Genome-wide Identification of R2R3-MYB Gene Family and Response to Stress in Zingiber officinale Roscoe

doi:10.21203/rs.3.rs-1661228/v1

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Genome-wide Identification of R2R3-MYB Gene Family and Response to Stress in Zingiber officinale Roscoe

https://doi.org/10.21203/rs.3.rs-1661228/v1

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Background: Zingiber officinale Roscoe (ginger), as a plant used for medicine and food, has a pivotal position all over the world. The R2R3-MYB transcription factor family is one of the largest, and it plays a significant role in plant growth, development, and stresses resistance. According to existing reports, the amounts of R2R3-MYB genes varies greatly among all kinds of plants. However, genome-wide discovery of ZoMYBs family and its corresponding stresses have not been reported in Z. officinale.

Results: The genome-wide analysis of R2R3-MYB genes in Z. officinale was carried out in this study. Protein analysis included phylogenetic analysis and conserved motifs analysis, while gene analysis included chromosome localization and gene replication analysis, gene structure analysis, cis-regulatory element analysis and gene expression pattern analysis under two different stresses. A total of 299 candidate ZoMYB genes were discovered in Z. officinale. Based on the grouping of R2R3-MYB genes in a model plant Arabidopsis thaliana, ZoMYBs was divided into 8 groups and distributed across 22 chromosomes at uneven densities. Through gene duplication analysis, a total of 28 segmental duplication were identified in ginger genome. In addition, the expression pattern of 10 chosen ZoMYBs in leaves of Z. officinale were detected under ABA and low temperature stress treatments. The result revealed that there were differences in gene expression of these ten MYB genes in ginger under ABA treatment and low temperature treatment.

Conclusions: Our results revealed the characteristics of ZoMYB genes family and provided valuable information for improving the ability of Z. officinale to cope with environmental changes during growth and development.

ginger

R2R3-MYB

phylogenetic analysis

gene structures

expression analysis

Ginger, as the rhizome of perennial herb ginger (Zingiber officinale Roscoe), appears in front of human beings with traditional Chinese medicine and condiment [1]. Ginger is grown all over the world, mostly distributed in tropical and subtropical regions, with the most diverse species in tropical Asia [2]. Since ginger has regular homologous attributes of medication and food, its applications are increasing diverse [3]. Ginger is subjected to a variety of challenges throughout its growth and development, so the optimization of genetic engineering brings hope for the improvement of ginger crops.

Transcription factors (TFs) are proteins binding to certain cis-acting regions and regulating the target genes [4]. The binding domains of TFs in the promoters of the target genes can be divided into several families [5]. MYB TFs consist of 1–4 adjacent repeat sequences (R) with a highly conserved domain, which containing approximately 52 amino acids. MYB TFs belong to one of most large gene family of proteins that have homologs in animals, plants, and yeasts [6, 7]. The characteristics of the repeats are spaced tryptophan residues at interval, which cover two а-helices helix-turn-helix (HTH), and the third helix-bound DNA main groove [8]. According to the number of neighboring repetitions, MYB TFs are categorized into four types, 1R (R1/2, R3), 2R (R2R3), 3R (R1R2R3), and 4R, respectively [6, 9]. The proteins of 3R-MYB exist in animals with limited amounts as usual, while a bulk amounts of 2R-MYB proteins contained in plants. The proteins of R2R3-MYB have been further divided into 25 subgroups in A. thaliana ([7].

