Genome-Wide Identification of YABBY Gene Family in Cucurbitaceae and Expression Analysis in Cucumber (Cucumis sativus L.)

YABBY transcription factors play important roles in plant growth and development. However, little is known about YABBY genes in Cucurbitaceae. Here, we identified 59 YABBY genes from eight cucurbit species, including cucumber (C. sativus L.), melon (C. melon L.), watermelon (C. lanatus), wax gourd (B. hispida), pumpkin (C. maxima), zucchini (C. pepo L.), silver-seed gourd (C. argyrosperma), and bottle gourd (L. siceraria). The 59 YABBY genes were clustered into five subfamilies wherein the gene structures and motifs are conserved, suggesting similar functions within each subfamily. Different YABBY gene numbers in eight cucurbit species indicated that gene loss or duplication events exist in an evolutionary process across Cucurbitaceae. The cis-acting elements analysis implied that the YABBYs may be involved in plant development, and phytohormone, stress, and light responses. Importantly, YABBY genes exhibited organ-specific patterns in expression in cucumber. Furthermore, a gene CsaV3_6G038650 was constitutively expressed at higher levels at different fruit development stages and might play a crucial role in cucumber fruit development. Collectively, our work will provide a better understanding for further function identifications of YABBY genes in Cucurbitaceae.

Based on evolutionary relationships, the angiosperm YABBY genes can be classified into five subfamilies-YAB1, CRC, INO, YAB2, and YAB5 [10,11]. Substantial evidence has demonstrated that the YABBY genes play important roles in many aspects of plant

Phylogenetic and Gene Duplication Analysis
Multiple sequence alignment of all identified YABBYs in Cucurbitaceae was carried out using ClustalW, and a phylogenetic tree was generated by neighbor-joining (NJ) method with default parameters: bootstrap method setting to 1000, Poisson model, and complete deletion in MEGA 11. The tree was visualized and optimized via Interactive Tree Of Life (iTOL) (https://itol.embl.de/ (accessed on 7 October 2021)). To explore the gene duplication events, the collinearity analysis was performed with Multiple Collinearity Scan toolkit (MCScanX) [51].

Plant Materials
The cucumber cultivar ("Xintaimici") was grown in a greenhouse in Beijing, China. Roots and tender stems of two-week-old seedlings, the third true leaves, tender tendrils, male and female buds at 8 DBF (days before flowering), and ovaries/fruits at different growth stages were selected as samples, frozen in liquid nitrogen, and stored at −80 • C.

RNA Extraction and qRT-PCR Analysis
The sample RNA was extracted using the Quick RNA Isolation Kit (Huayueyang, Beijing, China). The FastKing gDNA Dispelling RT SuperMix (TianGen Biotech, Beijing, China) was applied to synthesize the first-strand cDNA with the extractive RNA template. qRT-PCR was performed using the UltraSYBR Mixture (Low ROX) (Cwbio, Beijing, China) on an Applied Biosystems 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The UBIQUITIN EXTENSION PROTEIN (UBI-EP) gene [56] was used as a reference gene. Three biological and three technical replicates were carried out for expression dynamics analysis. The significant differences were analyzed by Student's t-tests (p ≤ 0.05). The primers were listed in Table S6.

Transcriptome Analysis of YABBY Genes in Cucumber
For the expression patterns of CsYABBY genes at 4 DBF, ovaries from two near isogenic lines with different fruit lengths were obtained from publicly available transcriptomic data, which were downloaded from Gene Expression Omnibus (GEO) and analyzed to reveal the genes and gene networks that regulate fruit length in cucumber (GSE60346) [57]. Clean tags were remapped to the cucumber v3 genome sequence (http://cucurbitgenomics.org (accessed on 20 October 2021)) by Hisat2, and the TPM values were recalculated. Every line had two biological replicates. The p-value ≤ 0.05 and fold-change ≥ 1.5 were used to define differential expression genes. The expression pattern of the YABBY genes was shown on a heatmap using TBtools software [54].

