Genome-Wide Analysis of R2R3-MYB Transcription Factors Family in The Autopolyploid Saccharum Spontaneum: An Exploration of Dominance Expression and Stress Response 

 Genome-Wide Analysis of R2R3-MYB Transcription Factors Family in The Autopolyploid Saccharum Spontaneum: An Exploration of Dominance Expression and Stress Response  | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-Wide Analysis of R2R3-MYB Transcription Factors Family in The Autopolyploid Saccharum Spontaneum: An Exploration of Dominance Expression and Stress Response Yuan Yuan, Xiping Yang, Mengfang Feng, Hongyan Ding, Khan Muhammad Tahir, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-199103/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Aug, 2021 Read the published version in BMC Genomics → Version 1 posted 8 You are reading this latest preprint version Abstract Background: Sugarcane ( Saccharum ) is the most important sugar crop in the world. As one of the most enriched transcription factor families in plants, MYB genes display a great potential to contribute to sugarcane improvement by trait modification. We have identified the sugarcane MYB gene family at a whole-genome level through systematic evolution analyses and expression profiling. R2R3-MYB is a large subfamily involved in many plant-specific processes. Results: A total of 202 R2R3-MYB genes (356 alleles) were identified in the polyploid Saccharum spontaneum genome and classified into 15 subgroups by phylogenetic analysis. The sugarcane MYB family had more members by a comparative analysis in sorghum and significant advantages among most plants, especially grasses. Collinearity analysis revealed that 70% of the SsR2R3-MYB genes had experienced duplication events, logically suggesting the contributors to the MYB gene family expansion. Functional characterization was performed to identify 56 SsR2R3-MYB genes involved in various plant bioprocesses with expression profiling analysis on 60 RNA-seq databases. We identified 22 MYB genes specifically expressed in the stem, of which MYB43 , MYB53 , MYB65 , MYB78 , and MYB99 were validated by qPCR. Allelic expression dominance in the stem was more significant than that in the leaf, implying the differential expression of alleles may be responsible for the high expression of MYB in the stem. MYB169 , MYB181 , MYB192 were identified as candidate C 4 photosynthetic regulators by C 4 expression pattern and robust circadian oscillations. Furthermore, stress expression analysis showed that MYB 36, MYB 48, MYB 54, MYB 61 actively responded to drought treatment; 19 and 10 MYB genes were involved in response to the sugarcane pokkah boeng and mosaic disease, respectively. Conclusions: A Genome-wide expression analysis demonstrated that SsMYB genes were involved in stem development and stress response. This study largely contributed to understanding the extent to which MYB transcription factors investigate regulatory mechanisms and functional divergence in sugarcane. Epigenetics & Genomics MYB Sugarcane Expression analysis of stress Allelic diversity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Modern cultivated sugarcane ( Saccharum spp. ) is the major source of sugar for the world. It is the topmost crop concerning total biomass production and is listed among the ten most valuable crops [1]. Sugarcane, having a complex genetic background resulting from polyploid interspecific hybrids, was first domesticated approximately10,000 years ago in New Guinea. Saccharum spontaneum contributes about 10%-15% genome to the modern sugarcane cultivars, endowing the characteristics such as disease resistance and ratooning capacity [2]. The genome of haploid S. spontaneum has been assembled to the chromosome level and used as the reference genome of sugarcane [3]. Because of the development of multiple transcriptome models in recent times, including those for different tissues, developmental stages, and under various stress treatments, huge RNA-seq data has become available and provides detailed insights and rich resources for studying gene functions of sugarcane. Transcription factors recognize specific DNA motifs in upstream regions of the genes to regulate their expression. MYB genes constitute one of the largest families of plant transcription factors and characteristically possess highly conserved Myb DNA-binding domains, forming a helix-turn-helix structure of about 52 amino acids [4]. MYB genes can be divided into four categories, including MYB -related, R2R3-MYB , R1R2R3-MYB , and atypical MYB , depending on the number of adjacent MYB repeats (R). Proteins with a single or a partial MYB repeat, generally located at either ends or middle of the peptide chain, are MYB-related. MYB-related proteins include important telomere binding proteins in maintaining the integrity of the chromosome structure [5]. Moreover, they also play an important role in regulating gene transcription, e.g. , the GARP family of plant Myb-related DNA binding motifs is involved in organ polarity in Arabidopsis[6]. Further, CIRCADIAN CLOCK ASSOCIATED 1 ( CCA 1) and LATE ELONGATED HYPOCOTYL ( LHY ) genes regulate the plant circadian clock [7]. A small number of members of R1R2R3-MYB genes are found in higher plants. Interestingly, plant R1R2R3-MYB genes share a similar function of regulating the cell cycle control with the animals [8]. 3R-MYB has also been involved in cell differentiation [9] and plant stress tolerance [10]. Atypical MYB proteins contain four or more adjacent MYB repeats (R). These proteins have been found to encode in a few plants, e.g., Arabidopsis thaliana, Oryza sativa , Vitis vinifera , Glycine max , Physcomitrella patens (data sources displayed in Materials and Methods 2.1), as shown in Figure 1. Only a few reports have been published about atypical MYB proteins by now, and the role of these proteins in the plant bioprocesses is largely unknown. MYB transcription factors binding specific DNA sequence (CAACG/TG) result from domain structure that is formed by two closely packed amino acid sequence repeats(R) [11]. When the MYB gene contains at least two MYB repeats (R), it has transcription factor characteristics and specifically recognizes the DNA motifs to regulate the gene transcription. R2R3-MYB proteins are the largest subfamily of MYB transcription factors in plants, as well as in S. spontaneum (Figure 1). R2R3-MYB is characterized by two MYB repeats and the presence of a single amino acid (Leu) in the first (R2) repeat [12]. R2R3-MYB has two MYB repeats and a single amino acid (Leu) inserted in the first (R2) repeat. The R2R3-MYB family's expansion originated from the R1R2R3-MYB gene ancestor when losing the R1 repeat sequences during evolution [13] and benefiting from gene duplication events [14]. MYB genes are widely involved in plant-specific processes, such as differentiation [15], hormone response [16], secondary metabolism [17], environmental stress tolerance [18], and diseases resistance [19][20]. At least four MYB genes are involved in lignin biosynthesis in Arabidopsis by activating key regulator genes related to secondary cell wall formation [21-23]. Under environmental stress, MYB genes have been reported to function in response to adverse stress in Arabidopsis . Moreover, AtMYB2 and AtMYB96 function as transcriptional activators in ABA-inducible gene expression under drought stress [24]. AtMYB96 mediates abscisic acid signaling, induces pathogen resistance response by promoting salicylic acid biosynthesis, and provides drought tolerance via controlling the cuticular wax biosynthesis [20, 25]. This study focused on the R2R3-MYB gene family in the S. spontaneum published sugarcane genome. We provided a detailed overview of phylogenetic relationship, gene structure, regulatory elements, expression profiles, allelic evolution, and functional characterization based on abundant transcriptome data. Taken together, our study systematically explored the evolutionary dynamics and functional diversification of SsR2R3-MYB genes and could hence facilitate future research on sugarcane MYB transcription factors. Materials And Methods Obtainment of MYB genes The autopolyploid sugarcane Saccharum spontaneum L. genome was published in 2018 and is available online ( http://www.life.illinois.edu/ming/downloads/Spontaneum_genome/ ). The Hidden Markov Model (HMM) profile of the MYB DNA-binding domain (PF00249) downloaded from Pfam database ( http://pfam.xfam.org/ ) [62] was used to search protein sequences containing MYB domain by hmmsearch program (HMM3.0) [63]. Then, putative MYB proteins were further screened through the NCBI-CDD database to investigate the former protein sequences and delete the proteins with incomplete domains. SbR2R3-MYB genes were obtained by performing the same sugarcane method without publicly available data for sorghum MYB genes. Data for sorghum protein sequences (the newest version of Sbicolor_454_v3.1.1.) were downloaded from the plant genome website Phytozome ( https://phytozome.jgi.doe.gov/ ). Finally, we identified 418 (695) SsMYB genes and 252 SbMYB genes (Table S1), including 202 SsR2R3-MYB genes and 125 SbR2R3-MYB genes, belonging to haplotype genes. A plant phylogeny tree was constructed by the TimeTree Database ( http://www.timetree.org )[64]. The distribution of MYB family genes in 19 plant species were demonstrated on the previously published reports: Ostreococcus lucimarinus , Volvox carteri and Chlamydomonas reinhardtii from PlantTFDB ( http://planttfdb.cbi.pku.edu.cn/ ), and a public plant transcription factor database [65], including Physcomitrella patens [66], Oryza sativa [29], Brachypodium distachyon [28], Zea mays [45], Ananas comosus [33], Vitis vinifera [68], Arabidopsis thaliana [29], Brassica napus [69], Glycine max [70], Medicago truncatula [71], Pyrus bretschneideri [72], Rosa chinensis [73], Populus trichocarpa [74], Beta vulgaris [75], Solanum tuberosum [76], and Solanum lycopersicum [77]. Phylogenetic analysis To generate the phylogenetic trees of MYB transcription factor family genes, multiple protein sequence alignment was performed through ClustalW [78] program using the reported 88 rice MYB proteins [29], and further phylogenetic trees were constructed via the neighbor-joining (NJ) method using software MEGA7.0 [79]. The consistency of the phylogenetic estimates was evaluated through several models as well as pairwise deletion treatment. NJ based phylogenetic tree of sugarcane and sorghum was performed as the same method. Naming R2R3-MYB genes and gene structure Because of the autopolyploid nature of sugarcane ( S. spontaneum ), the identified SsR2R3-MYB genes partly possessed several alleles. The representative gene models for different alleles were screened by comparing the phylogenetic relationship and protein identity with sorghum homology protein and paralogs. Tandem replication genes and paralogs were regarded as new, which gene IDs were followed by P and T, respectively. The 202 representative SsR2R3-MYB genes were named from SsMYB1 to SsMYB202 according to their physical position on the chromosomes. Subsequently, allele names were supplemented with numbers (e.g., The Sspon.01G0002470-1A gene located at the top of chromosome 1A is MYB1-1, and Sspon.01G0002470-2D is named as MYB1-2 ). In general, MYB1-1 as a representative gene model was directly regarded as MYB1 . The naming method of sorghum MYB genes was also treated like that of S. spontaneum. SsR2R3-MYB genes and CDS sequences come from the newest version of Sspon.v20190103. The domain location was derived from the previous hmmsearch results. Gene structures were displayed using the Gene Structure Display Server (GSDS2.0) [80], consisting of the CDS region, intron region, and MYB domain. Each gene structure was arranged according to the phylogenetic location. Collinearity analysis Utilizing MCScanX analysis [78], collinearity relationships of SsR2R3-MYB genes and classifier program were used to sort gene duplication types. The identified collinear gene pairs were mapped to their respective locus in the S. spontaneum genome in a circular diagram using Circos 0.69 [81]. Regulatory element of upstream sequences The 2000 bp upstream sequences were extracted from SsR2R3-MYB genes to the PlantCARE website, plant promoter, and cis-element database [82]. Then, we used them to predict regulatory motifs and estimate potentially related functions. Abundant RNA-seq data showing gene expression To analyze SsR2R3-MYB gene expression profiles thoroughly, 60 RNA-seq data were conducted to decipher their expressions from our lab and cooperative labs. Tissue and development transcriptome contained RNA-seq data of 16 samples, including leaf, stem, three different development stages viz . seeding (35-day-old), pre-maturity (9-month-old), and maturity (12-month-old) stages in S. spontaneum [83]. The leaf development transcriptome was derived from the second leaf alone, the ligule on 11-day-old seedlings; 15 cm leaves were selected and cut into 15 pieces with one segment per centimeter [84]. Mature leaves corresponding to ligule in S. spontaneum , over 12-month-old, were selected to supply circadian rhythm transcriptome using 19-time points, i.e. , 2 hours apart from 6:00 am to the second day 4:00 am, and 4 hours apart from 6:00 am to the third day 6:00 am. RNA-seq were extracted from the drought-treatment sugarcane of FN95-1702, a new sugarcane variety for both sugar and energy, bred by Fujian Agriculture and Forestry University. Sugarcane grown to 4-5 leaves was subjected to the natural drought stress treatment in the greenhouse. The mild drought was characterized by soil relative water content of about 55%~60% after six days, and severe drought by 25%~30% after twelve days. After a severe drought, rehydration was done, and relative water content was kept around 75%～85%, and then leave samples were retaken (5 days later). The R2R3-MYB gene expression profiles were obtained by Blast mapping to express data with transcripts of unreferenced genomes. RNA-seq for pokkah boeng disease were extracted from hybrid sugarcane ZZ1, which is highly resistant to smut disease but highly susceptible to pokkah boeng disease. According to the severity of the diseased leaves, pokkah boeng disease was divided into five grades from 0-5. The mildly diseased leaves (1 or 2 grades) and severely diseased leaves (4 or 5 grades) were selected for analysis, while healthy leaves were used as control (CK). Three samples were extracted for RNA-seq for sugarcane mosaic disease transcriptome analysis. For the infection experiment, sugarcane grown through virus-free tissue culture was used, and then leaves corresponding to ligule were collected one month after the infection, while the control plants were not infected. An expression ratio >2 (adjusted p-value<0.05) was considered statistically significant for evaluating differentially expressed genes. Quantitative RT-PCR spontaneum was planted in Multifunctional Specimen Garden, Institute of Agriculture, Guangxi University. The stem-3 at the third internode and mature leaves were collected for comparing the difference of relative expression between stem and leaf. Pro-stem is short for prophase stem, in which the samples were taken from the stem precursor tissue wrapped in the leaf sheath and is located on the upper part of the stem with obvious stem nodes. Combining with stem-3, stem-6, stem-9, and mature leaves were used to verify the expression during the prophase of stem formation. The extractions of samples total RNA was carried out using TRIZOL reagent (Takara), employing the corresponding protocol. The qualified RNA was reverse transcribed to produce cDNA using PrimeScript TM RT reagent Kit with gDNA Eraser reagent (Takara, Japan). Primers were designed by qPCR-PrimerQuest Tool, and qPCR primers were shown in Table S8. Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was selected as a reference gene [85]. The real-time qPCR with three biological replications were performed with SYBR green on Roche Lightcyler® 480 instrument using 2×TB Green Mix (Takara). The reaction profile was as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, 60°C for 30s, and 95°C for 10s. The relative expression levels were calculated by the 2 - △△ CT method. Results Genome-wide identification of R2R3-MYB genes and classification in S. spontaneum genome Based on the functional annotation of the Myb_DNA-binding domain (PF00249), a total of 418 MYB genes (695 alleles) were identified in the S. spontaneum genome by combining the HMMER program and NCBI-CDD database (Figure 1). The SsMYB gene family was classified into four distinct subfamilies, including 207 MYB-related (329 alleles), 202 R2R3-MYB (356 alleles), 3 R1R2R3-MYB (3 alleles), and 5 Atypical MYB (7 alleles) genes (detailed data presented in supplementary Table S1). Total 122 SbMYB-related, 125 SbR2R3-MYB, 3 SbR1R2R3-MYB, and 2 Atypical MYB genes were also identified to increase the understanding of SsR2R3-MYB genes (Table S3). To analyze the plant MYB genes thoroughly, twenty species in 11 lineages were screened to construct a plant phylogenetic tree with S. spontaneum , including Green algae, Bryophyta, Gramineae, Cruciferous, Leguminous, Rosaceae, Solanaceae, and others. The tree topology reflected the phylogenetic relationship of these species and divergence time (Figure 1). Plant phylogeny showed that the higher plants possessed more MYB genes than the lower plants, such as green algae (e.g., Ostreococcus lucimarinus, Volvox carteri, and Chlamydomonas reinhardtii ). A significant expansion of MYB genes was observed after the Cambrian (about 540~480MYA), demonstrating an explosive biological diversification episode near the early period [26]. Most of the phylogenetic nodes of plant species were observed in the Cretaceous, a geological period when a typical global warming climate contributed to the diversity of the terrestrial species [27]. Compared with the other four kinds of grasses, S. spontaneum had one of the largest MYB genes as predicted by PlantTFDB. One reason is the tetraploid nature of the autopolyploid S. spontaneum (mainly octoploid). However, when corrected for ploidy level, the number of SsR2R3-MYB genes in S. spontaneum was still significantly higher than most of the species, including Arabidopsis and other grass species. From green algae to bryophyte and land plants, the number of MYB genes increased. The phylogenetic analysis of the plant species using the number of MYB genes indicated the extending of MYB genes from lower to higher plants, consistent with previous reports [28]. A neighbor-joining phylogenetic tree of R2R3-MYB genes from O. sativa and S. spontaneum showed that the sugarcane genome contained 15 subgroups (G1-G15) (Figure 2, Table S2) with OsR2R3-MYB genes [29]. Sugarcane and rice diverged in the Paleogene (67-26MYA) (Figure 1); the