Genome-Wide Analysis and the Expression Pattern of the MADS-Box Gene Family in Bletilla striata

Bletilla striata (Thunb. ex A. Murray) Rchb. f., a species of the perennial herb Orchidaceae, has potent anti-inflammatory and antiviral biological activities. MADS-box transcription factors play critical roles in the various developmental processes of plants. Although this gene family has been extensively investigated in many species, it has not been analyzed for B. striata. In total, 45 MADS-box genes were identified from B. striata in this study, which were classified into five subfamilies (Mδ, MIKC, Mα, Mβ, and Mγ). Meanwhile, the highly correlated protein domains, motif compositions, and exon–intron structures of BsMADSs were investigated according to local B. striata databases. Chromosome distribution and synteny analyses revealed that segmental duplication and homologous exchange were the main BsMADSs expansion mechanisms. Further, RT-qPCR analysis revealed that BsMADSs had different expression patterns in response to various stress treatments. Our results provide a potential theoretical basis for further investigation of the functions of MADS genes during the growth of B. striata.

All MADS-box gene family members contain a DNA-binding domain of~60 amino acids, known as the MADS-box domain, located at the N-terminal region of the protein [18]. In A. thaliana, the gene family can be sub-divided into two classes based on gene structure: type I (Mα, Mβ, Mγ, and Mδ) and type II (MIKC) [19]. This diversity is primarily caused by a gene duplication event [20]. The plant type II genes contain highly conserved MADS domains-of which the K-domain is essential for functional diversity and protein-protein interactions [21]-while type I MADS-box genes have a relatively simple structure and lack a K-domain. Although the MADS-box genes in type II have been well documented and extensively studied, few reports exist that describe type I MADS-box genes in plants [22][23][24].
Bletilla striata (Thunb.) Rchb. f. is a perennial medicinal herb of the Orchidaceae family that is extensively distributed across China, Korea, Japan, and Myanmar [25]. According A total of 45 presumed MADS proteins were searched from the B. striata protein databases using the BLASTP program with 146 AtMADS (70 M-type MADS and 76 MIKC MADS) proteins as queries. All B. striata MADS proteins (designated BsMADS01 to BsMADS45) were confirmed to contain a single SRF-TF domain by InterProscan and SMART (Supplementary Table S1). The number of BsMADS genes was less than the identified MADS genes in Arabidopsis (146) and rice (75).
The basic features of the BsMADSs were predicted, and the coding sequence (CDS) lengths of 45 BsMADS genes ranged from 438 bp (BsMADS41) to 8612 bp (BsMADS14); thus, the proteins encoded would potentially include from 75 (BsMADS45) to 433 amino acids (BsMADS20). The molecular weight (MW) ranged from 8713.2 to 49280.86 Da. The isoelectric point (pI) ranged from 4.66 (BsMADS23) to 10.43 (BsMADS45). The prediction of subcellular localization revealed that all BsMADS proteins resided in the nuclear region (Supplementary Table S2).

Analysis of Multiple Sequence Alignments and Cis-Acting Elements
Multiple sequence alignments were performed based on the conserved SRF-TF domain of BsMADSs (Figure 1). The results revealed some highly conserved amino acids in the SRF-TF domain, including the 15th arginine (R), 21st lysine (K), 25th glutamic acid (E), 26th leucine (L), etc. (Figure 1a), which verified that the domain was a highly conserved DNA-binding/dimerization region [38]. To comprehensively investigate the function and regulatory roles we analyzed the various cis-acting elements of the promoter regions. acting elements were classified into five main functional classes: transc hormone, abiotic or biotic stress, and development. A total of 103 types found, most of which were associated with different stress conditions, nal responses such as ABA, GA, MeJA, SA, and auxin, as well as abio as wound, low temperature, drought, etc. (Figure 2, Supplementary Tab To comprehensively investigate the function and regulatory roles of the BsMADSs, we analyzed the various cis-acting elements of the promoter regions. The identified cis-acting elements were classified into five main functional classes: transcription, cell cycle, hormone, abiotic or biotic stress, and development. A total of 103 types of elements were found, most of which were associated with different stress conditions, including hormonal responses such as ABA, GA, MeJA, SA, and auxin, as well as abiotic responses such as wound, low  Table S3). We focused on drought inducibility and MeJA-related elements to predict the functions of the BsMADS genes. Figure 2. Distribution of various cis-acting elements in the promoter regions of B. striata MADS genes (BsMADSs). The cis-elements of the BsMADSs were searched using the online PlantCARE website. Special cis-acting elements were selected (e.g., auxin-responsive elements), and TBtools was used to create the diagrams.

