Genome-Wide Identification of the YABBY Gene Family in Dendrobium Orchids and Its Expression Patterns in Dendrobium chrysotoxum

The small plant-specific YABBY gene family plays key roles in diverse developmental processes in plants. Dendrobium chrysotoxum, D. huoshanense, and D. nobile are perennial herbaceous plants belonging to Orchidaceae with a high ornamental value. However, the relationships and specific functions of the YABBY genes in the Dendrobium species remain unknown. In this study, six DchYABBYs, nine DhuYABBYs, and nine DnoYABBYs were identified from the genome databases of the three Dendrobium species, which were unevenly distributed on five, eight, and nine chromosomes, respectively. The 24 YABBY genes were classified into four subfamilies (CRC/DL, INO, YAB2, and FIL/YAB3) based on their phylogenetic analysis. A sequence analysis showed that most of the YABBY proteins contained conserved C2C2 zinc-finger and YABBY domains, while a gene structure analysis revealed that 46% of the total YABBY genes contained seven exons and six introns. All the YABBY genes harbored a large number of Methyl Jasmonate responsive elements, as well as anaerobic induction cis-acting elements in the promoter regions. Through a collinearity analysis, one, two, and two segmental duplicated gene pairs were identified in the D. chrysotoxum, D. huoshanense, and D. nobile genomes, respectively. The Ka/Ks values of these five gene pairs were lower than 0.5, indicating that the Dendrobium YABBY genes underwent negative selection. In addition, an expression analysis revealed that DchYABBY2 plays a role in ovary and early-stage petal development, while DchYABBY5 is essential for lip development and DchYABBY6 is crucial for early sepal formation. DchYABBY1 primarily regulates sepals during blooming. Furthermore, there is the potential involvement of DchYABBY2 and DchYABBY5 in gynostemium development. The results of a comprehensive genome-wide study would provide significant clues for future functional investigations and pattern analyses of YABBY genes in different flower parts during flower development in the Dendrobium species.

YABBY genes have been well identified and analyzed in the model plant Arabidopsis thaliana. AtFIL/AtYAB3, AtYAB2, and AtYAB5 are highly expressed in vegetative tissues, whereas AtCRC and AtINO are restricted to the reproductive organs, and are thus called floral-specific YABBY genes [12][13][14]. AtFIL is necessary for floral meristem identity establishment and flower development [15]. AtCRC is involved in the development of nectaries [8] and the apical end of the carpel [11,16], while AtINO expresses in the outer integument of ovules [17]. Isolated on the basis of homology to AtCRC, three YABBY genes (AtFIL, AtYAB2, and AtYAB3) play important roles in the specification of abaxial cell fate in lateral organs [18]. Furthermore, YABBY genes have been found to be involved in the developmental processes of many core eudicots. SlCRCa, a CRC homolog from the tomato (Solanum lycopersicum), has high expression in the petals and stamens [19], while VvYAB1, 2, 3, and 5 have high expression levels in the vegetative organs of grapes (Vitis vinifera) [20]. As for monocots, members of the YABBY family have been well reported in rice (Oryza sativa). Molecular cloning has revealed that OsDL (DROOPING LEAF), closely related to AtCRC, is a member of the YABBY gene family, which regulates midrib formation by promoting cell proliferation in the central region of the rice leaf [21]. OsYABBY1 is involved in regulating the differentiation of a few specific cell types, rather than the polar regulation of lateral organ development [10]. OsYABBY4, belonging to the FIL/YAB3 subfamily, expresses in the meristems and developing vascular tissue of rice, predominantly in the phloem tissues [9].
Orchidaceae is one of the largest and most widespread families in the flowering plant kingdom, with more than 28,000 species [22]. As the second-largest genus of Orchidaceae, Dendrobium has been extensively studied for its medicinal properties and ornamental value, and has deeply fascinated botanists and plant enthusiasts over the centuries [23]. Although YABBY genes have been widely reported in model plants and main crops, nothing is known about the relationship and specific functions of the YABBY genes in the Dendrobium species during flower development. In recent years, some species of Dendrobium, including D. chrysotoxum [24], D. huoshanense [25], and D. nobile [26], have been sequenced. A highquality, chromosomal-level assembly of the whole genome of these Dendrobium species would provide a valuable database resource, facilitating the systematic exploration of the YABBY gene family in orchids.
In this study, 24 Dendrobium YABBY genes were identified using a bioinformatics analysis method, including gene structure, motif composition, chromosomal localization, phylogenetic tree analysis, and segmental duplication analysis. The expression patterns of D. chrysotoxum were analyzed. The results provide useful information on the biological function of YABBY genes in Dendrobium and the molecular mechanisms underlying floral morphogenesis in D. chrysotoxum.

