Characterization and Divergence Analysis of Duplicated R2R3-MYB Genes in Watermelon

in Journal of the American Society for Horticultural Science
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  • 1 Key Laboratory of Horticultural Plant Biology (MOE), College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China; and College of Horticulture, Nanjing Agricultural University, Nanjing 210000, China
  • 2 Key Laboratory of Horticultural Plant Biology (MOE), College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China

Watermelon (Citrullus lanatus) is an economically important cucurbit (Cucurbitaceae) crop cultivated globally for its nutritional benefits. Fruit development in watermelon is characterized by fast fruit expansion attributed to unique biological processes. Myeloblastosis (MYB) family genes play important roles in plant growth and development, especially R2R3-MYB-type genes. However, the evolution of R2R3-MYB family genes in the watermelon genome and whether they participate in the regulation of watermelon fruit development remain unknown. To address these questions, duplication modes of R2R3-MYB family genes were identified and their expression profiles were investigated during watermelon fruit development. A total of 48 duplicated gene pairs were identified among the 89 R2R3-MYBs in watermelon. Segmental and transposed duplication events play major roles in the R2R3-MYB family gene expansion process. The ratio of nonsynonymous mutation and synonymous mutation analysis indicated that all the duplicated R2R3-MYBs experienced negative selection. Gene structures and cis-element compositions in promoter sequences exhibited abundant divergences between the R2R3-MYB duplicated genes. Transcriptome analyses of seed, rind, and flesh during fruit development showed that only two duplicated gene pairs had significantly similar expression patterns, whereas divergent expression profiles were found between the remaining duplicated gene pairs. Tissue-specific and development stage-specific divergent expression patterns demonstrated that neo-functionalization occurred between watermelon R2R3-MYB duplicated genes. The current study provides valuable information for further functional analyses of R2R3-MYBs in watermelon.

Abstract

Watermelon (Citrullus lanatus) is an economically important cucurbit (Cucurbitaceae) crop cultivated globally for its nutritional benefits. Fruit development in watermelon is characterized by fast fruit expansion attributed to unique biological processes. Myeloblastosis (MYB) family genes play important roles in plant growth and development, especially R2R3-MYB-type genes. However, the evolution of R2R3-MYB family genes in the watermelon genome and whether they participate in the regulation of watermelon fruit development remain unknown. To address these questions, duplication modes of R2R3-MYB family genes were identified and their expression profiles were investigated during watermelon fruit development. A total of 48 duplicated gene pairs were identified among the 89 R2R3-MYBs in watermelon. Segmental and transposed duplication events play major roles in the R2R3-MYB family gene expansion process. The ratio of nonsynonymous mutation and synonymous mutation analysis indicated that all the duplicated R2R3-MYBs experienced negative selection. Gene structures and cis-element compositions in promoter sequences exhibited abundant divergences between the R2R3-MYB duplicated genes. Transcriptome analyses of seed, rind, and flesh during fruit development showed that only two duplicated gene pairs had significantly similar expression patterns, whereas divergent expression profiles were found between the remaining duplicated gene pairs. Tissue-specific and development stage-specific divergent expression patterns demonstrated that neo-functionalization occurred between watermelon R2R3-MYB duplicated genes. The current study provides valuable information for further functional analyses of R2R3-MYBs in watermelon.

Transcription factors (TFs) are extensively involved in the regulation of plant growth and development. MYB is an important TF gene family with the largest members (Dubos et al., 2010). Its N-terminal region has a highly conserved DNA binding domain, which is called MYB domain. A MYB gene usually contains one to four incomplete MYB domain repeats (R) of ≈52 amino acid residues. Based on the number of MYB repeats, the MYB family gene can be divided into four types, namely, 4R-MYB-type with four repeats, 3R-MYB-type with three repeats, R2R3-MYB-type with two repeats, and MYB-related-type with a single repeat or partial MYB-related repeats (Jia et al., 2004). R2R3-MYB is dominant and has the largest members in most plants (Rosinski and Atchley, 1998), and it has a wide range of functions, including the regulation of plant growth and development, primary and secondary metabolism, and response to biotic and abiotic stresses (Stracke et al., 2001).

Gene duplication has an important role in the expansion of the gene family and functional diversity. Large-scale gene duplication and single-gene duplication are two types of gene duplication models that widely exist in eukaryotic genomes (Wang et al., 2013b). Large-scale gene duplication includes whole-genome duplication (WGD) and segmental duplication (Salman-Minkov et al., 2016). Single-gene duplication is also prevalent in plant genomes and consists of three duplication models, including transposed duplication, proximal duplication, and tandem duplication (Freeling, 2009). Transposed duplication generates a duplicated gene on a new chromosome position via replicative transposition (Panchy et al., 2016). Tandem duplication results in a gene pair produced by an unequal cross between alleles (Hahn, 2009). The proximal gene pairs are two duplicated genes separated by a small number of genes (Wang et al., 2012). The WGD in watermelon (Citrullus lanatus) has seven major triplications dating back to between 76 and 130 million years ago and generated 302 paralogous relationships covering 29% of the genome (Guo et al., 2013). The segmental duplication event reportedly has a major role in the expansion of the MYB gene family in pineapple (Ananas comosus) (Liu et al., 2017).