Phylogenetic Analysis of the MADS-box Family between B. striata and Arabidopsis
To clarify the phylogenetic relationships of the MADS family proteins between B. striata and Arabidopsis, all whole-length amino acid sequences of 45 BsMADS and 146 AtMADS were employed to perform a multiple sequence alignment (MSA) protein analysis. An unrooted phylogenetic tree was developed using the neighbor-joining (NJ) method according to the similarity and topology of sequences, and the 45 BsMADS were distributed into five subgroups: A to E (Figure 3). The results could be highly useful for The cis-elements of the BsMADSs were searched using the online PlantCARE website. Special cis-acting elements were selected (e.g., auxin-responsive elements), and TBtools was used to create the diagrams.

Phylogenetic Analysis of the MADS-Box Family between B. striata and Arabidopsis
To clarify the phylogenetic relationships of the MADS family proteins between B. striata and Arabidopsis, all whole-length amino acid sequences of 45 BsMADS and 146 AtMADS were employed to perform a multiple sequence alignment (MSA) protein analysis. An unrooted phylogenetic tree was developed using the neighbor-joining (NJ) method according to the similarity and topology of sequences, and the 45 BsMADS were distributed into five subgroups: A to E (Figure 3). The results could be highly useful for predicting the various roles of unknown BsMADS according to the functionality confirmed in AtMADS or subgroups in Arabidopsis, which might contribute to the selection of target BsMADS for further functional analysis.

Gene Structure, Motif Composition, and Ka/Ks Analysis of BsMADSs
To glean insights into the similarities and diversity of gene structures and c motifs in BsMADSs, a phylogenetic tree was constructed based on multiple alignments (Figure 4a), which clustered almost identically to the result shown in indicating good consistency. The genomic DNA and CDS sequences were em analyze the intron and exon structures of the BsMADSs (Figure 4c). The results o structure analysis showed that the number of introns varied from zero to ten. In cluster of the phylogenetic tree, most genes possessed similar exon-intron s where among them, 26.7% of the BsMADSs contained only one exon without in A total of eight conserved motifs were detected in the BsMADSs (Figure each motif sequence logo displayed (Figure 4d). Most of the BsMADSs contained Motif-2, and Motif-4. Motif-7 was involved in 19 genes, while Motif-3 existed in Motif-8 was found in five genes, whereas only three genes contained Motif-6 found that the MADS genes with close evolutionary relationships shared the sa compositions, suggesting that the function of MADS proteins is similar.
We discovered that there were 14 pairs of homologous genes in the BsMADS 4a), which suggested that ~62.2% BsMADSs were replicated and that the BsMAD had experienced evolutionary gene expansion. To explore the influence of evo

Gene Structure, Motif Composition, and Ka/Ks Analysis of BsMADSs
To glean insights into the similarities and diversity of gene structures and conserved motifs in BsMADSs, a phylogenetic tree was constructed based on multiple sequence alignments (Figure 4a), which clustered almost identically to the result shown in Figure 3, indicating good consistency. The genomic DNA and CDS sequences were employed to analyze the intron and exon structures of the BsMADSs (Figure 4c). The results of the gene structure analysis showed that the number of introns varied from zero to ten. In the same cluster of the phylogenetic tree, most genes possessed similar exon-intron structures, where among them, 26.7% of the BsMADSs contained only one exon without introns.
A total of eight conserved motifs were detected in the BsMADSs (Figure 4b), with each motif sequence logo displayed ( Figure 4d). Most of the BsMADSs contained Motif-1, Motif-2, and Motif-4. Motif-7 was involved in 19 genes, while Motif-3 existed in 25 genes. Motif-8 was found in five genes, whereas only three genes contained Motif-6. We also found that the MADS genes with close evolutionary relationships shared the same motif compositions, suggesting that the function of MADS proteins is similar. orthologous gene pairs were no more than 1, which implied that a strong purifying selective pressure existed in the homologous genes during the course of their evolution (Supplementary Table S4).