Screening of DchYABBY, DhuYABBY, and DnoYABBY Transcription Factors
Using six Arabidopsis YABBY protein sequences as the queries, 26 YABBY genes were initially identified by BLAST searches. These candidate genes were uploaded to Simple HMM search, NCBI, and SMART for further confirmation. Finally, six DchYABBYs, nine DhuYABBYs, and nine DnoYABBYs were obtained as YABBY family members. According to the order distribution on the chromosomes, a total of 24 YABBY genes were named DchYABBY1-6, DhuYABBY1-9, and DnoYABBY1-9. In addition, an encoded protein sequence analysis indicated that the physicochemical properties, including aa, pI, MW, GRAVY, II, and AI, of the YABBY genes in Dendrobium were considerably different. The shortest was DhuYABBY6 with 95 aa and the longest were DhuYABBY5 and DnoY-ABBY2 with 233 aa. The pI values of the 24 YABBY genes in Dendrobium ranged from 5.74 (DnoYABBY3) to 10.00 (DhuYABBY6). Among them, eight YABBY proteins had an acidic pI below seven, while sixteen YABBY proteins with a pI higher than seven were alkaline. The MW values ranged from 10,677. 48

Phylogeny and Classification of YABBY Genes
To analyze the deeper relationships and roles of YABBY family members in Dendrobium, we used 56 YABBY protein sequences from D. chrysotoxum, D. huoshanense, D. nobile, A. thaliana, O. sativa subsp. Indica, A. shenzhenica, D. catenatum, and P. equestris to construct a neighbor-joining (NJ) phylogenetic tree ( Figure 1). Based on the classification of AtYABBYs, the 24 YABBY genes in Dendrobium were divided into four subfamilies, named CRC/DL, INO, YAB2, and FIL/YAB3. The CRC/DL subfamily had the largest number of members (a total of eight members, three DnoYABBYs, three DhuYABBYs, and two DchYABBYs), while the INO subfamily harbored the least members (three members, DnoYABBY1, DhuYABBY7, and DchYABBY1). Seven Dendrobium YABBY proteins were exhibited in the YAB2 subfamily, followed by the FIL/YAB3 subfamily (six members).
GRAVY, II, and AI, of the YABBY genes in Dendrobium were considerably different. The shortest was DhuYABBY6 with 95 aa and the longest were DhuYABBY5 and DnoYABBY2 with 233 aa. The pI values of the 24 YABBY genes in Dendrobium ranged from 5.74 (DnoY-ABBY3) to 10.00 (DhuYABBY6). Among them, eight YABBY proteins had an acidic pI below seven, while sixteen YABBY proteins with a pI higher than seven were alkaline. The MW values ranged from 10,677. 48

Phylogeny and Classification of YABBY Genes
To analyze the deeper relationships and roles of YABBY family members in Dendrobium, we used 56 YABBY protein sequences from D. chrysotoxum, D. huoshanense, D. nobile, A. thaliana, O. sativa subsp. Indica, A. shenzhenica, D. catenatum, and P. equestris to construct a neighbor-joining (NJ) phylogenetic tree ( Figure 1). Based on the classification of AtYABBYs, the 24 YABBY genes in Dendrobium were divided into four subfamilies, named CRC/DL, INO, YAB2, and FIL/YAB3. The CRC/DL subfamily had the largest number of members (a total of eight members, three DnoYABBYs, three DhuYABBYs, and two DchYABBYs), while the INO subfamily harbored the least members (three members, DnoYABBY1, DhuYABBY7, and DchYABBY1). Seven Dendrobium YABBY proteins were exhibited in the YAB2 subfamily, followed by the FIL/YAB3 subfamily (six members).

