Exploring the PpEXPs Family in Peach: Insights into Their Role in Fruit Texture Development through Identification and Transcriptional Analysis

: Expansins (EXPs) loosen plant cell walls and are involved in diverse developmental processes through modifying cell-walls; however, little is known about the role of PpEXPs in peach fruit. In this study, 26 PpEXP genes were identified in the peach genome and grouped into four subfamilies, with 20 PpEXPAs, three PpEXPBs, one PpEXPLA and two PpEXPLBs. The 26 PpEXPs were mapped on eight chromosomes. The primary mode of gene duplication of the PpEXPs was dispersed gene duplication (DSD, 50%). Notably, cis -elements involved in light responsiveness and MeJA-responsiveness were detected in the promoter regions of all PpEXPs, while ethylene responsive elements were observed in 12 PpEXPs. Transcript profiling of PpEXPs in the peach fruit varieties of MF (melting), NMF (non-melting) and SH (stony hard) at different stages showed that PpEXPs displayed distinct expression patterns. Among the 26 PpEXPs, 15 PpEXPs were expressed in the fruit. Combining the expressing patterns of PpEXPs in fruits with different flesh textures, PpEXPA7, PpEXPA13 and PpEXPA15 were selected as candidate genes, as they were highly consistent with the patterns of previous reported key genes (PpPGM, PpPGF and PpYUC11) in regard to peach fruit texture. The genes with different expression patterns between MF and NMF were divided into 16 modules, of which one module, with pink and midnightblue, negatively correlated with the phenotype of fruit firmness and was identified as PpEXPA1 and PpEXPA7, while the other module was identified as PpERF in the pink module, which might potentially effect fruit texture development by regulating PpEXPs. These results provide a foundation for the functional characterization of PpEXPs in peach.


Introduction
The peach (Prunus persica) is an important economic tree species that originated in China.Fruit texture is a vital characteristic of quality in ripe fruit.In peaches, the fruit texture can be classified into three types: melting flesh (MF), non-melting flesh (NMF) and stony hard flesh (SH) [1,2].Two genes encoding endopolygalacturonase (PG) were responsible for the MF/NMF flesh type [3], and PpYUC11 controls the stony hard phenotype in peaches [4].As a climacteric fruit, peaches generally soften rapidly during maturation and especially after harvest, which causes difficulties in postharvest handling and quality maintenance during storage and transportation.Improving the fruit texture quality of peaches is one of the most important goals of cultivar development.
Biochemical and molecular studies have revealed that fruit softening is a consequence of cell wall disassembly, including decline in cell wall strength and cell-to-cell adhesion [5,6].Numerous genes have been verified as being critical to cell wall disassembly in tomatoes (Solanum lycopersicum) [7], apples (Malus domestica) [8], strawberries (Fragaria vesca) and others [9][10][11].These genes were mainly related to primary cell wall (PCW) degrading and modifying, such as PG, pectin methylesterases (PME), pectate lyases (PL) and so on.In strawberries, the ripened fruits of FaPG1 antisense transgenic plants were on average 163% firmer than that of the control [9].Similarly, downregulation of MdPG1 increased the firmness and intercellular adhesion in apples [8].Numerous studies regarding fruit softening have focused on tomatoes as a model system [12].There is increasing evidence that softening is a complex process that involves multiple genes.PG, PME, PL, β-galactosidase arabinofuranosidase and expansin have been proven to be involved in the softening of tomatoes [13][14][15][16][17][18].
In recent years, a few studies have indicated that EXPs play a critical role in fruit firmness and ripening.SlExp1 loss-of-function mutants showed firmer and later ripening fruits [27,28].Overexpressing MdEXLB1 lines in tomatoes showed lower fruit firmness and an accelerated fruit ripening process [29].Four EXPs in strawberry were selected as candidate genes involved in fruit softening [30].However, the functions of EXPs in regulating of peach fruit development remains unknown.
In this study, PpEXPs were identified in the peach genome, and their molecular and physiological characters, phylogenetic relationships and expression profiles were further examined to better understand the PpEXPs family.To probe the potential role of PpEXPs in flesh phenotypes, the expression of PpEXPs among different flesh phenotypes (MF, NMF and SH) was examined.The results provide a functional basis for further investigation of PpEXPs in fruit textures.

