Genome-Wide Identification of Expansins in Rubus chingii and Profiling Analysis during Fruit Ripening and Softening

Improving fruit size or weight, firmness, and shelf life is a major target for horticultural crop breeding. It is associated with the depolymerization and rearrangement of cell components, including pectin, hemicellulose, cellulose, and other structural (glyco)proteins. Expansins are structural proteins to loosen plant cell wall polysaccharides in a pH-dependent manner and play pivotal roles in the process of fruit development, ripening, and softening. Rubus chingii Hu, a unique Chinese red raspberry, is a prestigious pharmaceutical and nutraceutical dual-function food with great economic value. Thirty-three RchEXPs were predicted by genome-wide identification in this study, containing twenty-seven α-expansins (EXPAs), three β-expansins (EXPBs), one expansin-like A (EXPLA), and two expansin-like B (EXPLBs). Subsequently, molecular characteristics, gene structure and motif compositions, phylogenetic relationships, chromosomal location, collinearity, and regulatory elements were further profiled. Furthermore, transcriptome sequencing (RNA-seq) and real-time quantitative PCR assays of fruits from different developmental stages and lineages showed that the group of RchEXPA5, RchEXPA7, and RchEXPA15 were synergistically involved in fruit expanding and ripening, while another group of RchEXPA6 and RchEXPA26 might be essential for fruit ripening and softening. They were regulated by both abscisic acid and ethylene and were collinear with phylogenetic relationships in the same group. Our new findings laid the molecular foundation for improving the fruit texture and shelf life of R. chingii medicinal and edible fruit.


Introduction
Rubus chingii Hu is an economically important Eastern Chinese raspberry with a prestigious pharmaceutical and nutraceutical dual function [1,2].The unripe fruit harvested at the stages of big green (BG) or green-to-yellow (GY) has been used in traditional Chinese medicine for tonifying kidneys and eyesight from the ancient Qin and Han Dynasties [1,3].It has been widely used as a key ingredient in some Chinese Proprietary Medicines, such as Wuzi Yanzong Pill, Guilu Bushen Pill, Shenbao Mixture, and others, or soaked in white wine for medicinal liquor in folk [1].More than 235 chemical constituents, including terpenoids, flavonoids, phenolics, polysaccharides, and others, have been identified and isolated in recent years [4][5][6].The pharmacological studies indicated the therapeutical effects of antiinflammation, antioxidantion, antidiabetic, anti-thrombosis, anti-tumor, neuroprotective, and liver protection of R. chingii fruit and leaves [1,4,[7][8][9][10][11].Simultaneously, the ripe fruit of R. chingii is popular for pleasant fresh fruit, like raspberries and blackberries, and has rich nutrients, including amino acids, phenolic acids, vitamins C and PP, ellagitannins, aromatics, and minerals, such as magnesium, zinc, and potassium, and it is beneficial Plants 2024, 13, 431 3 of 18 hensive understanding of fruit development and softness for these attractive berries.In this study, we identified 33 expansins encoding genes from R. chingii and systematically analyzed the bioinformation of RchEXPs and their roles in fruit ripening and softening.The results provided valuable insights into the characterization and function of the expansin genes family in R. chingii, an important Chinese herb and red raspberry.