Synteny Analysis and Chromosomal Distribution of MADS Genes
Gene duplication events may have given rise to new functionalities that assisted B. striata in adapting to changing environments, thus enriching gene family members. Three comparative synteny maps were constructed, including B. striata, A. thaliana, Vanilla fragrans, and Vitis vinifera ( Figure 5). A total of 17 MADS genes showed a syntenic relationship with those of V. fragrans, followed by A. thaliana (4) and V. vinifera (10). We discovered that there were 14 pairs of homologous genes in the BsMADSs (Figure 4a), which suggested that~62.2% BsMADSs were replicated and that the BsMADS family had experienced evolutionary gene expansion. To explore the influence of evolutionary factors on the BsMADS family, the Ka/Ks ratios of 14 BsMADS gene pairs were computed based on the phylogenetic tree ( Figure 4). The results showed that the ratios of 92.9% orthologous gene pairs were no more than 1, which implied that a strong purifying selective pressure existed in the homologous genes during the course of their evolution (Supplementary  Table S4).

Synteny Analysis and Chromosomal Distribution of MADS Genes
Gene duplication events may have given rise to new functionalities that assisted B. striata in adapting to changing environments, thus enriching gene family members. Three comparative synteny maps were constructed, including B. striata, A. thaliana, Vanilla fragrans, and Vitis vinifera ( Figure 5). A total of 17 MADS genes showed a syntenic relationship with those of V. fragrans, followed by A. thaliana (4) and V. vinifera (10). To gain insights into the evolution of the 45 B. striata MADS-box genes, we analyzed their genomic distribution and discovered that they were unevenly distributed on chromosomes 01-10 and 12-14 (except for five members that were found within repeat sequences or unassembled scaffolds) ( Figure 6). Chr01 and Chr03 contained six MADS-box genes, while Chr08, Chr09, and Chr14 had only one. The results of genomic distribution proved that some B. striata MADS-box genes of type I or type II were located in the same chromosomal region. A similar previously reported situation regarding the MADS-box genes of Arabidopsis [39] and rice [40] suggested that they were widely distributed across the genomes of the common monocotyledon and dicotyledon ancestors.  To gain insights into the evolution of the 45 B. striata MADS-box genes, we analyzed their genomic distribution and discovered that they were unevenly distributed on chromosomes 01-10 and 12-14 (except for five members that were found within repeat sequences or unassembled scaffolds) ( Figure 6). Chr01 and Chr03 contained six MADS-box genes, while Chr08, Chr09, and Chr14 had only one. The results of genomic distribution proved that some B. striata MADS-box genes of type I or type II were located in the same chromosomal region. A similar previously reported situation regarding the MADS-box genes of Arabidopsis [39] and rice [40] suggested that they were widely distributed across the genomes of the common monocotyledon and dicotyledon ancestors. To gain insights into the evolution of the 45 B. striata MADS-box genes, we analyzed their genomic distribution and discovered that they were unevenly distributed on chromosomes 01-10 and 12-14 (except for five members that were found within repeat sequences or unassembled scaffolds) ( Figure 6). Chr01 and Chr03 contained six MADS-box genes, while Chr08, Chr09, and Chr14 had only one. The results of genomic distribution proved that some B. striata MADS-box genes of type I or type II were located in the same chromosomal region. A similar previously reported situation regarding the MADS-box genes of Arabidopsis [39] and rice [40] suggested that they were widely distributed across the genomes of the common monocotyledon and dicotyledon ancestors.  (BsMADS18, BsMADS19, BsMADS21, BsMADS22, and BsMADS35) could not be anchored on a specific chromosome. The pink genes belong to group A (Mα), the yellow genes belong to group B (Mβ), the green genes belong to group C (Mγ), the blue genes belong to group D (Mδ), and the purple genes belong to group E (MIKC).