Plant Materials
The peach cultivars 'HuangShuimi' (HSM, MF) and 'Zhongyoutao14' (CN14, NMF) were planted in the experimental station of the College of Horticulture, Henan Agricultural University (Zhengzhou, China, 34.86 • N, 113.60 • E).Samples of each fruits' flesh was collected for transcriptome and qRT-PCR analysis at four critical growth stages, namely S1 (fruit setting stage), S2 (fruit expansion period), S3 (fruit color change period) and S4 (ripening and softening stage).All samples were quickly frozen in liquid nitrogen and stored at −80 • C.

Measurement of Peach Fruit Firmness
The peach fruit firmness was assessed using the TA.XT Plus Texture Analyzer (Stable Micro Systems Ltd., Godalming, UK) by inserting a P/2 probe into the fruit at a depth of 5 mm, with each fruit being pierced in three places.The data on firmness were collected by selecting and assessed 10 fruits in each period.

Identification of PpEXPs in Peach Genome
The hidden markov model (HMM) of DPBB domain (PF03330) and Pollen_allergens domain (PF01357), two conserved domains in expansin proteins, were downloaded from the Pfam website (http://pfam.xfam.org,accessed on 6 June 2023).The hmmsearch was performed against the peach genome protein sequences (v2.1) with a threshold of e < 1 × 10 −5 .Candidate proteins were further identified by Pfam analysis, with each protein containing both conserved domains.

Chromosome Location, Gene Structure and cis-Elements Analysis of the PpEXP Genes
The chromosomal physical position of each expansin gene was mapped by TBtools.The gene duplication of all the PpEXPs was analyzed using MCScanX and drawn by TBtools [31].The exon−intron structure of the PpEXP genes were graphically displayed by TBtools.The conserved motif of each PpEXPs protein was pretreated by MEME online software (https://meme-suite.org/meme/ accessed on 22 February 2024).The up-stream 2-Kb genomic DNA sequences of PpEXPs genes were used to predict the cis-element in the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/accessed on 22 February 2024).

Transcriptomic Analysis
Total RNA was extracted using the Total RNA Rapid Extraction Kit (Sangon, Shanghai, China).For each sample, 2 µg of high-quality RNA was used for library construction and sequencing.Eight libraries, each generated from a sample of CN14 and HSM fruit at S1-4 stages, were sequenced on the BGI-SEQ-500 platform in paired-end 100 bp mode.
The low-quality reads and adapters of the RNA-Seq raw data were removed for all samples using Trimmomatic-0.38.Clean reads were mapped to the CN14 reference genome using hisat2 (v2.1), and transcript abundance was estimated using HTSeq (v0.12.3) based on the alignments.

Expression Pattern Analysis of PpEXP Genes in Peach Fruit and qRT-PCR Analysis
The expression levels of the PpEXP genes were shown via a heatmap generated by TBtools [31].To validate the results of RNA sequencing, the expression level of six PpEXP genes was detected using qRT-PCR.The RNA was used to transcribe cDNA with a Prime-Script RT kit (TaKaRa, Kusatsu, Japan), then the cDNA was subjected to qRT-PCR using a Step One Plus Real-Time PCR System (Applied Biosystems, Foster, CA, USA).PpGAPDH (Prupe.1G234000)was used as the endogenous control gene.The primer sequences were listed in Table S7, and we also produced another heatmap through the melting flesh of CN13 and stony hard flesh of CN16 at four development stages (S3, S4I, S4II, S4III), as obtained from our previous study [32].