Identification of Expansin Family Members in Rubus chingii
A total of 39 genes and their proteins were initially obtained by the HMM search.Among these, thirty proteins consisting of two conserved domains (DPBB_1 and Ex-pansin_C domains) were selected as expansins candidates in R. chingii.In addition, proteins with one complete DPBB_1 domain, such as LG04.3299,LG04.3300, and LG04.3301, were also retained according to strawberry expansins [21,25].Therefore, 33 RchEXPs were identified in this study (Tables 1 and S1).Furthermore, expansin genes in its sister plants, red raspberry (Rubus idaeus) and black raspberry (Rubus occidentalis), were also extracted from the genomic data, and a similar number of members were obtained (Tables S1 and S2), indicating the similarity of the same Rubus.The amino acids of RchEXPs ranged from 125 aa (RchEXPA8, molecular weight MW = 12,970.17)to 482 aa (RchEXPA4, MW = 53,196.43)and the isoelectric point (pI) of most members (26/33) were greater than 7, indicating these members might be rich in alkaline amino acids.The prediction of the subcellular location showed that RchEXPs were all located on the cell wall.Phylogenetic analyses showed RchEXPs had relatively distant relatives with AtEXPs (Figure S1a), while they were more closely related to FvEXPs, RiEXPs, and RoEXPs, which belonged to the same Rosaceae (Figure 1).Based on the phylogenetic relationships with FvEXPs, all RchEXPs were divided into four subfamilies, including twenty-seven (81.8%) members of EXPA, three members of EXPB, one member of EXPLA, and two members of EXPLB (Tables 1 and S1).Twenty-three orthologous pairs in R. chingii and F. vesca, and five paralogous pairs in R. chingii, were identified based on the high bootstrap values (>90%) (Figures 2 and S1b).

Gene Structures and Protein Motifs of RchEXPs
The analyses of gene structures of RchEXPs exhibited the members of exons and varied from two to six, and introns varied from one to five (Figure 2).All RchEXPBs and RchEXPLB1 had four exons, whereas RchEXPLA and RchEXPLB2 contained five exons.Among the EXPA subfamily, 37% of the members contained two exons and 40.7% of the members contained three exons, whereas the others had four, five, or six exons.They could be divided into six clusters, and most members in the same cluster showed consistent exon numbers.All these results indicated the similarities of the same subfamily in structure.

Cis-Regulatory Elements and Interaction Analyses of RchEXPs
Analysis of cis-acting elements on the promoter sequences (2 kb upstream of ATG) could predict the regulatory factors and temporal-spatial expression patterns of RchEXPs (Figure 5a).First, cis-regulatory elements of a phytohormone response, such as abscisic acidresponsive elements (ABREs and AAGAA-motif), ethylene-responsive elements (EREs), auxin-responsive elements (AuxRR-core and TGA-element), gibberellin (GA)-responsive elements (GARE-motif, P-box and CARE), salicylic acid (SA)-responsive element (TCAelement) SA, auxin-response element (as-1), and jasmonic acid methyl ester (MeJA) responsive elements (TGACG-motif and CGTCA-motif) were detected in this study.The results showed promoters of most RchEXPs possessed ABRE sites, except for EXPA1, A8, A22, and A24.Moreover, the majority of them contained multiple ABRE sites, such as nine sites of the RchEXPA13 promoter, six sites of the RchEXPA4 promoter, and four sites of the RchEXPA5, RchEXPA6, RchEXPA26, RchEXPA3, RchEXPA9, RchEXPA14, RchEXPA20, RchEXPB1, and RchEXPLB1 promoters.In addition, promoters of major RchEXPs had 1-4 AAGAA-motif sites.Both ABRE and AAGAA-motif sites indicated the possible ABA regulation of RchEXPs.Promoters of some RchEXPs had ERE sites, such as RchEXPA26, RchEXPA1-2, RchEXPA7-9, RchEXPA11-12, and others.There was an auxin-responsive element (TGA-element) in the promoter of RchEXPA5, RchEXPA6, and RchEXPA7.Most RchEXPs were associated with SA and MeJA, but may not respond to GA.Taken together, the five genes that were highly expressed in fruit (Figure 6), including