Expression Profile of BsMADS Genes under Non-Stressed Growth Conditions
To explore the tissue-specific expression of BsMADSs, we examined their expression profile in roots, stems, leaves, and flowers under non-stressed growth conditions based on quantitative real-time PCR (RT-qPCR) analysis (Figure 7, Supplementary Table S6). The expression levels of diverse BsMADSs changed significantly between different tissues. From the heatmap, BsMADS01, BsMADS05, BsMADS06, BsMADS07, BsMADS11, BsMADS27, and BsMADS40 exhibited a higher transcript accumulation in flowers, and they all belonged to group E (MIKC) (Figure 3). This result verified that the MADS-box genes described in the ABCDE model belonged to the MIKC subclass [41,42].
The expression levels of diverse BsMADSs changed significantly between d sues. From the heatmap, BsMADS01, BsMADS05, BsMADS06, BsMADS07, BsMADS27, and BsMADS40 exhibited a higher transcript accumulation in f they all belonged to group E (MIKC) (Figure 3). This result verified that the genes described in the ABCDE model belonged to the MIKC subclass [41,42].

Analysis of Expression Patterns under Different Stress Treatments
Eight BsMADS genes were randomly selected to further analyze their level patterns under different abiotic stressors ( Figure 8) and hormone treatm 9) at five time points (0, 1, 3, 6, and 12 h). BsMADS03 belonged to group A, belonged to group B, BsMADS18 belonged to group C, BsMADS10 and BsM longed to group D, and BsMADS01, BsMADS07, and BsMADS11 belonged Genetic patterns with significantly different expression levels were detected b As shown in Figs. 7 and 8, BsMADS33 had robust responses to all treatmen expressions of BsMADS01 and BsMADS07 were not obvious under the abiotic and BsMADS43 was not induced by NaCl.
The expression levels of two MADS genes (BsMADS03 and BsMADS33) d

Analysis of Expression Patterns under Different Stress Treatments
Eight BsMADS genes were randomly selected to further analyze their expression level patterns under different abiotic stressors ( Figure 8) and hormone treatments (Figure 9) at five time points (0, 1, 3, 6, and 12 h). BsMADS03 belonged to group A, BsMADS43 belonged to group B, BsMADS18 belonged to group C, BsMADS10 and BsMADS33 belonged to group D, and BsMADS01, BsMADS07, and BsMADS11 belonged to group E. Genetic patterns with significantly different expression levels were detected by RT-qPCR. As shown in Figures 7 and 8, BsMADS33 had robust responses to all treatments, while the expressions of BsMADS01 and BsMADS07 were not obvious under the abiotic treatments, and BsMADS43 was not induced by NaCl. 9 binding of ABRE (ABA response elements) to ABFs (downstream targets) [43], thus regulating the increased gene expression and contributing to the protein products conducive to plant resistance to stress.