Weighted Gene Co-Expression Network Analysis (WGCNA)
A total of 17,395 genes with FPKM values >1 was selected to perform WGCNA on ImageGP (https://www.bic.ac.cn/ImageGP/, accessed on 22 February 2024).Modules were identified using default settings, with the soft power being 30, the minimal module size being 25, and only select top genes with maximum mean absolute deviation (MAD) reaching 2000 [33].In addition, the expression of the genes of each module were correlated with the change in fruit firmness at four stages of CN14 and HSM.

Identification and Phylogenetic Analysis of the PpEXPs Family in Peaches
A total of 26 PpEXP genes containing two conserved domains (DPBB_1 and Pollen_allerg) were identified in the peach genome.The PpEXP protein showed divergent lengths (241-288 amino acids, aa), molecular weights (26.12-31.56kDa) and isoelectric points (4.69 to 9.83) (Table 1).Phylogenetic analysis showed that 26 PpEXPs were grouped into four subfamilies based on the full length protein sequences, including expansins from Arabidopsis (35) and rice (58).EXPA, as the largest subfamily, contained 79 genes, including 20 PpEXPAs, 25 AtEXPAs and 34 OsEXPAs (Figure 1).The second largest subfamily, EXPB, had 28 genes, with three PpEXPBs, six AtEXPBs and 19 OsEXPBs.There were four, three and one EXPLAs in rice, Arabidopsis and peach, respectively.In peach, two EXPLBs were identified, while only one EXPLB was found in rice and Arabidopsis, respectively.

Gene Structure and Motif Analysis of PpEXPs
The PpEXPs that clustered in the same subfamily showed similar gene structures and motif composition (Figure 2).All the members of the PpEXPB subfamily had four exons (Figure 2A,B).Most PpEXPA genes (80%) contained three exons, while others had two exons (20%).The PpEXPLB1 had five exons and PpEXPLB2 had four exons (Figure 2B).This indicated that PpEXPA and PpEXPB were more conserved in an exon−intron structure than PpEXPLB and reflected the gene structure diversity of different subfamilies.

Gene Structure and Motif Analysis of PpEXPs
The PpEXPs that clustered in the same subfamily showed similar gene structures and motif composition (Figure 2).All the members of the PpEXPB subfamily had four exons (Figure 2A,B).Most PpEXPA genes (80%) contained three exons, while others had two exons (20%).The PpEXPLB1 had five exons and PpEXPLB2 had four exons (Figure 2B).This indicated that PpEXPA and PpEXPB were more conserved in an exon−intron structure than PpEXPLB and reflected the gene structure diversity of different subfamilies.S1).All the PpEXPBs contained the same motifs, including Motif 1, Motif 4, Motif 7, Motif 9 and Motif 10.The motif analysis was consistent with the phylogenetic analyses, showing similar motif composition in members in the same subfamily.

Cis-Acting Elements Detection in the Promoter Regions of PpEXPs
Twenty-one types of cis-elements were identified in the 2-kb upstream regions of PpEXPs (Figure 4A, Table S3).The elements involved in light responsiveness and MeJAresponsiveness presented in the promoters of all PpEXPs, especially light responsive elements appearing at least two times in each promoter (Figure 4B).The copies of elements in the promoter region might enhance the regulating effects of their corresponding factors.In addition, elements related to the MYB binding site, abscisic acid responsiveness and anaerobic induction elements were apparently abundant, being detected in most PpEXPs promoters.These elements were related to many processes, such as plant hormone (MeJA respone, auxin response, abscisic acid response, ethylene response), environmental factors (light response, low-temperature response) and stress (wound, cold and defense responses).The different cis-elements in the promoters of PpEXPs suggested that PpEXPs may participate in various processes.