There were 126 AtMYB TFs discovered in A. thaliana [7], and 244 GmMYB TFs identified in Glycine max [10], 205 GrMYB TFs in Gossypium raimondii [11], 166 MeMYB TFs in Manihot esculenta [12], 157 ZmMYB TFs in Zea mays [13], and 122 SlMYB TFs in Solanum lycopersicum [14]. The most fully explored functions of R2R3-MYB TFs were their regulatory roles in four key plant-specific processes, involving the response to various stresses and regulation of plant defense [15, 16], primary and secondary metabolic processes [17, 18], developmental processes, and cell fate and identity [19, 20]. For example, AtMYB41 from A. thaliana is induced when the plants were exposed to high salt, abscisic acid (ABA), drought, and cold [21]. VcMYB from Vaccinium corymbosum may be involved in the regulation of drought stress [22]. AtMYB102 is dramatically up-regulated under osmotic stress and ABA treatment, either [23]. The expression level of OsMYB55 increased gradually in rice treated by high temperature, which led to improving plant growth [24]. Moreover, OsMYB91 contributes a lot to plant growth regulation and salinity stress response in rice [25]. Mechanisms of JrMYB transcription factors in response to drought and ABA treatment in Juglans regia [26]. TaMYB4 in Triticum aestivum responds to some biological stresses, such as salicylic acid, ABA, MeJA and ethylene [27]. GuMYB from Glycyrrhiza uralensis regulates flavonoid accumulation under MeJA induction [28]. GbMYBFL, also related to flavonoid biosynthesis accumulation in Ginkgo biloba, is the most strongly linked to R2R3-MYB TFs [29].

R2R3-MYBs have been identified clearly and their functions have been validated in most plants, which offered us a better view of the functional classification of the TF subfamily. In ginger, nevertheless, little is known about this gene subfamily. The identification and analysis of 299 ZoMYBs were performed at the genome-wide level of Z. officinale in this article. A comparison with data from known MYB TFs in A. thaliana was also conducted, which resulted in a systematic genome-wide identification and comprehensive evolutionary analysis of these R2R3-MYB TFs. In addition, comparative investigation with the MYB genes of A. thaliana predicted the roles of protein in every subcategory of the ZoMYB family. We used quantitative real-time reverse transcription (qRT-PCR) analysis to quantify and compare the expression level of chosen MYBs under two stress treatments of ABA (abscisic acid) and cold treatment.

R2R3-MYB transcriptional factors in Z. officinale

Totally, 299 R2R3-MYBs were discovered in Z. officinale based on their similarity to the R2R3-MYBs of A. thaliana and the MYB consensus sequence existing as well as the HMM profile of MYB-DNA binding domain. They were named ZoMYB1-ZoMYB299 (Table S1) successively. Chemical properties analysis of these Z. officinale R2R3 MYB proteins revealed that their length ranged from 103 to 1651 amino acids, molecular weight ranged from 11.87 kDa to 182.93 kDa, and their isoelectric point (PI) ranged from 4.44 to 10.67 (Table 1). The subcelluar localization of these MYBs were predicted, most of them were in nucleus (89.97%), while a few were in cytoplasmic (5.02%), mitochondrial (4.01%) and endoplasmic reticulum (1.00%) (Table S2).

Phylogenetic evolution and classification analysis of R2R3 MYBs in Z. officinale

For further analysis the phylogenetic evolutionary relationship of these R2R3-MYBs in ginger and other model plants, all ZoMYBs proteins discovered in ginger and 126 existing MYBs (AtMYBs) proteins in A. thaliana were chosen to construct a phylogenetic tree. The amino acid sequence alignment of R2R3-MYB proteins of the two species were analyzed by bootstrap at 1000 replicates. Through the comparative phylogenetic analysis of Z. officinale and A. thaliana, the evolutionary relationship in interspecies of the R2R3-MYB gene family of the two plants can be constructed, and the function of the unknown target gene in ginger can also be predicted. The resulting phylogenetic tree was divided into 8 groups according to the known clustering of AtMYBs from A. thalian, and that named as group G1 to Group G8 successively (Fig. 1). Seven of these groups contained R2R3 MYB from both ginger and A. thaliana. However, ZoMYB086 was not clustered with all of other R2R3-MYBs, so we divided it into a group (G4). Among the groups containing two species, The G1 group had the largest number of members, with a total of 106, of which 84 members were identified from Z. officinale and 22 members were discovered from A. thaliana. In comparison, the G3 group had the second smallest number of members, with 17 members, including 4 from A. thaliana and 13 from Z. officinale. In the remaining groups, group G2, G5, G6, G7 and G8 contain 86, 18, 30, 74 and 93 R2R3-MYB members, respectively.