After duplication, mutations increasingly accumulate in the regulatory and coding regions of duplicated genes during the course of evolution (Zhu et al., 2014). Different regulatory regions may lead to changes in expression levels, and different coding regions may result in new functions (Long and Thornton, 2001). The function-differentiated duplicated genes may undergo positive or negative selection. The ratio of nonsynonymous mutation (Ka) to synonymous mutation (Ks) is used to estimate the selection pressure of duplicated genes (Zhang et al., 2006). Many evolutionary models have been proposed to elucidate the gene status after duplication, including the short- and long-term retention or loss function (Qiao et al., 2018). Furthermore, three functional models are used to describe the functions of the retained duplicated genes, including gene dosage, sub-functionalization, and neo-functionalization (Flagel and Wendel, 2009). Gene dosage is one mechanism that affects the functions by changing the genes number of copies after duplication, and recent research has suggested that the presence of gene dosage may be beneficial to gene duplication (Panchy et al., 2016). The sub-functionalization model suggests that each duplicated gene retains its original ancestral function during the natural mutational process (Freeling et al., 2015). Sub-functionalization was found between the duplicated MYB genes ScAN2 and ScAN1 in potato (Solanum commersonii) in the response to cold stress (D'Amelia et al., 2018). The phenomenon that one duplicated gene keeps the ancestral function while the other gene acquires a new function is called neo-functionalization (Duarte et al., 2006). Neo-functionalization has been reported for the B-class MADS-box duplicated genes of PFGLO1 and PFDEF in Physalis floridana (Zhang et al., 2015).

Watermelon is an economically important cucurbit crop widely grown for its large, edible fruit. The watermelon planted area and total yield are ranked among the world’s top 10 for fresh fruits (Food and Agriculture Organization of the United Nations, 2018). Because a whole genome sequence of the crop is available, watermelon has become a crop widely researched by plant breeders and plant physiologists. A total of 162 MYB genes have been identified in the watermelon genome, and expression patterns of 25 MYB genes were measured in watermelon seedlings under abiotic stresses (Xu et al., 2018). This study provides a fundamental understanding of the possible roles of MYB family genes in watermelon stress tolerance. Fruit development is one of the most important biological processes of watermelon; it determines the economic value of watermelon production. MYB genes have been reported to widely participate in the regulation of fruit development (Machemer et al., 2011). However, the roles R2R3-MYB genes in regulating watermelon fruit development and whether the functions of duplicated R2R3-MYB genes are different during fruit development are still unknown.

In this study, duplicated R2R3-MYB gene pairs were identified in the watermelon genome. Their expression profiles in rind, flesh, and seeds were determined during fruit development. Expression patterns of duplicated genes were associated with their Ka/Ks values, gene structures, and cis-element compositions. This study aimed to identify the R2R3-MYB genes involved in watermelon fruit development and clarify the function divergences of duplicated R2R3-MYB genes.

Materials and Methods

Identification of R2R3-MYB genes.

The genome of watermelon cultivar 97103 (v1) was used as the reference genome (Guo et al., 2013). All protein sequences and coding sequences were downloaded from the Cucurbit Genomics Database (Zheng et al., 2019). The hidden Markov model (HMM) profile of the MYB DNA-binding domain (PF00249) was downloaded from the Pfam database (El-Gebali et al., 2019). HMMER (Finn et al., 2011) was applied to identify the MYB family members with a cutoff of E ≤ 0.01. Protein domains of R2R3-MYB were validated by SMART (Letunic and Bork, 2018). Gene structures were analyzed using the Gene Structure Display Server (Hu et al., 2015). The MEME online tool (version 5.1.0; National Institutes of Health, Bethesda, MD) was used to investigate conserved domains. Weblogo (Crooks et al., 2004) was used to draw the sequence logos. Protein modeling was predicted using SWISS-MODEL (Waterhouse et al., 2018).

Identification of duplicated genes and cis-element analysis.

MCScanX-transposed was used to identify and classify gene duplication modes (Wang et al., 2013a). Circos (Krzywinski et al., 2009) was used to display the synteny relationships. KaKs_Calculator1.2 (Zhang et al., 2006) was applied to estimate the Ks and Ka values. Bedtools (Quinlan and Hall, 2010) was used to obtain the 1-kb upstream sequence for each gene. Cis-elements were predicted using PlantCARE (Lescot et al., 2002). The cis-elements index value of the duplicated genes were calculated using the following equation: [1−(common cis-elements/all cis-elements)]. This is used to measure the dissimilarity of cis-elements between two duplicated genes. The value of the cis-element index ranged from 0 to 1, and the larger value indicated greater divergence.

Transcriptome data analysis.

The transcriptome data of watermelon seeds sampled at 25, 31, 37, and 49 d after pollination (DAP) under BioProject ID PRJNA319011 (University of Nebraska, Lincoln, NE) and transcriptome data of watermelon fruit flesh and rind sampled at 10, 18, 26, and 34 DAP under BioProject ID SRP012849 (Boyce Thompson Institute, Ithaca, NY) were downloaded from the National Center for Biotechnology Information Sequence Read Archive (Bethesda, MD) database. All the annotated gene sequences of the watermelon cultivar 97103 reference genome were used as the reference transcriptome. Kallisto (Bray et al., 2016) was used to calculate the transcript per million (TPM) value for each gene. RStudio (version 1.0.136; RStudio, Boston, MA) was used to conduct the data analysis and draw the plots.

Results

Identification of watermelon R2R3-MYB genes.