short divergence time indicated relative conservatism of the ortholog genes. As expected, two species of R2R3-MYB genes were evenly distributed in the tree, and most genes in rice clustered with sugarcane, except for LOC_Os03g14100 . However, the number of genes in each clade varied greatly; for instance, the biggest group, G4, contained 26 genes while the group G13 comprised just one SsMYB gene Sspon.02G0044740-1B . Twenty SsMYB genes from three unique subgroups, G7, G10, and G15, did not contain rice genes, indicating the species' genetic divergence. Besides, the clusters depicted that the sugarcane MYB family exhibited a greater number of genes than that in rice, showing a significant expansion of the SsMYB family. Analysis of genomic location, gene structure, and regulatory elements A total of 202 SsR2R3-MYB genes were named in turn according to their physical position on the chromosomes. MYB genes were distributed throughout all 32 chromosomes (Figure 3C); the autopolyploid S. spontaneum genome comprised of 8 homologous groups of 4 members each [3]. The chromosome distribution map showed that the location of the MYB genes was not evenly distributed. Most of the SsMYB genes were located on Chr3A and Chr7A, encompassing 19 and 16 genes, respectively. About 11 enrichment clusters, tiny fragments on genomic regions containing 3 MYB genes, were detected, and half of these genes contained MYB-binding sites (MBS) depicting potential interaction among each cluster. However, some chromosomes only contained a few MYB genes. For instance, five chromosomes, including Chr2C, Chr2D, Chr6C, Chr8B, and Chr8D, had only one MYB gene. S. bicolor is one of the closest lineages of sugarcane, possessing relatively perfect genome data [30-31]. Total 125 SbR2R3-MYB genes were identified from the available sorghum genome using a similar method (Figure 1, Table S3). The diversity of the gene structure might be a shred of evidence regarding the evolution of gene families. The phylogenetically and gene structure analysis were performed by the Neighbor-Joining method using diverse gene information (Figure 3A and Figure S1). The distribution of the tree branches was basically consistent with the structural features of the genes. In many clusters, various sorghum genes were clustered with highly similar SsR2R3-MYB genes, e.g. , SbMYB92 clustered with SsMYB149 and SsMYB156 while SbMYB27 was clustered with SsMYB30 and SsMYB44 . These results sharpened our understanding of the evolution of gene events during sugarcane polyploidization. A total of 19 SsR2R3-MYB genes did not show the presence of intron, including SsMYB154, SsMYB188, SsMYB194, SsMYB170 , SsMYB122 , SsMYB182 , and SsMYB189 . Many MYB genes demonstrated a domain with a cross-intron structure. Cis-elements in promoter regions play an essential role in controlling transcription and expression, and hence they can deepen the understanding of the regulatory function of MYB genes. Total 2000 bp upstream of transcription initiation site (ATG) was regarded as MYB gene promoters and submitted to the PlantCARE for predicting the motifs. Various motifs from 202 SsR2R3-MYB gene promoters were involved in various plant bioprocesses (Figure 3B). These diversified cis-regulatory elements could be divided into four main categories in terms of function: stress response, hormone response, light response, and plant growth and metabolism. A high percentage of MYB genes in the anaerobic induction (92%) and drought elements (58.9%) indicated that the MYB genes were more likely to function under these stresses. Moreover, a notable gene, MYB88, was found to have 10 LTR motifs, which is a cis-acting element involved in low-temperature responsiveness. The significantly enriched LTR elements (5'-CCG AAA-3') suggested that the MYB88 gene might be involved in plant metabolic response to cold stress. Many of the MYB genes regulate the plant hormone response, especially methyl jasmonate (MeJA) and abscisic acid (ABA) responsiveness. A total of 75 genes promoters enriched regulatory elements TGACG-motif (5'-TGACG-3') and CGTCA-motif (5'-CGTCA-3') involved in MeJA-responsiveness, while 38 gene promoters enriched regulatory elements ABRE involved in abscisic acid responsiveness. These MYB genes were predicted to regulate MeJA and ABA signaling in plants and function in plant defense and leaf abscission. Furthermore, more than thirty light response-related elements were predicted; for instance, conservative light element G-box was widely present in the upstream sequence of genes. Several regulatory elements were also associated with other functions in plant growth and development and regulation of seed growth and meristem development. Genes involved in seed-specific regulation contained the same RY-element (5'-CATGCATG-3'), and the elements involved in meristem expression demonstrated CAT-box (5'-GCC ACT-3') and NON-box (5'-AGATCGACG-3') in promoter regions. Finally, 119 genes were detected to be scattered on MYB binding sites, and 49 genes showed more than one binding site, suggesting that these genes probably interacted with other MYB genes. Four MYB binding elements were found in 202 SsR2R3-MYB promoters, including CCAAT-box (5'-CAACGG-3'), MBS (5'-CAACTG-3'), MBSI (5'-aaaAaaC(G/C)GTTA-3'), and MRE (5'-AACCTAA-3'). There was only one base difference between the former two elements, which accounted for 80% of the total MYB binding elements, suggesting the conservative nature of the sequence CAACG/TG of the MYB binding site. The autoregulation of plant transcription factors is common in one family, which showed sequence-specific interactions of the family [32-33]. Dof1 binds the PEPC1 promoter, but Dof2 blocks the transactivation of Dof1 [34]. Hence, these MYB genes with MYB binding site indicated the potential interaction effects. Pervasive gene duplications Duplication is a striking feature of the plant genome. Gene duplication in the R2R3-MYB gene family occurred during earlier evolution in land plants and contributed to its amplification [35]. We estimated gene duplication events in the S. spontaneum genome by collinearity analysis. A total of 274 collinearity pairs of SsR2R3-MYB genes were identified by Blastp for all protein sequences and evaluated with MCScanX, including 144 allelic pairs and 130 non-allelic pairs (Figure 4, Table S4). The collinearity relationships revealed that over half of the collinearity genes were concentrated in Chr 3 and Chr 7. The duplication events for MYB genes were predicted. Total 91 (25.84%) genes were tandem repeats, of which one-quarter of genes were located on Chr 7. Furthermore, 146 (39.88%) genes were identified to derive from segmental duplication events; 28.1% genes on Chr 2 and 33.5% on Chr 3 evolved from segmental duplication (Figure 4, Table S5). Segmental duplication played a critical role in the evolution of S. spontaneum MYB genes, similar as in the other species. Totally, 66.5% of the R2R3-MYB genes derived from gene duplication events, driving the MYB gene family expansion. Temporal and spatial expression of the R2R3-MYB gene family To characterize the expression profiles of MYB transcription factors, the temporally and spatially expression profiles of 202 SsR2R3-MYB genes were analyzed using a total of 50 RNA-seq data among three transcriptome models, including tissue and developmental stages, leaf developmental gradient, and circadian rhythm. The expression heatmap showed that most of the MYB genes had low expression levels, but 71% of gene expression values were greater than 1 (FPKM) in at least one RNA-seq sample (Figure 5A, Table S6). Expression values of 15 MYB groups were presented in Table S6, and G14 genes seemed to be expressed greater than the other groups. Five different expression patterns, i.e., C1-C5, were investigated on the tissue and developmental stages transcriptome by K-means (Figure 5B). A total of 85 SsR2R3-MYB genes belonging to the C1 and C3 clusters had low expression value, particularly C1 genes with almost no expression. On the contrary, C2 cluster genes displayed a relatively higher expression level in all developmental periods of leaf and stem. Interestingly, 37 genes of the C4 cluster were highly expressed in the stem during the seedling stage, the early stage of the stem formation (Figure S2A). Moreover, in the C5 cluster, 35 genes were highly expressed in the stem during each period, probably playing a regulatory role in the stem development (Figure S2B). The clusters indicated that the gene expression levels in the stem as a whole were significantly higher than those in the leaves, suggesting SsR2R3-MYB genes might play an important role in stem tissue. The relative expression of SsMYB43 , SsMYB52 , SsMYB65 , SsMYB78 , and SsMYB99 were quantified by qPCR, verifying the results of RNA-seq data (Figure S3B); additionally, SsMYB3, SsMYB15, and SsMYB157 predominant expressed in the early stage of stem formation depicted as prophase of the stem (Pro-stem), which was much higher than other stem nodes and leaf tissues (Figure S3A). Sugarcane is a typical C 4 plant with high light use efficiency. The developmental gradient model of grass leaves could be used to study C 4 photosynthesis and its regulatory factors [36-38]. The regulatory role of SsMYB genes on C 4 photosynthesis was investigated on the developmental dynamical transcriptome of sugarcane leaf. As suggested by the C 4 photosynthetic development model, leaves are gradually differentiated for active photosynthesis [36]. A total of 27 differentially expressed SsR2R3-MYB genes were detected by the leaf developmental gradient alone, and most of the genes (class I) showed an expression profile, illustrating high value in the early stage of leaf development (Figure S4). Only three genes SsMYB169 , SsMYB181 , and SsMYB192 in class II (Figure 5C, Figure S4), were identified as putative C 4 -related transcription factors using the method that associated the co-expression pattern with the photosynthetic activity [37]. The expression increased with the development of C 4 photosynthesis and displayed the highest accumulation at the leaf mature zone. Interestingly, SsMYB181 and SsMYB192 shared one haplotype gene Sspon.07G0015250 with SsMYB169 , as the tandem genes SsMYB181 and SsMYB192 derived from a gene duplication event. Circadian rhythm is another module to study photosynthesis, in which previously identified C 4 -related regulators could also be verified. Nine SsR2R3-MYB genes showed a significant association of expression profile with the light-dark cycle (Figure 5D). These genes were divided into three types, containing three genes each type. The expression level of SsMYB169 , SsMYB159 , and SsMYB153 tailed off during the daytime until around 6:00 pm, and then it gradually recovered till the next cycle. However, the expression profiles of SsMYB48 , SsMYB57 , and SsMYB158 were just opposite to the expression pattern of the former, rising during the day and falling at night. Unexpected but reasonable, the preliminarily identified three C 4 -related regulators, SsMYB169 , SsMYB181 , and SsMYB192 , also showed daylight expression pattern, hinting at their involvement in the regulation of circadian rhythm. This strong evidence showed that the three candidate MYB transcription factors were associated with C 4 photosynthesis. MYB genes involved in response to drought and disease-induced stress. The expression patterns of SsR2R3-MYB genes were evaluated under environmental stress (biotic and abiotic stress). Six SsR2R3-MYB genes with significantly differentially expressed genes (SDEGs) were responsive to drought induction (Figure 6A, Table S7). The transcripts of four genes, SsMYB54 , SsMYB36 , SsMYB61 , and SsMYB48 , rapidly accumulated after drought treatment, but their expression reduced to normal after rewatered. On the other hand, SsMYB29 and SsMYB166 showed the opposite trend. Further, the upstream regulatory elements of these six genes contained the MBS element (5’-CAACTG-3’), which was identified as MYB binding site involved in drought-inducibility. Half of these genes retained more than one MBS. Pokkah boeng disease of sugarcane (PBD) is one of the most serious and devastating diseases caused by the Fusarium species complex, a fungal pathogen [39-40]. Nineteen19 different MYBs were associated with sugarcane PBD-infection and response (Figure 6B, Table S7). According to the gene expression trends, these genes could be divided into 14 genes with increased expression in defense response and the other 5 genes with reduced expression. Sugarcane mosaic disease is a highly transmissible viral disease present in the cane-growing regions worldwide. Sugarcane mosaic virus (SCMV), belonging to the positive-sense single-stranded RNA viruses, reduces yields by damaging chloroplast and blocking photosynthesis [41-42]. After SCMV infection, 10 SsR2R3-MYB genes expression increased, and one gene, MYB176 , decreased, suggesting that these MYB genes were involved in defense against SCMV infection (Figure 6C, Table S7). We discovered that these MYB genes were unique to sugarcane diseases, indicating the defense specificity of MYB genes for conferring the resistance of sugarcane pokkah boeng and mosaic disease. Functional characterization The potential function of SsR2R3-MYB genes was predicted on the identified genes with significantly specific expression. Fifty-six SsMYB genes were involved in seven plant bioprocesses (Figure 6D), of which six MYB genes only expressed during seeding stem and were possibly involved in stem differentiation and formation (Figure 6A). Three MYB genes were identified as candidate C 4 photosynthesis regulators, and nine genes responded in the circadian clock. Under diverse stresses, it was seen that six, nineteen, and ten SsR2R3-MYB genes responded to drought, pokkah boeng disease, and mosaic disease, respectively. Notably, SsMYB51 and SsMYB162 illustrated different expression changes between two sugarcane diseases (pokkah boeng and mosaic disease). SsMYB162 significantly accumulated, actively responding to the infection of two diseases (Table S7). However, SsMYB51 showed a different expression pattern, negatively responding to pokkah boeng but positively answering SCMV. Moreover, 13 MYB genes had more than one putative function, indicating their role in diverse plant bioprocesses (Figure 6D). Allelic expression dominance drove SsMYB to function in stem The transcriptional levels of R2R3-MYB allelic genes were compared among different tissues and different developmental stages to investigate the transcriptome dynamics of R2R3-MYB genes in the allopolyploid across eight homoeologous chromosome pairs, of which 25% of the R2R3-MYB genes displayed allelic expression dominance in all samples. The number of expression dominant genes in the A, B, C, and D genomes was 84, 93, 82, and 79, respectively. Further, the allelic genes were compared in pairs, including A-B, A-C, A-D, B-C, B-D, and C-D (Figure 7a). Both the number of dominant genes in a single set of homoeologous chromosomes and the pairwise comparison of alleles showed no significant allelic dominance. Captivatingly, the number of dominant genes in the stem was more than that in the leaf in each allelic pair comparison. For four sets of homoeologous chromosomes, the percentages increase was 46.5%, 90.6%, 10.2%, 143.4%, corresponding to A, B, C, and D genomes, respectively, and the overall average rise was 64.5%. The transcriptional expression of allelic genes in the stem tissues showed significant differences among different alleles than those in the leaf tissues. Allelic expression dominant genes derived predominantly from stem transcriptomes. Selective pressure analysis demonstrated that Ka/Ks values of expression dominance MYB genes in the stem were higher than in the leaf, indicating tissue specificity (Figure 7B). In contrast with the neutral genes, the Ka/Ks values of differential expression genes were higher, while the subordinate genes exhibited top Ka/Ks values (Figure 7C). Discussion Gene duplication played an important role in gene expansion and functional diversification in the genetic revolution and phenotypic evolution [43]. A total of 202 SsR2R3-MYB genes were identified, the second-highest number of these genes among the 21 important plant species (displayed in Figure 1). The number of R2R3-MYB in sugarcane was far higher than the other members of the grass family. Nevertheless, sugarcane with octoploid nature had a higher number of MYB genes compared with the other species. The significant enrichment of SsMYB genes probably was affected by the two rounds of whole-genome duplication, including allopolyploidization followed by autopolyploidization [44], or two rounds of autopolyploidization [3]. In grasses, 11 (7.09%) genes in O. sativa were derived from tandem duplications, 26 (21.31%) in B. distachyon , and 24 (15%) in Z. mays , while 44 (28.38%) segmental gene pairs were derived from segmental duplications in O. sativa , 34 (45.08%) in B. distachyon , and 19 (24%) in Z. mays , respectively [28-29, 45]. The duplication of genes distribution indicated that the MYB genes family expansion in S. spontaneum could be attributed to these duplication events. The large R2R3-MYB gene family resulted from duplication events and autopolyploidization, demonstrated diverse functions in plant-specific processes. Some genes specially expressed in stem tissues were concentrated in the stem prophase, indicating that these MYB genes might regulate biological processes related to stem development. Stem morphogenesis is tightly associated with a secondary wall (the major mechanical tissue in the stems of grass species) formation and lignification [46]. Indeed, some MYB transcription factors are identified to be involved in sugarcane stem development. A previous study revealed that 7 ScMYB genes were correlated with lignin content and biosynthesis [47]. ShMYB78 has been recognized as an activator of suberin biosynthesis and regulates suberin deposition [48]. In Arabidopsis , the asymmetric leaves1 ( as 1) gene encoding an MYB protein-mediated stem cell function and interacted with meristematic genes to regulate the shoot morphogenesis [49]. Furthermore, a group of rice and maize MYB genes ( OsMYB46 and ZmMYB46 ) activated the transcription of secondary cell wall biosynthesis and probably interacted with secondary wall-associated NAC genes[46]. The stem is the main storage organ of sugarcane. The role of these SsMYB genes in stem development might provide potential genetic resources for sugarcane breeding. MYB genes also play an important role in leaf development in grasses. In maize, a group of MYB was recognized to be involved in leaf development, as indicated by expression gradients. Myb-ZmRS2 , MYB60, and MYB61 influence adaxial/abaxial polarity and stomata patterning [36, 50-51]. Moreover, some