Gene duplication and chromosomal location of R2R3-MYBs in Z. officinale

The physical locations of these ZoMYB TF genes were localized to the chromosomes of Z. officinale (ZoChr) by Mapgene2chrom online tool (Fig. 2). Among the 299 R2R3-MYB genes discovered, 293 MYB genes were distributed on 22 chromosomes. ZoChr08A and ZoChr08B contained 21 ZoMYB genes, which is the largest number in chromosomes, followed by 20 ZoMYB genes on chromosome of ZoChr01A. Most of the chromosomes contained 10 to 19 R2R3 MYB TFs, except ZoChr22A and ZoChr10A, they were five and four this kind of TFs, respectively. The above results show that the distribution of genes on chromosomes is uneven. Obviously, the majority of ZoMYB genes are found at the top and bottom of chromosomes, and less in the central position of chromosomes, such as ZoChr01A.

Gene duplication events play an important role in promoting plant evolution, that is, new gene functions are derived with plant evolution [30]. The gene family expands through the segmental and tandem duplication events mainly [30]. Here, collinearity of R2R3-MYB TF family in ginger genome was analyzed, as shown in Fig. 3 and Table 1. A total of 28 segmental duplication events were identified in ginger genome.

Table 1

Duplication models for *ZoMYB* gene pairs in *Z. officinale*.
Seq_1	Seq_2	Ka	Ks	Ka/Ks	Effective Length	Average S-sites	Average N-sites
ZoMYB007	ZoMYB027	0.024980233	0.0181278	1.37800908	867	195.4166667	671.5833333
ZoMYB019	ZoMYB036	0.03799668	0.0428178	0.887403642	918	216.25	701.75
ZoMYB048	ZoMYB060	0.038458337	0.1232118	0.31213203	615	143.75	471.25
ZoMYB066	ZoMYB082	0.088251239	0.1346333	0.655493596	831	195.4166667	635.5833333
ZoMYB069	ZoMYB084	0.015136163	0.0582714	0.259752685	753	163.5	589.5
ZoMYB091	ZoMYB105	0.067654496	0.0780992	0.866263133	615	128.0833333	486.9166667
ZoMYB093	ZoMYB113	0.014229676	0.1015634	0.140106374	276	63.16666667	212.8333333
ZoMYB094	ZoMYB111	0.02641193	0.0578003	0.456951786	786	188.75	597.25
ZoMYB095	ZoMYB110	0.008699532	0.0313362	0.27761968	741	162.9166667	578.0833333
ZoMYB097	ZoMYB108	0.075502439	0.1554003	0.48585789	942	223.8333333	718.1666667
ZoMYB115	ZoMYB132	0.016040386	0.0518246	0.309512718	2973	725.5833333	2247.416667
ZoMYB117	ZoMYB133	0.069518092	0.131514	0.528598424	1053	259.75	793.25
ZoMYB124	ZoMYB138	0.013781611	0.0175276	0.786281678	759	173.1666667	585.8333333
ZoMYB144	ZoMYB156	0.011592567	0.0588176	0.19709339	663	141.4166667	521.5833333
ZoMYB147	ZoMYB159	0.026184478	0.0224841	1.164576521	1527	361.1666667	1165.833333
ZoMYB150	ZoMYB160	0.01779943	0.0306339	0.581037658	690	149.9166667	540.0833333
ZoMYB153	ZoMYB164	0.069929281	0.1109741	0.630140699	1278	293.25	984.75
ZoMYB174	ZoMYB192	0.050252249	0.0629373	0.798449193	684	149.0833333	534.9166667
ZoMYB198	ZoMYB218	0.05024913	0.0888539	0.56552526	1191	287.4166667	903.5833333
ZoMYB204	ZoMYB221	0.05076618	0.1588057	0.319674818	615	136.25	478.75
ZoMYB207	ZoMYB227	0.073245268	0.0975543	0.750815495	681	160.3333333	520.6666667
ZoMYB208	ZoMYB231	0.054867763	0.1352741	0.405604428	483	119.1666667	363.8333333
ZoMYB209	ZoMYB232	0.062485297	0.0740966	0.843294525	1647	401.5833333	1245.416667
ZoMYB210	ZoMYB230	0.051619265	0.0622201	0.829623784	1359	326.5833333	1032.416667
ZoMYB210	ZoMYB233	0.039384048	0.0680599	0.578667794	1791	422.6666667	1368.333333
ZoMYB239	ZoMYB254	0.067049697	0.1004926	0.667210203	633	147.0833333	485.9166667
ZoMYB245	ZoMYB259	0.005828101	0.0083888	0.694750988	1101	239.75	861.25
ZoMYB266	ZoMYB272	0.086966689	0.1668916	0.521096878	879	189.9166667	689.0833333