Gene Structure and Motif Analysis of YABBY Genes
To observe the gene structure of the YABBY genes in Dendrobium, ten conserved motifs were predicted through the MEME program, and the exon-intron structures were exhibited using Tbtools ( Figure 2). The results show that most of the YABBY genes had four conserved motifs in the order of motifs 8, 2, 3 and 1. In addition, YABBY family members in the same subfamily had similar conserved motifs, and had certain specific motifs which were not found in the other subfamilies. Motif 9 was observed only in FIL/YAB3 and motif 6 was peculiar to YAB2. The INO subfamily possessed motif 10 exclusively and motif 7 only existed in the CRC/DL clade. Interestingly, motif 5 was present in two clades (YAB2 and CRC/DL), while motif 4 appeared in all clades except the YAB2 clade. Further, almost all the YABBY genes harbored at least four conserved motifs, while DchYABBY4 and DhuYABBY9 contained three conserved motifs, and DchYABBY5 and DhuYABBY6 had only two motifs.
was constructed with the neighbor-joining (NJ) method in MEGA 7.0 software and was divided into four subfamilies according to the classification of AtYABBYs.

Gene Structure and Motif Analysis of YABBY Genes
To observe the gene structure of the YABBY genes in Dendrobium, ten conserved motifs were predicted through the MEME program, and the exon-intron structures were exhibited using Tbtools ( Figure 2). The results show that most of the YABBY genes had four conserved motifs in the order of motifs 8, 2, 3 and 1. In addition, YABBY family members in the same subfamily had similar conserved motifs, and had certain specific motifs which were not found in the other subfamilies. Motif 9 was observed only in FIL/YAB3 and motif 6 was peculiar to YAB2. The INO subfamily possessed motif 10 exclusively and motif 7 only existed in the CRC/DL clade. Interestingly, motif 5 was present in two clades (YAB2 and CRC/DL), while motif 4 appeared in all clades except the YAB2 clade. Further, almost all the YABBY genes harbored at least four conserved motifs, while DchYABBY4 and Dhu-YABBY9 contained three conserved motifs, and DchYABBY5 and DhuYABBY6 had only two motifs. Subsequently, all 24 YABBY genes possessed introns ranging from one to six. Among them, DnoYABBY8 had the longest intron, followed by DhuYABBY3. In general, 46% of the total YABBY genes (11 members) contained seven exons and six introns, while 33% (eight genes) had six exons and five introns.
Sequence logos of the C2C2 zinc-finger and YABBY domains in the three Dendrobium species were generated by multiple sequence alignments. As shown in Figure 3, these two domains were highly conserved. The C2C2 zinc-finger domain ( Figure 3A) had serval significantly conserved amino acid residues, including cysteine residues (C), glycine residues (G), histidine residues (H), and valine residues (V). In the YABBY domain ( Figure 3B), most of the amino acid residues were extremely conserved. Subsequently, all 24 YABBY genes possessed introns ranging from one to six. Among them, DnoYABBY8 had the longest intron, followed by DhuYABBY3. In general, 46% of the total YABBY genes (11 members) contained seven exons and six introns, while 33% (eight genes) had six exons and five introns.
Sequence logos of the C2C2 zinc-finger and YABBY domains in the three Dendrobium species were generated by multiple sequence alignments. As shown in Figure 3, these two domains were highly conserved. The C2C2 zinc-finger domain ( Figure 3A) had serval significantly conserved amino acid residues, including cysteine residues (C), glycine residues (G), histidine residues (H), and valine residues (V). In the YABBY domain ( Figure 3B), most of the amino acid residues were extremely conserved.