ZmMYBs are highly expressed in the transition zone, affecting secondary cell wall and lignin production [36]. The Class I containing 24 SsR2R3-MYB genes were also inferred to have similar functions. Furthermore, a few MYB genes were identified as C 4 regulators and correlated with C 4 photosynthetic cell type-specific gene expression. An MYB gene encoding GRMZM2G130149, which apparently regulates the transcription of phosphoenolpyruvate carboxykinase (PEPCK) in Z. mays , was categorized as a C 4 transcription factor [38]. Similarly, three putative C 4 transcription factors ( SsMYB169 , SsMYB181 , and SsMYB192 ) identified in this study might play a potential role in forming photosynthetic organs and regulating the C 4 photosynthetic pathway. The LATE ELONGATED HYPOCOTYL ( LHY ) gene, encoding an MYB transcription factor, regulated circadian rhythms in Arabidopsis , and MYB-LHY was involved in circadian photoperiod [52]. In sugarcane, nine candidate MYB genes with high expression were associated with the circadian cycle and therefore performed similar functions. The genes associated with leaf development showed relatively low expression levels (FPKM<10) than those linked with the stem tissues, hinting at a stem-related expression dominance for most SsMYB genes. Drought is one of the main factors restricting sugarcane growth and sugar production [53]. Identifying special and novel candidate genes is a great strategy to improve stress tolerance in sugarcane in this context. Certain MYB transcription factors, for instance, MYB_2 [54], SoMYB18 [55], ScMYB2S1 & 2 [56], and ScMYBAS1 [57], have been associated with the response to drought-induced stress in sugarcane. Six differentially expressed MYB genes were predicted in this study, helping understand the sugarcane drought tolerance mechanism. Following pathogen invasions, plants turn on a series of plant defense mechanisms. MYB transcription factors play a facilitating role in disease resistance by regulating plant hormone metabolism and mediating systemic resistance [58]. AtMYB30 [59], AtMYB96 [20], and SpMYB [60] have already been reported to be involved in disease resistance. Several defense-related MYB candidate genes were identified against pokkah boeng disease and mosaic disease of sugarcane. Hence, MYB genes are a component of plant defense mechanisms against fungal and viral pathogens. Polyploids are widely distributed among plants, and about 70% of the angiosperms have experienced one or more polyploidization events during their evolution. As a plant genome evolutionary force, polyploidization plays an essential role in speciation and genomic plasticity. In this study, homologous expression dominant genes Ka/Ks of autopolyploid sugarcane were higher than those of neutral genes, consistent with the allopolyploid of B.juncea [61]. Besides, MYB homologous expression of dominant genes was greater in number in stem tissues than those in leaves, and the Ka/Ks ratio was also higher, implying that MYB stems dominant genes intensified selection in sugarcane. Not surprisingly, the transcription level of MYB genes more significantly enriched in the stem. The transcriptional advantages of these MYB homologous expression dominant genes in stem tissues might provide new insights for facilitating polyploid crop breeding, including sugarcane. Conclusions It is the first time deciphering the phylogeny, gene structure, and expression of the MYB family in S. spontaneum . Genome-wide expression analysis demonstrated that SsMYB genes were involved in the stem development and stress response. The MYB genes might be engineered to adjust important sugarcane traits, and therefore, these genes would be a promising target for sugarcane genetic improvement. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in supplementary information files. Genomic data of sugarcane and sorghum for testing were obtained from the autopolyploid Saccharum spontaneum L. genome ( http://www.life.illinois.edu/ming/downloads/Spontaneum_genome/ ) and Sorghum bicolor genome ( https://phytozome-next.jgi.doe.gov/info/Sbicolor_v3_1_1 ). The domain architecture of the MYB genes was downloaded from Pfam database ( http://pfam.xfam.org/family/PF00249/hmm ). The sequencing data of Sugarcane pokkah boeng disease: SRP127969 ( https://www.ncbi.nlm.nih.gov/sra/SRP127969 ); and Sugarcane mosaic virus disease: SRR10058145, SRR10058144 in the GenBank database. RNA-seq of tissues and development stage, leaf segments and circadian rhythms were downloaded from sugarcane public database ( http://sugarcane.zhangjisenlab.cn/sgd/html/mRNA.html ). Competing interests The authors declare that they have no competing interests. Funding This work was funded by the National Natural Science Foundation of China (31660420), the Key Project of Science and Technology of Guangxi (AA17202042-7), and the earmarked fund for the Modern Agriculture Technology of China (CARS-170190) and the Innovation Project of Guangxi Graduate Education (YCBZ2020031). The funding body only provided the funds and didn't have any role in the study's design, sample collection, data analysis and interpretation, and writing of the manuscript. Authors’ contributions YY and MQZ conceived and designed this study. XPY and JSZ made guidance during the experiment. YY performed the most analysis, including identifying the MYB family, phylogenetic analysis, collinearity analysis, and expression analysis. MFF assisted in allelic differential expression analysis and Ka/Ks calculation. HYD completed the qPCR experiment together. YY prepared the manuscript. MQZ, XPY and MTK advised on the revised manuscript, providing valuable comments. All authors reviewed and approved the final manuscript. Acknowledgements Not Applicable. Author Details 1 State Key Laboratory for Conservation and Utilization of Agro Bioresources; Guangxi Key Laboratory for Sugarcane Biology, Guangxi University, Nanning 530005, China. 2 Nuclear Institute of Agriculture (NIA), Tando Jam, 70060, Pakistan. 3 Fujian Agricultural and Forestry University, Fuzhou, 350002, China References FAO F. Food and Agriculture Organization of the United Nations - Statistic Divisionhttps://www.fao.org/faostat/en/#data/QC. 2019. D’Hont A, Grivet L, Feldmann P, Rao S, Berding N, Glaszmann JC. Characterisation of the double genome structure of modern sugarcane cultivars (. MGG Mol Gen Genet. 1996;250:405. Saccharum L, Zhang J, Zhang X, Tang H, Zhang Q, Hua X, et al. Allele-defined genome of the autopolyploid. Nat Genet. 2018;50 November:1565–73. Stracke R, Werber M, Weisshaar B. The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol. 2001;4:447–56. Bilaud T, Koering CE, Binet-Brasselet E, Ancelin K, Pollice A, Gasser SM, et al. The Telobox, a Myb-Related Telomeric DNA Binding Motif Found in Proteins from Yeast, Plants, and Human. Nucleic Acids Res. 1996;24:1294–303. Kerstetter RA, Bollman K, Taylor RA, Bomblies K, Poethig RS. KANADI regulates organ polarity in Arabidopsis. Nature. 2001;411:706–9. Yakir E, Hilman D, Kron I, Hassidim M, Melamed-Book N, Green RM. Posttranslational Regulation of CIRCADIAN CLOCK ASSOCIATED1 in the Circadian Oscillator of Arabidopsis. Plant Physiol. 2009;150:844–57. Ito M, Araki S, Matsunaga S, Itoh T, Nishihama R, Machida Y, et al. G2/M-Phase-Specific Transcription during the Plant Cell Cycle Is Mediated by c-Myb-Like Transcription Factors. Plant Cell. 2001;13:1891–905. Haga N, Kato K, Murase M, Araki S, Kubo M, Demura T, et al. R1R2R3-Myb proteins positively regulate cytokinesis through activation of KNOLLE transcription in Arabidopsis thaliana Development. 2007;134:1101 LP – 1110. Dai X, Xu Y, Ma Q, Xu W, Wang T, Xue Y, et al. Overexpression of an R1R2R3 MYB gene, OsMYB3R-2, increases tolerance to freezing, drought, and salt stress in transgenic Arabidopsis. Plant Physiol. 2007;143:1739–51. Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. MYB transcription factors in Arabidopsis. Trends Plant Sci. 2010;15:573–81. Dias AP, Braun EL, McMullen MD, Grotewold E. Recently Duplicated Maize R2R3 Myb Genes Provide Evidence for Distinct Mechanisms of Evolutionary Divergence after Duplication. Plant Physiol. 2003;131:610 LP – 620. Rosinski JA, Atchley WR. Molecular Evolution of the Myb Family of Transcription Factors: Evidence for Polyphyletic Origin. J Mol Evol. 1998;46:74–83. Zhang P, Chopra S, Peterson T. A segmental gene duplication generated differentially expressed myb-homologous genes in maize. Plant Cell. 2000;12:2311–22. Waites R, Selvadurai HRN, Oliver IR, Hudson A. The PHANTASTICA Gene Encodes an MYB Transcription Factor Involved in Growth and Dorsoventrality of Lateral Organs in Antirrhinum. Cell. 1998;93:779–89. Jin H, Martin C. Multifunctionality and diversity within the plant MYB-gene family. Plant Mol Biol. 1999;41:577–85. Bender J, Fink GR. A Myb homolog, ATR1, activates tryptophan gene expression in Arabidopsis. Proc Natl Acad Sci U S A. 1998;95:5655–60. Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K. An Arabidopsis myb homolog is induced by dehydration stress, and its gene product binds to the conserved MYB recognition sequence. Plant Cell. 1993;5:1529 LP – 1539. Vos M De, Denekamp M, Dicke M, Vuylsteke M. The Arabidopsis thaliana Transcription Factor AtMYB102 Functions in Defense Against The Insect Herbivore Pieris rapae ND SC RIB. Plant Signal Behav. 2006; December:305–11. Seo PJ, Park C-M. MYB96-mediated abscisic acid signals induce pathogen resistance response by promoting salicylic acid biosynthesis in Arabidopsis. New Phytol. 2010;186:471–83. McCarthy RL, Zhong R, Ye Z-H. MYB83 Is a Direct Target of SND1 and Acts Redundantly with MYB46 in the Regulation of Secondary Cell Wall Biosynthesis in Arabidopsis. Plant Cell Physiol. 2009;50:1950–64. Zhou J, Lee C, Zhong R, Ye Z-H. MYB58 and MYB63 Are Transcriptional Activators of the Lignin Biosynthetic Pathway during Secondary Cell Wall Formation in Arabidopsis Plant Cell. 2009;21:248 LP – 266. Zhong R, Ye Z-H. MYB46 and MYB83 Bind to the SMRE sites and Directly Activate a Suite of Transcription Factors and Secondary Wall Biosynthetic Genes. Plant Cell Physiol. 2012;53:368–80. Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) Function as Transcriptional Activators in Abscisic Acid Signaling. Plant Cell. 2003;15:63 LP – 78. Seo PJ, Lee SB, Suh MC, Park M-J, Go YS, Park C-M. The MYB96 Transcription Factor Regulates Cuticular Wax Biosynthesis under Drought Conditions in Arabidopsis Plant Cell. 2011;23:1138 LP – 1152. Bowring SA, Grotzinger JP, Isachsen CE, Knoll AH, Pelechaty SM, Kolosov P. Calibrating rates of early Cambrian evolution. Science. 1993;261:1293–8. McInerney FA, Wing SL. The Paleocene-Eocene Thermal Maximum: A Perturbation of Carbon Cycle, Climate, and Biosphere with Implications for the Future. Annu Rev Earth Planet Sci. 2011;39:489–516. Chen S, Niu X, Guan Y, Li H. Genome-Wide Analysis and Expression Profiles of the MYB Genes in Brachypodium distachyon. Plant & cell Physiol. 2017;58:1777–88. Katiyar A, Smita S, Lenka SK, Rajwanshi R, Chinnusamy V, Bansal KC. Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis. BMC Genomics. 2012;13:544. Paterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, Gundlach H, et al. The Sorghum bicolor genome and the diversification of grasses. Nature. 2009;457:551–6. McCormick RF, Truong SK, Sreedasyam A, Jenkins J, Shu S, Sims D, et al. The Sorghum bicolor reference genome: improved assembly, gene annotations, a transcriptome atlas, and signatures of genome organization. Plant J. 2018;93:338–54. Kieffer M, Stern Y, Cook H, Clerici E, Maulbetsch C, Laux T, et al. Analysis of the transcription factor WUSCHEL and its functional homolog in Antirrhinum reveals a potential mechanism for their roles in meristem maintenance. Plant Cell. 2006;18:560–73. Sharma P, Lin T, Grandellis C, Yu M, Hannapel DJ. The BEL1-like family of transcription factors in potato. J Exp Bot. 2014;65:709–23. Yanagisawa S, Sheen J. Involvement of maize Dof zinc finger proteins in tissue-specific and light-regulated gene expression. Plant Cell. 1998;10:75–89. Rabinowicz PD, Braun EL, Wolfe AD, Bowen B, Grotewold E. Maize R2R3 Myb genes: Sequence analysis reveals amplification in the higher plants. Genetics. 1999;153:427–44. Li P, Ponnala L, Gandotra N, Wang L, Si Y, Tausta SL, et al. The developmental dynamics of the maize leaf transcriptome. Nat Genet. 2010;42:1060–7. Wang L, Czedik-Eysenberg A, Mertz RA, Si Y, Tohge T, Nunes-Nesi A, et al. Comparative analyses of C₄ and C₃ photosynthesis in developing leaves of maize and rice. Nat Biotechnol. 2014;32:1158–65. Studer AJ, Schnable JC, Weissmann S, Kolbe AR, McKain MR, Shao Y, et al. The draft genome of the C3 panicoid grass species Dichanthelium oligosanthes. Genome Biol. 2016;17:223. R Rott, RA Bailey, JC Comstock BC, and AS. A guide to sugarcane diseases. France: CIRAD; 2000. Singh A, Chauhan SS, Singh A, Singh SB. Deterioration in sugarcane due to pokkah boeng disease. Sugar Tech. 2006;8:187–90. Xu D-L, Park J-W, Mirkov TE, Zhou G-H. Viruses causing mosaic disease in sugarcane and their genetic diversity in southern China. Arch Virol. 2008;153:1031. Chauhan RP, Rajakaruna P, Verchot J. Complete genome sequence of nine isolates of canna yellow streak virus reveals its relationship to the sugarcane mosaic virus (SCMV) subgroup of potyviruses. Arch Virol. 2015;160:837–44. Flagel LE, Wendel JF. Gene duplication and evolutionary novelty in plants. New Phytol. 2009;183:557–64. Kim C, Wang X, Lee T-H, Jakob K, Lee G-J, Paterson AH. Comparative Analysis of Miscanthus and Saccharum Reveals a Shared Whole-Genome Duplication but Different Evolutionary Fates. Plant Cell. 2014;26:2420–9. Du H, Feng B-R, Yang S-S, Huang Y-B, Tang Y-X. The R2R3-MYB transcription factor gene family in maize. PLoS One. 2012;7:e37463. Zhong R, Lee C, McCarthy RL, Reeves CK, Jones EG, Ye Z-H. Transcriptional activation of secondary wall biosynthesis by rice and maize NAC and MYB transcription factors. Plant & cell Physiol. 2011;52:1856–71. Michael dos Santos Brito. Unraveling Two Ways of Lignin Biosynthesis in Sugarcane Culm: An Overview of Transcriptional Regulation. 2012; P0738. Figueiredo R, Portilla Llerena JP, Kiyota E, Ferreira SS, Cardeli BR, de Souza SCR, et al. The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana. Plant Mol Biol. 2020;104:411–27. Byrne ME, Barley R, Curtis M, Arroyo JM, Dunham M, Hudson A, et al. Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis. Nature. 2000;408:967–71. Husbands AY, Chitwood DH, Plavskin Y, Timmermans MCP. Signals and prepatterns: new insights into organ polarity in plants. Genes Dev. 2009;23:1986–97. Liu T, Ohashi-Ito K, Bergmann DC. Orthologs of Arabidopsis thaliana stomatal bHLH genes and regulation of stomatal development in grasses. Development. 2009;136:2265–76. Carré IA, Kim J-Y. MYB transcription factors in the Arabidopsis circadian clock. J Exp Bot. 2002;53:1551–7. Prabu G, Kawar PG, Pagariya MC, Prasad DT. Identification of Water Deficit Stress Upregulated Genes in Sugarcane. Plant Mol Biol Report. 2011;29:291–304. Silvana Creste Souza. Characterization of Transcription Factors Differentially Expressed Under Drought Conditions in Sugarcane (Saccharum spp). 2015; P0180. Shingote PR, Kawar PG, Pagariya MC, Kuhikar RS, Thorat AS, Babu KH. SoMYB18, a sugarcane MYB transcription factor improves salt and dehydration tolerance in tobacco. Acta Physiol Plant. 2015;37:217. Guo J, Ling H, Ma J, Chen Y, Su Y, Lin Q, et al. A sugarcane R2R3-MYB transcription factor gene is alternatively spliced during drought stress. Sci Rep. 2017;7:41922. Fávero Peixoto-Junior R, Mara de Andrade L, Dos Santos Brito M, Macedo Nobile P, Palma Boer Martins A, Domingues Carlin S, et al. Overexpression of ScMYBAS1 alternative splicing transcripts differentially impacts biomass accumulation and drought tolerance in rice transgenic plants. PLoS One. 2018;13:e0207534. Raffaele S, Rivas S, Roby D. An essential role for salicylic acid in AtMYB30-mediated control of the hypersensitive cell death program in Arabidopsis. FEBS Lett. 2006;580:3498–504. Vailleau F, Daniel X, Tronchet M, Montillet J-L, Triantaphylidès C, Roby D. A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack. Proc Natl Acad Sci U S A. 2002;99:10179–84. Liu Z, Luan Y, Li J, Yin Y. Expression of a tomato MYB gene in transgenic tobacco increases resistance to Fusarium oxysporum and Botrytis cinerea. Eur J Plant Pathol. 2016;144:607–17. Yang J, Liu D, Wang X, Ji C, Cheng F, Liu B, et al. The genome sequence of allopolyploid Brassica juncea and analysis of differential homolog gene expression influencing selection. Nat Genet. 2016;48:1225–32. Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 2016;44:D279-85. Wheeler TJ, Eddy SR. nhmmer: DNA homology search with profile HMMs. Bioinformatics. 2013;29:2487–9. Hedges SB, Dudley J, Kumar S. TimeTree: a public knowledge-base of divergence times among organisms. Bioinformatics. 2006;22:2971–2. Guo A-Y, Chen X, Gao G, Zhang H, Zhu Q-H, Liu X-C, et al. PlantTFDB: a comprehensive plant transcription factor database. Nucleic Acids Res. 2008;36 Database issue: D966-9. Pu X, Yang L, Liu L, Dong X, Chen S, Chen Z, et al. Genome-Wide Analysis of the MYB Transcription Factor Superfamily in Physcomitrella patens. Int J Mol Sci. 2020;21. Liu C, Xie T, Chen C, Luan A, Long J, Li C, et al. Genome-wide organization and expression profiling of the R2R3-MYB transcription factor family in pineapple (Ananas comosus). BMC genomics. 2017;18:503. Matus JT, Aquea F, Arce-Johnson P. Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related clades and conserved gene structure organization across Vitis and Arabidopsis genomes. BMC Plant Biol. 2008;8:83. Chen B, Niu F, Liu W-Z, Yang B, Zhang J, Ma J, et al. Identification, cloning and characterization of R2R3-MYB gene family in canola (Brassica napus L.) identify a novel member modulating ROS accumulation and hypersensitive-like cell death. DNA Res. 2016;23:101–14. Du H, Yang S-S, Liang Z, Feng B-R, Liu L, Huang Y-B, et al. Genome-wide analysis of the MYB transcription factor superfamily in soybean. BMC Plant Biol. 2012;12:106. ZHENG X, YI D, SHAO L, LI C. In silico genome-wide identification, phylogeny and expression analysis of the R2R3-MYB gene family in Medicago truncatula. J Integr Agric. 2017;16:1576–91. Feng S, Xu Y, Yang L, Sun S, Wang D, Chen X. Genome-wide identification and characterization of R2R3-MYB transcription factors in pear. Sci Hortic (Amsterdam). 2015;197:176–82. Han Y, Yu J, Zhao T, Cheng T, Wang J, Yang W, et al. Dissecting the Genome-Wide Evolution and Function of R2R3-MYB Transcription Factor Family in Rosa chinensis. Genes (Basel). 2019;10:823. Wilkins O, Nahal H, Foong J, Provart NJ, Campbell MM. Expansion and Diversification of the Populus R2R3-MYB Family of Transcription Factors. Plant Physiol. 