Ks and Ka mutations occur in the open reading frame region after gene duplication, leading to new gene functions. Consequently, the rate of nonsynonymous (Ka) and synonymous (Ks) substitution (Ka/Ks) between target gene and its duplicated gene was determined whether there is selective pressure on the protein coding gene. The calculation results of Ka, Ks, and Ka/Ks for 28 pairs of repeated pairings were shown in Table 1. The Ka values of 28 ZoMYB duplicated genes were at the range of 0.005828–0.088251 and their Ks value were at 0.0083888–0.1668916. The Ka/Ks value of 26 paralogous pairs of ZoMYB is < 1, which suggested that purifying selection with the segmental replication was the major driving force for the evolution of ZoMYBs. However, the Ka / Ks value of 2 paralogous pairs of ZoMYB is > 1, indicating that positive selection with the segmental duplication occurred during the evolution of ZoMYB family genes, for example, ZoMYB007-ZoMYB027 and ZoMYB147-ZoMYB159.

Motifs of the R2R3 MYB TF genes in Z. officinale

All the amino acid sequences in full-length of 299 ZoMYBs were studied by the Multiple Em for Motif Elicitation (MEME Suite) program tool. There were 20 motifs identified in these ZoMYBs, and the length of them is between 6–50 amino acids (Figure S1). The results show that the number of conservative motifs in each ZoMYB is ranged from 1 to 10. Motifs 1, 2, 3, 7 and 20 are most likely to appear in ZoMYBs, and there is more than one same motif in most ZoMYBs. Except ZoMYB063, ZoMYB188, and ZoMYB203. As shown in Fig. 1 and Figure S1, most closed members belonging to the same subgroup exhibited similar motif compositions and exons position, which indicating that the members belonging to the same subgroup may perform the similar functions [11].

Exon–intron organization analysis of ZoMYB genes

The gene structure information is considered to provide a new source of evolutionary data for plant evolution [31]. In order to deeply comprehend the structural characteristics of genes encoding ZoMYBs, we studied the distribution of introns and exons of these 299 ZoMYB TF genes (Figure S2). R2R3 MYB had at least one exon in the DNA binding domain. Among these 299 ZoMYBs, 288 of them had 1–12 intron(s), accounting for 96.32%. In contrast, 11 ZoMYBs lacked introns. Among the 299 ZoMYB genes, 148 of them contain 3 exons and 2 introns. Obviously, ZoMYB162 and ZoMYB236 contain the largest number of introns and exons in their CDS, they all contain 13 exons and 12 introns. Remarkably, the gene structure analysis of these ZoMYBs indicated that those genes which were in the same subgroups commonly had nearly the same number of exon-intron pattern, with fully conserved position(s) of the intron(s). These results indicated the existence of a structure that is highly conserved within the ZoMYBs family and provided important evidence for the family nomenclature.