Promoter Analysis of YABBY Genes
A large number of cis-acting regulatory elements in the promoters of DchYABBYs, DhuYABBYs, and DnoYABBYs were obtained. The most frequently occurring responsive elements in the upstream sequence of D. chrysotoxum included Methyl Jasmonate (MeJA)responsive elements (8 times), anaerobic induction (ARE)-responsive elements (8 times), and abscisic acid-responsive elements (7 times) ( Figure 4A). However, a low temperatureresponsive element only appeared once in DchYABBY2. It is noteworthy that all four seed-specific regulation elements appeared in the 400-600 bp upstream CDS of DchYABBY1. In Figure 4B, similar to the cis-acting regulatory elements in D. chrysotoxum, both the MeJAresponsive elements and ARE elements were observed most frequently. DhuYABBY8 had the largest number of cis-acting regulatory elements (14), followed by DhuYABBY4 (12). DhuYABBY2 contained the least number with only four elements. A wound-responsive element was solely present in DhuYABBY1. The largest number of elements were found in DnoYABBY5 (fourteen elements), while DnoYABBY8 had the fewest (six elements) ( Figure 4C). Interestingly, low temperature-responsive elements mainly appeared in the 0-500 bp upstream CDS of DnoYABBY2. Generally, all three Dendrobium species had AREs, MeJA-responsive elements, gibberellin-responsive elements, abscisic acid-responsive ele-ments, meristem expression, zein metabolism regulation, and low temperature-responsive elements, suggesting the wide functional variability of the YABBY genes in Dendrobium.

Promoter Analysis of YABBY Genes
A large number of cis-acting regulatory elements in the promoters of DchYABBYs, DhuYABBYs, and DnoYABBYs were obtained. The most frequently occurring responsive elements in the upstream sequence of D. chrysotoxum included Methyl Jasmonate (MeJA)responsive elements (8 times), anaerobic induction (ARE)-responsive elements (8 times), and abscisic acid-responsive elements (7 times) ( Figure 4A). However, a low temperatureresponsive element only appeared once in DchYABBY2. It is noteworthy that all four seedspecific regulation elements appeared in the 400-600 bp upstream CDS of DchYABBY1. In Figure 4B, similar to the cis-acting regulatory elements in D. chrysotoxum, both the MeJAresponsive elements and ARE elements were observed most frequently. DhuYABBY8 had the largest number of cis-acting regulatory elements (14), followed by DhuYABBY4 (12). DhuYABBY2 contained the least number with only four elements. A wound-responsive element was solely present in DhuYABBY1. The largest number of elements were found in DnoYABBY5 (fourteen elements), while DnoYABBY8 had the fewest (six elements) (Figure 4C). Interestingly, low temperature-responsive elements mainly appeared in the 0-500 bp upstream CDS of DnoYABBY2. Generally, all three Dendrobium species had AREs, MeJA-responsive elements, gibberellin-responsive elements, abscisic acid-responsive elements, meristem expression, zein metabolism regulation, and low temperature-responsive elements, suggesting the wide functional variability of the YABBY genes in Dendrobium.

Collinearity Analysis and Ka/Ks Value of YABBY Genes
The D. chrysotoxum genome harbored one pair of segment duplicated genes, which were DchYABBY2 on Chr04 and DchYABBY3 on Chr05 ( Figure 6A). Two segmental duplicated gene pairs were identified in the D. huoshanense genome, which were DhuYABBY1 on Chr2 and DhuYABBY4 on Chr9, and DhuYABBY2f on Chr6 and DhuYABBY8 on Chr15 ( Figure 6B). Similarly, the D. nobile genome also contained two pairs of segmental duplicated genes ( Figure 6C), DnoYABBY2 and DnoYABBY3, and DnoYABBY4 and DnoYABBY6, which showed similar conserved motifs and gene arrangements.
The Ka/Ks values of all five gene pairs were lower than 0.5, ranging between 0.1 and 0.46. The average Ka/Ks value of D. huoshanense was 0.24, lower than that of D. nobile (0.27) (Supplement Table S3