Identification of YABBY Genes in Eight Cucurbit Species and Their Chromosomal Distribution
To identify YABBY members in Cucurbitaceae, we performed a Hidden Markov Model (HMM) search using YABBY domain (PF04690) across eight cucurbit species. Based on a further confirmation of conserved C2C2 domain and YABBY domain by CDD and SMART analysis, 59 YABBYs were finally obtained in Cucurbitaceae, including 8 from cucumber (C. sativus L.), 4 from melon (C. melon L.), 9 from watermelon (C. lanatus), 5 from wax gourd (B. hispida), 11 from pumpkin (C. maxima), 9 from zucchini (C. pepo L.), 10 from silver-seed gourd (C. argyrosperma), and 3 from bottle gourd (L. siceraria) (Tables 1 and S1 ). These gene coding sequence lengths varied from 471 to 1179 bp and the encoded protein length ranged from 156 to 392 amino acids along with the predicted protein molecular weight (MW) ranging from 17.05 to 43.64 KDs. The isoelectric point (pI) values of the YABBYs were 4.23 to 9.62 (Table S2). Next, we mapped the YABBY genes on chromosomes of seven cucurbit species (except silver-seed gourd with a lower quality genome draft), respectively. The YABBY genes of each cucurbit species were randomly distributed on their chromosomes and distribution results were shown in Figure S1. For example, the chromosome 1, 2, 3, 5, and 6-but not chromosome 4 and 7-harbored the eight YABBY genes in cucumber, the nine YABBYs were distributed on chromosome 1, 2, 5, 6, 8, 10, and 11 other than chromosome 3, 4, 7, and 9 in watermelon ( Figure S1).