Cis-Acting Elements Detection in the Promoter Regions of PpEXPs
Twenty-one types of cis-elements were identified in the 2-kb upstream regions of PpEXPs (Figure 4A, Table S3).The elements involved in light responsiveness and MeJAresponsiveness presented in the promoters of all PpEXPs, especially light responsive elements appearing at least two times in each promoter (Figure 4B).The copies of elements in the promoter region might enhance the regulating effects of their corresponding factors.In addition, elements related to the MYB binding site, abscisic acid responsiveness and anaerobic induction elements were apparently abundant, being detected in most Pp-EXPs promoters.These elements were related to many processes, such as plant hormone (MeJA respone, auxin response, abscisic acid response, ethylene response), environmental factors (light response, low-temperature response) and stress (wound, cold and defense responses).The different cis-elements in the promoters of PpEXPs suggested that PpEXPs may participate in various processes.
Ethylene responsive elements were identified in the promoter regions of 12 PpEXPs, such as PpEXPA1, PpEXPA3, PpEXPA7, PpEXPA15, etc.Since ethylene plays an important role in fruit ripening [34], the expression of these genes might be involved in regulating peach fruit ripening.Ethylene responsive elements were identified in the promoter regions of 12 PpEXPs, such as PpEXPA1, PpEXPA3, PpEXPA7, PpEXPA15, etc.Since ethylene plays an important role in fruit ripening [34], the expression of these genes might be involved in regulating peach fruit ripening.

Expression Analysis of PpEXPs during Fruit Ripening in MF, NMF and SH Peach
To comprehensively reveal the expression patterns of PpEXPs in peach fruit, the transcriptome of the fruit was performed in HSM (MF) and CN14 (NMF) cultivars at four different stages (Figure 5A, Table S4).Among 26 PpEXP genes, 14 PpEXPs were expressed in the fruit of either MF or NMF during fruit ripening, including PpEXPA1, PpEXPA6-8, PpEXPA10, PpEXPA13-16, PpEXPA20, PpEXPB2-3, PpEXPLA1 and PpEXPLB2.Six PpEXPs were used to confirm the expression levels by quantitative real-time PCR (qRT-PCR) in CN14 and HSM at four stages.The results of the qRT-PCRs were consistent with those of the RNA-seq (Figure S1).PpEXPA1, PpEXPA6-8, PpEXPA15-16 and PpEXPLA1 showed high expression levels during the development and ripening stages in all samples.This suggested that these genes might be related to fruit development.The PpPGM and PpPGF were responsible

Expression Analysis of PpEXPs during Fruit Ripening in MF, NMF and SH Peach
To comprehensively reveal the expression patterns of PpEXPs in peach fruit, the transcriptome of the fruit was performed in HSM (MF) and CN14 (NMF) cultivars at four different stages (Figure 5A, Table S4).Among 26 PpEXP genes, 14 PpEXPs were expressed in the fruit of either MF or NMF during fruit ripening, including PpEXPA1, PpEXPA6-8, PpEXPA10, PpEXPA13-16, PpEXPA20, PpEXPB2-3, PpEXPLA1 and PpEXPLB2.Six PpEXPs were used to confirm the expression levels by quantitative real-time PCR (qRT-PCR) in CN14 and HSM at four stages.The results of the qRT-PCRs were consistent with those of the RNA-seq (Figure S1).Ethylene responsive elements were identified in the promoter regions of 12 PpEXPs, such as PpEXPA1, PpEXPA3, PpEXPA7, PpEXPA15, etc.Since ethylene plays an important role in fruit ripening [34], the expression of these genes might be involved in regulating peach fruit ripening.