Discussion
The MADS family is one of the largest plant transcription factor families, playing a critical role in plant growth and development and in the regulation of plant responses to both abiotic and biotic stresses [44,45]. Although this family has been widely researched for many plants, including Arabidopsis [4,7], rice [34], maize [46,47], Erycina pusilla [36], and Phalaenopsis [37], the identification, expression patterns, and functions of MADSs based on the whole-genome sequence of B. striata have remained poorly understood. Therefore, we conducted a comprehensive and systematic analysis of the MADS-box gene family in B. striata.
For our study, a total of 45 MADS-box genes were identified in B. striata, which was lower than that of both Arabidopsis (146 genes) [19] and rice (75 genes) [1], implying that the MADS family had diminished over the course of evolution. The number of genes varied for different species, which may have been caused by gene duplication (e.g., tandem and segmental duplication) [48,49]; however, members of the RsMADS may not have undergone such a process [50]. It was also revealed that gene function was a major determinant of gene family size, thus further suggesting that natural stressors were the major driving evolutionary forces [51].
To further explore the restrictive conditions during the evolution of the BsMADS family, the Ka/Ks ratios of the 28 paralogous BsMADS gene pairs were computed. The smaller the value of Ka/Ks between gene pairs, the more severe the selective constraint they evolved. The Ka/Ks ratios of most duplicated BsMADS gene pairs were less than 1, which indicated that the purification selection pressure played a dominant role in the evolutionary process of this family (Supplementary Table S4). The results revealed that the MADS-box gene family was currently being purified under B. striata selection.
According to the phylogenetic tree between B. striata and Arabidopsis, the BsMADS genes were divided into five subgroups, which also provided a reliable reference for the prediction of functional target BsMADS genes (Figure 3). Some BsMADSs were clustered in the same branch as the known AtMADSs, suggesting the conservation of MADS protein functions between the two species. For example, AGL6 (AGAMOUS-LIKE6, AT2G456501) is involved in the development of bractless flowers in Arabidopsis [7], while the expression level of AGL12 contributes to the regulation and development of root structures (AGA-MOUS-LIKE12, AT1G716921) [52], both of which belong to group E (MIKC).
Consequently, BsMADSs in the MIKC subgroups might be regarded as prime candidate genes for the development of plant organs. The results of the sequence alignments revealed characteristic amino acids (e.g., the majority of the BsMADSs contained one critical amino acid residue, which was the 25th glutamic acid (E)). The secondary structural elements of the SRF-TF domain were found to contain two α-helix and three β-sheets (Figure S1). SRF had been reported to activate transcription by interacting with the B-box [53]. The expression levels of two MADS genes (BsMADS03 and BsMADS33) dramatically increased under the CuSO 4 treatments. Five genes upregulated under AgNO 3 stress; among them, BsMADS10 was mostly upregulated~60-fold at 1 h, which then decreased, and BsMADS33 continued to increase after being upregulated~60-fold at 3 h. BsMADS10 downregulated following the ABA and SA treatments. All genes in the ABA treatment group were initially significantly downregulated and then upregulated, which indicated that the ABA treatment inhibited gene expression within 1 h.
Taking BsMADS33 stimulated by external ABA as an example, we hypothesized that the phosphorylation of SnRK2 protein kinase caused by the ABA signal promotes the binding of ABRE (ABA response elements) to ABFs (downstream targets) [43], thus regulating the increased gene expression and contributing to the protein products conducive to plant resistance to stress.