Stress-related elements in the candidate promoters of ZoMYB genes

Cis-regulatory elements are commonly restricted to the promoters of genes at 5’ upstream areas. They are the DNA-binding sites of transcription factors, and they are responsible for regulating the target genes at transcriptional level [32]. Thus, the putative promoters of these 299 identified ZoMYB genes were used to excavate stress-related regulatory elements. As expected, there were abundant light-responsive elements in 298 of 299 ZoMYB gene promoters, except ZoMYB298 (Figure S3). MeJA is one of the most important phytohormaones that plays a key role in plant responses to stress [33]. Interestingly, MeJA-responsive elements were the second largest element group of ZoMYB gene promoters, in which 256 ZoMYB genes respond to light response. The content of ABA- responsive elements in the whole ZoMYB genes was also very high.

ABA is considered as an important mediator in the plants answering to different adverse environmental conditions, including low temperature, drought, and salinity [34]. Among all promoters in these ZoMYB genes, 166 ZoMYBs contain ABA-responsive elements, followed by drought-responsive element and low-temperature-responsive element, 109 and 78, respectively.

Expression of ZoMYB genes under ABA and Cold stress in Z. officinale

There is always association between gene expression pattern and its function. MYB TFs have been claimed to regulate gene expression when plants suffered from environmental changes in previous studies [351, 36]. As have been reported, quite a lot MYB genes occupied a place in response to various abiotic stresses, for instance drought and salt stress [21, 23, 37, 38]. Ten ZoMYB genes, from 299 ZoMYBs, were selected to study their gene expression patterns under between abiotic stresses and hormone treatments. These ten ZoMYBs were clustered together in the phylogenetic tree with known stress-related AtMYB. RT-qPCR was carried out at 5 time points: 0, 1, 3, 6 and 12 h after ABA (Figure. 4A) and low temperature (Figure. 4B) treatment.

As indicated in Figure. 4, the expression of many ZoMYB genes exhibited different trends after the treatments of ABA and low temperature. By treated with ABA, the transcriptional level of 5 genes (ZoMYB26, ZoMYB46, ZoMYB61, ZoMYB84, ZoMYB157) arrived at the peak value after 6 h treatment, and the relative expressional level of the remaining 5 MYB genes exhibited roughly increasing patterns during pulp development. Under low temperature treatment, there is significant difference among ten MYB genes in transcriptional level. For example, ZoMYB46 and ZoMYB148 reached the highest value at 12 h. However, ZoMYB84 and ZoMYB263 highly expressed at 6 h after treatment. The relative expression patterns of ZoMYB61 and ZoMYB157 tended to be the same at 3 h and 6 h.

MYB TFs encoded by genes constitute a fairly large transcriptional factor family in higher plants. Of the four different types, type R2R3 is the more common type in the MYB family. Different plant species have different amounts of R2R3-MYB transcription factors in genome-wide. It is the first time to discover, identify and describe the 299 ZoMYB TF genes in Z. officinale, and which were categorized into 8 subfamilies (Fig. 1) in this study. Among them, the number of 299 ZoMYBs subfamily members in Z. officinale were far more than that the number of 126 AtMYBs members in A. thaliana, thus suggesting that the gene duplication occurring during genetic evolution may lead to the increase of R2R3-MYB gene abundance in ginger plants. Gene duplications are acted as the primary driving force underlying new gene functions [39, 40]. Here, the different expression patterns of ten ZoMYB genes under ABA stress and low temperature stress were observed, showing that this category of MYB transcription factors made a significant contribution in plant growth and development in Z. officinale.