Evolutionary Relationship and Synteny Analysis of YABBYs in Cucurbitaceae
To better explore the evolutionary relationship of YABBYs, six Arabidopsis YABBYs, eight rice YABBYs and 59 YABBY members in Cucurbitaceae were used to construct an evolutionary tree using MEGA 11 with ClustalW and NJ methods. As reported in Arabidopsis, the Cucurbitaceae YABBYs were also divided into five subfamilies, YAB1, YAB2, CRC, INO, and YAB5 ( Figure 1 and Table 1). The YAB1 and YAB5 subfamilies had the larger numbers of YABBYs, in which the YAB1 subfamily contained 14 members while YAB5 subfamily contained 19 members. The two subfamilies of YAB2 and CRC shared the smallest YABBYs with seven members, respectively. The four cucurbit species cucumber, watermelon, pumpkin, and silver-seed gourd YABBYs could be divided into all five subfamilies. However, the other four cucurbit species lack one or two subfamilies, such as melon lacking YAB1 subfamily, wax gourd lacking INO subfamily, zucchini lacking CRC subfamily, and bottle gourd lacking CRC and YAB2 subfamilies ( Figure 1 and Table 1).
Taken together, these results suggested that there are evolutionary splits and diversifications of YABBYs among different cucurbit species.
YAB5 subfamily contained 19 members. The two subfamilies of YAB2 and CRC shared the smallest YABBYs with seven members, respectively. The four cucurbit species cucumber, watermelon, pumpkin, and silver-seed gourd YABBYs could be divided into all five subfamilies. However, the other four cucurbit species lack one or two subfamilies, such as melon lacking YAB1 subfamily, wax gourd lacking INO subfamily, zucchini lacking CRC subfamily, and bottle gourd lacking CRC and YAB2 subfamilies ( Figure 1 and Table 1). Taken together, these results suggested that there are evolutionary splits and diversifications of YABBYs among different cucurbit species. Figure 1. Phylogenetic tree of the YABBY proteins from Arabidopsis, rice, and eight cucurbit species. Red star, red star filled with pink color, yellow star, green star filled with light purple color, black star filled with dark purple color, bluish grey star, red star filled with blue color, black star, black triangle filled with red color and black circle filled with green color represent the YABBYs from cucumber, melon, watermelon, wax gourd, pumpkin, zucchini, silver-seed gourd, bottle gourd, Arabidopsis, and rice, respectively. Cs, cucumber (C. sativus L.); MELO, melon (C. melo L.); Cla, watermelon (C. lanatus); Bhi, wax gourd (B. hispida); Cma, pumpkin (C. maxima); Cp, zucchini (C. pepo L.); Carg, silver-seed gourd (C. argyrosperma); Lsi, bottle gourd (L. siceraria); AT, Arabidopsis (Arabidopsis thaliana); Os, rice (Oryza sativa L.).
Generally, gene duplications contribute to novel gene function generation and gene family expansion [58]. Here, we performed a synteny analysis of YABBYs between cucumber and other cucurbit species (except silver-seed gourd with a lower quality genome draft) by MCScanX. The collinear gene pairs between cucumber and melon, wax gourd, watermelon, bottle gourd, pumpkin, and zucchini were 10,9,14,14,19, and 21, respectively ( Figure 2 and Table S3). The all eight CsYABBYs showed a syntenic relationship (at least two syntenic gene pairs of each CsYABBY gene) with zucchini YABBYs, 75% of CsYABBYs (except CsaV3_5G033400 and CsaV3_6G038650 possessing one syntenic gene pair, respectively) shared two, or more than two, syntenic pairs with the YABBYs in watermelon, pumpkin, and bottle gourd ( Figure 2 and Table S3), indicating that the YABBY genes in cucumber, watermelon, pumpkin, bottle gourd, and zucchini evolved from the same ancestral gene. In addition, six YABBY genes had only one syntenic gene pair between cucumber and melon, respectively. However, CsaV3_1G030340 and CsaV3_2G002960 were sisters to two melon YABBY genes, respectively. The same case was observed between cucumber and wax gourd (only CsaV3_2G002960 and CsaV3_2G024750 have two sisters in wax gourd, respectively) ( Figure 2 and Table S3), suggesting that there are no apparent gene family expansion events between cucumber and melon/wax gourd. Notably, when we identified YABBY genes, the truncated genes, lacking either a C2C2 domain and/or a YABBY domain, were excluded. This is possibly responsible for why the final number of YABBY genes of each cucurbit species (except cucumber and watermelon) is less than that obtained by synteny analysis (Table S3).  The MEME analysis was carried out to predict the conserved motifs of protein sequence to better understand the conservation and diversification of YABBYs in Cucurbitaceae. We observed that the YABBYs in same subfamily share highly similar motif compositions (Figures 3 and S2). For instance, motif 9 was unique to YAB1 subfamily, whereas motif 5 was lacking in YAB2 subfamily. Specially, motif 10 only existed in a subgroup of INO subfamily (Figure 3). These results suggested the possible functional diversification of YABBYs across Cucurbitaceae. Finally, given that the YABBYs are plant-specific proteins containing conserved C2C2 domain and YABBY domain, we aligned the 59 YABBY protein sequences. The alignment results exhibited that all 59 YABBYs contain the two conserved domains (Figure 4), indicating the conservation in Cucurbitaceae YABBYs evolution.

Gene Structure and Conserved Motifs Analysis of YABBYs in Cucurbitaceae
The evolutionary history of gene families can be reflected by gene structural diversity [59]. Thus, we analyzed the gene structure that are characterized by the exon-intron organization of YABBYs open reading fame (ORF) from eight cucurbit species to explore the YABBYs evolution in Cucurbitaceae. An unrooted evolutionary tree was conducted by MEGA 11 and used for distinguishing different YAB subfamilies. As shown in Figure 3 and Table S2, the 59 YABBYs contained exon numbers ranging from 5 to 12 and intron number varying from 4 to 11. Among them, all YAB2 subfamily members had six exons and five introns, most members of the CRC subfamily (except Carg17317 with nine exons and eight introns in silver-seed gourd) shared seven exons and six introns ( Figure 3 and Table S2). However, we found that the INO subfamily and the two larger subfamilies, YAB1 and YAB5, harbor different numbers of exon and intron, 5 to 11 exons and 4 to 10 introns in the Genes 2022, 13, 467 8 of 16 YAB1 subfamily, 5 to 9 exons and 4 to 8 introns in the YAB5 subfamily, and 6 to 12 exons and 5 to 11 introns in the INO subfamily ( Figure 3 and Table S2).
The MEME analysis was carried out to predict the conserved motifs of protein sequence to better understand the conservation and diversification of YABBYs in Cucurbitaceae. We observed that the YABBYs in same subfamily share highly similar motif compositions (Figures 3 and S2). For instance, motif 9 was unique to YAB1 subfamily, whereas motif 5 was lacking in YAB2 subfamily. Specially, motif 10 only existed in a subgroup of INO subfamily (Figure 3). These results suggested the possible functional diversification of YABBYs across Cucurbitaceae. Finally, given that the YABBYs are plantspecific proteins containing conserved C2C2 domain and YABBY domain, we aligned the 59 YABBY protein sequences. The alignment results exhibited that all 59 YABBYs contain the two conserved domains (Figure 4), indicating the conservation in Cucurbitaceae YABBYs evolution.