Expression Analysis of PpEXPs during Fruit Ripening in MF, NMF and SH Peach
To comprehensively reveal the expression patterns of PpEXPs in peach fruit, the transcriptome of the fruit was performed in HSM (MF) and CN14 (NMF) cultivars at four different stages (Figure 5A, Table S4).Among 26 PpEXP genes, 14 PpEXPs were expressed in the fruit of either MF or NMF during fruit ripening, including PpEXPA1, PpEXPA6-8, PpEXPA10, PpEXPA13-16, PpEXPA20, PpEXPB2-3, PpEXPLA1 and PpEXPLB2.Six PpEXPs were used to confirm the expression levels by quantitative real-time PCR (qRT-PCR) in CN14 and HSM at four stages.The results of the qRT-PCRs were consistent with those of the RNA-seq (Figure S1).PpEXPA1, PpEXPA6-8, PpEXPA15-16 and PpEXPLA1 showed high expression levels during the development and ripening stages in all samples.This suggested that these genes might be related to fruit development.The PpPGM and PpPGF were responsible PpEXPA1, PpEXPA6-8, PpEXPA15-16 and PpEXPLA1 showed high expression levels during the development and ripening stages in all samples.This suggested that these genes might be related to fruit development.The PpPGM and PpPGF were responsible for the flesh texture of MF and NMF fruit, promoting fruit softening [3].According to the expression patterns, PpEXPA1, PpEXPA6-7, PpEXPA13-16, PpEXPB2 and PpEXPLB2 showed similar expression patterns with PpPGM and PpPGF.Moreover, PpEXPA1, Pp-EXPA7, PpEXPA13 and PpEXPA15 showed higher expression levels than the other genes, indicating that these four genes might regulate flesh texture.
In addition, the expression pattern of PpEXPs in CN13 (MF) and CN16 (SH) were also analyzed across four stages of ripening.The results showed that 15 PpEXPs were expressed in CN13 and CN16, including PpEXPA1, PpEXPA6-8, PpEXPA10, PpEXPA13-16, PpEXPA20, PpEXPB1-3, PpEXPLA1 and PpEXPLB1.PpYUC11 was a candidate gene for the control of the SH phenotype, of which the expression pattern was associated with flesh texture of MF, not expressed in SH [4].PpEXPA7, PpEXPA13, PpEXPA15, PpEXPB1 and PpEXPLB1 showed a clustered expression pattern with PpYUC11.Combining the above results of the expression patterns of PpEXPs in the two sets of transcriptome data, PpEXPA7, PpEXPA13 and PpEXPA15 were the common candidate genes, suggesting that they might participate in fruit texture development.

Weighted Gene Co-Expression Network Analysis (WGCNA) of Fruit Development
The development of fruit texture is a complex and dynamic process consisting of a diverse collection of genes with a variety of functional relationships.In order to detect relationships underlying modules and fruit texture development, we performed a weighted gene co-expression network analysis (WGCNA).As a result, 2000 genes (FPKM > 1) in eight samples of four stages were clustered into 16 modules (Figure 6A, Table S5).The module-trait association showed that pink and midnightblue modules were negatively correlated with fruit firmness (Figure 6B, Table S6).There were 89 genes in the pink module and 36 genes were in the midnightblue module.These genes were annotated in the three GO (Gene ontology) categories (biological process, cellular component and molecular function) (Table S5).In the biological process category, two genes, annotated as cell wall organization (GO:0009664), encoded the PpEXPA1 and PpEXPA7.Meanwhile, PpEXPA7 was identified as the hub gene.Notably, a transcription factor, PpERF (Ethylene response factor, Prupe.1G130300), was grouped into the pink module.Based on the results, PpEXPS, PpEXPA1 and PpEXPA7, play a critical role in fruit texture, which might be regulated by the PpERF transcription factor.
for the flesh texture of MF and NMF fruit, promoting fruit softening [3].According to the expression patterns, PpEXPA1, PpEXPA6-7, PpEXPA13-16, PpEXPB2 and PpEXPLB2 showed similar expression patterns with PpPGM and PpPGF.Moreover, PpEXPA1, PpEXPA7, PpEXPA13 and PpEXPA15 showed higher expression levels than the other genes, indicating that these four genes might regulate flesh texture.
In addition, the expression pattern of PpEXPs in CN13 (MF) and CN16 (SH) were also analyzed across four stages of ripening.The results showed that 15 PpEXPs were expressed in CN13 and CN16, including PpEXPA1, PpEXPA6-8, PpEXPA10, PpEXPA13-16, PpEXPA20, PpEXPB1-3, PpEXPLA1 and PpEXPLB1.PpYUC11 was a candidate gene for the control of the SH phenotype, of which the expression pattern was associated with flesh texture of MF, not expressed in SH [4].PpEXPA7, PpEXPA13, PpEXPA15, PpEXPB1 and PpEXPLB1 showed a clustered expression pattern with PpYUC11.Combining the above results of the expression patterns of PpEXPs in the two sets of transcriptome data, PpEXPA7, PpEXPA13 and PpEXPA15 were the common candidate genes, suggesting that they might participate in fruit texture development.