Discussion
The MADS family is one of the largest plant transcription factor families, playing a critical role in plant growth and development and in the regulation of plant responses to both abiotic and biotic stresses [44,45]. Although this family has been widely researched for many plants, including Arabidopsis [4,7], rice [34], maize [46,47], Erycina pusilla [36], and Phalaenopsis [37], the identification, expression patterns, and functions of MADSs based on the whole-genome sequence of B. striata have remained poorly understood. Therefore, we conducted a comprehensive and systematic analysis of the MADS-box gene family in B. striata.
For our study, a total of 45 MADS-box genes were identified in B. striata, which was lower than that of both Arabidopsis (146 genes) [19] and rice (75 genes) [1], implying that the MADS family had diminished over the course of evolution. The number of genes varied for different species, which may have been caused by gene duplication (e.g., tandem and segmental duplication) [48,49]; however, members of the RsMADS may not have undergone such a process [50]. It was also revealed that gene function was a major determinant of gene family size, thus further suggesting that natural stressors were the major driving evolutionary forces [51].
To further explore the restrictive conditions during the evolution of the BsMADS family, the Ka/Ks ratios of the 28 paralogous BsMADS gene pairs were computed. The smaller the value of Ka/Ks between gene pairs, the more severe the selective constraint they evolved. The Ka/Ks ratios of most duplicated BsMADS gene pairs were less than 1, which indicated that the purification selection pressure played a dominant role in the evolutionary process of this family (Supplementary Table S4). The results revealed that the MADS-box gene family was currently being purified under B. striata selection.
According to the phylogenetic tree between B. striata and Arabidopsis, the BsMADS genes were divided into five subgroups, which also provided a reliable reference for the prediction of functional target BsMADS genes (Figure 3). Some BsMADSs were clustered in the same branch as the known AtMADSs, suggesting the conservation of MADS protein functions between the two species. For example, AGL6 (AGAMOUS-LIKE6, AT2G456501) is involved in the development of bractless flowers in Arabidopsis [7], while the expression level of AGL12 contributes to the regulation and development of root structures (AGAMOUS-LIKE12, AT1G716921) [52], both of which belong to group E (MIKC).
Consequently, BsMADSs in the MIKC subgroups might be regarded as prime candidate genes for the development of plant organs. The results of the sequence alignments revealed characteristic amino acids (e.g., the majority of the BsMADSs contained one critical amino acid residue, which was the 25th glutamic acid (E)). The secondary structural elements of the SRF-TF domain were found to contain two α-helix and three β-sheets (Figure S1). SRF had been reported to activate transcription by interacting with the B-box [53].
Structural analysis determined that 26.7% of the BsMADSs contained just one exon ( Figure 4). The close BsMADSs had similar structures and conserved motif complements, which also confirmed the impregnable evolutionary conservation. Conserved motifs might serve as potential DNA binding sites to regulate the expression of specific genes [54,55]. The transcriptional activities and specificity of DNA combinations depended on unique structures and motifs. Therefore, based on the phylogenetic tree (Figure 3), motif composition analysis (Figure 4b), and gene structures (Figure 4c), we speculated that the BsMADSs with the same subgroups might have similar functions, which were highly conserved.
The results of the cis-elements analysis suggested the responses of the BsMADS genes to various biotic and abiotic stressors. There have been many studies of plant MADS transcription factors involved in plant growth and development. For example, phytohormone abscisic acid (ABA) was observed to inhibit the expression of FAMADS1a in strawberries and promote fruit ripening [56]. TaMADS2 isolated from wheat was useful for the treatment of wheat-stripe rust interactions [57]. The yields of transgenic rice that overexpressed JCMADS40 were reduced [58]. CpMADS3 in cyclamen had a novel function in flowering [59]. Furthermore, MADS genes participate in multiple abiotic stress responses [60][61][62].
This suggested that BsMADS genes were likely to be involved in these physiological processes. To further investigate the functionality of MADS-box genes, we analyzed the expression levels of eight randomly selected BsMADSs under different treatments (Figures 8 and 9). BsMADS07, BsMADS10, and BsMADS11 had no significant differences following the MeJA treatment, while the expression of the BsMADS33 gene significantly increased, suggesting that it may regulate the transcription of related genes. Under Cu 2+ and Ag + stress conditions, both BsMADS33 and BsMADS10 were upregulated and belonged to group D (Mδ).
Therefore, Mδ MADS genes in B. striata may play critical roles in responding to abiotic stresses. According to the phylogeny analysis, the selected BsMADS genes exhibited similar stress-responsive expression patterns to model species within the same clade or subgroup. Among them, BsMADS33 was upregulated under all treatments; thus, we predicted that it could be employed as a candidate gene for later functional research. In brief, the results of our study might provide a credible reference for further research on the functions of type I MADS genes in B. striata.

Plant Material and Treatment
In order to perform a genome-wide analysis of B. striata, we planted germplasm resources at the greenhouse in the National Engineering Laboratory for Resource Development of Endangered Chinese Crude Drugs, Northwest China. Seeds (2n = 2x = 32) were collected and sprouted on a seedling bed at 25 • C under natural lighting (16 h light/8 h dark) at 60-80% humidity. Two-month-old seedlings were employed for stress-related expression profile analysis. For the abiotic stress treatments, the seedlings were treated with NaCl (200 mM), AgNO 3 (200 mM), and CuSO 4 (200 mM) solutions, respectively. The above aseptic seedlings were subjected differently in an MS liquid medium, which contained methyl jasmonate (MeJA, 200 µM), abscisic acid (100 µM), and salicylic acid (10 mM) for the hormonal treatment. The stress samples were collected at 0, 1, 3, 6, and 12 h with three biological replicates. Each sample was instantly frozen in liquid nitrogen and stored at −80 • C for further RNA isolation.