Cis-Acting Elements Analysis of Cucurbitaceae YABBY Genes Promoter Regions
The 2000 bp upstream sequences from the transcription start site of 59 YABBYs were extracted by TBtools from the genome of the eight cucurbit species and used to analyze the cis-acting elements in these promoters using the online PlantCARE database. The predicted cis-acting elements were mainly associated with phytohormone response, plant development, stress, and light responses ( Figure 5 and Table S4). We noted that the light response elements exist in most YABBYs promoters. The phytohormone response elements mainly involved in abscisic acid (ABRE) and methyl jasmonate (CGTCA-motif, TGACG-motif) were identified, while fewer response elements associated with auxin (TGA-element), cytokinin (O2-site), gibberellin (P-box), and salicylic acid (TCA-element) were discovered. Moreover, the stress response elements-such as TC-rich repeats for defense and stress, MBS for drought stress, and LTR for low temperature stress-were also found. Notably, we detected some development-related elements but with fewer numbers, including CAT-box (meristem), circadian (circadian control), GCN4_motif (endosperm), MSA-like (cell cycle), and RY-element (seed-specific) ( Figure 5 and Table S4). Taken together, these results suggested possible roles of YABBYs in plant development, stress, and phytohormone responses across Cucurbitaceae.

Cis-Acting Elements Analysis of Cucurbitaceae YABBY Genes Promoter Regions
The 2000 bp upstream sequences from the transcription start site of 59 YABBYs were extracted by TBtools from the genome of the eight cucurbit species and used to analyze the cis-acting elements in these promoters using the online PlantCARE database. The predicted cis-acting elements were mainly associated with phytohormone response, plant development, stress, and light responses ( Figure 5 and Table S4). We noted that the light response elements exist in most YABBYs promoters. The phytohormone response elements mainly involved in abscisic acid (ABRE) and methyl jasmonate (CGTCA-motif, TGACGmotif) were identified, while fewer response elements associated with auxin (TGA-element), cytokinin (O2-site), gibberellin (P-box), and salicylic acid (TCA-element) were discovered. Moreover, the stress response elements-such as TC-rich repeats for defense and stress, MBS for drought stress, and LTR for low temperature stress-were also found. Notably, we detected some development-related elements but with fewer numbers, including CAT-box (meristem), circadian (circadian control), GCN4_motif (endosperm), MSA-like (cell cycle), and RY-element (seed-specific) ( Figure 5 and Table S4). Taken together, these results suggested possible roles of YABBYs in plant development, stress, and phytohormone responses across Cucurbitaceae.

Expression Pattern of YABBYs in Cucumber
To explore the detailed expression profiles of YABBYs in Cucurbitaceae, we selected cucumber as a representative species to examine YABBY gene expression patterns in different organs including root, stem, leaf, tendril, male bud, female bud, and different development stages of ovary/fruit. As shown in Figure 6, the CsYABBYs exhibited various expression patterns in test materials. The four genes CsaV3_1G030340, CsaV3_2G002960, CsaV3_3G003040, and CsaV3_5G003950 had high expression levels in leaf. We noticed that CsaV3_2G002960 also expressed higher in tendrils and CsaV3_2G024750 shows high expression level in stems. Moreover, we found that CsaV3_5G003950 and CsaV3_5G033400 are highly expressed in floral organs. In addition, CsaV3_2G002960, CsaV3_3G003040, and CsaV3_6G038650 also had higher expression levels in male and female buds. However, the expression of CsaV3_5G031440 was mostly undetectable in all organs ( Figure 6). Given the higher expression levels of CsaV3_2G002960, CsaV3_3G003040, CsaV3_5G003950, CsaV3_5G033400 and CsaV3_6G038650 in floral organs, we further explore the expression patterns of these five genes in different development stages of ovary/fruit. We found that CsaV3_2G002960, CsaV3_3G003040, CsaV3_5G003950, and CsaV3_5G033400 are mainly expressed at all ovary development stages (12,9,6, 3, and 0 DBF) but not fruit development stages, whereas CsaV3_6G038650 shows high expression at all fruit development stages (3, 6, and 12 DAF) other than ovary development stages ( Figure 7A). The similar expression patterns were also observed in an RNA-seq data wherein a transcriptome analysis of 4 DBF ovary from two near-isogenic lines 408 and 409 was performed [57] (Figure 7B and Table S5). Four YABBY genes (except CsaV3_2G002960 with no differential expression)