Gene structure analysis of these 299 ZoMYB TF genes indicated that most coding sequences of ZoMYBs contained introns, except 11 genes (ZoMYB002, ZoMYB023, ZoMYB096, ZoMYB109, ZoMYB117, ZoMYB141, ZoMYB184, ZoMYB228, ZoMYB242, ZoMYB270, ZoMYB289) From Figure S2. The most of the ZoMYBs (96.32%) contained more than one intron, and the number of introns seems to be limited, which is similar to the corresponding gene structures of from A. thaliana [41]. It is worth noting that genes in the same subgroup usually showed similar gene structure profiles, including the position, distribution and phases of introns [10]. The main members of the same subfamily often have common conserved motifs and similar gene structure, but there are great differences among different subfamilies. In the same evolutionary branch, ZoMYBs and AtMYBs share same gene structures, and conserved motifs, and are classified in the same branch, strongly supported the result of our subfamily classification.

In present work, almost all ZoMYB TFs had many cis-acting elements connected to presses, such as light, ABA, drought, cold, MeJA, gibberellin, Auxin, salicylic acid, and wound stress, indicating that these ZoMYB genes TF from Z. officinale involved in various abiotic presses and hormone responses. All of 166 genes from this kind of MYB are regulated by ABA-responsive elements in their promoter regions, and 78 corresponding genes are regulated by low-temperature-responsive elements in their promoter regions to adapt low temperature stress (Figure S3). We selected ten genes to analyze their expression patterns under low temperature (cold) and hormone treatments (ABA) at five time points. Ten chosen genes were expressed in all samples (despite varying levels of expression), suggesting that these genes play a regulatory role in different abiotic stresses and hormone treatments by signal transduction pathway.

Our results laid a basic foundation for the identification and functional analysis of R2R3-MYB TFs on genome-wide in Z. officinale. A total of 299 ZoMYB TF genes were characterized from the genome of Z. officinale and analyzed by their related proteins and genes, such as phylogenic relationship, conserved domain, gene structure analysis, collinearity analysis, etc. Furthermore, the potential functions of 299 ZoMYB TFs were predicted by Cis-regulatory Elements Analysis and gene expression patterns under various stresses were detected. These results revealed the characteristics of ZoMYB genes family and provided valuable information for improving the ability of Z. officinale to cope with environmental changes during growth and development.

Plant materials

The ginger raw materials were obtained from Laiwu city, Shandong province, and were cultivated in Anhui University of Chinese Medicine (Hefei, Anhui). The ginger samples were stored in the Herbarium of Anhui University of Chinese Medicine (code: 20211105). The seedlings of 45 days were chosen for related-stress and expression pattern analysis. For the ABA stress treating, the seedlings were suffered from 100 mM. For the treatment of low temperature treatment, the tested plants were kept in an incubator at 4°C. All ginger samples were gathered at 0, 1, 3, 6, 12 h after two types of treatment.

Identification of R2R3-MYBs in Z. officinale

The genomic database, protein database and their annotated documents of Z. officinale were extracted from the famous database of NCBI, which assembly accession is GCA-018446385.1 in GenBank. The R2R3-MYB TF genes of A. thaliana were identified subjecting to the Arabidopsis Information Resource, and the database is used as the query sequence for local BLASTp search, E-value is set to 0.00001. In addition, the hidden Markov model profile of MYB with accession number PF00249 [38](Finn et al., 2016) was downloaded from the Pfam database. These sequences were aligned and the producing file was used to build the HMM profile of R2R3-MYB domain using HMMER (http://hmmer.org/) and this profile was used to identify the R2R3-MYB domain containing protein sequences amongst these MYBs in Z. officinale. The resulting sequences were combined, and redundant sequences were deleted by CD-HIT online analysis software [43]. R2R3-MYB sequences were manually screened according to their structural characteristics. Chemical properties of these protein sequences were evaluated with the ProtParam tool of ExPASy [44] and protein subcellular localization predictions were made by the BUSCA online site (http://busca.biocomp.unibo.it/) [45].

Phylogenetic Evolution Analysis of R2R3-MYBs in Z. officinale

To analyze the evolutionary of conserved protein motifs, a phylogenetic tree was constructed based on the obtained protein sequences of R2R3-MYB TFs from ginger and Arabidopsis by MEGA X software [46]. The sequences were aligned by ClustalW and the neighbor-joining method was used to generate evolutionary tree, which bootstrap value was set at 1000.