RchEXPA5, Rch-EXPA6, RchEXPA7, RchEXPA15, and RchEXPA26, all could respond to the ABA signal, while only RchEXPA7 and RchEXPA26 were putatively regulated by Eth (Figure 5a).These results predicted the main regulation of ABA in non-climacteric fruits [14].Protein-protein interaction networks (PPIs) were constructed by a cytoscape tool based on transcriptome data (Figure 5b).RchEXPA6 was predicted to strongly interact with cell wall hydrolases, including PME and PG.It also could interact with an ethylene receptor (ETR) and ethylene-insensitive protein 2 (EIN2) and then result in ethylene-induced responses.Meanwhile, RchEXPA26 also could tightly interact with PG.These results suggest that RchEXPA6 and RchEXPA26 were apt in their involvement in fruit softening.RchEXPA5 was predicted to interact with PME, xyloglucan/xyloglucosyl transferase (XET), sucrose synthase (SS), and others.Hence, it might work earlier in fruit development.No PPI was found between RchEXPA7 and other proteins.Only UDP glucose 6dehydrogenase (UGDH) was found to interact with RchEXPA15.Secondly, cis-regulatory elements of plant growth and development were analyzed, including elements involved in light responsiveness (MRE, Sp1, TCCC-motif, ATCT-motif, GT1-motif, I-box, TCT-motif, A-box, AE-box and GATA-motif), meristem expression (CATbox), circadian control (circadian), seed-specific regulation (RY-element), zein metabolism regulation (O2-site), endosperm expression (GCN4_motif), and MYBHv1 and AT-rich DNA binding protein (ATBP-1) binding sites (CCAAT-box and AT-rich element).The results indicated the action of RchEXPs was mainly regulated by light in divergent ways (Figure 5a).Finally, RchEXPs might actively participate in stress responses because of the most abundant sites in their promoters, including cis-acting elements of STRE (woundresponsive element), ARE (for anaerobic induction), LTR (involved in low-temperature responsiveness), MBS (involved in drought inducibility), TC-rich repeats (involved in defense and stress responsiveness), WUN-motif (wound-responsive element), DRE core, W box, and WRE3, especially MYB and MYC.Five genes, RchEXPA5, RchEXPA6, RchEXPA7, RchEXPA15, and RchEXPA26, all could respond to anaerobic and wound stress and could be regulated by MYBs and MYCs, whereas RchEXPA6, RchEXPA15, and RchEXPA26 could respond to drought (Figure 5a).
Protein-protein interaction networks (PPIs) were constructed by a cytoscape tool based on transcriptome data (Figure 5b).RchEXPA6 was predicted to strongly interact with cell wall hydrolases, including PME and PG.It also could interact with an ethylene receptor (ETR) and ethylene-insensitive protein 2 (EIN2) and then result in ethylene-induced responses.Meanwhile, RchEXPA26 also could tightly interact with PG.These results suggest that RchEXPA6 and RchEXPA26 were apt in their involvement in fruit softening.RchEXPA5 was predicted to interact with PME, xyloglucan/xyloglucosyl transferase (XET), sucrose synthase (SS), and others.Hence, it might work earlier in fruit development.No PPI was found between RchEXPA7 and other proteins.Only UDP glucose 6-dehydrogenase (UGDH) was found to interact with RchEXPA15.

Expression Profiles of RchEXPs in Different Fruit Ripening and Softening Stages of R. chingii
The fresh weight of the eight stages (SG-small green, MG-middle green, BGIbig green I, BGII-big green II, BGIII-big green III, GY-green to yellow, YO-yellow to orange, Re-red) during R. chingii fruit development were 0.25, 0.48, 0.97, 1.02, 1.02, 1.43, 1.80, and 4.54 g, respectively [5].Fruit firmness before the 60% red stages was all >15 kg/cm 2 , whereas fruit firmness of the 70% red, 80% red, and 90% red stages were 10.81, 8.47, 6.48 kg/cm 2 , respectively, indicating that the firmness was gradually reduced with fruit ripening.The data of transcriptomic sequencing showed the divergent expression pattern of all the expansin genes in R. chingii.Most RchEXPs were not detected or had very low expression levels in fruit, such as