Expression Pattern of YABBYs in Cucumber
To explore the detailed expression profiles of YABBYs in Cucurbitaceae, we selected cucumber as a representative species to examine YABBY gene expression patterns in different organs including root, stem, leaf, tendril, male bud, female bud, and different development stages of ovary/fruit. As shown in Figure 6, the CsYABBYs exhibited various expression patterns in test materials. The four genes CsaV3_1G030340, CsaV3_2G002960, CsaV3_3G003040, and CsaV3_5G003950 had high expression levels in leaf. We noticed that CsaV3_2G002960 also expressed higher in tendrils and CsaV3_2G024750 shows high expression level in stems. Moreover, we found that CsaV3_5G003950 and CsaV3_5G033400 are highly expressed in floral organs. In addition, CsaV3_2G002960, CsaV3_3G003040, and CsaV3_6G038650 also had higher expression levels in male and female buds. However, the expression of CsaV3_5G031440 was mostly undetectable in all organs ( Figure 6). Given the higher expression levels of CsaV3_2G002960, CsaV3_3G003040, CsaV3_5G003950, CsaV3_5G033400 and CsaV3_6G038650 in floral organs, we further explore the expression patterns of these five genes in different development stages of ovary/fruit. We found that CsaV3_2G002960, CsaV3_3G003040, CsaV3_5G003950, and CsaV3_5G033400 are mainly expressed at all ovary development stages (12,9,6,3, and 0 DBF) but not fruit development stages, whereas CsaV3_6G038650 shows high expression at all fruit development stages (3,6, and 12 DAF) other than ovary development stages ( Figure 7A). The similar expression patterns were also observed in an RNA-seq data wherein a transcriptome analysis of 4 DBF ovary from two near-isogenic lines 408 and 409 was performed [57] (Figure 7B and Table S5). Four YABBY genes (except CsaV3_2G002960 with no differential expression) were higher expressed in line 408 which is characterized by long fruit compared to the line 409 which has a short fruit [57] (Figure 7B and Table S5). Hence, the results indicated that the YABBYs may play various and important roles during plant growth and development in cucumber, such as leaf, tendril, stem, male flower, and ovary/fruit development.
Genes 2022, 13, x FOR PEER REVIEW 11 of 17 were higher expressed in line 408 which is characterized by long fruit compared to the line 409 which has a short fruit [57] ( Figure 7B and Table S5). Hence, the results indicated that the YABBYs may play various and important roles during plant growth and development in cucumber, such as leaf, tendril, stem, male flower, and ovary/fruit development.