R2R3-MYBs’ gene duplication and chromosomal location in Z. officinale

The location information and chromosome length of the putative genes of R2R3-MYB were downloaded from the General Characteristic Format (GFF) file of ginger genome. Based on the result of Wang et al. [47], collinear analysis was carried out by the Multiple Collinear Scan Toolkit (MCScanX) and applied to analyze the duplication patterns of those acquired ZoMYB genes. The circular gene viewer software package of TBtools was used for integrating data and displaying the collinear relationship between the chromosome position of ZoMYBs and parahomologous MYBs in ginger. Synonymous (Ks) and nonsynonymous (Ka) substitution nucleotide ratios of ZoMYB gene pairs were also assessed by software of TBtools.

R2R3-MYBs’ conserved motifs identification in Z. officinale

All full-length protein sequences of 299 ZoMYBs were analyzed by the Multiple Em for Motif Elicitation program (MEME; v4.9.0) to get conserved motifs except MYB repeats. The following parameters were set as follow: the motif sites are expected to be sequentially distributed, a contributing motif site per sequence; maximum number of motifs to find, 20; the minimum width of motif and maximum width of motif are between 6 and 50 (to identify long R2R3 domains). The motif values less than E-20 were retained, and the default values were used for other options, and further analysis was conducted.

Gene Structure analysis of Z. officinale R2R3-MYB genes family

The whole genome sequence of Z. officinale was downloaded from the NCBI and the corresponding ZoMYB genes sequence were obtained from the genome sequence of Z. officinale were used for gene structure analysis. The gene structure schematic diagram is mapped through the Gene Structure Display Server (GSDS, http://gsds.gao-lab.org/index.php) after integrating genome and CDS sequence [48].

Cis-regulatory elements identification of ZoMYB genes promoter

Regulatory region sequence of ZoMYB genes at 1-K (Kilobase) upstream was extracted from ginger CDS sequence by TBtool software [49]. The software of PlantCARESearch for CARE website4. Plots was used to examine the cis-regulatory elements of the promoter sequence in TBtools software, and they are presented using the same software [50].

Real-time Florescence Quantitative Pcr (Rt-qpcr) Analysis

Total RNA from ginger was obtained using the TransZol kit (TransGen Biotech, Inc., Beijing, China). The obtained RNAs were synthesized into the cDNA using FastKing RT Kit (Tiangen, China) by reverse transcription reaction. The real-time quantitative PCR (RT-qPCR) was carried out on a LightCycle480 machine (Roche, Switzerland). SuperReal PreMix Plus (Tiangen, China) and a 20 µL reaction system were used [51]. Primer sequences were designed by software of Primer Premier. The primer sequences of genes of ZoMYBs were designed by primer design company (Sangon Biotech, Shanghai) (Table S3). And the data from qRT-PCR amplification were counted by the 2^−ΔΔCT method with ‘housekeeping’ gene actin as the internal reference gene. Thermal cycling conditions were set as per the manufacturer’s instructions for SYBR Green qPCR Master Mix (Vazyme). The cycling conditions included denaturation at 95°C for 30 seconds, 40 cycles of denaturation at 95°C for 5 seconds and annealing at 56°C for 30 seconds, and followed by a final melting curve analysis is performed. Each reaction was carried out in three independent biological replicates with a negative control group.

ABA, abscisic acid; HTH, helix-turn-helix; Ka, nonsynonymous; Ks, Synonymous; LT, low temperature; MeJA, methyl jasmonate; NCBI, National Center for Biotechnology; qRT-PCR, quantitative real-time reverse transcription; TFs, Transcription factors.

Acknowledgments

We would like to thank Dr. Shuai Liu from University of Hawaii Cancer Center for helping to revise the manuscript. We also thank Dr. Xiaoxi Meng from Department of Horticultural Science, University of Minnesota for helpful comments on an earlier version of the manuscript.