RchEXPA2-3, RchEXPA8, RchEXPA10-12, RchEXPA14, RchEXPA16-24, RchEXPA27, RchEXPB2, and RchEXPLB1.They might play vital roles in the root, stem, leaf, and flowers, or respond to different kinds of stresses.Nevertheless, some RchEXPs exhibited remarkable effects on fruit ripening and softening (Figure 6).Especially, RchEXPA6 and RchEXPA26 might be notably involved in fruit expanding and softening.The expression level of RchEXPA6 at the immature BGI stage was 25.66 (FPKM, fragments per kilobase per million mapped fragments), while it rapidly increased to 6774.05 at the turning stage of GY, decreased at the YO stage, and then finally dramatically elevated to 14360.67 at the red stage, which was 559.65-fold in comparison with BGI (Figure 6a).The qPCR result verified its strong expression in the red stage (Figure 6c).The expression levels of RcEXPA6 were related to fruit size, fresh weight, and firmness throughout the whole fruit development.In addition, RchEXPA6 kept the highest expression level through the softening process from 70% to 90% ripe (Figure 6b).Simultaneously, the expression pattern of RchEXPA26 was in line with RchEXPA6 (Figure 6a-d).Oppositely, the expression levels of RchEXPA5 and RchEXPA15 were at high values at the beginning of the fruit set and earlier stages and decreased at later stages (Figure 6a,b,e,f).The FPKM of RchEXPA5 and RchEXPA15 at the BGI stage were 348.06 (449.34/1.29)and 146.04 (186.93/1.28)folds in comparison with the red stage, respectively.The RchEXPA7 had relatively high expression levels from BG to 80% Re and then sharply reduced to a low level at 90% Re.Therefore, RchEXPA5, RchEXPA7, and RchEXPA15 mainly participated in the process of cell expansion and fruit ripening.Therefore, red fruit from different lineages with different fruit sizes and different hardness were further analyzed.The fresh weights and firmness of L1, L2, L3, L6, L7, L14, L20, L26, L20⊗ (selfing), L3 × L7, L3 × L20, and L14 × L3 were measured (Figure 7a,b).The fruit of L3 × L7 was the smallest, and the fruit of L20⊗ was the biggest (Figure 7a).The 90% red fruit of L26, L20⊗, L1, L3 × L20, and L2 were relative hard, while the fruit of L3 × L7, L3, L6, L7, and L14 × L3 were softer (Figure 7b).The expression levels of RchEXP6 and RchEXP26 were relatively higher in softer fruits, while the expression levels of RchEXP7 and RchEXP15 were relatively higher in bigger fruits (Figure 7c-f).Pearson correlation analysis showed that RchEXP6 and RchEXP26 had significantly negative correlations with hardness, while RchEXP7 and RchEXP15 positively correlated with fresh weight (Figure 7g).These results verified the essential functions of RchEXPA6 and RchEXPA26 for fruit expanding and softening, while RchEXP7 and RchEXP15 mainly participated in fruit expansion and ripening.These experimental results were greatly consistent with the bioinformatics analysis.In Figure 2, the phylogenetic relationships confirmed that RchEXPA6 and RchEXPA26 were in cluster 6, and this cluster was composed of only these two proteins.Moreover, in Figure 4, syntenic relationships also predicted the collinear and paralog gene pairs of RchEXPA6 and RchEXPA26, suggesting the synergistic and complementary of the two EXPs.Simultaneously, RchEXPA7 and RchEXPA15 were in cluster 7, whereas RchEXPA5 was in cluster 5.In Figure 1, these three proteins were gathered in the same upper cluster.RchEXPA7 and RchEXPA15 showed a syntenic relationship.PPI analysis showed the interaction among RchEXPA6, RchEXPA26, PME, PG, ETR, and EIN2 (Figure 5b).The consistent functions of the two expansin genes RchEXPA6 and RchEXPA26, or the three ex- These experimental results were greatly consistent with the bioinformatics analysis.In Figure 2, the phylogenetic relationships confirmed that RchEXPA6 and RchEXPA26 were in cluster 6, and this cluster was composed of only these two proteins.Moreover, in Figure 4, syntenic relationships also predicted the collinear and paralog gene pairs of RchEXPA6 and RchEXPA26, suggesting the synergistic and complementary of the two EXPs.