Discussion
The YABBYs, plant-specific transcription factors, play significant roles during different plant development processes [12,13]. So far, it is well known that Cucurbitaceae-such as cucumber, melon, watermelon, and pumpkin-are important horticultural crops with global economic value [31]. However, little is known about the characteristics and functions of YABBYs across Cucurbitaceae, except some studies of cucumber [44,45].
Recently, the whole-genome sequences of eight cucurbit species-including cucumber, melon, watermelon, wax gourd, pumpkin, zucchini, silver-seed gourd, and bottle gourd-were released or updated [34,[60][61][62][63][64][65][66], providing a useful strategy to deepen our understanding of genome-wide identification of YABBY family in Cucurbitaceae. In this study, we identified and characterized the YABBY genes in Cucurbitaceae based on these whole-genome sequences. Totally, we obtained 3,4,5,8,9,9,10, and 11 YABBY genes in bottle gourd, melon, wax gourd, cucumber, watermelon, zucchini, silver-seed gourd, and pumpkin, respectively (Tables 1 and S1). Like Arabidopsis YABBY genes [10,11], we found that the YABBY genes in eight cucurbit species are also divided into five subfamilies, YAB1, YAB2, CRC, INO, and YAB5 ( Figure 1 and Table 1). Notably, compared to Arabidopsis which has six YABBY genes [1][2][3], the number of YABBY genes in bottle gourd, melon, and wax gourd was less than six but more than six in the other five cucurbit species (Tables 1 and S1). We speculated that the differences of YABBY gene numbers among different cucurbit species may be explained by gene duplication or loss during evolutionary process, as the segment and tandem duplications contribute to the expansion of gene family [58]. The synteny analysis results showed that there are many more syntenic gene pairs between cucumber and watermelon, bottle gourd, pumpkin, and zucchini ( Figure 2 and Table S3). In addition, we observed that an obvious expansion of INO and YAB5 subfamilies among Cucurbitaceae, which possibly is owing to much more orthologs within the two subfamilies in watermelon, pumpkin, and zucchini except bottle gourd (Figure 1 and Table 1). These results suggested that gene duplication may contribute to the more YABBY genes in these three cucurbit species except bottle gourd. However, for melon and wax