Author contributions

SX, XY, CQ conceived, designed, and implemented the study; XY, XD, ZS, LW, SW analyzed the statistics; XY, SX, ZW, CQ and JZ performed the experiments; SX, XY, FM, XG, JW and XS drafted the manuscript; All authors participated in the editing of the manuscript and submitted the final version.

Funding

Collaborative Innovation Project of Colleges and Universities of Anhui Province (Grant No. GXXT-2019-043), the NSF of Anhui Province (Grant No. 1908085MH268), and University Natural Science Research Project of Anhui Province (Grant No. KJ2019A0453, Grant No. KJ2021A0676 and KJ2018A0275).

Availability of data and materials

The original contributions presented in the study included the article and Supplementary Material, further inquiries can be directed to the corresponding authors.

Ethics approval and consent to participate

This study include no human, animal or endangered plant samples, and the sample was legally collected in accordance with guidelines provided by the authors’ institution and national or international regulations. Field studies was complied with local legislation. No ethical approval/permission is required in this study. The samples were stored in the Herbarium of Anhui University of Chinese Medicine (code: 20211105).

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Author details

¹College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China.

² Institute of Traditional Chinese Medicine Resources Protection and Development, Anhui Academy of Chinese Medicine, Hefei 230012, China.

³ Anhui Province Key Laboratory of Environmental Hormone and Reproduction, Anhui Province Key Laboratory of Embryo Development and Reproductive Regulation, Fuyang Normal University, Fuyang 236037, China.

⁴Hunan Key Laboratory for Conservation and Utilization of Biological Resources in the Nanyue Mountainous Region, College of Life Sciences and Environment, Hengyang Normal University, Hengyang 421008, Hunan, China.

⁵ MOE-Anhui Joint Collaborative Innovation Center for Quality Improvement of Anhui Genuine Chinese Medicinal Materials, Hefei 230038, China.

⁶State Key Laboratory of Crop Biology, College of Agronomic Sciences, Shandong Agricultural University, Tai'an 271018, China.

⁷Anhui Province Key Laboratory of Research & Development of Chinese Medicine, Hefei 230012, China.

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No competing interests reported.

FigureS1.VisualizationoftheclassificationofZoMYBproteinsandthedistributionof20predictedmotifs.pdf
Figure S1. Visualization of the classification of ZoMYB proteins and the distribution of 20 predicted motifs. The phylogenetic tree was inferred using the Neighbor–Joining method and 1000 bootstrap replicates with full-length of ZoMYB amino acid sequences by Clustal X 2.0 software. The conserved motifs were investigated by the MEME program.
FigureS2.PhylogeneticanalysisandstructuresofZoMYBgenesinZ.officinale.pdf
Figure S2. Phylogenetic analysis and structures of ZoMYB genes in Z. officinale. The phylogenetic tree was constructed by Clustal X 2.0 with the Neighbor–Joining method and 1000 bootstrap replicates based on alignments of complete predicted ZoMYB protein sequences. In the gene structure diagram, black boxes and lines represent exons and introns, respectively.
FigureS3.Predictionofcisresponsiveelementsinthe1kupstreamregulatoryregionsofZoMYBgenes.pdf
Figure S3. Prediction of cis-responsive elements in the 1-k upstream regulatory regions of ZoMYB genes. Different cis-responsive elements are represented by different colored boxes.
TableS1.Theaminoacidsequencesof299R2R3MYBfromZ.officinale.xlsx
Table S1. The amino acid sequences of 299 R2R3-MYB genes form Z. officinale.
TableS2.ListofallZoMYBgenesidentifiedinZ.officinalegenome.xlsx
Table S2. List of all ZoMYB genes identified in Z. officinale genome.
TableS3.PCRprimersusedforquantitativerealtimePCRanalysis.xlsx
Table S3. PCR primers used for quantitative real-time PCR analysis.

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Genome-wide Identification of R2R3-MYB Gene Family and Response to Stress in Zingiber officinale Roscoe

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