2009;149:981 LP – 993. Stracke R, Holtgräwe D, Schneider J, Pucker B, Rosleff Sörensen T, Weisshaar B. Genome-wide identification and characterisation of R2R3-MYB genes in sugar beet (Beta vulgaris). BMC Plant Biol. 2014;14:249. Sun W, Ma Z, Chen H, Liu M. MYB Gene Family in Potato (Solanum tuberosum L.): Genome-Wide Identification of Hormone-Responsive Reveals Their Potential Functions in Growth and Development. Int J Mol Sci. 2019;20. Zhao P, Li Q, Li J, Wang L, Ren Z. Genome-wide identification and characterization of R2R3MYB family in Solanum lycopersicum. Mol Genet Genomics. 2014;289:1183–207. Thompson JD, Gibson TJ, Higgins DG. Multiple Sequence Alignment Using ClustalW and ClustalX. Curr Protoc Bioinforma. 2003;00:2.3.1-2.3.22. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33:1870–4. Hu B, Jin J, Guo A-Y, Zhang H, Luo J, Gao G. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2015;31:1296–7. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, et al. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19:1639–45. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002;30:325–7. Chen Y, Zhang Q, Hu W, Zhang X, Wang L, Hua X, et al. Evolution and expression of the fructokinase gene family in Saccharum. BMC Genomics. 2017;18:197. Li Z, Hua X, Zhong W, Yuan Y, Wang Y, Wang Z, et al. Genome-Wide Identification and Expression Profile Analysis of WRKY Family Genes in the Autopolyploid Saccharum spontaneum. Plant Cell Physiol. 2020;61:616–30. Ling H, Wu Q, Guo J, Xu L, Que Y. Comprehensive selection of reference genes for gene expression normalization in sugarcane by real-time quantitative rt-PCR. PLoS One. 2014;9:e97469. Additional Declarations No competing interests reported. Supplementary Files FigureS1.pdf Additional file 9: Figure S1. Comparison of phylogeny and gene structure of R2R3-MYB gene between S. spontaneum and S. bicolor. (A), (B), (C), (D) continue to supplement Figure 3A in turn. FigureS2.pdf Additional file 10: Figure S2. Expression heat map of special highly expressed in prophase of stem formation (A) and whole stem development period (B). FigureS3.pdf Additional file 11: Figure S3. Relative expression in prophase of stem formation (A) and whole stem development period (B) quantified by qPCR. FigureS4.pdf Additional file 12: Figure S4. Heatmap of gene-expression levels for co-expression modules alone leaf developmental. Each gene (row) in the two co-expression modules (I, II) was sorted according to the point of leaf segment point (column) at which peak expression occurred. TableS1.xlsx Additional file 1: Table S1. Members of identified MYB family in Sugarcane, including four subfamilies MYB-related, R2R3-MYB, R1R2R3-MYB, and Atypical family. TableS2.xlsx Additional file 2: Table S2. The 15 subgroups of the SsR2R3-MYB family by phylogenetic classification with rice. TableS3.xlsx Additional file 3: Table S3. Members of identified MYB family in Sorghum. TableS4.xlsx Additional file 4: Table S4. Collinear gene pairs in the SsR2R3-MYB gene family TableS5.xlsx Additional file 5: Table S5. Tandem duplication genes and segmental duplication genes TableS6.xlsx Additional file 6: Table S6. The expression value of SsR2R3-MYB genes in both temporal and spatial models, including tissue and developmental stages, leaf developmental gradient, circadian rhythm. TableS7.xlsx Additional file 7: Table S7. The expression value of DEGs of SsR2R3-MYB genes in stress response. TableS8.xlsx Additional file 8: Table S8. Gene primers of expression quantified by qPCR. Cite Share Download PDF Status: Published Journal Publication published 18 Aug, 2021 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Major revision 08 Mar, 2021 Reviews received at journal 01 Mar, 2021 Reviewers agreed at journal 19 Feb, 2021 Reviewers invited by journal 07 Feb, 2021 Editor assigned by journal 07 Feb, 2021 Editor invited by journal 07 Feb, 2021 Submission checks completed at journal 07 Feb, 2021 First submitted to journal 02 Feb, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-199103","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":10737225,"identity":"8bab16b5-0958-4df2-a75a-2770827cac08","order_by":0,"name":"Yuan Yuan","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Yuan","suffix":""},{"id":10737226,"identity":"3c6d3dcd-86fc-42d7-8ed6-f05361e25460","order_by":1,"name":"Xiping Yang","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiping","middleName":"","lastName":"Yang","suffix":""},{"id":10737227,"identity":"eb387b43-7134-4f17-aa2f-4caec082ba9b","order_by":2,"name":"Mengfang Feng","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mengfang","middleName":"","lastName":"Feng","suffix":""},{"id":10737228,"identity":"977495b7-d842-4c37-b0b4-93e779cb01fe","order_by":3,"name":"Hongyan Ding","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongyan","middleName":"","lastName":"Ding","suffix":""},{"id":10737229,"identity":"1fc5d2f3-68d9-42df-b618-fa00b0ded6e9","order_by":4,"name":"Khan Muhammad Tahir","email":"","orcid":"","institution":"Nuclear Institute of Agriculture","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Khan","middleName":"Muhammad","lastName":"Tahir","suffix":""},{"id":10737230,"identity":"55058695-76f9-4bf5-bd85-9ac7057f0cbf","order_by":5,"name":"Jisen Zhang","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jisen","middleName":"","lastName":"Zhang","suffix":""},{"id":10737231,"identity":"86942e7f-06c9-4271-9245-6ad1f262ebfb","order_by":6,"name":"Muqing Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYLCCBAYGOTb29gOkaTHm4zmTQJpFifMkHAyIUyo/7YzhjYc7atPbJICW/ajYRlgL4+wcY4vEM8dz26QbDzD2nLlNWAuzdI6ZRGLbsdw2mQMJzIxtRGhhg2pJZ5NIMCBOCw9ES00C8VokpNOKLRLbDhi2AQP5IFF+kZ+dvPHmz7Y6efn29oMPflQQoQVsEwPDYTDjAHHqIVrqiFY8CkbBKBgFIxAAABW5OPUMbDsiAAAAAElFTkSuQmCC","orcid":"","institution":"Guangxi University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Muqing","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2021-02-02 06:44:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-199103/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-199103/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-021-07689-w","type":"published","date":"2021-08-18T15:02:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":6027974,"identity":"65bbc4de-7fb9-4222-93eb-8ecd01243962","added_by":"auto","created_at":"2021-02-16 22:13:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52313,"visible":true,"origin":"","legend":"Phylogenetic tree of diverse species showing the number of MYB family. \nThe phylogenetic tree reflects the evolutionary relationship and divergence time of various species in plants through the TimeTree database (Hedges et al., 2006; http://www.timetree.org). Linear scale Time MYA (millions of years ago) and Geologic Timescale are shown at the tree's bottom. These species contain green algae, Grasses (red node), Cruciferous (green node), Leguminosae (orange node), Rosaceae (purple node), Chenopodiaceae (blue node), and others altogether 11 lineages. MYB gene family can be divided into four subfamilies according to the number of Myb domain. The available information of the MYB gene family was obtained from the reported literature, showing the table. The short line represents undetermined. The MYB families were estimated by performing profile searches using a combination of the HMMER3 program (PF00249) and NCBI-CDD database for S. spontaneum and S. bicolor. Myb_DNA-binding domain (PF00249) was downloaded from Pfam (Finn et al., 2010). S. spontaneum MYB gene in brackets are identified throughout the genome and contain alleles. A single set of genes are shown in outsides for each subfamily. Different alleles of one gene may be divided into different subfamilies. Thus, to better classification, a single set of genes does not distinguish this condition that one gene is in different subfamilies. Geologic Periods: C(Cambrian), O(Ordovician), D(Devonian), P(Permian), Tr(Triassic), J(Jurassic), K(Cretaceous), Pg(Paleogene).","description":"","filename":"Fig01.png","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/1d99f1f382f2ac20dc15fbe0.png"},{"id":6027479,"identity":"b6d54814-b6df-4af9-b849-c3f82568a4e5","added_by":"auto","created_at":"2021-02-16 22:07:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":123277,"visible":true,"origin":"","legend":"Phylogenetic relationships of R2R3-MYB subgroup members between S. spontaneum and O. sativa. \nA phylogenetic tree of R2R3-MYB proteins from Saccharum and rice was constructed using MEGA 7.0 with the Neighbor-Joining (NJ) method with the bootstrap test replicated 1000 times, the NO.of difference and Pairwise deletion. The sugarcane R2R3-MYB families are clustered into 18 subgroups (no containing the branches with rice gene only), named G1 to G15. The different clade of subgroups are marked as colorful lines, and their gene ID label was added with green and grey background in turn. The size of the grey point positively reflects phylogenetic bootstrap.","description":"","filename":"Fig02.png","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/e004af0737636709b40a56de.png"},{"id":6027972,"identity":"44efd2ea-ace5-49fb-aeb5-37a72b372ff3","added_by":"auto","created_at":"2021-02-16 22:13:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":115171,"visible":true,"origin":"","legend":"Collinearity relationships of SsR2R3-MYB genes on the S. spontaneum genome.\nSsR2R3-MYB collinear gene pairs were mapped to their respective locus in the S. spontaneum genome in a circular diagram. Genes located on the same chromosome (e.g., Chr1, Chr2, Chr3, Chr4, Chr5, Chr6, Chr7, Chr8) share one line, and the interchromosomal collinear genes pairs are linked with the former linear color. ","description":"","filename":"Fig03.png","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/e66341b8c28079e28b127130.png"},{"id":6027711,"identity":"c968892c-9f56-46b5-9882-7f7e4d2b44e0","added_by":"auto","created_at":"2021-02-16 22:10:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":304860,"visible":true,"origin":"","legend":"Structure, distribution, and regulatory elements of SsR2R3-MYB genes. \n(A) Comparison of gene structure between S. spontaneum and S. bicolor based on phylogenetic tree. The sequence alignment of SsR2R3-MYB and SbR2R3-MYB proteins were performed by ClustalX, and the Phylogenetic tree was constructed using MEGA 7.0 with Neighbor-Joining (NJ) method, 1,000 bootstrap replicates, Pairwise deletion, and Bootstrap values on the nodes. SsMYB gene names are marked black, and SbMYB gene names are marked red. Gene sequences were modified to start at the transcription initiation site (ATG), and gene structures were displayed using GSDS2.0 (http://gsds.cbi.pku.edu.cn/). The CDS sequence and intron are represented as fine lines and yellow cylinders, and the MYB domain were highlighted by green cylinders. One of the subgroups was showed in here when the estimated phylogenetic relationship of S. spontaneum and S. bicolor, and others were shown in Figure S1. (B) Cis-regulatory elements of SsR2R3-MYB gene promoters with diversified plant biological functions. The functions of the predicted cis-regulatory elements cover four main categories: stress response, hormone response, light response, plant growth, and metabolism. The x-axis shows divers plant biological functions, and the y-axis indicates the number of a specific category of genes in that main category. The red rectangle represents the genes containing more than six elements involved in regulating a certain plant function. (C) Distribution of SsR2R3-MYB gene members in S. spontaneum genome. 202 SsR2R3-MYB genes were named according to their physical position on the chromosome and tagged in red font. Yellow font indicated chromosome name, and chromosome is represented as hollow cylinders with length scale (bp) on the left. The green spots displayed a gene enrichment cluster.","description":"","filename":"Fig04.png","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/359edd2ae4b0cd9d5e329d1d.png"},{"id":6027460,"identity":"050b3bf0-392d-4fc2-899a-cb8140af1ac9","added_by":"auto","created_at":"2021-02-16 22:07:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":147564,"visible":true,"origin":"","legend":"Temporal and spatial expression dynamics of SsR2R3-MYB genes.\n(A) A heatmap showed the expression profile of SsR2R3-MYB genes. Columns showed 18 subfamilies, and rows showed developmental stages and tissues, leaf developmental gradient, and circadian rhythm. (B) K-means clustering showing the expression profile of the developmental stages and tissue transcriptome. Five clusters were identified as C1-C5, error bars showing standard deviation. (C) The expression of three genes identified as C4 regulator was shown along with the leaf development. (D) The expression of 9 genes showed a circadian cycle, and the x-axis indicates different time points on the second day. Developmental stages and tissues: s, seeding stage; pm, pre-mature stage; m, mature stage; r leaf, roll leaf; m leaf, mature leaf. Leaf developmental gradient showed 1-15 segment in one leaf blade from base to tip. Circadian rhythm showed 19-time point including first day 2h time span and second day 4h time span. Asterisk was used to distinguish the same time point on different days. ","description":"","filename":"Fig05.png","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/041320cc886275ee612672dd.png"},{"id":6027470,"identity":"088011df-ea0d-460c-b61e-d780d9e59c4a","added_by":"auto","created_at":"2021-02-16 22:07:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":66584,"visible":true,"origin":"","legend":"Heatmaps of differentially expressed MYB genes in response to stress conditions and presumed function.\nThe heatmaps of DEGs expression value were shown in (A) (B) (C) based on RNA-seq data from drought stress, Pokkah boeng disease, and sugarcane mosaic disease. DEGs were identified due to their expression having significant variation after suffering stress stimulation (FPKM \u003e2, fold change \u003e2, p-value \u003c0.05). Abbreviation: D1, CK; D2, mild; D3, severe; D4, rehydrate. P1, CK; P2, inchoate; P3, advanced. S1, CK; S2, CK detoxify; S3, post-infection. (D) 56 SsR2R3-MYB genes with specific expression patterns with putative functionalities. Blue boxes represent this function, and yellow with none.","description":"","filename":"Fig06.png","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/6b9470dc63fbb6322d94d96e.png"},{"id":6027473,"identity":"e9ac01f2-9f46-4b99-862b-581ebf50e6f8","added_by":"auto","created_at":"2021-02-16 22:07:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":104916,"visible":true,"origin":"","legend":"Allelic expression dominance and selective pressure analysis in SsR2R3-MYB family\n(A) Expression histograms of SsR2R3-MYB allelic genes among the tissue and development stage of S. spontaneum. N values indicate the number of dominant genes in allelic genes identified R2R3-MYB genes. (B) Boxplot of the distribution of Ka/Ks values of expression dominance genes in leaf and stem. Ka/Ks median values of leaf and stem are 0.455 and 0.482, respectively. (C) Boxplot of the distribution of Ka/Ks values among allelic expression dominance genes as dominant, subordinate, and neutral (non-dominance). Ka/Ks median values of dominant, subordinate, and neutral are 0.491, 0.506, and 0.485, respectively.","description":"","filename":"Fig07.png","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/ac84988a6ac7e4ea9ef11dff.png"},{"id":13660515,"identity":"eac2245a-f59a-4558-990e-830eab58a4eb","added_by":"auto","created_at":"2021-09-17 10:25:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1419165,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/e75be1df-6902-4537-a5fc-ce3c23793629.pdf"},{"id":6027468,"identity":"0ea88760-fdc9-4873-84d0-12b73847b9e1","added_by":"auto","created_at":"2021-02-16 22:07:11","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1617713,"visible":true,"origin":"","legend":"Additional file 9: Figure S1. Comparison of phylogeny and gene structure of R2R3-MYB gene between S. spontaneum and S. bicolor. (A), (B), (C), (D) continue to supplement Figure 3A in turn. ","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/99061978d56ead5b34d28230.pdf"},{"id":6027474,"identity":"9eb2e9ba-6091-4ef0-a4cb-dd6200bccb91","added_by":"auto","created_at":"2021-02-16 22:07:11","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":925468,"visible":true,"origin":"","legend":"Additional file 10: Figure S2. Expression heat map of special highly expressed in prophase of stem formation (A) and whole stem development period (B). ","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/551ab991018a29b5c4311597.pdf"},{"id":6027467,"identity":"c7fe9b97-664b-44ca-84ea-8384310b660a","added_by":"auto","created_at":"2021-02-16 22:07:11","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":827732,"visible":true,"origin":"","legend":"Additional file 11: Figure S3. Relative expression in prophase of stem formation (A) and whole stem development period (B) quantified by qPCR. ","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/3b8912847b478ec4c7977283.pdf"},{"id":6027718,"identity":"8dd09006-4065-45e8-99e4-147a1d7c96b9","added_by":"auto","created_at":"2021-02-16 22:10:11","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":508086,"visible":true,"origin":"","legend":"Additional file 12: Figure S4. Heatmap of gene-expression levels for co-expression modules alone leaf developmental. Each gene (row) in the two co-expression modules (I, II) was sorted according to the point of leaf segment point (column) at which peak expression occurred.","description":"","filename":"FigureS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/bdd7586180430243a7c275b8.pdf"},{"id":6027472,"identity":"395e8731-00bf-4acc-8071-6d2154b2d858","added_by":"auto","created_at":"2021-02-16 22:07:11","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":57709,"visible":true,"origin":"","legend":"Additional file 1: Table S1. Members of identified MYB family in Sugarcane, including four subfamilies MYB-related, R2R3-MYB, R1R2R3-MYB, and Atypical family. ","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/b12f25c3cbfc481d402a9eb3.xlsx"},{"id":6027462,"identity":"3d529fe0-a62b-4a37-9d0c-112b919c375d","added_by":"auto","created_at":"2021-02-16 22:07:11","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":13821,"visible":true,"origin":"","legend":"Additional file 2: Table S2. The 15 subgroups of the SsR2R3-MYB family by phylogenetic classification with rice. ","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/0596907feae6c90c61b463de.xlsx"},{"id":6027476,"identity":"7cbabda5-d768-464f-8bd7-8b734160feb7","added_by":"auto","created_at":"2021-02-16 22:07:11","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":26888,"visible":true,"origin":"","legend":"Additional file 3: Table S3. Members of identified MYB family in Sorghum.","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/3fe0d2714370898503d359f8.xlsx"},{"id":6027715,"identity":"c010931d-9219-4101-92dd-340dd82efc5a","added_by":"auto","created_at":"2021-02-16 22:10:11","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":47895,"visible":true,"origin":"","legend":"Additional file 4: Table S4. Collinear gene pairs in the SsR2R3-MYB gene family","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/d40cb913fad9d5e217b135e6.xlsx"},{"id":6027975,"identity":"ef2157e5-9067-47d6-812a-4c8a877cca0e","added_by":"auto","created_at":"2021-02-16 22:13:11","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":22773,"visible":true,"origin":"","legend":"Additional file 5: Table S5. Tandem duplication genes and segmental duplication genes ","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/92090009df6e0189c1939edf.xlsx"},{"id":6027973,"identity":"9d9ed3f5-2fda-4126-ac4c-76b2b186d22c","added_by":"auto","created_at":"2021-02-16 22:13:11","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":75217,"visible":true,"origin":"","legend":"Additional file 6: Table S6. The expression value of SsR2R3-MYB genes in both temporal and spatial models, including tissue and developmental stages, leaf developmental gradient, circadian rhythm.","description":"","filename":"TableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/94d42a356b7a10672fc8311a.xlsx"},{"id":6027466,"identity":"efdae783-88a5-4219-b7cc-cf43c25d8cdc","added_by":"auto","created_at":"2021-02-16 22:07:11","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":12235,"visible":true,"origin":"","legend":"Additional file 7: Table S7. The expression value of DEGs of SsR2R3-MYB genes in stress response. ","description":"","filename":"TableS7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/bd5ea3e4dcb376e001368501.xlsx"},{"id":6027713,"identity":"de40da01-78f9-4c7e-8488-7374eac2e453","added_by":"auto","created_at":"2021-02-16 22:10:11","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":9820,"visible":true,"origin":"","legend":"Additional file 8: Table S8. Gene primers of expression quantified by qPCR.","description":"","filename":"TableS8.