Simultaneously, RchEXPA7 and RchEXPA15 were in cluster 7, whereas RchEXPA5 was in cluster 5.In Figure 1, these three proteins were gathered in the same upper cluster.RchEXPA7 and RchEXPA15 showed a syntenic relationship.PPI analysis showed the interaction among RchEXPA6, RchEXPA26, PME, PG, ETR, and EIN2 (Figure 5b).The consistent functions of the two expansin genes RchEXPA6 and RchEXPA26, or the three expansin genes RchEXPA5, RchEXPA7, and RchEXPA15, were first identified in this study.Unveiling the action mechanism and regulatory pathways of these vital EXPs would be vital for genetic modification and biotechnological breeding for fruit quality improvement and softening delay.Unveiling the action mechanism and regulatory pathways of these vital EXPs would be vital for genetic modification and biotechnological breeding for fruit quality improvement and softening delay.

Discussion
The plant cell wall is pivotal for cell size, shape, and strength, and affects cell division and differentiation, growth rate, and functional realization [23,26].It can be hydrolyzed, modified, or reconstructed by a series of enzymic and non-enzymic proteins.Expansins (EXPs) are noncatalytic structure proteins that are predominately located in the cell wall.They are involved in almost all aspects of plant growth and development, from germination to fruiting, by loosening the cell walls [22,24,25,27].They also play vital roles in stress responses [22,28,29].In total, 35 and 114 EXPs have been identified in woodland strawberry Fragaria vesa and octoploid-cultivated strawberry F. ananassa with distinct expression patterns [21,25].In this study, we identified 33 EXPs from the R. chingii genome for the first time (Table 1, Figures 1-3).Transcriptomic and quantitative PCR profiles verified that RchEXPA5, RchEXPA7, and RchEXPA15 were synergistically involved in fruit expansion and ripening, while RchEXPA6 and RchEXPA26 might be essential for fruit ripening and softening (Figure 6).Our new findings laid a molecular foundation for improving fruit quality, controlling fruit firmness, and extending the storage life of R. chingii edible red fruit.

Discussion
The plant cell wall is pivotal for cell size, shape, and strength, and affects cell division and differentiation, growth rate, and functional realization [23,26].It can be hydrolyzed, modified, or reconstructed by a series of enzymic and non-enzymic proteins.Expansins (EXPs) are noncatalytic structure proteins that are predominately located in the cell wall.They are involved in almost all aspects of plant growth and development, from germination to fruiting, by loosening the cell walls [22,24,25,27].They also play vital roles in stress responses [22,28,29].In total, 35 and 114 EXPs have been identified in woodland strawberry Fragaria vesa and octoploid-cultivated strawberry F. ananassa with distinct expression patterns [21,25].In this study, we identified 33 EXPs from the R. chingii genome for the first time (Table 1, Figures 1-3).Transcriptomic and quantitative PCR profiles verified that RchEXPA5, RchEXPA7, and RchEXPA15 were synergistically involved in fruit expansion and ripening, while RchEXPA6 and RchEXPA26 might be essential for fruit ripening and softening (Figure 6).Our new findings laid a molecular foundation for improving fruit quality, controlling fruit firmness, and extending the storage life of R. chingii edible red fruit.
The expansins can be divided into four subfamilies according to the phylogenetic analysis.EXPA members are the main components of expansins in all kinds of plants, while EXPB members might mainly exist in Gramineae monocotyledons.In dicotyledonous plants, Arabidopsis has twenty-six, six, three, and one members of EXPA, EXPB, EXLA, and EXLB, respectively; polar (Populus) contains twenty-seven, three, two, and four members of the four subfamilies, respectively; and F. vesa consists of twenty-seven EXPAs, five EXPBs, one EXPLA, and two EXPLBs, respectively [25].The proportion of EXPs in R. chingii is similar to F. vesa.RchEXPs are composed of twenty-seven EXPAs, three EXPBs, one EXPLA, and two EXPLBs (Table 1, Figure 2).These results suggest that the EXPA subfamily in dicotyledons might be expanded far more than the other three subfamilies.Nevertheless, Gramineae plants contain higher numbers of EXPB members.There are 34 EXPAs and 19 EXPBs in rice, 36 EXPAs and 48 EXPBs in maize, and 45 EXPAs and 29 EXPBs in moso bamboo [22,25].The cell wall is fundamentally made up of different groups of polysaccharides, such as cellulose, hemicellulose, and pectin.Loosening of the polysaccharides network is the direct cause of cell wall looseness and cell expansions [30].Xyloglucan is the main hemicellulose in the primary wall of dicotyledonous and nongramineous monocotyledonous plants, comprising up to 20% of the wall dry matter [31].However, the content of xyloglucan in gramineous plants was very low.EXPAs promote movement and separation of microfibrils by means of molecular creep for the dissociation and slippage of xyloglucans, while EXPBs work on another glycan, maybe xylan [19, 23,32].Among the xylans, arabinoxylan (AX) mainly exists in the grain of gramineous plants, glucuronoarabinoxylan (GAX) exists in vegetative tissues of gramineous plants and the primary cell wall of dicotyledonous, and glucuronoxylan (GX) mainly exists in the secondary wall of dicotyledonous and non-gramineous monocotyledonous plants [33].Therefore, the various compositions of plant cell wall polysaccharides and the distinct action modes of EXPs might determine the proportion and numbers of EXP subfamilies.