Identification of MADS-Box Gene in B. striata
A total of 146 previously identified Arabidopsis MADS-domain protein sequences were used to query the B. striata database. Further, a BLASTP search of the B. striata database was performed using the conserved MADS-domain HMM profile (PF00319) obtained from the Pfam website (Pfam 32.0, http://pfam.xfam.org/, accessed on 9 October 2021) with an e-value (expected value) cut-off set to 0.01. All potential proteins were confirmed through the SMART service (http://smart.embl-heidelberg.de/, accessed on 9 October 2021). The relative molecular mass and theoretical isoelectric point of all confirmed Bs-MADS proteins were predicted by ExPASy ProtParam tool (http://expasy.org/, accessed on 9 October 2021).

Multiple Alignment and Phylogenetic Analysis
For multiple alignment analysis, the InterPro program (http://www.ebi.ac.uk/interpro/, accessed on 9 October 2021) was used to acquire the core sequence of the SRF-TF domain, which was further analyzed via the DNAMAN and SMART software programs. The Weblogo (http://weblog.berkeley.edu/logo.cgi, accessed on 9 October 2021) online program was employed to reveal the characteristics of the domain.
MADS protein sequences from Arabidopsis were downloaded from the PlantTFDB (http://planttfdb.gao-lab.org/, accessed on 9 October 2021). The ClustalX (1.83) program was utilized to perform the alignment of multiple amino acid sequences. The neighborjoining (NJ) method was employed to construct a phylogenetic tree using MEGA v 7.0 with 1000 bootstraps and default parameters of ClusterW.

Prediction of Conserved Motifs and Gene Structure Analysis
The online MEME (http://meme-suite.org/tools/meme, accessed on 9 October 2021) program was utilized to search and detect the protein motifs of 45 BsMADSs with expected e-values of less than 2 × 10 −30 [63]. The following default parameter setting changed the number of motifs into eight. Next, the result of the XML file obtained from MEME was displayed using TBtools v 0.58 [64]. The exon-intron structures of the BsMADSs were displayed using GSDS (http://gsds.cbi.pku.edu.cn, accessed on 9 October 2021) [65].

Cis-Elements and Ka/Ks Analysis
The 2500 bp promoter sequences located upstream of the gene start codon were obtained from the whole-genome sequences of B. striata using BioEdit software. The potential cis-elements of BsMADSs were searched in the PlantCARE database [66]. With the purpose of examining whether positive selection existed in the evolution of BsMADS genes, the synonymous substitution rate (Ks) and non-synonymous substitution rate (Ka) values of homologous gene pairs, with their amino acid sequences, were calculated by the online software Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 9 October 2021) and PAL2NAL (http://www.bork.embl.de/pal2nal/, accessed on 9 October 2021) [67].

RT-qPCR Analysis
Four different tissue (root, stem, leaf, and flower) samples of two-year-old B. striata were individually collected during its flowering development stage. These samples were used to conduct RT-qPCR to detect the tissue-specific BsMADSs expression patterns. The PCR conditions were set at 95 • C for 30 s, 95 • C for 5 s, and 60 • C for 30 s with 45 cycles. Each reaction had three biological and technical replicates, which used 30-fold diluted cDNA as a template. The 2 − CT method was employed to calculate the corresponding expression values of the BsMADSs. According to our laboratory studies, we selected BsGAPDH as the internal reference gene in this work, with the primer sequences listed in Supplementary Table S5.

Conclusions
In summary, we identified 45 BsMADS genes in B. striata, and the phylogenetic relationships, gene structures, conserved motifs, cis-acting elements, and expression profiles of this family were comprehensively analyzed for the first time. This revealed that BsMADS had a significant role in response to ABA, SA, MeJA, NaCl, CuSO 4 , and AgNO 3 stressors. Based on the analysis of the phylogenetic tree and expression patterns, we predicted and verified the potential functions of the BsMADSs in our study, which provided a credible reference for further exploring the regulatory mechanisms and stress resistance in the development of B. striata.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/plants10102184/s1: Table S1, List of the 45 BsMADS genes identified in this study; Table S2, Physicochemical property; Table S3, The promoter cis-elements analysis of the MADS genes in B. striata; Table S4, Ka/Ks analysis for the duplicated BsMADS paralogs.; Table S5, Analysis and distribution of conserved motifs of MADS proteins in B. striata; Table S6, Primers of sequences; and Figure S1. SRF-TF.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest:
All authors declare that there are no conflict of interest regarding the publication of this paper.