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-199103/v1/cbe950fccf27c130cec9b8f8.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u0026nbsp;Genome-Wide Analysis of R2R3-MYB Transcription Factors Family in The Autopolyploid Saccharum Spontaneum: An Exploration of Dominance Expression and Stress Response\u0026nbsp;\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eModern cultivated sugarcane (\u003cem\u003eSaccharum spp.\u003c/em\u003e) is the major source of sugar for the world. It is the topmost crop concerning total biomass production and is listed among the ten most valuable crops [1]. Sugarcane, having a complex genetic background resulting from polyploid interspecific hybrids, was first domesticated approximately10,000 years ago in New Guinea. \u003cem\u003eSaccharum spontaneum \u003c/em\u003econtributes about 10%-15% genome to the modern sugarcane cultivars, endowing the characteristics such as disease resistance and ratooning capacity [2]. The genome of haploid \u003cem\u003eS. spontaneum\u003c/em\u003e has been assembled\u0026nbsp;to\u0026nbsp;the chromosome\u0026nbsp;level and used as the reference genome of sugarcane [3]. Because of the development of multiple transcriptome models in recent times, including those for different tissues, developmental stages, and under various stress treatments, huge RNA-seq data has become available and provides detailed insights and rich resources for studying gene functions of sugarcane.\u003c/p\u003e\n\u003cp\u003eTranscription factors recognize specific DNA motifs in upstream regions of the genes to regulate their expression. \u003cem\u003eMYB \u003c/em\u003egenes constitute one of the largest families of plant transcription factors and characteristically possess highly conserved Myb DNA-binding domains, forming a helix-turn-helix structure of about 52 amino acids [4]. \u003cem\u003eMYB \u003c/em\u003egenes can be divided into four categories, including \u003cem\u003eMYB\u003c/em\u003e-related, \u003cem\u003eR2R3-MYB\u003c/em\u003e, \u003cem\u003eR1R2R3-MYB\u003c/em\u003e, and atypical \u003cem\u003eMYB\u003c/em\u003e, depending on the number of adjacent \u003cem\u003eMYB\u003c/em\u003e repeats (R). Proteins with a single or a partial MYB repeat, generally located at either ends or middle of the peptide chain, are MYB-related.\u003c/p\u003e\n\u003cp\u003eMYB-related proteins include important telomere binding proteins in maintaining the integrity of the chromosome structure [5]. Moreover, they also play an important role in regulating gene transcription, \u003cem\u003ee.g.\u003c/em\u003e, the GARP family of plant Myb-related DNA binding motifs is involved in organ polarity in Arabidopsis[6]. Further, \u003cem\u003eCIRCADIAN CLOCK ASSOCIATED\u003c/em\u003e1 (\u003cem\u003eCCA\u003c/em\u003e1) and \u003cem\u003eLATE ELONGATED HYPOCOTYL\u003c/em\u003e (\u003cem\u003eLHY\u003c/em\u003e) genes regulate the plant circadian clock [7]. A small number of members of \u003cem\u003eR1R2R3-MYB\u003c/em\u003e genes are found in higher plants. Interestingly, plant \u003cem\u003eR1R2R3-MYB\u003c/em\u003e genes share a similar function of regulating the cell cycle control with the animals [8]. \u003cem\u003e3R-MYB\u003c/em\u003e has also been involved in cell differentiation [9] and plant stress tolerance [10].\u003c/p\u003e\n\u003cp\u003eAtypical MYB proteins contain four or more adjacent MYB repeats (R). These proteins have been found to encode in a few plants, e.g., \u003cem\u003eArabidopsis thaliana,\u003c/em\u003e \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eVitis vinifera\u003c/em\u003e, \u003cem\u003eGlycine max\u003c/em\u003e, \u003cem\u003ePhyscomitrella patens\u003c/em\u003e (data sources displayed in Materials and Methods 2.1), as shown in Figure 1. Only a few reports have been published about atypical MYB proteins by now, and the role of these proteins in the plant bioprocesses is largely unknown. MYB transcription factors binding specific DNA sequence (CAACG/TG) result from domain structure that is formed by two closely packed amino acid sequence repeats(R) [11]. When the MYB gene contains at least two MYB repeats (R), it has transcription factor characteristics and specifically recognizes the DNA motifs to regulate the gene transcription. R2R3-MYB proteins are the largest subfamily of MYB transcription factors in plants, as well as in \u003cem\u003eS. spontaneum \u003c/em\u003e(Figure 1). R2R3-MYB is characterized by two MYB repeats and the presence of a single amino acid (Leu) in the first (R2) repeat [12]. R2R3-MYB has two MYB repeats and a single amino acid (Leu) inserted in the first (R2) repeat. The R2R3-MYB family's expansion originated from the R1R2R3-MYB gene ancestor when losing the R1 repeat sequences during evolution [13] and benefiting from gene duplication events [14].\u003c/p\u003e\n\u003cp\u003eMYB genes are widely involved in plant-specific processes, such as differentiation [15], hormone response [16], secondary metabolism [17], environmental stress tolerance [18], and diseases resistance [19][20]. At least four MYB genes are involved in lignin biosynthesis in \u003cem\u003eArabidopsis\u003c/em\u003e by activating key regulator genes related to secondary cell wall formation [21-23]. Under environmental stress, MYB genes have been reported to function in response to adverse stress in \u003cem\u003eArabidopsis\u003c/em\u003e. Moreover,\u003cem\u003e AtMYB2\u003c/em\u003e and \u003cem\u003eAtMYB96\u003c/em\u003e function as transcriptional activators in ABA-inducible gene expression under drought stress [24]. \u003cem\u003eAtMYB96\u003c/em\u003e mediates abscisic acid signaling, induces pathogen resistance response by promoting salicylic acid biosynthesis, and provides drought tolerance \u003cem\u003evia\u003c/em\u003e controlling the cuticular wax biosynthesis [20, 25].\u003c/p\u003e\n\u003cp\u003eThis study focused on the R2R3-MYB gene family in the \u003cem\u003eS. spontaneum \u003c/em\u003epublished sugarcane genome. We provided a detailed overview of phylogenetic relationship, gene structure, regulatory elements, expression\u0026nbsp;profiles, allelic evolution, and functional characterization based on abundant transcriptome data. Taken together, our study systematically explored the evolutionary dynamics and functional diversification of SsR2R3-MYB genes and could hence facilitate future research on sugarcane MYB transcription factors.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003ch1\u003eObtainment of MYB genes\u003c/h1\u003e\n\u003cp\u003eThe autopolyploid sugarcane \u003cem\u003eSaccharum spontaneum\u003c/em\u003e L. genome was published in 2018 and is available online (\u003ca href=\"http://www.life.illinois.edu/ming/downloads/Spontaneum_genome/\"\u003ehttp://www.life.illinois.edu/ming/downloads/Spontaneum_genome/\u003c/a\u003e). The Hidden Markov Model (HMM) profile of the MYB DNA-binding domain (PF00249) downloaded from Pfam database (\u003ca href=\"http://pfam.xfam.org/\"\u003ehttp://pfam.xfam.org/\u003c/a\u003e) [62] was used to search protein sequences containing MYB domain by hmmsearch program (HMM3.0) [63]. Then, putative MYB proteins were further screened through the NCBI-CDD database to investigate the former protein sequences and delete the proteins with incomplete domains. SbR2R3-MYB genes were obtained by performing the same sugarcane method without publicly available data for sorghum MYB genes. Data for sorghum protein sequences (the newest version of Sbicolor_454_v3.1.1.) were downloaded from the plant genome website Phytozome (\u003ca href=\"https://phytozome.jgi.doe.gov/\"\u003ehttps://phytozome.jgi.doe.gov/\u003c/a\u003e). Finally, we identified 418 (695) \u003cem\u003eSsMYB\u003c/em\u003e genes and 252 \u003cem\u003eSbMYB\u003c/em\u003e genes (Table S1), including 202 \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes and 125 \u003cem\u003eSbR2R3-MYB\u003c/em\u003e genes, belonging to haplotype genes. A plant phylogeny tree was constructed by the TimeTree Database (\u003ca href=\"http://www.timetree.org\"\u003ehttp://www.timetree.org\u003c/a\u003e)[64]. The distribution of MYB family genes in 19 plant species were demonstrated on the previously published reports: \u003cem\u003eOstreococcus lucimarinus\u003c/em\u003e, \u003cem\u003eVolvox carteri\u003c/em\u003e and \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e from PlantTFDB (\u003ca href=\"http://planttfdb.cbi.pku.edu.cn/\"\u003ehttp://planttfdb.cbi.pku.edu.cn/\u003c/a\u003e), and a public plant transcription factor database [65], including \u003cem\u003ePhyscomitrella patens \u003c/em\u003e[66], \u003cem\u003eOryza sativa \u003c/em\u003e[29], \u003cem\u003eBrachypodium distachyon \u003c/em\u003e[28], \u003cem\u003eZea mays \u003c/em\u003e[45], \u003cem\u003eAnanas comosus\u003c/em\u003e [33], \u003cem\u003eVitis vinifera \u003c/em\u003e[68], \u003cem\u003eArabidopsis thaliana\u003c/em\u003e[29], \u003cem\u003eBrassica napus \u003c/em\u003e[69], \u003cem\u003eGlycine max \u003c/em\u003e[70], \u003cem\u003eMedicago truncatula \u003c/em\u003e[71], \u003cem\u003ePyrus bretschneideri \u003c/em\u003e[72], \u003cem\u003eRosa chinensis\u003c/em\u003e [73], \u003cem\u003ePopulus trichocarpa\u003c/em\u003e [74], \u003cem\u003eBeta vulgaris\u003c/em\u003e [75], \u003cem\u003eSolanum tuberosum \u003c/em\u003e[76], and \u003cem\u003eSolanum lycopersicum\u003c/em\u003e [77].\u003c/p\u003e\n\u003ch1\u003ePhylogenetic analysis\u003c/h1\u003e\n\u003cp\u003eTo generate the phylogenetic trees of MYB transcription factor family genes, multiple protein sequence alignment was performed through ClustalW [78] program using the reported 88 rice MYB proteins [29], and further phylogenetic trees were constructed \u003cem\u003evia\u003c/em\u003e the neighbor-joining (NJ) method using software MEGA7.0 [79]. The consistency of the phylogenetic estimates was evaluated through several models as well as pairwise deletion treatment. NJ based phylogenetic tree of sugarcane and sorghum was performed as the same method.\u003c/p\u003e\n\u003ch1\u003eNaming R2R3-MYB genes and gene structure\u003c/h1\u003e\n\u003cp\u003eBecause of the autopolyploid nature of sugarcane (\u003cem\u003eS. spontaneum\u003c/em\u003e), the identified SsR2R3-MYB genes partly possessed several alleles. The representative gene models for different alleles were screened by comparing the phylogenetic relationship and protein identity with sorghum homology protein and paralogs. Tandem replication genes and paralogs were regarded as new, which gene IDs were followed by P and T, respectively. The 202 representative SsR2R3-MYB genes were named from SsMYB1 to SsMYB202 according to their physical position on the chromosomes. Subsequently, allele names were supplemented with numbers (e.g., The Sspon.01G0002470-1A gene located at the top of chromosome 1A is MYB1-1, and Sspon.01G0002470-2D is named as \u003cem\u003eMYB1-2\u003c/em\u003e). In general, \u003cem\u003eMYB1-1 \u003c/em\u003eas a representative gene model was directly regarded as \u003cem\u003eMYB1\u003c/em\u003e. The naming method of sorghum MYB genes was also treated like that of \u003cem\u003eS. spontaneum.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSsR2R3-MYB genes and CDS sequences come from the newest version of Sspon.v20190103. The domain location was derived from the previous hmmsearch results. Gene structures were displayed using the Gene Structure Display Server (GSDS2.0) [80], consisting of the CDS region, intron region, and MYB domain. Each gene structure was arranged according to the phylogenetic location.\u003c/p\u003e\n\u003ch1\u003eCollinearity analysis\u003c/h1\u003e\n\u003cp\u003eUtilizing MCScanX analysis [78], collinearity relationships of \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes and classifier program were used to sort gene duplication types. The identified collinear gene pairs were mapped to their respective locus in the \u003cem\u003eS. spontaneum\u003c/em\u003e genome in a circular diagram using Circos 0.69 [81].\u003c/p\u003e\n\u003ch1\u003eRegulatory element of upstream sequences\u003c/h1\u003e\n\u003cp\u003eThe 2000 bp upstream sequences were extracted from \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes to the PlantCARE website, plant promoter, and cis-element database [82]. Then, we used them to predict regulatory motifs and estimate potentially related functions.\u003c/p\u003e\n\u003ch1\u003eAbundant RNA-seq data showing gene expression\u003c/h1\u003e\n\u003cp\u003eTo analyze \u003cem\u003eSsR2R3-MYB\u003c/em\u003e gene expression profiles thoroughly, 60 RNA-seq data were conducted to decipher their expressions from our lab and cooperative labs. Tissue and development transcriptome contained RNA-seq data of 16 samples, including leaf, stem, three different development stages \u003cem\u003eviz\u003c/em\u003e. seeding (35-day-old), pre-maturity (9-month-old), and maturity (12-month-old) stages in \u003cem\u003eS.\u003c/em\u003e\u003cem\u003espontaneum \u003c/em\u003e[83]. The leaf development transcriptome was derived from the second leaf alone, the ligule on 11-day-old seedlings; 15 cm leaves were selected and cut into 15 pieces with one segment per centimeter [84]. Mature leaves corresponding to ligule in \u003cem\u003eS.\u003c/em\u003e\u003cem\u003espontaneum\u003c/em\u003e, over 12-month-old, were selected to supply circadian rhythm transcriptome using 19-time points, \u003cem\u003ei.e.\u003c/em\u003e, 2 hours apart from 6:00 am to the second day 4:00 am, and 4 hours apart from 6:00 am to the third day 6:00 am.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRNA-seq were extracted from the drought-treatment sugarcane of FN95-1702, a new sugarcane variety for both sugar and energy, bred by Fujian Agriculture and Forestry University. Sugarcane grown to 4-5 leaves was subjected to the natural drought stress treatment in the greenhouse. The mild drought was characterized by soil relative\u0026nbsp;water\u0026nbsp;content of about 55%~60% after six days, and severe drought by 25%~30% after twelve days. After a severe drought, rehydration was done, and relative\u0026nbsp;water\u0026nbsp;content was kept around 75%～85%, and then leave samples were retaken (5 days later). The\u003cem\u003e R2R3-MYB\u003c/em\u003e gene expression profiles were obtained by Blast mapping to express data with transcripts of unreferenced genomes. RNA-seq for pokkah boeng disease were extracted from hybrid sugarcane ZZ1, which is highly resistant to smut disease but highly susceptible to pokkah boeng disease. According to the severity of the diseased leaves, pokkah boeng disease was divided into five grades from 0-5. The mildly diseased leaves (1 or 2 grades) and severely diseased leaves (4 or 5 grades) were selected for analysis, while healthy leaves were used as control (CK). Three samples were extracted for RNA-seq for sugarcane mosaic disease transcriptome analysis. For the infection experiment, sugarcane grown through virus-free tissue culture was used, and then leaves corresponding to ligule were collected one month after the infection, while the control plants were not infected. An expression ratio \u0026gt;2 (adjusted p-value\u0026lt;0.05) was considered statistically significant for evaluating differentially expressed genes.\u003c/p\u003e\n\u003ch1\u003eQuantitative RT-PCR\u003c/h1\u003e\n\u003col start=\"8\"\u003e\n\u003cli\u003e\u003cem\u003e spontaneum\u003c/em\u003e was planted in Multifunctional Specimen Garden, Institute of Agriculture, Guangxi University. The stem-3 at the third internode and mature leaves were collected for comparing the difference of relative expression between stem and leaf. Pro-stem is short for prophase stem, in which the samples were taken from the stem precursor tissue wrapped in the leaf sheath and is located on the upper part of the stem with obvious stem nodes. Combining with stem-3, stem-6, stem-9, and mature leaves were used to verify the expression during the prophase of stem formation. The extractions of samples total RNA was carried out using TRIZOL reagent (Takara), employing the corresponding protocol. The qualified RNA was reverse transcribed to produce cDNA using PrimeScript\u003csup\u003eTM\u003c/sup\u003e RT reagent Kit with gDNA Eraser reagent (Takara, Japan). Primers were designed by qPCR-PrimerQuest Tool, and qPCR primers were shown in Table S8. Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was selected as a reference gene [85]. The real-time qPCR with three biological replications were performed with SYBR green on Roche Lightcyler\u0026reg; 480 instrument using 2\u0026times;TB Green Mix (Takara). The reaction profile was as follows: 95\u0026deg;C for 30 s, followed by 40 cycles of 95\u0026deg;C for 10 s, 60\u0026deg;C for 30s, and 95\u0026deg;C for 10s. The relative expression levels were calculated by the 2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e△△\u003c/sup\u003eCT method.