Chromosomal Localization of YABBY Genes
As shown in Figure 5A, six DchYABBYs were distributed on five chromosomes of D. chrysotoxum (Chr02, 04, 05, 06, and 17). Chromosome 06 had two genes (DchYABBY4 and DchYABBY5), while the other chromosomes each contained one gene. The chromosome mapping results for D. huoshanense indicated that eight DhuYABBYs were unevenly distributed on Chromosomes 2, 6, 7, 9, 10, 11, 12, and 15 ( Figure 5B). DhuYABBY9 was  localized to the unanchored scaffold, named Scaffold2022. Nine DnoYABBYs were present on nine chromosomes. Only DnoYABBY5 was localized on the top of CM039729.1 ( Figure  5C). were DchYABBY2 on Chr04 and DchYABBY3 on Chr05 ( Figure 6A). Two segmental duplicated gene pairs were identified in the D. huoshanense genome, which were DhuYABBY1 on Chr2 and DhuYABBY4 on Chr9, and DhuYABBY2f on Chr6 and DhuYABBY8 on Chr15 ( Figure 6B). Similarly, the D. nobile genome also contained two pairs of segmental duplicated genes ( Figure 6C), DnoYABBY2 and DnoYABBY3, and DnoYABBY4 and DnoYABBY6, which showed similar conserved motifs and gene arrangements.  Table S3).

Expression Patterns of YABBY Genes in D. chrysotoxum
According to the FPKM values, the expression levels of six DchYABBYs in different parts and developmental periods (Figure 7) suggest the higher expression of DchYABBY2, 3, 5, and 6 in the S1 stage than in the other two stages. DchYABBY1 showed high expression in the ovary in the S2 stage, and DchYABBY4 was only expressed in the lip in the S3 stage. DchYABBY2 and DchYABBY3, both belonging to the CRC/DL subfamily, exhibited similar expression patterns throughout flower development. DchYABBY5 was highly expressed in the lip and gynostemium in the S1 stage, while DchYABBY6 showed high expression in the ovary, sepal, and petal in the S1 stage.

Expression Patterns of YABBY Genes in D. chrysotoxum
According to the FPKM values, the expression levels of six DchYABBYs in different parts and developmental periods (Figure 7) suggest the higher expression of DchYABBY2, 3, 5, and 6 in the S1 stage than in the other two stages. DchYABBY1 showed high expression in the ovary in the S2 stage, and DchYABBY4 was only expressed in the lip in the S3 stage. DchYABBY2 and DchYABBY3, both belonging to the CRC/DL subfamily, exhibited similar expression patterns throughout flower development. DchYABBY5 was highly expressed in the lip and gynostemium in the S1 stage, while DchYABBY6 showed high expression in the ovary, sepal, and petal in the S1 stage.