Discussion
The YABBYs, plant-specific transcription factors, play significant roles during different plant development processes [12,13]. So far, it is well known that Cucurbitaceae-such as cucumber, melon, watermelon, and pumpkin-are important horticultural crops with global economic value [31]. However, little is known about the characteristics and functions of YABBYs across Cucurbitaceae, except some studies of cucumber [44,45].
Recently, the whole-genome sequences of eight cucurbit species-including cucumber, melon, watermelon, wax gourd, pumpkin, zucchini, silver-seed gourd, and bottle gourd-were released or updated [34,[60][61][62][63][64][65][66], providing a useful strategy to deepen our understanding of genome-wide identification of YABBY family in Cucurbitaceae. In this study, we identified and characterized the YABBY genes in Cucurbitaceae based on these whole-genome sequences. Totally, we obtained 3,4,5,8,9,9,10, and 11 YABBY genes in bottle gourd, melon, wax gourd, cucumber, watermelon, zucchini, silver-seed gourd, and pumpkin, respectively (Tables 1 and S1). Like Arabidopsis YABBY genes [10,11], we found that the YABBY genes in eight cucurbit species are also divided into five subfamilies, YAB1, YAB2, CRC, INO, and YAB5 ( Figure 1 and Table 1). Notably, compared to Arabidopsis which has six YABBY genes [1][2][3], the number of YABBY genes in bottle gourd, melon, and wax gourd was less than six but more than six in the other five cucurbit species (Tables 1 and S1). We speculated that the differences of YABBY gene numbers among different cucurbit species may be explained by gene duplication or loss during evolutionary process, as the segment and tandem duplications contribute to the expansion of gene family [58]. The synteny analysis results showed that there are many more syntenic gene pairs between cucumber and watermelon, bottle gourd, pumpkin, and zucchini ( Figure 2 and Table S3). In addition, we observed that an obvious expansion of INO and YAB5 subfamilies among Cucurbitaceae, which possibly is owing to much more orthologs within the two subfamilies in watermelon, pumpkin, and zucchini except bottle gourd ( Figure 1 and Table 1). These results suggested that gene duplication may contribute to the more YABBY genes in these three cucurbit species except bottle gourd. However, for melon and wax gourd which had less YABBY genes (Tables 1 and S1), we noticed that 75% (6 of 8) of CsYABBY genes share only one syntenic gene pair between cucumber and melon/wax gourd ( Figure 2 and Table S3), and the loss events also exist in melon and wax gourd, including no YAB1 subfamily in melon and no INO subfamily in wax gourd ( Figure 1 and Table 1). Furthermore, although more syntenic gene pairs between cucumber and bottle gourd were found (Figure 2 and Table S3), bottle gourd contained only three YABBY genes and lacked CRC and YAB2 subfamilies ( Figure 1 and Table 1). Since the YABBY gene contains a conserved C2C2 domain and a conserved YABBY domain, the truncated genes, which lack either a C2C2 domain and/or a YABBY domain, were excluded when we identified the YABBY genes, which might account for the final number of identified YABBY genes in Cucurbitaceae (except cucumber and watermelon) being lower than that obtained by synteny analysis and one and/or two YABBY subfamilies are lacking in melon, wax gourd, zucchini, and bottle gourd. Possibly due to the low quality of silver-seed gourd genome draft, we could not perform the synteny analysis between cucumber and silver-seed gourd. Hence, further studying is recommended to explore whether lower quality of genome draft or gene loss events during evolutionary process are the cause of fewer YABBY genes and the lacking of some subfamilies in these cucurbit species.
To better understand the conservation and diversification of YABBYs in Cucurbitaceae, we performed an analysis of gene structures and conserved motifs/domains. The 59 YAB-BYs shared a conserved C2C2 domain and a conserved YABBY domain ( Figure 4 and Table  S1). However, there were some differences among different subfamilies. For instance, all YAB2 subfamily members had six exons and five introns, whereas CRC subfamily (except Carg17317 with nine exons and eight introns in silver-seed gourd) members contained seven exons and six introns. YAB2 and CRC subfamilies commonly contained motif 1, motif 2, motif 4, and motif 6; meanwhile, the YAB2 subfamily had motif 8 but lost motif 5, whereas the CRC subfamily had motif 5 but lacked motif 8 ( Figure 3). These results hinted that the similar characteristics of YABBY genes within the same subfamily may indicate conserved function, and the differences of YABBY gene characteristics among various subfamilies possibly contribute to functional differentiation across Cucurbitaceae. Generally, transcription factors bind to specific cis-acting elements of targeted genes to regulate their expression. The cis-acting elements in promoters of the 59 YABBY genes suggested that they may be involved in plant development, phytohormone, stress, and light responses in Cucurbitaceae.
Importantly, the expression patterns of CsYABBY genes in cucumber were analyzed to explore the potential functions of different YABBY subfamilies across Cucurbitaceae. Previous reports found that YAB1 (FIL)/YAB3, YAB2, and YAB5 are associated with leaf and floral organ development in Arabidopsis and rice [2,14,16,18,19,29,30,67], the similar higher expression levels of their homologous genes (except YAB2 homolog) CsaV3_5G003950, CsaV3_3G003040, CsaV3_2G002960, and CsaV3_1G030340 in leaf and floral organs suggested the functional conservation of these genes between cucumber, Arabidopsis, and rice ( Figure 6). Meanwhile, the special expression of CsaV3_5G033400, the homolog of CRC which functions in carpel development in Arabidopsis [1,20,21] and rice [23], in female bud hinted the underlying role in carpel development of cucumber ( Figure 6). However, we observed that the YAB2 homolog CsaV3_6G038650 expression level is higher only in floral organs, but not leaf or other tissues. CsaV3_2G002960 was also highly expressed in tendrils. Additionally, there are two homologs of INO in cucumber. According to the tissue-specific feature with low levels in various tissues of INO expression in Arabidopsis [3], we detected almost no expression of CsaV3_5G031440 in cucumber tissues. However, the other gene CsaV3_2G024750 were expressed in different tissues, especially in stem (Figure 6), although CsaV3_2G024750 (gene ID in paper: Csa011583) was reported in involved with integument of ovules but not with functional verification [44]. Thus, the different or specific expression patterns of these four genes suggested their function differentiations in cucumber. Further studies are needed to verify our speculation by their functional identifications.
Furthermore, four genes-CsaV3_2G002960, CsaV3_3G003040, CsaV3_5G003950, and CsaV3_5G033400-showed higher expression levels at all ovary development stages but not in fruits ( Figure 7A), suggesting their possible roles in ovary development of cucumber. Particularly, CsaV3_5G003950-the homolog of YAB1-had very high transcriptional level and thus might mainly regulate ovary development of cucumber, this is in line with the function of YAB1 which is involved in abaxial cell type specification in leaves and fruits in Arabidopsis [2,14,16]. In rice, the YAB1 clade genes-TOB1, TOB2, and TOB3-are required to maintain proper function of the spikelet and branch meristems [29,30]. It is likely that the function of YAB1 clade genes has been conserved between cucumber, rice, and Arabidopsis. For the CRC homolog in cucumber, CsaV3_5G033400, was significantly expressed in the early development stage of ovary at 12DBF and 9DBF ( Figure 7A), indicating its possible role in carpel development of cucumber and is similar with CRC function involved in carpel development in Arabidopsis [1,20,21] and rice [23]. Interestingly, we noted that the YAB2 homolog CsaV3_6G038650 is more highly expressed in fruit than ovary ( Figure 7A). Although YAB2 is involved in polarity development of leaf and flower [2], the function in regulating fruit development is not proved in Arabidopsis. The specific expression pattern of CsaV3_6G038650 in fruit implied its prominent function in cucumber fruit development. Coincidentally, fas-a YABBY-like transcription factor homologous to Arabidopsis YAB2-regulated fruit development by controlling carpel number in tomato, resulting in an extreme fruit size [68]. Additionally, in cereals, SH1 or ObSH3-which are closely related to Arabidopsis YAB2-is required for seed shattering during domestication [26,27]. These results suggested the functional differentiation of YAB2 gene between cucumber, tomato, cereals, and Arabidopsis, and CsaV3_6G038650 is a possibly major regulatory factor of fruit development in cucumber. Taken together, the results implied that the YABBY genes possibly play vital roles in many aspects of plant growth and development in cucumber-such as leaf, tendril, and ovary/fruit-which will contribute to the applications of YABBY genes in breeding of cucumber, even Cucurbitaceae.