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Results","content":"\u003ch1\u003eGenome-wide identification of R2R3-MYB genes and classification in S. spontaneum genome\u003c/h1\u003e\n\u003cp\u003eBased on the functional annotation of the \u003cem\u003eMyb_DNA-binding\u003c/em\u003e domain (PF00249), a total of 418 \u003cem\u003eMYB\u003c/em\u003e genes (695 alleles) were identified in \u003cem\u003ethe S. spontaneum \u003c/em\u003egenome by combining the HMMER program and NCBI-CDD database (Figure 1). The SsMYB gene family was classified into four distinct subfamilies, including 207 MYB-related (329 alleles), 202 R2R3-MYB (356 alleles), 3 R1R2R3-MYB (3 alleles), and 5 Atypical MYB (7 alleles) genes (detailed data presented in supplementary Table S1). Total 122 SbMYB-related, 125 SbR2R3-MYB, 3 SbR1R2R3-MYB, and 2 Atypical MYB genes were also identified to increase the understanding of SsR2R3-MYB genes (Table S3).\u003c/p\u003e\n\u003cp\u003eTo analyze the plant MYB genes thoroughly, twenty species in 11 lineages were screened to construct a plant phylogenetic tree with \u003cem\u003eS. spontaneum\u003c/em\u003e, including Green algae, Bryophyta, Gramineae, Cruciferous, Leguminous, Rosaceae, Solanaceae, and others. The tree topology reflected the phylogenetic relationship of these species and divergence time (Figure 1). Plant phylogeny showed that the higher plants possessed more MYB genes than the lower plants, such as green algae (e.g., \u003cem\u003eOstreococcus lucimarinus, Volvox carteri, \u003c/em\u003eand\u003cem\u003e Chlamydomonas reinhardtii\u003c/em\u003e). A significant expansion of MYB genes was observed after the Cambrian (about 540~480MYA), demonstrating an explosive biological diversification episode near the early period [26]. Most of the phylogenetic nodes of plant species were observed in the Cretaceous, a geological period when a typical global warming climate contributed to the diversity of the terrestrial species [27]. Compared with the other four kinds of grasses, \u003cem\u003eS. spontaneum \u003c/em\u003ehad one of the largest MYB genes as predicted by PlantTFDB. One reason is the tetraploid nature of the autopolyploid \u003cem\u003eS. spontaneum\u003c/em\u003e (mainly octoploid). However, when corrected for ploidy level, the number of SsR2R3-MYB genes in \u003cem\u003eS. spontaneum\u003c/em\u003e was still significantly higher than most of the species, including \u003cem\u003eArabidopsis\u003c/em\u003e and other grass species. From green algae to bryophyte and land plants, the number of MYB genes increased. The phylogenetic analysis of the plant species using the number of MYB genes indicated the extending of MYB genes from lower to higher plants, consistent with previous reports [28].\u003c/p\u003e\n\u003cp\u003eA neighbor-joining phylogenetic tree of \u003cem\u003eR2R3-MYB\u003c/em\u003e genes from \u003cem\u003eO. sativa \u003c/em\u003eand \u003cem\u003eS. spontaneum \u003c/em\u003eshowed that the sugarcane genome contained 15 subgroups (G1-G15) (Figure 2, Table S2) with \u003cem\u003eOsR2R3-MYB \u003c/em\u003egenes [29]. Sugarcane and rice diverged in the Paleogene (67-26MYA) (Figure 1); the short divergence time indicated relative conservatism of the ortholog genes. As expected, two species of R2R3-MYB genes were evenly distributed in the tree, and most genes in rice clustered with sugarcane, except for \u003cem\u003eLOC_Os03g14100\u003c/em\u003e. However, the number of genes in each clade varied greatly; for instance, the biggest group, G4, contained 26 genes while the group G13 comprised just one SsMYB gene \u003cem\u003eSspon.02G0044740-1B\u003c/em\u003e. Twenty \u003cem\u003eSsMYB \u003c/em\u003egenes from three unique subgroups, G7, G10, and G15, did not contain rice genes, indicating the species' genetic divergence. Besides, the clusters depicted that the sugarcane MYB family exhibited a greater number of genes than that in rice, showing a significant expansion of the SsMYB family.\u003c/p\u003e\n\u003ch1\u003eAnalysis of genomic location, gene structure, and regulatory elements\u003c/h1\u003e\n\u003cp\u003eA total of 202 \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes were named in turn according to their physical position on the chromosomes. \u003cem\u003eMYB\u003c/em\u003e genes were distributed throughout all 32 chromosomes (Figure 3C); the autopolyploid \u003cem\u003eS. spontaneum \u003c/em\u003egenome comprised of 8 homologous groups of 4 members each [3]. The chromosome distribution map showed that the location of the \u003cem\u003eMYB\u003c/em\u003e genes was not evenly distributed. Most of the \u003cem\u003eSsMYB\u003c/em\u003e genes were located on Chr3A and Chr7A, encompassing 19 and 16 genes, respectively. About 11 enrichment clusters, tiny fragments on genomic regions containing 3 \u003cem\u003eMYB\u003c/em\u003e genes, were detected, and half of these genes contained MYB-binding sites (MBS) depicting potential interaction among each cluster. However, some chromosomes only contained a few \u003cem\u003eMYB\u003c/em\u003e genes. For instance, five chromosomes, including Chr2C, Chr2D, Chr6C, Chr8B, and Chr8D, had only one \u003cem\u003eMYB \u003c/em\u003egene.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. bicolor \u003c/em\u003eis one of the closest lineages of sugarcane, possessing relatively perfect genome data [30-31]. Total 125 \u003cem\u003eSbR2R3-MYB\u003c/em\u003e genes were identified from the available sorghum genome using a similar method (Figure 1, Table S3). The diversity of the gene structure might be a shred of evidence regarding the evolution of gene families. The phylogenetically and gene structure analysis were performed by the Neighbor-Joining method using diverse gene information (Figure 3A and Figure S1). The distribution of the tree branches was basically consistent with the structural features of the genes. In many clusters, various sorghum genes were clustered with highly similar \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes, \u003cem\u003ee.g.\u003c/em\u003e, \u003cem\u003eSbMYB92 \u003c/em\u003eclustered with \u003cem\u003eSsMYB149 \u003c/em\u003eand\u003cem\u003e SsMYB156\u003c/em\u003e while \u003cem\u003eSbMYB27\u003c/em\u003e was clustered with \u003cem\u003eSsMYB30 \u003c/em\u003eand \u003cem\u003eSsMYB44\u003c/em\u003e. These results sharpened our understanding of the evolution of gene events during sugarcane polyploidization. A total of 19 \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes did not show the presence of intron, including \u003cem\u003eSsMYB154, SsMYB188, SsMYB194, SsMYB170\u003c/em\u003e,\u003cem\u003e SsMYB122\u003c/em\u003e,\u003cem\u003e SsMYB182\u003c/em\u003e, and\u003cem\u003e SsMYB189\u003c/em\u003e. Many MYB genes demonstrated a domain with a cross-intron structure.\u003c/p\u003e\n\u003cp\u003eCis-elements in promoter regions play an essential role in controlling transcription and expression, and hence they can deepen the understanding of the regulatory function of MYB genes. Total 2000 bp upstream of transcription initiation site (ATG) was regarded as MYB gene promoters and submitted to the PlantCARE for predicting the motifs. Various motifs from 202 \u003cem\u003eSsR2R3-MYB\u003c/em\u003e gene promoters were involved in various plant bioprocesses (Figure 3B). These diversified cis-regulatory elements could be divided into four main categories in terms of function: stress response, hormone response, light response, and plant growth and metabolism. A high percentage of \u003cem\u003eMYB\u003c/em\u003e genes in the anaerobic induction (92%) and drought elements (58.9%) indicated that the MYB genes were more likely to function under these stresses. Moreover, a notable gene, \u003cem\u003eMYB88, \u003c/em\u003ewas found to have 10 LTR motifs, which is a cis-acting element involved in low-temperature responsiveness. The significantly enriched LTR elements (5'-CCG AAA-3') suggested that the\u003cem\u003e MYB88 \u003c/em\u003egene might be involved in plant metabolic response to cold stress. Many of the \u003cem\u003eMYB\u003c/em\u003e genes regulate the plant hormone response, especially methyl jasmonate (MeJA) and abscisic acid (ABA) responsiveness. A total of 75 genes promoters enriched regulatory elements TGACG-motif (5'-TGACG-3') and CGTCA-motif (5'-CGTCA-3') involved in MeJA-responsiveness, while 38 gene promoters enriched regulatory elements ABRE involved in abscisic acid responsiveness. These MYB genes were predicted to regulate MeJA and ABA signaling in plants and function in plant defense and leaf abscission. Furthermore, more than thirty light response-related elements were predicted; for instance, conservative light element G-box was widely present in the upstream sequence of genes. Several regulatory elements were also associated with other functions in plant growth and development and regulation of seed growth and meristem development. Genes involved in seed-specific regulation contained the same RY-element (5'-CATGCATG-3'), and the elements involved in meristem expression demonstrated CAT-box (5'-GCC ACT-3') and NON-box (5'-AGATCGACG-3') in promoter regions. Finally, 119 genes were detected to be scattered on MYB binding sites, and 49 genes showed more than one binding site, suggesting that these genes probably interacted with other \u003cem\u003eMYB\u003c/em\u003e genes. Four MYB binding elements were found in 202 \u003cem\u003eSsR2R3-MYB\u003c/em\u003e promoters, including CCAAT-box (5'-CAACGG-3'), MBS (5'-CAACTG-3'), MBSI (5'-aaaAaaC(G/C)GTTA-3'), and MRE (5'-AACCTAA-3'). There was only one base difference between the former two elements, which accounted for 80% of the total MYB binding elements, suggesting the conservative nature of the sequence CAACG/TG of the MYB binding site. The autoregulation of plant transcription factors is common in one family, which showed sequence-specific interactions of the family [32-33]. Dof1 binds the PEPC1 promoter, but Dof2 blocks the transactivation of Dof1 [34]. Hence, these MYB genes with MYB binding site indicated the potential interaction effects.\u003c/p\u003e\n\u003ch1\u003ePervasive gene duplications\u003c/h1\u003e\n\u003cp\u003eDuplication is a striking feature of the plant genome. Gene duplication in the \u003cem\u003eR2R3-MYB\u003c/em\u003e gene family occurred during earlier evolution in land plants and contributed to its amplification [35]. We estimated gene duplication events in the\u003cem\u003e S. spontaneum \u003c/em\u003egenome by collinearity analysis. A total of 274 collinearity pairs of SsR2R3-MYB genes were identified by Blastp for all protein sequences and evaluated with MCScanX, including 144 allelic pairs and 130 non-allelic pairs (Figure 4, Table S4). The collinearity relationships revealed that over half of the collinearity genes were concentrated in Chr 3 and Chr 7. The duplication events for MYB genes were predicted. Total 91 (25.84%) genes were tandem repeats, of which one-quarter of genes were located on Chr 7. Furthermore, 146 (39.88%) genes were identified to derive from segmental duplication events; 28.1% genes on Chr 2 and 33.5% on Chr 3 evolved from segmental duplication (Figure 4, Table S5). Segmental duplication played a critical role in the evolution of \u003cem\u003eS. spontaneum\u003c/em\u003e\u003cem\u003eMYB\u003c/em\u003e genes, similar as in the other species. Totally, 66.5% of the \u003cem\u003eR2R3-MYB \u003c/em\u003egenes derived from gene duplication events, driving the MYB gene family expansion.\u003c/p\u003e\n\u003ch1\u003eTemporal and spatial expression of the R2R3-MYB gene family\u003c/h1\u003e\n\u003cp\u003eTo characterize the expression profiles of MYB transcription factors, the temporally and spatially expression profiles of 202 \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes were analyzed using a total of 50 RNA-seq data among three transcriptome models, including tissue and developmental stages, leaf developmental gradient, and circadian rhythm. The expression heatmap showed that most of the MYB genes had low expression levels, but 71% of gene expression values were greater than 1 (FPKM) in at least one RNA-seq sample (Figure 5A, Table S6). Expression values of 15 MYB groups were presented in Table S6, and G14 genes seemed to be expressed greater than the other groups.\u003c/p\u003e\n\u003cp\u003eFive different expression patterns, i.e., C1-C5, were investigated on the tissue and developmental stages transcriptome by K-means (Figure 5B). A total of 85 \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes belonging to the C1 and C3 clusters had low expression value, particularly C1 genes with almost no expression. On the contrary, C2 cluster genes displayed a relatively higher expression level in all developmental periods of leaf and stem. Interestingly, 37 genes of the C4 cluster were highly expressed in the stem during the seedling stage, the early stage of the stem formation (Figure S2A). Moreover, in the C5 cluster, 35 genes were highly expressed in the stem during each period, probably playing a regulatory role in the stem development (Figure S2B). The clusters indicated that the gene expression levels in the stem as a whole were significantly higher than those in the leaves, suggesting \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes might play an important role in stem tissue. The relative expression of \u003cem\u003eSsMYB43\u003c/em\u003e, \u003cem\u003eSsMYB52\u003c/em\u003e, \u003cem\u003eSsMYB65\u003c/em\u003e, \u003cem\u003eSsMYB78\u003c/em\u003e, and \u003cem\u003eSsMYB99\u003c/em\u003e were quantified by qPCR, verifying the results of RNA-seq data (Figure S3B); additionally, \u003cem\u003eSsMYB3, SsMYB15, and SsMYB157 \u003c/em\u003epredominant\u0026nbsp;expressed in the early stage of stem formation depicted as prophase of the stem (Pro-stem), which was much higher than other stem nodes and leaf tissues (Figure S3A).\u003c/p\u003e\n\u003cp\u003eSugarcane is a typical C\u003csub\u003e4\u003c/sub\u003e plant with high light use efficiency. The developmental gradient model of grass leaves could be used to study C\u003csub\u003e4\u003c/sub\u003e photosynthesis and its regulatory factors [36-38]. The regulatory role of \u003cem\u003eSsMYB\u003c/em\u003e genes on C\u003csub\u003e4\u003c/sub\u003e photosynthesis was investigated on the developmental dynamical transcriptome of sugarcane leaf. As suggested by the C\u003csub\u003e4\u003c/sub\u003e photosynthetic development model, leaves are gradually differentiated for active photosynthesis [36]. A total of 27 differentially expressed \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes were detected by the leaf developmental gradient alone, and most of the genes (class I) showed an expression profile, illustrating high value in the early stage of leaf development (Figure S4). Only three genes \u003cem\u003eSsMYB169\u003c/em\u003e, \u003cem\u003eSsMYB181\u003c/em\u003e, and \u003cem\u003eSsMYB192\u003c/em\u003e in class II (Figure 5C, Figure S4), were identified as putative C\u003csub\u003e4\u003c/sub\u003e-related transcription factors using the method that associated the co-expression pattern with the photosynthetic activity [37]. The expression increased with the development of C\u003csub\u003e4\u003c/sub\u003e photosynthesis and displayed the highest accumulation at the leaf mature zone. Interestingly, \u003cem\u003eSsMYB181\u003c/em\u003e and \u003cem\u003eSsMYB192\u003c/em\u003e shared one haplotype gene Sspon.07G0015250 with \u003cem\u003eSsMYB169\u003c/em\u003e, as the tandem genes \u003cem\u003eSsMYB181\u003c/em\u003e and \u003cem\u003eSsMYB192\u003c/em\u003e derived from a gene duplication event. Circadian rhythm is another module to study photosynthesis, in which previously identified C\u003csub\u003e4\u003c/sub\u003e-related regulators could also be verified. Nine \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes showed a significant association of expression profile with the light-dark cycle (Figure 5D). These genes were divided into three types, containing three genes each type. The expression level of \u003cem\u003eSsMYB169\u003c/em\u003e, \u003cem\u003eSsMYB159\u003c/em\u003e, and \u003cem\u003eSsMYB153\u003c/em\u003e tailed off during the daytime until around 6:00 pm, and then it gradually recovered till the next cycle. However, the expression profiles of \u003cem\u003eSsMYB48\u003c/em\u003e, \u003cem\u003eSsMYB57\u003c/em\u003e, and \u003cem\u003eSsMYB158\u003c/em\u003e were just opposite to the expression pattern of the former, rising during the day and falling at night. Unexpected but reasonable, the preliminarily identified three C\u003csub\u003e4\u003c/sub\u003e-related regulators, \u003cem\u003eSsMYB169\u003c/em\u003e, \u003cem\u003eSsMYB181\u003c/em\u003e, and \u003cem\u003eSsMYB192\u003c/em\u003e, also showed daylight expression pattern, hinting at their involvement in the regulation of circadian rhythm. This strong evidence showed that the three candidate MYB transcription factors were associated with C\u003csub\u003e4\u003c/sub\u003e photosynthesis.\u003c/p\u003e\n\u003ch1\u003eMYB genes involved in response to drought and disease-induced stress.\u003c/h1\u003e\n\u003cp\u003eThe expression patterns of SsR2R3-MYB genes were evaluated under environmental stress (biotic and abiotic stress). Six \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes with significantly differentially expressed genes (SDEGs) were responsive to drought induction (Figure 6A, Table S7). The transcripts of four genes, \u003cem\u003eSsMYB54\u003c/em\u003e, \u003cem\u003eSsMYB36\u003c/em\u003e, \u003cem\u003eSsMYB61\u003c/em\u003e, and \u003cem\u003eSsMYB48\u003c/em\u003e, rapidly accumulated after drought treatment, but their expression reduced to normal after rewatered. On the other hand, \u003cem\u003eSsMYB29\u003c/em\u003e and \u003cem\u003eSsMYB166 \u003c/em\u003eshowed the opposite trend. Further, the upstream regulatory elements of these six genes contained the MBS element (5\u0026rsquo;-CAACTG-3\u0026rsquo;), which was identified as MYB binding site involved in drought-inducibility. Half of these genes retained more than one MBS.\u003c/p\u003e\n\u003cp\u003ePokkah boeng disease of sugarcane (PBD) is one of the most serious and devastating diseases caused by the \u003cem\u003eFusarium\u003c/em\u003e species complex, a fungal pathogen [39-40]. Nineteen19 different MYBs were associated with sugarcane PBD-infection and response (Figure 6B, Table S7). According to the gene expression trends, these genes could be divided into 14 genes with increased expression in defense response and the other 5 genes with reduced expression.