The HMM profile of MYB was used to search for watermelon genome-wide annotated proteins; 164 MYB genes were identified. These MYB genes were further validated and classified by searching both the Pfam and SMART databases, resulting in 89 R2R3-type genes, 5 R3-type genes, and 70 MYB-related-type genes. No R4-type genes were found in the watermelon genome. The R2R3-type genes were selected for further analysis and named ClR2R3-MYB. Information regarding these genes is listed in Supplemental Table 1. Sequence logo was used to exhibit the characteristics of ClR2R3-MYB conserved motifs (Fig. 1). Results revealed 53 amino acid residues in the R2 repeat and 71 amino acid residues in the R3 repeat. Highly conserved Trp residues were found on positions 6, 26, and 46 of the R2 repeat and 25, 45, 64 on the R3 repeat, respectively. The three-dimensional protein structure models showed that each of the two domains build an HTH structure, which is the main characterization of R2R3-MYB family genes. Amino acids such as Glu-10, Gly-22, Arg-37, Lys-40, Ser-41, Lys-45, Cys-42, Leu-44, Asn-48, and Leu-50 in the R2 repeat and Glu-10, Ala-29, Thr-56, Asp-57, Asn-58, Lys-61, and Asn-62 in the R3 repeat were also conserved among watermelon R2R3-MYB genes.

Fig. 1.
Fig. 1.

Domains of ClR2R3-MYB family genes and protein three-dimensional (3D) structure models of R2 and R3 MYB repeats. (A) R2 domain. (B) R3 domain. The bit score indicates the information content for each position in the sequence and the purple asterisks indicate the conserved tryptophan residues (Trp, W). (C) R2 repeats 3D structure model. (D) R3 repeats 3D structure models.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 5; 10.21273/JASHS04849-19

Duplication events of ClR2R3-MYB genes.

A total of 48 ClR2R3-MYB duplicated gene pairs were identified, including 27 segmental duplicated pairs, 19 transposed duplicated pairs, 1 proximal duplicated pair, and 1 tandem duplicated pair (Supplemental Table 2). Duplicated gene pairs and their genome locations are displayed in the Circos plot (Fig. 2). The duplicated genes were distributed on all 11 watermelon chromosomes and tended to cluster at the two ends of each chromosome. Chromosome 5 harbored the largest number of duplicated genes. Specially, mutually segmental duplicated events were found among the triple genes of Cla013979-Cla020663-Cla022601, Cla005982-Cla007586-Cla021520, Cla010332-Cla019115-Cla018610, and Cla007715-Cla019451-Cla001341.

Fig. 2.
Fig. 2.

The positions of ClR2R3-MYB genes in the watermelon genome and duplication events. Blue lines indicate transposed duplication gene pairs. Orange lines indicate segmental duplication gene pairs. Red line represents the tandem duplication gene pair. Green line represents the proximal duplication gene pair. Chr = chromosome.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 5; 10.21273/JASHS04849-19

Gene structure divergences of duplicated ClR2R3-MYB genes.

To compare gene structures of duplicated genes, DNA and mRNA sequences of each gene were aligned to analyze the exon–intron composition (Fig. 3). Exons ranged from one to four in the duplicated genes. The duplicated gene pair Cla017337-Cla015165 had the highest number of exons, whereas duplicated gene pairs Cla020702-Cla022542, Cla020633-Cla022601, Cla004362-Cla013979, Cla013979-Cla020633, and Cla013979-Cla022601 harbored only one exon in each gene. A total of 36 duplicated gene pairs comprised similar exon–intron compositions. The remaining 12 duplication gene pairs had different gene structures, including three segmental duplicated gene pairs and nine transposed duplicated gene pairs. The different gene structures probably lead to functional divergences between the duplicated genes.

Fig. 3.
Fig. 3.

Gene structures of duplicated R2R3-MYB gene pairs. Green boxes represent the coding sequence (CDS) regions. Black lines show the intron regions. Yellow indicates transposed duplicated gene pairs. Purple indicates segmental duplicated gene pairs. The proximal and tandem duplicated gene pairs each have a pair of duplicated genes.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 5; 10.21273/JASHS04849-19

Cis-elements divergences of duplicated ClR2R3-MYB genes.

To compare the cis-regulatory element composition in the promoter region between the duplicated gene pair, a 1-kb upstream sequence of each gene was selected and the putative cis-regulatory elements were identified (Supplemental Table 3). To quantify the dissimilarity of cis-element composition between duplicated genes, the cis-element index was calculated for each gene pair. The density distribution of the cis-element index for segmental and transposed duplicated genes is shown in Fig. 4A. The cis-element index of segmental and transposed duplicated genes ranged from 0.55 to 0.85 and 0.54 to 0.8, respectively, demonstrating that wide divergences occurred in the promoter regions of duplicated gene pairs.

Fig. 4.
Fig. 4.

The cis-element summary and density distribution of the cis-element index. (A) The cis-elements index density of duplicated gene pairs. (B) The cis-element summary. Annotations of cis-elements are listed in Supplemental Table 3.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 5; 10.21273/JASHS04849-19

The cis-element types were summarized. The top 30 cis-element types are shown in Fig. 4B. Function annotations of these cis-elements were related to hormone regulations, cellular development, and stress response. For hormone regulation, the functions of cis-elements were mainly involved in responses related to salicylic acid, auxin, and abscisic acid metabolism. Cellular development mainly included meristem-specific activation, endosperm expression, palisade mesophyll cells, leaf morphology development, shoot-specific expression, and cell-cycle regulation. The remaining cis-elements were related to stress physiology, such as responses to light, low-temperature, heat, drought, dehydration, anaerobic induction, wound, and anoxic-specific inducibility. The cis-element sequences CAAT-box and TATA-box were found in all ClR2R3-MYB genes. The light response of cis-elements Box-I, Box-4, and GAG-motif occurred in the promoter regions of most duplicated ClR2R3-MYBs. A total of 59 duplicated ClR2R3-MYB genes had the ARE cis-acting regulatory element, which is essential for anaerobic induction. Skn-1motif was found in 62 duplicated genes, suggesting these genes are putatively involved in the regulation of seed development. The 5UTRPy-rich stretch and G-box were also identified in more than 50 ClR2R3-MYB promoter sequences, suggesting that these genes probably have high expression levels. TC-rich repeats, which are involved in defense and the stress response, were also found in the promotor regions of 54 ClR2R3-MYB genes.