The qRT-PCR Analysis of YABBY Genes in D. chrysotoxum
To investigate the expression patterns of the DchYABBY genes from the different subfamilies in five flower parts during flower development, DchYABBY1 (INO), DchYABBY2 (CRC/DL), DchYABBY5 (FIL/YAB3), and DchYABBY6 (YAB2) were selected for qRT-PCR analysis (Figure 8). The results show that the four genes were involved in ovary growth during the S1 stage, with DchYABBY2 showing a significantly higher expression compared to the other three genes. Moreover, DchYABBY2 exhibited sustained expression in the ovary during the subsequent two stages, highlighting its vital role in governing ovary  (CRC/DL), DchYABBY5 (FIL/YAB3), and DchYABBY6 (YAB2) were selected for qRT-PCR analysis (Figure 8). The results show that the four genes were involved in ovary growth during the S1 stage, with DchYABBY2 showing a significantly higher expression compared to the other three genes. Moreover, DchYABBY2 exhibited sustained expression in the ovary during the subsequent two stages, highlighting its vital role in governing ovary development. The highest expression of DchYABBY5 was found to be in the lip during the S1 stage, followed by a decrease in the S2 stage, and then an increase in the S3 stage. DchYABBY2, 5 exhibited continuous expression throughout the development of the gynostemium. During the S1 stage, DchYABBY5,6 exhibited predominant expression in the sepal, and DchYABBY2 displayed high expression in the petal. DchYABBY1 showed significant expression in the petal in the S3 stage.  Table S5.
In this study, the phylogenetic tree was constructed using six orchid species (four  Supplementary  Table S5.
In this study, the phylogenetic tree was constructed using six orchid species (four Dendrobium species, A. shenzenica, and P. equestris) and two model plants (A. thaliana and O. sativa). The results show a total of 43 orchid YABBY genes are classified into four subfamilies (CRC/DL, INO, YAB2, and FIL/YAB3), and no orchid YABBY gene was found in the YAB5 subfamily. For further confirmation, we combined the classification of other orchid YABBY genes, including V. shenzhenica, V. pompona, C. ensifolium, C.goeringii, and C.sinense [27,28], and found that none of these YABBY genes belonged to the YAB5 clade. Moreover, the results are consistent with some other monocot species. In Phyllostachys edulis, PeYABBYs were classified into four subfamilies (except YAB5) [31], while the AcYABBYs from pineapple were divided to three subfamilies-FIL/YAB3, CRC, and YAB2 [32]. Based on a phylogenetic analysis, all 20 TaYABBYs from wheat were classified into four clades: FIL, YAB2, INO, and CRC [33]. Due to the low homology of YAB2 and YAB5 in the Zingiberales, De Almeida believed that YAB2 and YAB5 were separated after monocots and eudicots were differentiated [34]. Further, a previous discovery held that the YAB5 gene was lost in monocot plants, and only occurred in basal angiosperms and eudicot. On the basis of these results, we believe that monocot plants might lack the YAB5 clade. It is noteworthy that each of the three Dendrobium species contained only one member in the INO subfamily, which is consistent with other orchids [27]. Thus, the INO subfamily is considered to be highly conserved in Orchidaceae.
The gene structure results show that each subfamily contains a unique conserved motif. Motifs 9, 6, 10, and 7 were specific to FIL/YAB3, YAB2, INO, and CRC/DL, respectively, implying that each subfamily might have certain special functions which set them apart from other subfamilies. The number of introns and exons in the same clade were similar. Almost all the YABBY members in the YAB2 clade had six exons and five introns, while all the other three subgroups had seven exons and six introns. In addition, FIL/YAB3-like, INOlike, and CRC/DL-like genes exhibited a higher number of exons and introns compared to YAB2-like genes, which revealed that YABBY genes from the former three clades were more conserved than those in the YAB2 clade. As shown in Figure 3, it is obvious that the YABBY domain is more conserved than the C2C2 domain in the three Dendrobium species. Overall, these findings strongly suggest that YABBY genes are relatively conserved during evolution.
Gene duplication has contributed significantly to the novelty and diversification of plants and has emerged as strong force for gene family expansion [35][36][37]. D. chrysotoxum has experienced two whole-genome duplication (WGD) events [24] and at least two WGD events have occurred in D. huoshanense and D. nobile since ancient times [26,38]. These events have resulted in a big difference in the distribution of YABBY genes on chromosomes and have led to differences in the number of YABBY genes among the three Dendrobium species. There were one, two, and two pairs of segmentally duplicated YABBY genes in D. chrysotoxum, D. huoshanense, and D. nobile, respectively, and one pair of tandem duplicated genes on Chr06 in D. chrysotoxum, suggesting that gene duplication is instrumental in the YABBY gene family in Dendrobium. In order to gain a deeper understanding of the evolutionary dynamics within the Dendrobium species, we calculated Ka (nonsynonymous substitution), Ks (synonymous substitution), and Ka/Ks (evolutionary selection pressure) values. The result of the Ka/Ks value revealed that five pairs of YABBY genes were lower than one and had experienced purifying selection.
The prediction of cis-acting elements at the transcriptional level is conductive to regulating gene expression [39]. In this study, we found that in the promoter regions of Dendrobium YABBY genes, MeJA-responsive elements (46/241) occurred most frequently, followed by ARE elements (39/241). As an important cellular regulator, Methyl jasmonate (MeJA) can alleviate environmental stresses, such as salt stress, drought, and low tempera-ture, during plant developmental processes [40,41]. Anaerobiosis in plants plays a vital role in overcoming the oxygen deficits caused by flooding [42]. Thus, we suggest that YABBY genes in Dendrobium may be involved in defense responses against variable environments.
Combining the results of the FPKM values and qRT-PCR analysis, we found that DchYABBYs were strongly expressed in the unpigmented bud stage, which was basically consistent with the expression levels of YABBY genes of D. officinale [43]. Furthermore, DchYABBY2 from the CRC subfamily exhibited remarkably high expression levels in the ovary across all three stages. Its expression surpassed all the rest of the genes expressing during the same developmental stage, indicating the crucial role of DchYABBY2 in the formation and development of the ovary. This finding aligns with the established understanding that CRC serves as a vital regulatory factor in carpel development in Arabidopsis [44]. Previous studies have provided evidence that FIL plays a crucial role in regulating anthocyanin synthesis in Arabidopsis [45]. Moreover, the lip of D. chrysotoxum exhibited the accumulation of purple pigments [46], which correlates with the elevated expression of DchYABBY5 in the lip, and is a member of FIL subfamily. Interestingly, the expression pattern of DchYABBY5 showed a distinctive "high-low-high" pattern. We believe that DchYABBY5 might participate in lip development during the non-pigmented bud stage and undergo a functional transition to contribute to the formation of purple spots during the pigmented bud stage and fully opened flower stage. According to the floral mRNA expression patterns of YAB2 in Arabidopsis, the expression of the YAB2 family was found in sepal, petal, and carpel primordia, which is consistent with our results for DchYABBY6. During the unpigmented bud stage, DchYABBY6 participated in sepal formation and DchYABBY2 had a significant regulatory role in petal development. However, DchYABBY1 from the INO subfamily emerged as the dominant factor in regulating petal development during the fully opened flower period. Therefore, we believe that the petal is successively regulated by DchYABBY2, 1 during flower development. DchYABBY2 and DchYABBY5 exhibited sustained expression during gynostemium development, indicating their potential involvement in this process.