Weighted Gene Co-Expression Network Analysis (WGCNA) of Fruit Development
The development of fruit texture is a complex and dynamic process consisting of a diverse collection of genes with a variety of functional relationships.In order to detect relationships underlying modules and fruit texture development, we performed a weighted gene co-expression network analysis (WGCNA).As a result, 2000 genes (FPKM >1) in eight samples of four stages were clustered into 16 modules (Figure 6A, Table S5).The module-trait association showed that pink and midnightblue modules were negatively correlated with fruit firmness (Figure 6B, Table S6).There were 89 genes in the pink module and 36 genes were in the midnightblue module.These genes were annotated in the three GO (Gene ontology) categories (biological process, cellular component and molecular function) (Table S5).In the biological process category, two genes, annotated as cell wall organization (GO:0009664), encoded the PpEXPA1 and PpEXPA7.Meanwhile, PpEXPA7 was identified as the hub gene.Notably, a transcription factor, PpERF (Ethylene response factor, Prupe.1G130300), was grouped into the pink module.Based on the results, PpEXPS, PpEXPA1 and PpEXPA7, play a critical role in fruit texture, which might be regulated by the PpERF transcription factor.

Discussion
EXPs, as plant cell-wall loosening proteins, participate in cell growth and other development processes by modifying cell-walls [35].EXPs in peach were identified as having 26 members, of which all contained two conserved domains (DPBB and Pollen_allerg).Canonical expansin proteins in the plant were displayed as torpedo-shaped and consisted of the two domains (DPBB and Pollen_allerg) [20,36].There were proteins, such as G2As, p12 and plant natriuretic peptide, which contained a domain homologous only to expansin and one domain in the plant [37,38], while they lacked the wall-extension activity of expansins and could not be considered part of the expansin superfamily [39,40].In this study, we identified 27 genes featuring a DPBB domain or Pollen_allerg domain (Prupe.7G203100only with Pollen_allergens domain).Hence, 26 genes were confirmed as expansins, containing both of these two domains.
The PpEXPs within the subfamily contained a similar gene structure and motif composition, indicating that PpEXPs in the same subfamily were highly conserved.Motif 1 and Motif 2 were present only in PpEXPAs, while all PpEXPBs and PpEXPLs contained Motif 7 and Motif 10.Gene duplication analysis showed that DSD was the major pattern, which was consistent with the other gene families in peaches, such as E3 ubiquitin ligase genes [32], HSF [44] and GRAS [45].The model of gene duplication might furnish evidence for the evolution of species and was considered a driving force in evolution [46].The peach has not undergone a recent whole-genome duplication [47], which might lead to the duplication pattern of gene families, including PpEXPs.
The expression of gene response to developmental or environmental signals were mainly regulated through cis-acting elements.The divergence of the promoter regions was associated with the expression pattern of genes [48].In this study, the potential cis-acting elements in the 2 Kb region upstream of the start codons for each PpEXP were detected, with 21 types of cis-elements.It was found that light responsive elements and hormone responsive elements were apparently abundant for the PpEXPs, similar to other studies of EXPs [26,49,50].In soybeans, four GmEXPBs were induced by IAA and eight GmEXPs responded to 6-BA correspondingly, with the promoters containing hormone-related ciselements [51].The wheat expansin gene TaEXPB23 only showed improved water-stress tolerance with the stress-inducible promoter RD29A while also showing negative effects on plant growth and development under the 35S promoter, such as earlier flowering and shorter plant height [48].The results showed a diversity of cis-acting regulatory elements in the promoter region of PpEXPs, which might participate in various processes.