\u003c/p\u003e\n\u003cp\u003eSugarcane mosaic disease is a highly transmissible viral disease present in the cane-growing regions worldwide. Sugarcane mosaic virus (SCMV), belonging to the positive-sense single-stranded RNA viruses, reduces yields by damaging chloroplast and blocking photosynthesis [41-42]. After SCMV infection, 10 \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes expression increased, and one gene, \u003cem\u003eMYB176\u003c/em\u003e, decreased, suggesting that these MYB genes were involved in defense against SCMV infection (Figure 6C, Table S7). We discovered that these MYB genes were unique to sugarcane diseases, indicating the defense specificity of MYB genes for conferring the resistance of sugarcane pokkah boeng and mosaic disease.\u003c/p\u003e\n\u003ch1\u003eFunctional characterization\u003c/h1\u003e\n\u003cp\u003eThe potential function of\u003cem\u003e SsR2R3-MYB\u003c/em\u003e genes was predicted on the identified genes with significantly specific expression. Fifty-six \u003cem\u003eSsMYB\u003c/em\u003e genes were involved in seven plant bioprocesses (Figure 6D), of which six MYB genes only expressed during seeding stem and were possibly involved in stem differentiation and formation (Figure 6A). Three MYB genes were identified as candidate C\u003csub\u003e4\u003c/sub\u003e photosynthesis regulators, and nine genes responded in the circadian clock. Under diverse stresses, it was seen that six, nineteen, and ten SsR2R3-MYB genes responded to drought, pokkah boeng disease, and mosaic disease, respectively. Notably, \u003cem\u003eSsMYB51\u003c/em\u003e and \u003cem\u003eSsMYB162\u003c/em\u003e illustrated different expression changes between two sugarcane diseases (pokkah boeng and mosaic disease). \u003cem\u003eSsMYB162\u003c/em\u003e significantly accumulated, actively responding to the infection of two diseases (Table S7). However, \u003cem\u003eSsMYB51\u003c/em\u003e showed a different expression pattern, negatively responding to pokkah boeng but positively answering SCMV. Moreover, 13 MYB genes had more than one putative function, indicating their role in diverse plant bioprocesses (Figure 6D).\u003c/p\u003e\n\u003ch1\u003eAllelic expression dominance drove SsMYB to function in stem\u003c/h1\u003e\n\u003cp\u003eThe transcriptional levels of \u003cem\u003eR2R3-MYB\u003c/em\u003e allelic genes were compared among different tissues and different developmental stages to investigate the transcriptome dynamics of \u003cem\u003eR2R3-MYB\u003c/em\u003e genes in the allopolyploid across eight homoeologous chromosome pairs, of which 25% of the R2R3-MYB genes displayed allelic expression dominance in all samples. The number of expression dominant genes in the A, B, C, and D genomes was 84, 93, 82, and 79, respectively. Further, the allelic genes were compared in pairs, including A-B, A-C, A-D, B-C, B-D, and C-D (Figure 7a). Both the number of dominant genes in a single set of homoeologous chromosomes and the pairwise comparison of alleles showed no significant allelic dominance. Captivatingly, the number of dominant genes in the stem was more than that in the leaf in each allelic pair comparison. For four sets of homoeologous chromosomes, the percentages increase was 46.5%, 90.6%, 10.2%, 143.4%, corresponding to A, B, C, and D genomes, respectively, and the overall average rise was 64.5%. The transcriptional expression of allelic genes in the stem tissues showed significant differences among different alleles than those in the leaf tissues. Allelic expression dominant genes derived predominantly from stem transcriptomes. Selective pressure analysis demonstrated that Ka/Ks values of expression dominance \u003cem\u003eMYB\u003c/em\u003e genes in the stem were higher than in the leaf, indicating tissue specificity (Figure 7B). In contrast with the neutral genes, the Ka/Ks values of differential expression genes were higher, while the subordinate genes exhibited top Ka/Ks values (Figure 7C).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGene duplication played an important role in gene expansion and functional diversification in the genetic revolution and phenotypic evolution [43]. A total of 202 \u003cem\u003eSsR2R3-MYB\u003c/em\u003e genes were identified, the second-highest number of these genes among the 21 important plant species (displayed in Figure 1). The number of \u003cem\u003eR2R3-MYB\u003c/em\u003e in sugarcane was far higher than the other members of the grass family. Nevertheless, sugarcane with octoploid nature had a higher number of MYB genes compared with the other species. The significant enrichment of \u003cem\u003eSsMYB\u003c/em\u003e genes probably was affected by the two rounds of whole-genome duplication, including allopolyploidization followed by autopolyploidization [44], or two rounds of autopolyploidization [3]. In grasses, 11 (7.09%) genes in \u003cem\u003eO.\u0026nbsp;sativa\u003c/em\u003e were derived from tandem duplications, 26 (21.31%) in \u003cem\u003eB.\u0026nbsp;distachyon\u003c/em\u003e, and 24 (15%) in \u003cem\u003eZ.\u0026nbsp;mays\u003c/em\u003e, while 44 (28.38%) segmental gene pairs were derived from segmental duplications in \u003cem\u003eO.\u0026nbsp;sativa\u003c/em\u003e, 34 (45.08%) in \u003cem\u003eB.\u0026nbsp;distachyon\u003c/em\u003e, and 19 (24%) in \u003cem\u003eZ.\u0026nbsp;mays\u003c/em\u003e, respectively [28-29, 45]. The duplication of genes distribution indicated that the MYB genes family expansion in \u003cem\u003eS. spontaneum\u003c/em\u003e could be attributed to these duplication events.\u003c/p\u003e\n\u003cp\u003eThe large \u003cem\u003eR2R3-MYB\u003c/em\u003e gene family resulted from duplication events and autopolyploidization, demonstrated diverse functions in plant-specific processes. Some genes specially expressed in stem tissues were concentrated in the stem prophase, indicating that these \u003cem\u003eMYB\u003c/em\u003e genes might regulate biological processes related to stem development. Stem morphogenesis is tightly associated with a secondary wall (the major mechanical tissue in the stems of grass species) formation and lignification [46]. Indeed, some MYB transcription factors are identified to be involved in sugarcane stem development. A previous study revealed that 7 ScMYB genes were correlated with lignin content and biosynthesis [47]. \u003cem\u003eShMYB78\u003c/em\u003e has been recognized as an activator of suberin biosynthesis and regulates suberin deposition [48]. In \u003cem\u003eArabidopsis\u003c/em\u003e, the asymmetric leaves1 (\u003cem\u003eas\u003c/em\u003e1) gene encoding an MYB protein-mediated stem cell function and interacted with meristematic genes to regulate the shoot morphogenesis [49]. Furthermore, a group of rice and maize MYB genes (\u003cem\u003eOsMYB46\u003c/em\u003e and \u003cem\u003eZmMYB46\u003c/em\u003e) activated the transcription of secondary cell wall biosynthesis and probably interacted with secondary wall-associated \u003cem\u003eNAC\u003c/em\u003e genes[46]. The stem is the main storage organ of sugarcane. The role of these \u003cem\u003eSsMYB\u003c/em\u003e genes in stem development might provide potential genetic resources for sugarcane breeding.\u003c/p\u003e\n\u003cp\u003eMYB genes also play an important role in leaf development in grasses. In maize, a group of MYB was recognized to be involved in leaf development, as indicated by expression gradients. \u003cem\u003eMyb-ZmRS2\u003c/em\u003e, \u003cem\u003eMYB60, \u003c/em\u003eand \u003cem\u003eMYB61 \u003c/em\u003einfluence adaxial/abaxial polarity and stomata patterning [36, 50-51]. Moreover, some \u003cem\u003eZmMYBs\u003c/em\u003e are highly expressed in the transition zone, affecting secondary cell wall and lignin production [36]. The Class I containing 24 SsR2R3-MYB genes were also inferred to have similar functions. Furthermore, a few MYB genes were identified as C\u003csub\u003e4\u003c/sub\u003e regulators and correlated with C\u003csub\u003e4\u003c/sub\u003e photosynthetic cell type-specific gene expression. An \u003cem\u003eMYB\u003c/em\u003e gene encoding GRMZM2G130149, which apparently regulates the transcription of phosphoenolpyruvate carboxykinase (PEPCK) in \u003cem\u003eZ. mays\u003c/em\u003e, was categorized as a C\u003csub\u003e4\u003c/sub\u003e transcription factor [38]. Similarly, three putative C\u003csub\u003e4\u003c/sub\u003e transcription factors (\u003cem\u003eSsMYB169\u003c/em\u003e, \u003cem\u003eSsMYB181\u003c/em\u003e, and \u003cem\u003eSsMYB192\u003c/em\u003e) identified in this study might play a potential role in forming photosynthetic organs and regulating the C\u003csub\u003e4\u003c/sub\u003e photosynthetic pathway. The \u003cem\u003eLATE ELONGATED HYPOCOTYL\u003c/em\u003e (\u003cem\u003eLHY\u003c/em\u003e) gene, encoding an \u003cem\u003eMYB\u003c/em\u003e transcription factor, regulated circadian rhythms in \u003cem\u003eArabidopsis\u003c/em\u003e, and \u003cem\u003eMYB-LHY\u003c/em\u003e was involved in circadian photoperiod [52]. In sugarcane, nine candidate MYB genes with high expression were associated with the circadian cycle and therefore performed similar functions. The genes associated with leaf development showed relatively low expression levels (FPKM\u0026lt;10) than those linked with the stem tissues, hinting at a stem-related expression dominance for most \u003cem\u003eSsMYB\u003c/em\u003e genes.\u003c/p\u003e\n\u003cp\u003eDrought is one of the main factors restricting sugarcane growth and sugar production [53]. Identifying special and novel candidate genes is a great strategy to improve stress tolerance in sugarcane in this context. Certain MYB transcription factors, for instance, MYB_2 [54], SoMYB18 [55], ScMYB2S1 \u0026amp; 2 [56], and ScMYBAS1 [57], have been associated with the response to drought-induced stress in sugarcane. Six differentially expressed MYB genes were predicted in this study, helping understand the sugarcane drought tolerance mechanism.\u003c/p\u003e\n\u003cp\u003eFollowing pathogen invasions, plants turn on a series of plant defense mechanisms. MYB transcription factors play a facilitating role in disease resistance by regulating plant hormone metabolism and mediating systemic resistance [58]. \u003cem\u003eAtMYB30 \u003c/em\u003e[59], \u003cem\u003eAtMYB96 \u003c/em\u003e[20], and \u003cem\u003eSpMYB \u003c/em\u003e[60] have already been reported to be involved in disease resistance. Several defense-related MYB candidate genes were identified against pokkah boeng disease and mosaic disease of sugarcane. Hence, MYB genes are a component of plant defense mechanisms against fungal and viral pathogens.\u003c/p\u003e\n\u003cp\u003ePolyploids are widely distributed among plants, and about 70% of the angiosperms have experienced one or more polyploidization events during their evolution. As a plant genome evolutionary force, polyploidization plays an essential role in speciation and genomic plasticity. In this study, homologous expression dominant genes Ka/Ks of autopolyploid sugarcane were higher than those of neutral genes, consistent with the allopolyploid of \u003cem\u003eB.juncea \u003c/em\u003e[61]. Besides, MYB homologous expression of dominant genes was greater in number in stem tissues than those in leaves, and the Ka/Ks ratio was also higher, implying that MYB stems dominant genes intensified selection in sugarcane. Not surprisingly, the transcription level of MYB genes more significantly enriched in the stem. The transcriptional advantages of these MYB homologous expression dominant genes in stem tissues might provide new insights for facilitating polyploid crop breeding, including sugarcane.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIt is the first time deciphering the phylogeny, gene structure, and expression of the MYB family in \u003cem\u003eS. spontaneum\u003c/em\u003e. Genome-wide expression analysis demonstrated that\u003cem\u003e SsMYB \u003c/em\u003egenes were involved in the stem development and stress response. The MYB genes might be engineered to adjust important sugarcane traits, and therefore, these genes would be a promising target for sugarcane genetic improvement.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in supplementary information files. Genomic data of sugarcane and sorghum for testing were obtained from the autopolyploid \u003cem\u003eSaccharum spontaneum\u003c/em\u003e L. genome (\u003ca href=\"http://www.life.illinois.edu/ming/downloads/Spontaneum_genome/\"\u003ehttp://www.life.illinois.edu/ming/downloads/Spontaneum_genome/\u003c/a\u003e) and \u003cem\u003eSorghum bicolor\u003c/em\u003e genome (\u003ca href=\"https://phytozome-next.jgi.doe.gov/info/Sbicolor_v3_1_1\"\u003ehttps://phytozome-next.jgi.doe.gov/info/Sbicolor_v3_1_1\u003c/a\u003e ). The domain architecture of the MYB genes was downloaded from Pfam database (\u003ca href=\"http://pfam.xfam.org/family/PF00249/hmm\"\u003ehttp://pfam.xfam.org/family/PF00249/hmm\u003c/a\u003e). The sequencing data of Sugarcane pokkah boeng disease: SRP127969 (\u003ca href=\"https://www.ncbi.nlm.nih.gov/sra/SRP127969\"\u003ehttps://www.ncbi.nlm.nih.gov/sra/SRP127969\u003c/a\u003e); and Sugarcane mosaic virus disease: SRR10058145, SRR10058144 in the GenBank database. RNA-seq of tissues and development stage, leaf segments and circadian rhythms were downloaded from sugarcane public database (\u003ca href=\"http://sugarcane.zhangjisenlab.cn/sgd/html/mRNA.html\"\u003ehttp://sugarcane.zhangjisenlab.cn/sgd/html/mRNA.html\u003c/a\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the National Natural Science Foundation of China (31660420), the Key Project of Science and Technology of Guangxi (AA17202042-7), and the earmarked fund for the Modern Agriculture Technology of China (CARS-170190) and the Innovation Project of Guangxi Graduate Education (YCBZ2020031). The funding body only provided the funds and didn't have any role in the study's design, sample collection, data analysis and interpretation, and writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYY and MQZ conceived and designed this study. XPY and JSZ made guidance during the experiment. YY performed the most analysis, including identifying the MYB family, phylogenetic analysis, collinearity analysis, and expression analysis. MFF assisted in allelic differential expression analysis and Ka/Ks calculation. HYD completed the qPCR experiment together. YY prepared the manuscript. MQZ, XPY and MTK advised on the revised manuscript, providing valuable comments. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e State Key Laboratory for Conservation and Utilization of Agro Bioresources; Guangxi Key Laboratory for Sugarcane Biology, Guangxi University, Nanning 530005, China. \u003csup\u003e2\u003c/sup\u003e Nuclear Institute of Agriculture (NIA), Tando Jam, 70060, Pakistan. \u003csup\u003e3\u003c/sup\u003e Fujian Agricultural and Forestry University, Fuzhou, 350002, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFAO F. Food and Agriculture Organization of the United Nations - Statistic Divisionhttps://www.fao.org/faostat/en/#data/QC. 2019.\u003c/li\u003e\n\u003cli\u003eD\u0026rsquo;Hont A, Grivet L, Feldmann P, Rao S, Berding N, Glaszmann JC. Characterisation of the double genome structure of modern sugarcane cultivars (. MGG Mol Gen Genet. 1996;250:405.\u003c/li\u003e\n\u003cli\u003eSaccharum L, Zhang J, Zhang X, Tang H, Zhang Q, Hua X, et al. Allele-defined genome of the autopolyploid. Nat Genet. 2018;50 November:1565\u0026ndash;73.\u003c/li\u003e\n\u003cli\u003eStracke R, Werber M, Weisshaar B. The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol. 2001;4:447\u0026ndash;56.\u003c/li\u003e\n\u003cli\u003eBilaud T, Koering CE, Binet-Brasselet E, Ancelin K, Pollice A, Gasser SM, et al. The Telobox, a Myb-Related Telomeric DNA Binding Motif Found in Proteins from Yeast, Plants, and Human. Nucleic Acids Res. 1996;24:1294\u0026ndash;303.\u003c/li\u003e\n\u003cli\u003eKerstetter RA, Bollman K, Taylor RA, Bomblies K, Poethig RS. KANADI regulates organ polarity in Arabidopsis. Nature. 2001;411:706\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eYakir E, Hilman D, Kron I, Hassidim M, Melamed-Book N, Green RM. Posttranslational Regulation of CIRCADIAN CLOCK ASSOCIATED1 in the Circadian Oscillator of Arabidopsis. Plant Physiol. 2009;150:844\u0026ndash;57.\u003c/li\u003e\n\u003cli\u003eIto M, Araki S, Matsunaga S, Itoh T, Nishihama R, Machida Y, et al. G2/M-Phase-Specific Transcription during the Plant Cell Cycle Is Mediated by c-Myb-Like Transcription Factors. Plant Cell. 2001;13:1891\u0026ndash;905.\u003c/li\u003e\n\u003cli\u003eHaga N, Kato K, Murase M, Araki S, Kubo M, Demura T, et al. R1R2R3-Myb proteins positively regulate cytokinesis through activation of \u0026amp;lt;em\u0026amp;gt;KNOLLE\u0026amp;lt;/em\u0026amp;gt; transcription in \u0026amp;lt;em\u0026amp;gt;Arabidopsis thaliana\u0026amp;lt;/em\u0026amp;gt; Development. 2007;134:1101 LP \u0026ndash; 1110.\u003c/li\u003e\n\u003cli\u003eDai X, Xu Y, Ma Q, Xu W, Wang T, Xue Y, et al. Overexpression of an R1R2R3 MYB gene, OsMYB3R-2, increases tolerance to freezing, drought, and salt stress in transgenic Arabidopsis. Plant Physiol. 2007;143:1739\u0026ndash;51.\u003c/li\u003e\n\u003cli\u003eDubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. MYB transcription factors in Arabidopsis. Trends Plant Sci. 2010;15:573\u0026ndash;81.\u003c/li\u003e\n\u003cli\u003eDias AP, Braun EL, McMullen MD, Grotewold E. Recently Duplicated Maize \u0026amp;lt;em\u0026amp;gt; R2R3 Myb\u0026amp;lt;/em\u0026amp;gt; Genes Provide Evidence for Distinct Mechanisms of Evolutionary Divergence after Duplication. Plant Physiol. 2003;131:610 LP \u0026ndash; 620.\u003c/li\u003e\n\u003cli\u003eRosinski JA, Atchley WR. Molecular Evolution of the Myb Family of Transcription Factors: Evidence for Polyphyletic Origin. J Mol Evol. 1998;46:74\u0026ndash;83.\u003c/li\u003e\n\u003cli\u003eZhang P, Chopra S, Peterson T. A segmental gene duplication generated differentially expressed myb-homologous genes in maize. Plant Cell. 2000;12:2311\u0026ndash;22.\u003c/li\u003e\n\u003cli\u003eWaites R, Selvadurai HRN, Oliver IR, Hudson A. The PHANTASTICA Gene Encodes an MYB Transcription Factor Involved in Growth and Dorsoventrality of Lateral Organs in Antirrhinum. Cell. 1998;93:779\u0026ndash;89.\u003c/li\u003e\n\u003cli\u003eJin H, Martin C. Multifunctionality and diversity within the plant MYB-gene family. Plant Mol Biol. 1999;41:577\u0026ndash;85.\u003c/li\u003e\n\u003cli\u003eBender J, Fink GR. A Myb homolog, ATR1, activates tryptophan gene expression in Arabidopsis. Proc Natl Acad Sci U S A. 1998;95:5655\u0026ndash;60.\u003c/li\u003e\n\u003cli\u003eUrao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K. An Arabidopsis myb homolog is induced by dehydration stress, and its gene product binds to the conserved MYB recognition sequence. Plant Cell. 1993;5:1529 LP \u0026ndash; 1539.