Identification and Physicochemical Properties of the YABBY Gene Family
To identify potential YABBY genes in Dendrobium, six Arabidopsis YABBY genes were used as probes in a BLAST search in TBtools v1.120 software [47]. The Hidden Markov Model (HMM) profile of the YABBY conserved domain (PF04690) from InterPro (https://www.ebi.ac.uk/interpro/, accessed on 23 November 2022) was used to further identify YABBY genes in Dendrobium using the Simple HMM Search in Tbtools. All the potential YABBY genes were confirmed with the NCBI CD-Search (https://www.ncbi. nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 24 November 2022) and SMART program (https://smart.embl.de/, accessed on 24 November 2022). Incomplete and redundant protein sequences were removed manually. Protein analyses of amino acid (aa), isoelectric point (pI), molecular weight (MW), hydrophilic large average (GRAVY), instability index (II), and fat index (AI) were performed through the online software ExPASy 3.0 (https://www.expasy.org/, accessed on 28 November 2022) [48].

Phylogenetic Analysis of YABBY Genes
The amino acid sequences of six YABBY proteins of D. chrysotoxum, nine YABBY proteins of D. huoshanense, nine YABBY proteins of D. nobile, six YABBY proteins of A. thaliana, seven YABBY proteins of O. sativa, four YABBY proteins of A. shenzhenica, seven YABBY proteins of D. catenatum, and eight YABBY proteins of P. equestris were introduced into the MEGA 7.0 software. The total 56 sequences were aligned using ClustalW with the default parameters. To construct a neighbor-joining (NJ) phylogenetic tree of the YABBYs, the bootstrap method was performed with 500 replicates and the partial deletion was set to 50%. For better visualization, the NJ phylogenetic tree was processed using the online software Evolview 3.0 (http://www.evolgenius.info/evolview/#/treeview, accessed on 12 December 2022).

Gene Structure and Conserved Motif Analysis of YABBY Genes
The conserved domains of the YABBY genes were predicted using NCBI's conserved domain database CDD (https://www.ncbi.nlm.nih.gov/cdd, accessed on 20 November 2022). Moreover, Multiple Em for Motif Elicitation (MEME, https://meme-suite.org/ meme/tools/meme, accessed on 20 November 2022) was used to analyze the conserved motifs of the YABBY genes in Dendrobium. Except for the maximum number of motifs, which was set at 10, the other parameters of MEME were kept at the defaults. The overall comparative maps of the NJ phylogenetic tree, conserved protein motifs, and gene structure were integrated through Tbtools.