Conclusions
In this study, 59 YABBY genes were identified from eight cucurbit species. A systematic characterization study was performed for chromosomal location, gene structure, conserved motifs, cis-acting elements, evolutional relationship, and gene duplication. The evolutionary relationship showed that the YABBY genes from Cucurbitaceae are classified into five subfamilies. Gene duplication events occurred between cucumber and watermelon, bottle gourd, pumpkin, and zucchini, which contributes to YABBY gene family expansions in these four cucurbit species. However, we have not confirmed whether or not low quality of genome draft or gene loss events are the cause of fewer YABBY genes in bottle gourd, melon, and wax gourd-further verification is required. The expression patterns of most YABBY genes in cucumber were similar with that in Arabidopsis and rice, indicating the function conservation of these YABBY genes among Cucurbitaceae. However, the different expression patterns of several CsYABBY genes are needed to illuminate by their function verifications. Importantly, we identified CsaV3_6G038650 as a potential regulatory factor of fruit development in cucumber. In conclusion, our study provided a foundation for further research on YABBY gene functions which will facilitate breeding in Cucurbitaceae.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/genes13030467/s1. Figure S1: Chromosomal locations of YABBY genes in the genome of seven cucurbit species; Figure S2: Analysis of conserved motifs in YABBY proteins of eight cucurbit species; Table S1: List of YABBY genes identified in cucumber, melon, watermelon, wax gourd, pumpkin, zucchini, silver-seed gourd, and bottle gourd; Table S2: Information of YABBY genes identified in eight cucurbit species; Table S3: Orthologous relationships of YABBY genes between cucumber and other six cucurbit species; Table S4: Cis-acting elements of YABBY genes promoters in cucumber, melon, watermelon, wax gourd, pumpkin, zucchini, silver-seed gourd, and bottle gourd; Table S5: The TPM values of YABBY family genes in cucumber by RNA-Seq analysis; Table S6: Primers of cucumber YABBY genes used in qRT-PCR.
Author Contributions: S.Y., X.L. and H.R. conceived, designed, and assessed the experiments; E.S.B., R.W., H.Y., C.L., X.C. and Y.W. participated in the design of the experiments. S.Y. and S.L. performed the experiments; S.Y. and Y.G. analyzed the data; S.Y., X.L. and H.R. wrote the manuscript. All authors reviewed and modified the manuscript. All authors have read and agreed to the published version of the manuscript.