It has been widely reported that EXPs participate in root hair formation [52,53], biotic and abiotic stresses [54,55] and organ development [56].Cell wall loosening catalyzed by EXP was central during fleshy fruit ripening [7].To identify EXPs related to peach fruit development, we identified the expression pattern of PpEXPs in fruits of different flesh types, with MF, NMF and SH featuring at four stages.Only half of 26 PpEXPs were expressed in peach fruit (Table S4).The flesh of melting, non-melting and stony hard peaches showed significantly differences in regard to firmness and texture, and PpPGM, PGF and PpYUC11 showed positive correlations with fruit firmness [3,4].The expression of nine PpEXPs clustered with PpPGM and PpPGF in melting and non-melting peaches, and six PpEXPs showed similar expression patterns to PpYUC11 in melting and stony hard peaches.Combining the two pairs of expression patterns, three PpEXPs, PpEXPA7, PpEXPA13 and PpEXPA15, were highly expressed during ripening.The expression of the four candidate EXPs dramatically increased when firmness decreased in strawberries [30].In tomatoes, SlEXP1 promoted fruit softening cooperating with SlCel2 [18].It is worth noting that ethylene responsive elements and abscisic acid responsive elements were detected in the promoter region of PpEXPA7, PpEXPA13 and PpEXPA15; therefore, we propose that PpEXPs might be involved in regulating fruit textures in peaches.
In this study, we identified the gene modules co-expressed with fruit firmness.Of 16 modules, 2 modules, pink and midnightblue, showed significantly negatively correlations in terms of fruit firmness, including 125 genes.PpEXPA1 and PpEXPA7 were confirmed to be highly related to fruit texture based on the WGCNA.In addition, a transcription factor, PpERF, was identified in the correlation module, indicating a potential role for them in regulating fruit texture.WGCNA has been used to identify the coexpression modules related to volatile organic compounds (VOCs) in Osmanthus fragrans [57].It has been reported that three genes (Prupe.7G234800,Prupe.8G079200 and Prupe.8G082100) were obtained as candidate genes for single fruit weight traits based on the WGCNA in peaches [58].ERF has been demonstrated to activate the expression of genes involved in fruit softening, such as PG [59], PME [60], and EXP [61].In peaches, the PpEXP gene (ppa017982m) might control fruit weight [62].After a homologous comparison, ppa017982m was the PpEXPLB1 homologous gene.PpExp3, which is homologous with PpEXPA15, showed an increase in the expression of ethylene-treated 'Manami' (SH) [63].These findings implied that PpEXPs play an important role in fruit texture and might be regulated by the PpERF transcription factor.

Conclusions
In this study, we identified 26 PpEXP genes in the peach genome which can be divided into four subfamilies (PpEXPA, PpEXPB, PpEXPLA and PpEXPLB) based on the phylogenic analysis.The gene structure and motif of members proved to be highly diverse among different subfamilies.The model of gene duplication of PpEXPs were DSD (50%), WGD (35%) and TD (15%).Moreover, 15 PpEXP genes were expressed in peach fruit at four stages.Among them, PpEXPA7, PpEXPA13 and PpEXPA15 were coexpressions with PpPGM, PpPGF and PpYUC11, key genes for peach fruit texture.Meanwhile, the ciselement of ethylene responsiveness and abscisic acid responsiveness were detected in the promoter of these three genes.Based on a WGCNA, PpEXPA1, PpEXPA7 and PpERF were identified in the correlation module; thus, the results lay a functional foundation for the PpEXP gene family and for further exploring the roles of candidate PpEXPs in peach fruit texture and their regulatory work.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/horticulturae10040332/s1, Figure S1: The relative expression level of PpEXPs in the fruit of MF (HSM) and NMF (CN14); Table S1: Motif sequences identified by MEME tools in PpEXPs; Table S2: The gene duplication of EXP genes in peach; Table S3: The responsive elements of EXP genes in peach; Table S4: The expression of PpEXPs in fruit of melting, non-melting and stony hard peaches at differernt stages; Table S5: The number of genes in each module was identified by WGCNA method and gene annotation of GO; Table S6: Fruit firmness across fruit development of MF and NMF; Table S7: List of primers used in this study.