Analysis of YABBY Genes Promoter Sequences
Tbtools was used to obtain a 2000 bp sequence upstream of the YABBY genes in Dendrobium as the start codon. To predict the cis-acting elements, elements in the promoter region of the YABBY genes in Dendrobium were analyzed using PlantCARE (https:// bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 20 November 2022). Then Excel was used for data processing and Tbtools was used for visualization.

Chromosomal Localization and Synteny Analysis of YABBY Genes
According to the genome files and annotation files for D. chrysotoxum, D. huoshanense, and D. nobile, the chromosomal localizations of the YABBY genes in Dendrobium were visualized through Tbtools. Further, the genome data of the three Dendrobium species were compared to themselves for collinearity analysis using the One Step MCScanx program in Tbtools. The duplication patterns of DchYABBYs, DhuYABBYs, and DnoYABBYs were visualized using the Advance Circos in Tbtools. Meanwhile, the Ka, Ks, and Ka/Ks values were calculated using the Simple Ka/Ks Calculator in Tbtools.

Expression Analysis and RT-qPCR
The plant materials used in this study were from the National Orchid Germplasm Resources of Fujian Agriculture and Forestry University, Fuzhou, China. All the flower parts of D. chrysotoxum were sampled, frozen using liquid nitrogen, and stored in a refrigerator at −80 • C. The total RNA was extracted from the different parts (sepal, petal, lip, ovary, and gynostemium) of D. chrysotoxum in three developmental stages, including unpigmented bud (S1), pigmented bud (S2), and fully opened flower (S3), using a FastPure Plant Total RNA Isolation Kit (for polysaccharide-and polyphenol-rich tissues) (Vazyme Biotech Co, Ltd., Nanjing, China). Transcriptome sequencing and library construction were completed by Bgi Genomics Co., Ltd. (Shenzhen, China). Then, clean reads were aligned to the assembled genome using Bowtie2 2.2.9 software. Calculations of the gene expression level of each sample were performed using the software RSEMv1.2.8 to obtain the fragments per kilobase of transcript per million fragments (FPKM) values. Finally, a heatmap representing the expression levels was generated using Tbtools based on the FPKM values.
Then we employed a Reverse Transcript Kit PrimerScript ®® RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) for reverse transcription to remove the contaminated genomic DNA and generate cDNA. TB Green ®® Premix Ex Taq™ II (Tli RnaseH Plus) was used for a qRT-PCR analysis on an ABI 7500 Real-Time System. The RT-qPCR conditions were 20 s at 95 • C in the holding stage, and then 40 cycles of 3 s at 95 • C and 30 s at 60 • C in the cycling stage. The experimental setup utilized 96-well plates with a 20 µL reaction system in each well, and three biological replicates were performed in this study. Primers were designed using Primer Premier 5 software, and we selected Maker75111 as the reference gene (Supplementary Table S5). The relative expressions of the target genes were calculated using the 2 −∆∆CT method and Graphpad prism 7.0 was used for normalization.

Conclusions
A total of six DchYABBYs, nine DhuYABBYs, and nine DnoYABBYs were identified and classified into four subfamilies. The genome-wide identification, phylogeny, functional classification, gene structure, motif composition, and chromosomal localization of the YABBY genes of the three Dendrobium species were carried out. Our results illustrate the dynamic transcription patterns of DchYABBYs in different flower parts during flower development. Notably, DchYABBY2 played an indispensable role in both ovary development and early-stage petal development. DchYABBY5 was essential for lip development, while DchYABBY6 was crucial for sepal formation in the early stages. DchYABBY1 primarily regulated the blooming sepals. DchYABBY2 and DchYABBY5 were potentially involved in the development of the gynostemium. These findings provide crucial information for future studies on the functional roles and regulatory mechanisms of the YABBY genes in the Dendrobium species in different flower parts throughout the various stages of flower development.