Ka and Ks substitution rates of duplicated ClR2R3-MYB genes.

Ka, Ks, and their ratios were calculated. The density distribution of Ks values is presented in Fig. 5A. The Ks values ranged from 0.42 to 3.95. The smallest Ks value was observed in the tandem duplicated gene pair Cla014140-Cla014141. A peak appeared at ≈2.8 in the density distribution plot, indicating that the duplication events occurred at one major stage and varied with the time of evolutionary process.

Fig. 5.
Fig. 5.

Density distribution of synonymous mutation (Ks) and boxplot of nonsynonymous mutation (Ka)/Ks values: (A) density distribution of Ks for the ClR2R3-MYB duplicated gene pairs and (B) Ka/Ks value distribution of ClR2R3-MYB duplicated gene pairs.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 5; 10.21273/JASHS04849-19

The Ka/Ks value is an index estimating the selection pressure during the gene evolution process. The Ka/Ks values of the R2R3-MYB duplicated gene pairs ranged from 0.06 to 0.32, with an average of 0.15 (Fig. 5B). All the Ka/Ks values were less than 1.00, suggesting that ClR2R3-MYB duplicated genes had undergone purifying selection during the evolution process. Gene pairs with different duplication models tended to have different Ka/Ks value distributions. The segmental duplicated gene pairs had relatively low Ka/Ks values, whereas the transposed duplicated gene pairs displayed relatively high Ka/Ks values. A maximum Ka/Ks value of 0.32 occurred in the tandem duplicated gene pair Cla014140-Cla014141, suggesting that the two genes might experience more relaxed purifying selection.

Expression divergences of duplicated ClR2R3-MYB genes.

RNA-seq data derived from the fleshes and rinds of watermelon fruit at 10, 18, 26, and 34 DAP and from seeds at 25, 31, 37, and 49 DAP were used to determine the expression profiles of ClR2R3-MYB family genes. A total of 34 genes were not expressed during watermelon fruit development. The remaining 55 genes were involved in the regulation of fruit development and maturity. Among them, 10 genes including Cla006761, Cla021243, Cla011548, Cla013099, Cla007114, Cla020633, Cla013668, Cla022601, Cla010413, and Cla013979 were constitutively expressed in all tissues and at all developmental stages. However, some ClR2R3-MYB genes exhibited tissue-specific expression patterns. Twelve ClR2R3-MYB genes including Cla007815, Cla007846, Cla015834, Cla013461, Cla018610, Cla021474, Cla018967, Cla010415, Cla017337, Cla017267, Cla017012, and Cla016980 were only expressed in seeds. Cla021520, Cla019115, and Cla017797 were specifically expressed in fleshes, whereas Cla005982, Cla008278, Cla019115, and Cla021520 were specifically expressed in the rinds.

To compare expression patterns of the duplicated genes, pairwise transcript abundances were illustrated in the heatmap (Fig. 6). A total of eight duplicated gene pairs were not expressed during watermelon fruit development, including one tandem duplicated gene pair, one proximal duplicated gene pair, two transposed duplicated pairs, and four segmental duplicated gene pairs. Three segmental duplicated gene pairs of Cla013979-Cla022601, Cla020633-Cla022601, and Cla013979-Cla020633 were expressed in all tissues and at all stages of watermelon fruit development. To further quantify the expression similarity of duplicated gene pairs, Pearson’s correlation coefficient was calculated for each expressed duplicated gene pair during watermelon fruit development. The significant correlation coefficients were marked aside the corresponding gene pairs (P < 0.05). A transposed duplicated gene pair of Cla004362-Cla013979 and a segmental duplicated gene pair of Cla020702-Cla022542 exhibited significantly similar expression patterns, demonstrating that the duplicated gene pairs had putatively similar functions during watermelon fruit development. Although the expression patterns were significantly correlated, significantly different transcript abundances were also observed between the two duplicated gene pairs. The remaining 38 duplicated gene pairs exhibited divergent expression patterns, suggesting different regulatory roles during fruit development.

Fig. 6.
Fig. 6.

Expression profiles of duplicated ClR2R3-MYB gene pairs in seed, fruit flesh, and rind during watermelon fruit development. The color scale at the left of each dendrogram represents log2 expression values, with black indicating high levels and white indicating low levels of transcript abundance. r = correlation coefficient expression of two duplicated genes.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 5; 10.21273/JASHS04849-19

Tissue-specific divergent expression patterns were observed between duplicated gene pairs. Five transposed duplicated gene pairs exhibited tissue-specific divergent expression patterns. Cla019999 was expressed in rind and flesh, but the expression of its duplicated gene Cla009903 was not detected in the two tissues. Cla009263 was expressed in all tissues, but Cla000767 was not expressed in the fruit. Similarly, Cla015834 was specifically expressed in the seed, but its duplicated gene Cla016689 was not expressed in the fruit. Cla019223 was expressed in rind and flesh, but its duplicated gene Cla001702 was not expressed in the fruit. Cla013668 was expressed in all tissues and at all stages, but expression of its duplicated gene Cla005321 was not detected in the fruit. Tissue-specific divergent expression patterns were also found in the four segmental duplicated gene pairs. Cla019115 was only expressed in the rind, but its duplicated gene Cla010332 was not expressed in the fruit. Cla018501 was expressed in all tissues, whereas its duplicated gene Cla018967 was only expressed in the seed. Cla018610 was only expressed in the seed, but its duplicated gene Cla019115 was only expressed in the rind. Cla018610 was only expressed in the seed, but its duplicated gene Cla010332 was not expressed in the fruit.