Furthermore, it has been reported that xyloglucan disassembly might be an early event in fruit softening [24], and firmness is one of the decisive indexes of fruit quality and postharvest shelf life.The effects of expansins on fruit ripening and softening have been described in various horticultural plants.For example, the overexpression of tomato Slexp1 hastened the softening process, while Slexp1-6 and Slexp1-7 mutants enhanced fruit firmness and could be stored for longer periods [34].FaEXP2 and FaEXP5 were predominantly expressed in cultivated octaploid strawberry fruit, and the significant increase in the FaEXP5 expression level was in close correlation with the rapid decrease in fruit firmness [19,35].The above expression pattern of EXP5 was also observed in Chilean strawberries [36].In addition, FaEXP7, FaEXP88, and FaEXP114 were also candidate genes for softening activation [20,21].Our experiment evaluated the expression pattern of RchEXPs during fruit development and ripening and found that three expansin genes of RchEXPA5, RchEXPA7, and RchEXPA15 were highly expressed at earlier stages but decreased at later stages, suggesting that they were predominantly involved in cell expansions and fruit ripening; while the expressions of the two expansin genes, RchEXPA6 and RchEXPA26, were dramatically activated at red stage, indicating their essential role in fruit softening (Figure 6).In F. vesa, both Dong et al. [25] and Mu et al. [21] reported that three FvEXPs were highly expressed in ripening fruit.Among these, FvEXPA9 (FveEXP11) and FvEXPA12 had especially high expression levels in the turning stage, while FvEXPA27 (FveEXP33) had especially high expression levels in the red stage and over-ripening stage.Phylogenetic analysis and synteny profiles showed that different expansins from diverse species belonging to the same clade or collinearity contributed to similar functions [21].As expected, RchEXPA6 had collinearity with RchEXPA26 and FvEXPA27 (Figure 4, Table S3), and the encoded protein had high structural similarity with FvEXP27 (Figure 2, Figure S1).Therefore, they exhibited the same function (Figure 6).Simultaneously, there were collinear relationships and close evolutionary relationships between RchEXPA5 and FvEXPA9, RchEXPA15 and FvEXPA12, as well as RchEXPA15 and RchEXPA7 (Figure 4), resulting in their consistent function.Overall, our results elucidated the key expansin encoding genes in fruit ripening and softening.
The regulator of expansins in R. chingii was also predicted through promoter and PPI analyses.Promoters of most RchEXPs showed multiple ABA-responsive element sites, and some of them also possessed Eth-responsive elements (Figure 5).Numerous studies have revealed that ethylene and ABA both could regulate fruit ripening in strawberries [14,37].ERE was found upstream of FvEXPA9 and FvEXPA27 [25].In this study, ERE was found in the promoter region of RchEXPA7 and RchEXPA26, whereas it was absent in promoters of RchEXPA5, RchEXPA6, and RchEXPA15 (Figure 5).Therefore, the ripening of R. chingii might be controlled by both ABA and Eth, and the RchEXPs act coordinately for fruit texture.It has been reported that the high relative expression of FvEXP12 in the turning stage was attributed to the auxin-responsive element in its promoter [25].Interestingly, RchEXPA7 had collinearity with FvEXP12 (Table S3), and RchEXPA7 was also the orthologous gene for FvEXP12.Hence, the high expression level of RchEXPA7 in the turning stage consisted of strawberry expansin.
