP, and green leaf poplar, L2025, were cultivated in the experimental field of the Nanjing Botanical Garden Mem. Sun Yat-Sen (32°3′N, 118°49′E). To better identify the candidate PdeMYB genes associated with leaf color, the expression level of candidate PdeMYB genes in previous research that showed significant changes between QHP and L2025 were further evaluated. The leaves of CHP and L2025 were collected in August 2020, and the RNA was extracted using an RNA Aprep Pure Plant Kit (Tiangen, Beijing, China). The quality and concentration of each RNA sample was evaluated by gel electrophoresis and using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the higher quality RNA with a 260/280 ratio of 1.8-2.1, 260/230 ratio≥2.0, were stored at −80 °C for further analyses. cDNA was synthesized using a ReverTra Ace qPCR RT kit (TOYOBO, Osaka, Japan). The expression levels of candidate PdeMYB genes in the leaves of CHP and L2025 were evaluated by qRT-PCR using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA). Gene-specific primers were designed according to the sequence of candidate PdeMYB genes (Additional file 6), and the ACTIN2 gene was used as a control gene [29]. The thermal cycling conditions were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s, and products collected at 60 °C for 34 s. The relative expression levels of genes were evaluated by the 2 −ΔΔCt method [60], and analyzed using SPSS 17.0, with three biological replicates.

Conclusions
In the present study, 302 PdeMYB transcription factors identified in P. deltoids. Genomic localization and paralogs of PdeMYB genes mapped 289 genes on 19 chromosomes, with collinearity relationships among genes. The conserved domain, gene structure, and evolutionary relationships of PdeMYBs were also established and analyzed. The expression levels of some PdeMYB genes were significantly different among different coloredleaf poplar varieties, which provided valuable clues for further functional characterization, in addition to providing candidate genes for the future improvement of leaf colorization in P. deltoids.