Development stage-specific divergent expression patterns were also observed between duplicated gene pairs. Regarding transposed duplicated gene pairs, Cla018657 was not expressed in fruit, but its duplicated gene Cla017496 was expressed in rinds and fleshes at all stages, as well as in the seed at 10 DAP. Cla012519 was expressed in all tissues and at all stages, but its duplicated gene Cla007846 was only expressed in the seed at 25 and 31 DAP. Cla007815 was only expressed in seed at 25 DAP, whereas its duplicated gene Cla010193 was not expressed in fruit. Similar results were also observed in segmental duplicated gene pairs. For instance, Cla020702 was expressed in all tissues, but its duplicated gene Cla022542 was only expressed in the rinds at 10 and 18 DAP. Similarly, Cla017337 was only expressed in the seed at 25 and 49 DAP, but its duplicated gene Cla015165 was expressed in all tissues. Cla019199 was expressed in all tissues but not in the seed at 25 DAP, whereas its duplicated gene Cla010415 was only expressed in the seed at 25 DAP. Cla003441 was expressed in the rind and flesh at 10 DAP. However, Cla019223 was only expressed in the rind and flesh at 18, 26, and 34 DAP.

Discussion

R2R3-MYB transcription factors are widely involved in the regulations of plant morphogenesis, growth, metabolism, developmental processes, and response to biotic or abiotic stresses. However, duplication types of watermelon R2R3-MYB and their possible roles regulating watermelon fruit development have been ignored. In this study, the duplicated ClR2R3-MYB genes were identified, and their expression profiles were investigated during fruit development to understand their potential roles during watermelon fruit development.

A total of 89 R2R3-MYB genes were identified in the watermelon genome; this number was similar to that of previous reports (Xu et al., 2018) but larger than that reported for cucumber (Cucumis sativus) (Ren et al., 2009) and smaller than that identified in Arabidopsis thaliana (Stracke et al., 2001). All ClR2R3-MYBs have two incomplete R. Every R forms an HTH structure, which is consistent with R2R3-MYB family characteristics identified in the present study. The first Trp residue in the R3 repeat was replaced by some hydrophobic amino acids like Phe or Ile. Similar results were also observed in A. thaliana (Stracke et al., 2001) and cucumber (Li et al., 2012). However, some other residues, such as Glu, Gly, Lys, Ser, Cys, Leu, Thr, Asp, and Asn, were specifically conserved in watermelon R2R3-MYB family genes.

A total of 48 ClR2R3-MYB duplicated gene pairs were identified in this study. Segmental duplication accounted for 56.25% of the total duplication events, demonstrating that it has a major role in ClR2R3-MYB gene expansion. Similar results were also reported for pineapple (Liu et al., 2017). Results of the present study suggested that genes produced by segmental duplications were retained during evolution (Cannon et al., 2004). In addition, mutually segmental duplicated events were found among the triple genes of Cla013979-Cla020663-Cla022601, Cla005982-Cla007586-Cla021520, Cla010332-Cla019115-Cla018610, and Cla007715-Cla019451-Cla001341, which probably occurred along with the triplicate evolution of the watermelon genome (Guo et al., 2013). One proximal gene pair and one tandem gene pair were detected in ClR2R3-MYBs. Low frequencies of tandem and proximity duplicated gene pairs in the R2R3-MYB family were also found in A. thaliana (Cannon et al., 2004). Transposed duplication accounted for 41.7% of the total duplication events, which also had an important role in the expansion of ClR2R3-MYB genes. Similar results were also reported for family genes related to sorbitol metabolism pathways (Qiao et al., 2018).

Different cis-regulatory elements in the promoter sequences of duplicated genes may produce different expression patterns. In this study, most duplicated gene pairs had divergent regulatory elements. There were four transposed duplicated gene pairs with a cis-element index more than 0.8, suggesting that their expressions can be regulated by several different transacting factors (Qiao et al., 2018). Several studies have shown that the duplicated genes inherited the transcription regulatory models from their ancestors, but that their cis-regulatory regions were divergent after evolution (Haberer et al., 2004). The highly divergent cis-regulatory elements between ClR2R3-MYB gene pairs potentially lead to sub-functionalization or neo-functionalization between the duplicated genes.

Expression patterns of ClR2R3-MYB duplicated genes were measured in flesh, rind, and seed during watermelon fruit development. Only two duplicated gene pairs, Cla004362-Cla013979 and Cla020702-Cla022542, had similar expression patterns, whereas less nonsynonymous mutations, similar gene structures, and cis-element compositions were observed. This suggested that function redundancy or sub-functionalization probably occurred between the two genes. Similar results were also observed between GmPIN duplicated genes in soybean (Glycine max) (Liu and Wei, 2017). The other 38 duplicated gene pairs exhibited tissue-specific or development stage-specific divergent expression patterns during watermelon fruit development. Different gene structures and cis-element compositions, as well as relatively high nonsynonymous mutations, were observed between these duplicated pairs, suggesting that neo-functionalization probably occurred after duplication. Similar results were also reported between the segmental duplication of MYB homologous genes in maize (Zea mays) (Zhang et al., 2000). Neo-functionalization potentially leads to the importance of ClR2R3-MYB duplicated genes in the regulation of watermelon fruit development.