Figure 2 .
Figure 2. Gene structure and motif analysis of PpEXPs.(A): Phylogenetic relationships of PpEXPs; (B): Gene structure of PpEXPs.Yellow box, turquoise box and black line represent exon, UTR and intron, respectively; (C): Conserved motif of PpEXPs.Different color boxes represent different motifs, noted in upper-right.The subfamily of PpEXPA apparently differed from the other subfamilies in motif composition.All the PpEXPAs contained Motifs 1, 2, 4 and 6, and most of the PpEXPAs also contained Motifs 3, 5 and 8, except PpEXPA10, PpEXPA11, PpEXPA12 and PpEXPA20 (Figure 2C, TableS1).All the PpEXPBs contained the same motifs, including Motif 1, Motif 4, Motif 7, Motif 9 and Motif 10.The motif analysis was consistent with the phylogenetic analyses, showing similar motif composition in members in the same subfamily.

Figure 2 .
Figure 2. Gene structure and motif analysis of PpEXPs.(A): Phylogenetic relationships of PpEXPs; (B): Gene structure of PpEXPs.Yellow box, turquoise box and black line represent exon, UTR and intron, respectively; (C): Conserved motif of PpEXPs.Different color boxes represent different motifs, noted in upper-right.The subfamily of PpEXPA apparently differed from the other subfamilies in motif composition.All the PpEXPAs contained Motifs 1, 2, 4 and 6, and most of the PpEXPAs also contained Motifs 3, 5 and 8, except PpEXPA10, PpEXPA11, PpEXPA12 and PpEXPA20 (Figure 2C, TableS1).All the PpEXPBs contained the same motifs, including Motif 1, Motif 4, Motif 7, Motif 9 and Motif 10.The motif analysis was consistent with the phylogenetic analyses, showing similar motif composition in members in the same subfamily.

Figure 3 .
Figure 3. Chromosome distribution and gene duplication of PpEXPs.The genes with the same color represented a type of gene duplication: red, DSD; blue, TD; green, WGD.Syntenic pairs were linked with lines, with colors representing different pairs.

Figure 3 .
Figure 3. Chromosome distribution and gene duplication of PpEXPs.The genes with the same color represented a type of gene duplication: red, DSD; blue, TD; green, WGD.Syntenic pairs were linked with lines, with colors representing different pairs.

Figure 4 .
Figure 4.The cis-elements in the promoters of PpEXPs.(A): The different types of cis-elements, with different colors, were shown in the promoter region of each PpEXP.(B): The number of each ciselement in the promoter regions.

Figure 4 .
Figure 4.The cis-elements in the promoters of PpEXPs.(A): The different types of cis-elements, with different colors, were shown in the promoter region of each PpEXP.(B): The number of each cis-element in the promoter regions.

Figure 4 .
Figure 4.The cis-elements in the promoters of PpEXPs.(A): The different types of cis-elements, with different colors, were shown in the promoter region of each PpEXP.(B): The number of each ciselement in the promoter regions.

Figure 6 .
Figure 6.WGCNA of expressed genes at four stages of HSM and CN fruit.(A): Hierarchical cluster tree of WGCNA analysis (R 2 = 0.85).(B): Module-trait correlation analysis.Sixteen modules were labeled with different colors.The numbers in each row represent the correlation of every module, the numbers in parentheses indicate the p-values of correlations, and the gene numbers were displayed in the right of each module.

Table 1 .
The characteristics of EXP genes in peach.