\u003c/li\u003e\n\u003cli\u003eVos M De, Denekamp M, Dicke M, Vuylsteke M. The Arabidopsis thaliana Transcription Factor AtMYB102 Functions in Defense Against The Insect Herbivore Pieris rapae ND SC RIB. Plant Signal Behav. 2006; December:305\u0026ndash;11.\u003c/li\u003e\n\u003cli\u003eSeo PJ, Park C-M. MYB96-mediated abscisic acid signals induce pathogen resistance response by promoting salicylic acid biosynthesis in Arabidopsis. New Phytol. 2010;186:471\u0026ndash;83.\u003c/li\u003e\n\u003cli\u003eMcCarthy RL, Zhong R, Ye Z-H. MYB83 Is a Direct Target of SND1 and Acts Redundantly with MYB46 in the Regulation of Secondary Cell Wall Biosynthesis in Arabidopsis. Plant Cell Physiol. 2009;50:1950\u0026ndash;64.\u003c/li\u003e\n\u003cli\u003eZhou J, Lee C, Zhong R, Ye Z-H. MYB58 and MYB63 Are Transcriptional Activators of the Lignin Biosynthetic Pathway during Secondary Cell Wall Formation in \u0026amp;lt;em\u0026amp;gt; Arabidopsis\u0026amp;lt;/em\u0026amp;gt; Plant Cell. 2009;21:248 LP \u0026ndash; 266.\u003c/li\u003e\n\u003cli\u003eZhong R, Ye Z-H. MYB46 and MYB83 Bind to the SMRE sites and Directly Activate a Suite of Transcription Factors and Secondary Wall Biosynthetic Genes. Plant Cell Physiol. 2012;53:368\u0026ndash;80.\u003c/li\u003e\n\u003cli\u003eAbe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) Function as Transcriptional Activators in Abscisic Acid Signaling. Plant Cell. 2003;15:63 LP \u0026ndash; 78.\u003c/li\u003e\n\u003cli\u003eSeo PJ, Lee SB, Suh MC, Park M-J, Go YS, Park C-M. The MYB96 Transcription Factor Regulates Cuticular Wax Biosynthesis under Drought Conditions in \u0026amp;lt;em\u0026amp;gt;Arabidopsis\u0026amp;lt;/em\u0026amp;gt; Plant Cell. 2011;23:1138 LP \u0026ndash; 1152.\u003c/li\u003e\n\u003cli\u003eBowring SA, Grotzinger JP, Isachsen CE, Knoll AH, Pelechaty SM, Kolosov P. Calibrating rates of early Cambrian evolution. Science. 1993;261:1293\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eMcInerney FA, Wing SL. The Paleocene-Eocene Thermal Maximum: A Perturbation of Carbon Cycle, Climate, and Biosphere with Implications for the Future. Annu Rev Earth Planet Sci. 2011;39:489\u0026ndash;516.\u003c/li\u003e\n\u003cli\u003eChen S, Niu X, Guan Y, Li H. Genome-Wide Analysis and Expression Profiles of the MYB Genes in Brachypodium distachyon. Plant \u0026amp;amp; cell Physiol. 2017;58:1777\u0026ndash;88.\u003c/li\u003e\n\u003cli\u003eKatiyar A, Smita S, Lenka SK, Rajwanshi R, Chinnusamy V, Bansal KC. Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis. BMC Genomics. 2012;13:544.\u003c/li\u003e\n\u003cli\u003ePaterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, Gundlach H, et al. The Sorghum bicolor genome and the diversification of grasses. Nature. 2009;457:551\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eMcCormick RF, Truong SK, Sreedasyam A, Jenkins J, Shu S, Sims D, et al. The Sorghum bicolor reference genome: improved assembly, gene annotations, a transcriptome atlas, and signatures of genome organization. Plant J. 2018;93:338\u0026ndash;54.\u003c/li\u003e\n\u003cli\u003eKieffer M, Stern Y, Cook H, Clerici E, Maulbetsch C, Laux T, et al. Analysis of the transcription factor WUSCHEL and its functional homolog in Antirrhinum reveals a potential mechanism for their roles in meristem maintenance. Plant Cell. 2006;18:560\u0026ndash;73.\u003c/li\u003e\n\u003cli\u003eSharma P, Lin T, Grandellis C, Yu M, Hannapel DJ. The BEL1-like family of transcription factors in potato. J Exp Bot. 2014;65:709\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eYanagisawa S, Sheen J. Involvement of maize Dof zinc finger proteins in tissue-specific and light-regulated gene expression. Plant Cell. 1998;10:75\u0026ndash;89.\u003c/li\u003e\n\u003cli\u003eRabinowicz PD, Braun EL, Wolfe AD, Bowen B, Grotewold E. Maize R2R3 Myb genes: Sequence analysis reveals amplification in the higher plants. Genetics. 1999;153:427\u0026ndash;44.\u003c/li\u003e\n\u003cli\u003eLi P, Ponnala L, Gandotra N, Wang L, Si Y, Tausta SL, et al. The developmental dynamics of the maize leaf transcriptome. Nat Genet. 2010;42:1060\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eWang L, Czedik-Eysenberg A, Mertz RA, Si Y, Tohge T, Nunes-Nesi A, et al. Comparative analyses of C₄ and C₃ photosynthesis in developing leaves of maize and rice. Nat Biotechnol. 2014;32:1158\u0026ndash;65.\u003c/li\u003e\n\u003cli\u003eStuder AJ, Schnable JC, Weissmann S, Kolbe AR, McKain MR, Shao Y, et al. The draft genome of the C3 panicoid grass species Dichanthelium oligosanthes. Genome Biol. 2016;17:223.\u003c/li\u003e\n\u003cli\u003eR Rott, RA Bailey, JC Comstock BC, and AS. A guide to sugarcane diseases. France: CIRAD; 2000.\u003c/li\u003e\n\u003cli\u003eSingh A, Chauhan SS, Singh A, Singh SB. Deterioration in sugarcane due to pokkah boeng disease. Sugar Tech. 2006;8:187\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eXu D-L, Park J-W, Mirkov TE, Zhou G-H. Viruses causing mosaic disease in sugarcane and their genetic diversity in southern China. Arch Virol. 2008;153:1031.\u003c/li\u003e\n\u003cli\u003eChauhan RP, Rajakaruna P, Verchot J. Complete genome sequence of nine isolates of canna yellow streak virus reveals its relationship to the sugarcane mosaic virus (SCMV) subgroup of potyviruses. Arch Virol. 2015;160:837\u0026ndash;44.\u003c/li\u003e\n\u003cli\u003eFlagel LE, Wendel JF. Gene duplication and evolutionary novelty in plants. New Phytol. 2009;183:557\u0026ndash;64.\u003c/li\u003e\n\u003cli\u003eKim C, Wang X, Lee T-H, Jakob K, Lee G-J, Paterson AH. Comparative Analysis of Miscanthus and Saccharum Reveals a Shared Whole-Genome Duplication but Different Evolutionary Fates. Plant Cell. 2014;26:2420\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eDu H, Feng B-R, Yang S-S, Huang Y-B, Tang Y-X. The R2R3-MYB transcription factor gene family in maize. PLoS One. 2012;7:e37463.\u003c/li\u003e\n\u003cli\u003eZhong R, Lee C, McCarthy RL, Reeves CK, Jones EG, Ye Z-H. Transcriptional activation of secondary wall biosynthesis by rice and maize NAC and MYB transcription factors. Plant \u0026amp;amp; cell Physiol. 2011;52:1856\u0026ndash;71.\u003c/li\u003e\n\u003cli\u003eMichael dos Santos Brito. Unraveling Two Ways of Lignin Biosynthesis in Sugarcane Culm: An Overview of Transcriptional Regulation. 2012; P0738.\u003c/li\u003e\n\u003cli\u003eFigueiredo R, Portilla Llerena JP, Kiyota E, Ferreira SS, Cardeli BR, de Souza SCR, et al. The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana. Plant Mol Biol. 2020;104:411\u0026ndash;27.\u003c/li\u003e\n\u003cli\u003eByrne ME, Barley R, Curtis M, Arroyo JM, Dunham M, Hudson A, et al. Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis. Nature. 2000;408:967\u0026ndash;71.\u003c/li\u003e\n\u003cli\u003eHusbands AY, Chitwood DH, Plavskin Y, Timmermans MCP. Signals and prepatterns: new insights into organ polarity in plants. Genes Dev. 2009;23:1986\u0026ndash;97.\u003c/li\u003e\n\u003cli\u003eLiu T, Ohashi-Ito K, Bergmann DC. Orthologs of Arabidopsis thaliana stomatal bHLH genes and regulation of stomatal development in grasses. Development. 2009;136:2265\u0026ndash;76.\u003c/li\u003e\n\u003cli\u003eCarr\u0026eacute; IA, Kim J-Y. MYB transcription factors in the Arabidopsis circadian clock. J Exp Bot. 2002;53:1551\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003ePrabu G, Kawar PG, Pagariya MC, Prasad DT. Identification of Water Deficit Stress Upregulated Genes in Sugarcane. Plant Mol Biol Report. 2011;29:291\u0026ndash;304.\u003c/li\u003e\n\u003cli\u003eSilvana Creste Souza. Characterization of Transcription Factors Differentially Expressed Under Drought Conditions in Sugarcane (Saccharum spp). 2015; P0180.\u003c/li\u003e\n\u003cli\u003eShingote PR, Kawar PG, Pagariya MC, Kuhikar RS, Thorat AS, Babu KH. SoMYB18, a sugarcane MYB transcription factor improves salt and dehydration tolerance in tobacco. Acta Physiol Plant. 2015;37:217.\u003c/li\u003e\n\u003cli\u003eGuo J, Ling H, Ma J, Chen Y, Su Y, Lin Q, et al. A sugarcane R2R3-MYB transcription factor gene is alternatively spliced during drought stress. Sci Rep. 2017;7:41922.\u003c/li\u003e\n\u003cli\u003eF\u0026aacute;vero Peixoto-Junior R, Mara de Andrade L, Dos Santos Brito M, Macedo Nobile P, Palma Boer Martins A, Domingues Carlin S, et al. Overexpression of ScMYBAS1 alternative splicing transcripts differentially impacts biomass accumulation and drought tolerance in rice transgenic plants. PLoS One. 2018;13:e0207534.\u003c/li\u003e\n\u003cli\u003eRaffaele S, Rivas S, Roby D. An essential role for salicylic acid in AtMYB30-mediated control of the hypersensitive cell death program in Arabidopsis. FEBS Lett. 2006;580:3498\u0026ndash;504.\u003c/li\u003e\n\u003cli\u003eVailleau F, Daniel X, Tronchet M, Montillet J-L, Triantaphylid\u0026egrave;s C, Roby D. A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack. Proc Natl Acad Sci U S A. 2002;99:10179\u0026ndash;84.\u003c/li\u003e\n\u003cli\u003eLiu Z, Luan Y, Li J, Yin Y. Expression of a tomato MYB gene in transgenic tobacco increases resistance to Fusarium oxysporum and Botrytis cinerea. Eur J Plant Pathol. 2016;144:607\u0026ndash;17.\u003c/li\u003e\n\u003cli\u003eYang J, Liu D, Wang X, Ji C, Cheng F, Liu B, et al. The genome sequence of allopolyploid Brassica juncea and analysis of differential homolog gene expression influencing selection. Nat Genet. 2016;48:1225\u0026ndash;32.\u003c/li\u003e\n\u003cli\u003eFinn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 2016;44:D279-85.\u003c/li\u003e\n\u003cli\u003eWheeler TJ, Eddy SR. nhmmer: DNA homology search with profile HMMs. Bioinformatics. 2013;29:2487\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eHedges SB, Dudley J, Kumar S. TimeTree: a public knowledge-base of divergence times among organisms. Bioinformatics. 2006;22:2971\u0026ndash;2.\u003c/li\u003e\n\u003cli\u003eGuo A-Y, Chen X, Gao G, Zhang H, Zhu Q-H, Liu X-C, et al. PlantTFDB: a comprehensive plant transcription factor database. Nucleic Acids Res. 2008;36 Database issue: D966-9.\u003c/li\u003e\n\u003cli\u003ePu X, Yang L, Liu L, Dong X, Chen S, Chen Z, et al. Genome-Wide Analysis of the MYB Transcription Factor Superfamily in Physcomitrella patens. Int J Mol Sci. 2020;21.\u003c/li\u003e\n\u003cli\u003eLiu C, Xie T, Chen C, Luan A, Long J, Li C, et al. Genome-wide organization and expression profiling of the R2R3-MYB transcription factor family in pineapple (Ananas comosus). BMC genomics. 2017;18:503.\u003c/li\u003e\n\u003cli\u003eMatus JT, Aquea F, Arce-Johnson P. Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related clades and conserved gene structure organization across Vitis and Arabidopsis genomes. BMC Plant Biol. 2008;8:83.\u003c/li\u003e\n\u003cli\u003eChen B, Niu F, Liu W-Z, Yang B, Zhang J, Ma J, et al. Identification, cloning and characterization of R2R3-MYB gene family in canola (Brassica napus L.) identify a novel member modulating ROS accumulation and hypersensitive-like cell death. DNA Res. 2016;23:101\u0026ndash;14.\u003c/li\u003e\n\u003cli\u003eDu H, Yang S-S, Liang Z, Feng B-R, Liu L, Huang Y-B, et al. Genome-wide analysis of the MYB transcription factor superfamily in soybean. BMC Plant Biol. 2012;12:106.\u003c/li\u003e\n\u003cli\u003eZHENG X, YI D, SHAO L, LI C. In silico genome-wide identification, phylogeny and expression analysis of the R2R3-MYB gene family in Medicago truncatula. J Integr Agric. 2017;16:1576\u0026ndash;91.\u003c/li\u003e\n\u003cli\u003eFeng S, Xu Y, Yang L, Sun S, Wang D, Chen X. Genome-wide identification and characterization of R2R3-MYB transcription factors in pear. Sci Hortic (Amsterdam). 2015;197:176\u0026ndash;82.\u003c/li\u003e\n\u003cli\u003eHan Y, Yu J, Zhao T, Cheng T, Wang J, Yang W, et al. Dissecting the Genome-Wide Evolution and Function of R2R3-MYB Transcription Factor Family in Rosa chinensis. Genes (Basel). 2019;10:823.\u003c/li\u003e\n\u003cli\u003eWilkins O, Nahal H, Foong J, Provart NJ, Campbell MM. Expansion and Diversification of the \u0026amp;lt;em\u0026amp;gt;Populus\u0026amp;lt;/em\u0026amp;gt; R2R3-MYB Family of Transcription Factors. Plant Physiol. 2009;149:981 LP \u0026ndash; 993.\u003c/li\u003e\n\u003cli\u003eStracke R, Holtgr\u0026auml;we D, Schneider J, Pucker B, Rosleff S\u0026ouml;rensen T, Weisshaar B. Genome-wide identification and characterisation of R2R3-MYB genes in sugar beet (Beta vulgaris). BMC Plant Biol. 2014;14:249.\u003c/li\u003e\n\u003cli\u003eSun W, Ma Z, Chen H, Liu M. MYB Gene Family in Potato (Solanum tuberosum L.): Genome-Wide Identification of Hormone-Responsive Reveals Their Potential Functions in Growth and Development. Int J Mol Sci. 2019;20.\u003c/li\u003e\n\u003cli\u003eZhao P, Li Q, Li J, Wang L, Ren Z. Genome-wide identification and characterization of R2R3MYB family in Solanum lycopersicum. Mol Genet Genomics. 2014;289:1183\u0026ndash;207.\u003c/li\u003e\n\u003cli\u003eThompson JD, Gibson TJ, Higgins DG. Multiple Sequence Alignment Using ClustalW and ClustalX. Curr Protoc Bioinforma. 2003;00:2.3.1-2.3.22.\u003c/li\u003e\n\u003cli\u003eKumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33:1870\u0026ndash;4.\u003c/li\u003e\n\u003cli\u003eHu B, Jin J, Guo A-Y, Zhang H, Luo J, Gao G. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2015;31:1296\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eKrzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, et al. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19:1639\u0026ndash;45.\u003c/li\u003e\n\u003cli\u003eLescot M, D\u0026eacute;hais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002;30:325\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eChen Y, Zhang Q, Hu W, Zhang X, Wang L, Hua X, et al. Evolution and expression of the fructokinase gene family in Saccharum. BMC Genomics. 2017;18:197.\u003c/li\u003e\n\u003cli\u003eLi Z, Hua X, Zhong W, Yuan Y, Wang Y, Wang Z, et al. Genome-Wide Identification and Expression Profile Analysis of WRKY Family Genes in the Autopolyploid Saccharum spontaneum. Plant Cell Physiol. 2020;61:616\u0026ndash;30.\u003c/li\u003e\n\u003cli\u003eLing H, Wu Q, Guo J, Xu L, Que Y. Comprehensive selection of reference genes for gene expression normalization in sugarcane by real-time quantitative rt-PCR. PLoS One. 2014;9:e97469.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"MYB, Sugarcane, Expression analysis of stress, Allelic diversity","lastPublishedDoi":"10.21203/rs.3.rs-199103/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-199103/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Sugarcane (\u003cem\u003eSaccharum\u003c/em\u003e) is the most important sugar crop in the world. As one of the most enriched transcription factor families in plants, MYB genes display a great potential to contribute to sugarcane improvement by trait modification. We have identified the sugarcane MYB gene family at a whole-genome level through systematic evolution analyses and expression profiling. R2R3-MYB is a large subfamily involved in many plant-specific processes. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eA total of 202 R2R3-MYB genes (356 alleles) were identified in the polyploid \u003cem\u003eSaccharum spontaneum\u003c/em\u003e genome and classified into 15 subgroups by phylogenetic analysis. The sugarcane MYB family had more members by a comparative analysis in sorghum and significant advantages among most plants, especially grasses. Collinearity analysis revealed that 70%\u003cem\u003e \u003c/em\u003eof the SsR2R3-MYB genes had experienced duplication events, logically suggesting the contributors to the MYB gene family expansion. Functional characterization was performed to identify 56 SsR2R3-MYB genes involved in various plant bioprocesses with expression profiling analysis on 60 RNA-seq databases. We identified 22 MYB genes specifically expressed in the stem, of which \u003cem\u003eMYB43\u003c/em\u003e, \u003cem\u003eMYB53\u003c/em\u003e, \u003cem\u003eMYB65\u003c/em\u003e, \u003cem\u003eMYB78\u003c/em\u003e, and \u003cem\u003eMYB99\u003c/em\u003e were validated by qPCR. Allelic expression dominance in the stem was more significant than that in the leaf, implying the differential expression of alleles may be responsible for the high expression of MYB in the stem. \u003cem\u003eMYB169\u003c/em\u003e, \u003cem\u003eMYB181\u003c/em\u003e, \u003cem\u003eMYB192\u003c/em\u003e were identified as candidate C\u003csub\u003e4 \u003c/sub\u003ephotosynthetic regulators by C\u003csub\u003e4\u003c/sub\u003e expression pattern and robust circadian oscillations. Furthermore, stress expression analysis showed that \u003cem\u003eMYB\u003c/em\u003e36, \u003cem\u003eMYB\u003c/em\u003e48, \u003cem\u003eMYB\u003c/em\u003e54, \u003cem\u003eMYB\u003c/em\u003e61 actively responded to drought treatment; 19 and 10 MYB genes were involved in response to the sugarcane pokkah boeng and mosaic disease, respectively. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eA Genome-wide expression analysis demonstrated that \u003cem\u003eSsMYB \u003c/em\u003egenes were involved in stem development and stress response. This study largely contributed to understanding the extent to which MYB transcription factors investigate regulatory mechanisms and functional divergence in sugarcane.\u003c/p\u003e","manuscriptTitle":"\u0026nbsp;Genome-Wide Analysis of R2R3-MYB Transcription Factors Family in The Autopolyploid Saccharum Spontaneum: An Exploration of Dominance Expression and Stress Response\u0026nbsp;","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-16 22:07:08","doi":"10.21203/rs.3.rs-199103/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-03-08T08:25:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-03-01T15:15:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4b56b84c-fc42-4af5-85d9-3d0e8f964964","date":"2021-02-19T18:54:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-02-07T09:10:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-02-07T07:05:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-02-07T07:02:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-02-07T07:01:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2021-02-02T06:36:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52fd8884-e0c0-4a12-a0b7-09c8303532f2","owner":[],"postedDate":"February 16th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":2452057,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"2021-08-29T15:09:31+00:00","versionOfRecord":{"articleIdentity":"rs-199103","link":"https://doi.org/10.1186/s12864-021-07689-w","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2021-08-18 15:02:47","publishedOnDateReadable":"August 18th, 2021"},"versionCreatedAt":"2021-02-16 22:07:08","video":"","vorDoi":"10.1186/s12864-021-07689-w","vorDoiUrl":"https://doi.org/10.1186/s12864-021-07689-w","workflowStages":[]},"version":"v1","identity":"rs-199103","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-199103","identity":"rs-199103","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}