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  • Hahn, M.W. 2009 Distinguishing among evolutionary models for the maintenance of gene duplicates J. Hered. 100 605 617

  • Hu, B., Jin, J., Guo, A., Zhang, H., Luo, J. & Gao, G. 2015 GSDS 2.0: An upgraded gene feature visualization server Bioinformatics 31 1296 1297

  • Jia, L., Clegg, M.T. & Jiang, T. 2004 Evolutionary dynamics of the DNA-binding domains in putative R2R3-MYB genes identified from rice subspecies indica and japonica genomes Plant Phytol. 134 575 585

    • Search Google Scholar
    • Export Citation
  • Krzywinski, M., Schein, J.E., Birol, I., Connors, J.M., Gascoyne, R.D., Horsman, D., Jones, S.J.M. & Marra, M.A. 2009 Circos: An information aesthetic for comparative genomics Genome Res. 19 1639 1645

    • Search Google Scholar
    • Export Citation
  • Lescot, M., Dehais, P., Thijs, G., Marchal, K., Moreau, Y., De Peer, Y.V., Rouze, P. & Rombauts, S. 2002 PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences Nucleic Acids Res. 30 325 327

    • Search Google Scholar
    • Export Citation
  • Letunic, I. & Bork, P. 2018 20 years of the SMART protein domain annotation resource Nucleic Acids Res. 46 D493 D496

  • Li, Q., Zhang, C., Li, J., Wang, L. & Ren, Z. 2012 Genome-wide identification and characterization of R2R3-MYB family in Cucumis sativus PLoS One 7 e47576 doi: 10.1371/journal.pone.0047576

    • Search Google Scholar
    • Export Citation
  • Liu, C., Xie, T., Chen, C., Luan, A., Long, J., Li, C., Ding, Y. & He, Y. 2017 Genome-wide organization and expression profiling of the R2R3-MYB transcription factor family in pineapple (Ananas comosus) BMC Genomics 18 503

    • Search Google Scholar
    • Export Citation
  • Liu, Y. & Wei, H. 2017 Genome-wide identification and evolution of the PIN-FORMED (PIN) gene family in Glycine max Genome 60 564 571

  • Long, M. & Thornton, K.R. 2001 Gene duplication and evolution Science 293 5535 1551

  • Machemer, K., Shaiman, O., Salts, Y., Shabtai, S., Sobolev, I., Belausov, E., Grotewold, E. & Barg, R. 2011 Interplay of MYB factors in differential cell expansion, and consequences for tomato fruit development Plant J. 68 337 350

    • Search Google Scholar
    • Export Citation
  • Panchy, N., Lehti-Shiu, M.D. & Shiu, S. 2016 Evolution of gene duplication in plants Plant Phytol. 171 2294 2316

  • Qiao, X., Yin, H., Li, L., Wang, R., Wu, J., Wu, J. & Zhang, S. 2018 Different modes of gene duplication show divergent evolutionary patterns and contribute differently to the expansion of gene families involved in important fruit traits in pear (Pyrus bretschneideri) Front. Plant Sci. 9 161

    • Search Google Scholar
    • Export Citation
  • Quinlan, A.R. & Hall, I.M. 2010 BEDTools: A flexible suite of utilities for comparing genomic features Bioinformatics 26 841

  • Ren, Y., Zhang, Z., Liu, J., Staub, J.E., Han, Y., Cheng, Z., Li, X., Lu, J., Miao, H., Kang, H., Xie, B., Gu, X., Wang, X., Du, Y., Jin, W. & Huang, S. 2009 An integrated genetic and cytogenetic map of the cucumber genome PLoS One 4 e5795 doi: 10.1371/journal.pone.0005795

    • Search Google Scholar
    • Export Citation
  • Rosinski, J.A. & Atchley, W.R. 1998 Molecular evolution of the Myb family of transcription factors: Evidence for polyphyletic origin J. Mol. Evol. 46 74 83

    • Search Google Scholar
    • Export Citation
  • Salman-Minkov, A., Sabath, N. & Mayrose, I. 2016 Whole-genome duplication as a key factor in crop domestication Nat. Plants 2 16115

  • Stracke, R., Werber, M. & Weisshaar, B. 2001 The R2R3-MYB gene family in Arabidopsis thaliana Curr. Opin. Plant Biol. 4 447 456

  • Wang, Y., Li, J. & Paterson, A.H. 2013a MCScanX-transposed: Detecting transposed gene duplications based on multiple colinearity scans Bioinformatics 29 1458 1460

    • Search Google Scholar
    • Export Citation
  • Wang, Y., Tan, X. & Paterson, A.H. 2013b Different patterns of gene structure divergence following gene duplication in Arabidopsis BMC Genomics 14 652

    • Search Google Scholar
    • Export Citation
  • Wang, Y., Tang, H., Debarry, J.D., Tan, X., Li, J., Wang, X., Lee, T., Jin, H., Marler, B.S. & Guo, H. 2012 MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity Nucleic Acids Res. 40 e49 doi: 10.1093/nar/gkr1293

    • Search Google Scholar
    • Export Citation
  • Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., De Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R. & Schwede, T. 2018 SWISS-MODEL: Homology modelling of protein structures and complexes Nucleic Acids Res. 46 W296 W303

    • Search Google Scholar
    • Export Citation
  • Xu, Q., He, J., Dong, J., Hou, X. & Zhang, X. 2018 Genomic survey and expression profiling of the MYB gene family in watermelon Hort. Plant J. 4 5 19

  • Zhang, P., Chopra, S. & Peterson, T. 2000 A segmental gene duplication generated differentially expressed myb-homologous genes in maize Plant Cell 12 2311 2322

    • Search Google Scholar
    • Export Citation
  • Zhang, S., Zhang, J., Zhao, J. & He, C. 2015 Distinct subfunctionalization and neofunctionalization of the B-class MADS-box genes in Physalis floridana Planta 241 387 402

    • Search Google Scholar
    • Export Citation
  • Zhang, Z., Li, J., Zhao, X., Wang, J., Wong, G. & Yu, J. 2006 KaKs_Calculator: Calculating Ka and Ks through model selection and model averaging Genom. Proteom. Bioinfo. 4 259 263

    • Search Google Scholar
    • Export Citation
  • Zheng, Y., Xu, Y., Weng, Y., Mazourek, M., Reddy, U.K., Ando, K., Mccreight, J.D., Schaffer, A.A., Burger, J., Tadmor, Y., Katzir, N., Tang, X., Liu, Y., Giovannoni, J.J., Ling, K., Wechter, W.P., Levi, A., Garciamas, J., Grumet, R. & Fei, Z. 2019 Cucurbit Genomics Database (CuGenDB): A central portal for comparative and functional genomics of cucurbit crops Nucleic Acids Res. 47 D1128 D1136

    • Search Google Scholar
    • Export Citation
  • Zhu, Y., Wu, N., Song, W., Yin, G., Qin, Y., Yan, Y. & Hu, Y. 2014 Soybean (Glycine max) expansion gene superfamily origins: Segmental and tandem duplication events followed by divergent selection among subfamilies BMC Plant Biol. 14 93

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    • Export Citation
Supplemental Table 1.

Characteristics of watermelon R2R3-MYB genes.

Supplemental Table 1.Supplemental Table 1.
Supplemental Table 2.

Duplication information of R2R3-MYB gene pairs in watermelon.

Supplemental Table 2.
Supplemental Table 3.

Cis-regulatory elements annotations.

Supplemental Table 3.Supplemental Table 3.

If the inline PDF is not rendering correctly, you can download the PDF file here.

Contributor Notes

Q.K. is the corresponding author: E-mail: qskong@mail.hzau.edu.cn.

  • View in gallery

    Domains of ClR2R3-MYB family genes and protein three-dimensional (3D) structure models of R2 and R3 MYB repeats. (A) R2 domain. (B) R3 domain. The bit score indicates the information content for each position in the sequence and the purple asterisks indicate the conserved tryptophan residues (Trp, W). (C) R2 repeats 3D structure model. (D) R3 repeats 3D structure models.

  • View in gallery

    The positions of ClR2R3-MYB genes in the watermelon genome and duplication events. Blue lines indicate transposed duplication gene pairs. Orange lines indicate segmental duplication gene pairs. Red line represents the tandem duplication gene pair. Green line represents the proximal duplication gene pair. Chr = chromosome.

  • View in gallery

    Gene structures of duplicated R2R3-MYB gene pairs. Green boxes represent the coding sequence (CDS) regions. Black lines show the intron regions. Yellow indicates transposed duplicated gene pairs. Purple indicates segmental duplicated gene pairs. The proximal and tandem duplicated gene pairs each have a pair of duplicated genes.

  • View in gallery

    The cis-element summary and density distribution of the cis-element index. (A) The cis-elements index density of duplicated gene pairs. (B) The cis-element summary. Annotations of cis-elements are listed in Supplemental Table 3.

  • View in gallery

    Density distribution of synonymous mutation (Ks) and boxplot of nonsynonymous mutation (Ka)/Ks values: (A) density distribution of Ks for the ClR2R3-MYB duplicated gene pairs and (B) Ka/Ks value distribution of ClR2R3-MYB duplicated gene pairs.

  • View in gallery

    Expression profiles of duplicated ClR2R3-MYB gene pairs in seed, fruit flesh, and rind during watermelon fruit development. The color scale at the left of each dendrogram represents log2 expression values, with black indicating high levels and white indicating low levels of transcript abundance. r = correlation coefficient expression of two duplicated genes.

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  • Jia, L., Clegg, M.T. & Jiang, T. 2004 Evolutionary dynamics of the DNA-binding domains in putative R2R3-MYB genes identified from rice subspecies indica and japonica genomes Plant Phytol. 134 575 585

    • Search Google Scholar
    • Export Citation
  • Krzywinski, M., Schein, J.E., Birol, I., Connors, J.M., Gascoyne, R.D., Horsman, D., Jones, S.J.M. & Marra, M.A. 2009 Circos: An information aesthetic for comparative genomics Genome Res. 19 1639 1645

    • Search Google Scholar
    • Export Citation
  • Lescot, M., Dehais, P., Thijs, G., Marchal, K., Moreau, Y., De Peer, Y.V., Rouze, P. & Rombauts, S. 2002 PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences Nucleic Acids Res. 30 325 327

    • Search Google Scholar
    • Export Citation
  • Letunic, I. & Bork, P. 2018 20 years of the SMART protein domain annotation resource Nucleic Acids Res. 46 D493 D496

  • Li, Q., Zhang, C., Li, J., Wang, L. & Ren, Z. 2012 Genome-wide identification and characterization of R2R3-MYB family in Cucumis sativus PLoS One 7 e47576 doi: 10.1371/journal.pone.0047576

    • Search Google Scholar
    • Export Citation
  • Liu, C., Xie, T., Chen, C., Luan, A., Long, J., Li, C., Ding, Y. & He, Y. 2017 Genome-wide organization and expression profiling of the R2R3-MYB transcription factor family in pineapple (Ananas comosus) BMC Genomics 18 503

    • Search Google Scholar
    • Export Citation
  • Liu, Y. & Wei, H. 2017 Genome-wide identification and evolution of the PIN-FORMED (PIN) gene family in Glycine max Genome 60 564 571

  • Long, M. & Thornton, K.R. 2001 Gene duplication and evolution Science 293 5535 1551

  • Machemer, K., Shaiman, O., Salts, Y., Shabtai, S., Sobolev, I., Belausov, E., Grotewold, E. & Barg, R. 2011 Interplay of MYB factors in differential cell expansion, and consequences for tomato fruit development Plant J. 68 337 350

    • Search Google Scholar
    • Export Citation
  • Panchy, N., Lehti-Shiu, M.D. & Shiu, S. 2016 Evolution of gene duplication in plants Plant Phytol. 171 2294 2316

  • Qiao, X., Yin, H., Li, L., Wang, R., Wu, J., Wu, J. & Zhang, S. 2018 Different modes of gene duplication show divergent evolutionary patterns and contribute differently to the expansion of gene families involved in important fruit traits in pear (Pyrus bretschneideri) Front. Plant Sci. 9 161

    • Search Google Scholar
    • Export Citation
  • Quinlan, A.R. & Hall, I.M. 2010 BEDTools: A flexible suite of utilities for comparing genomic features Bioinformatics 26 841

  • Ren, Y., Zhang, Z., Liu, J., Staub, J.E., Han, Y., Cheng, Z., Li, X., Lu, J., Miao, H., Kang, H., Xie, B., Gu, X., Wang, X., Du, Y., Jin, W. & Huang, S. 2009 An integrated genetic and cytogenetic map of the cucumber genome PLoS One 4 e5795 doi: 10.1371/journal.pone.0005795

    • Search Google Scholar
    • Export Citation
  • Rosinski, J.A. & Atchley, W.R. 1998 Molecular evolution of the Myb family of transcription factors: Evidence for polyphyletic origin J. Mol. Evol. 46 74 83

    • Search Google Scholar
    • Export Citation
  • Salman-Minkov, A., Sabath, N. & Mayrose, I. 2016 Whole-genome duplication as a key factor in crop domestication Nat. Plants 2 16115

  • Stracke, R., Werber, M. & Weisshaar, B. 2001 The R2R3-MYB gene family in Arabidopsis thaliana Curr. Opin. Plant Biol. 4 447 456

  • Wang, Y., Li, J. & Paterson, A.H. 2013a MCScanX-transposed: Detecting transposed gene duplications based on multiple colinearity scans Bioinformatics 29 1458 1460

    • Search Google Scholar
    • Export Citation
  • Wang, Y., Tan, X. & Paterson, A.H. 2013b Different patterns of gene structure divergence following gene duplication in Arabidopsis BMC Genomics 14 652

    • Search Google Scholar
    • Export Citation
  • Wang, Y., Tang, H., Debarry, J.D., Tan, X., Li, J., Wang, X., Lee, T., Jin, H., Marler, B.S. & Guo, H. 2012 MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity Nucleic Acids Res. 40 e49 doi: 10.1093/nar/gkr1293

    • Search Google Scholar
    • Export Citation
  • Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., De Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R. & Schwede, T. 2018 SWISS-MODEL: Homology modelling of protein structures and complexes Nucleic Acids Res. 46 W296 W303

    • Search Google Scholar
    • Export Citation
  • Xu, Q., He, J., Dong, J., Hou, X. & Zhang, X. 2018 Genomic survey and expression profiling of the MYB gene family in watermelon Hort. Plant J. 4 5 19

  • Zhang, P., Chopra, S. & Peterson, T. 2000 A segmental gene duplication generated differentially expressed myb-homologous genes in maize Plant Cell 12 2311 2322

    • Search Google Scholar
    • Export Citation
  • Zhang, S., Zhang, J., Zhao, J. & He, C. 2015 Distinct subfunctionalization and neofunctionalization of the B-class MADS-box genes in Physalis floridana Planta 241 387 402

    • Search Google Scholar
    • Export Citation
  • Zhang, Z., Li, J., Zhao, X., Wang, J., Wong, G. & Yu, J. 2006 KaKs_Calculator: Calculating Ka and Ks through model selection and model averaging Genom. Proteom. Bioinfo. 4 259 263

    • Search Google Scholar
    • Export Citation
  • Zheng, Y., Xu, Y., Weng, Y., Mazourek, M., Reddy, U.K., Ando, K., Mccreight, J.D., Schaffer, A.A., Burger, J., Tadmor, Y., Katzir, N., Tang, X., Liu, Y., Giovannoni, J.J., Ling, K., Wechter, W.P., Levi, A., Garciamas, J., Grumet, R. & Fei, Z. 2019 Cucurbit Genomics Database (CuGenDB): A central portal for comparative and functional genomics of cucurbit crops Nucleic Acids Res. 47 D1128 D1136

    • Search Google Scholar
    • Export Citation
  • Zhu, Y., Wu, N., Song, W., Yin, G., Qin, Y., Yan, Y. & Hu, Y. 2014 Soybean (Glycine max) expansion gene superfamily origins: Segmental and tandem duplication events followed by divergent selection among subfamilies BMC Plant Biol. 14 93

    • Search Google Scholar
    • Export Citation
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