In addition, many pieces of evidence also supported the effects of expansins on stress resistance.TaEXPA2 regulated by MYB transcription factor enhanced drought tolerance in wheat [29].PeEXPA19, highly expressed in the leaves and roots of moso bamboo, had three MBS elements in its promoter and exhibited drought resistance, whereas PeEXPA44 showed downregulated expression under PEG stress [22].Therefore, the functions of other expansins in R. chingii need to be further investigated.All expansins are distributed in different plant tissues and have distinct roles, whereas they are synergistically involved in plant growth, development, maturity, senescence, and response to stresses.

Conclusions
In conclusion, this study performed the first genome-wide identification of 33 expansins and their encoding genes in the unique Chinese raspberry, Rubus chingii Hu.The molecular characterization, biological function, and evolutionary pattern were elucidated.The results confirmed that three RchEXPs, including RchEXP5, RchEXP7, and RchEXP15, and two RchEXPs, RchEXP6 and RchEXP26, played pivotal roles in fruit ripening and softening, respectively.Our study gives a comprehensive understanding of the expansin family in R. chingii and screens out the candidate genes for molecular breeding to slow down fruit softening and extend storage life.

Motifs, Gene Structures, Chromosomal Location, and Synteny Analysis of Expansins in R. chingii
The gene structures of exon/intron were mapped using TBtools according to the genome annotation "Rubus_chingii_Hu.gff"file.Conserved motifs were identified with the online Multiple Em for Motif Elicitation (MEME, https://meme-suite.org/meme/tools/meme, accessed on 27 May 2021) tool with the parameter setting of the maximum number of motifs equal to 10. Gene density was profiled, and gene locations were visualized by TBtools.The synteny analysis in R. chingii genome and synteny analysis between R. chingii and F. vesa genomes were calculated with MCScanX (CPU for BlastP: 2; E-value: 1 × 10 −10 ; Num of BlastHits:5) [38].The syntenic diagram was visualized by an advanced circos and dual synteny plot using TBtools.

Cis-Regulatory Elements Analysis and Protein-Protein Interaction Network Construction of Expansins in R. chingii
The 2000 bp upstream genomic sequences of the expansin genes were extracted as the putative promoters to examine the cis-elements by PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/,accessed on 27 May 2021).The predicted cis-acting elements and their numbers for phytohormone response, plant growth, and development, were summarized, respectively.The protein-protein interaction network (PPI) was analyzed based on transcriptome data, and the protein-protein interaction network (PPI) was constructed using cytoscape_v3.10.1.

Expression Analyses of RchExps during Developmental Stages
Transcriptomic sequencing of fruits from the four representative stages (BG, GY, YO, and Re) of L7 was carried out in 2017 using the Illumina (HiSeq X-Ten, San Diego, CA, USA) platform from the Beijing Genomics Institute (BGI, Wuhan, China).Approximately 536.05 Mb total clean reads with high quality (Q20 > 98.5%, Q30 > 82.5%) were obtained from all samples (4 stages in triplicate) [5].Then, the de nova sequencing data were reanalyzed in 2021, according to the later published R. chingii genome [6].The expression levels of RchEXPs were expressed by Fragments Per Kilobase per Million mapped fragments (FPKM) value (=10 6 C/(NL/10 3 ) and mapped as a heat map using TBtools.Moreover, RNA-seq of the ripening fruits of 70% red, 80% red, and 90% red from L26 was performed in 2021 using the DNBseq platform by the Beijing Genomics Institute (BGI, Wuhan, China).A total of 380.41 million total clean reads with high quality were obtained from all 9 samples (3 stages in triplicate), and the FPKM values of RchEXPs were extracted and mapped with the above method.
For a further real-time quantitative PCR (aPCR) assay, fruits of eight stages from L7 (SG, MG, BGI, BGII, BGIII, GY, YO, and Re) were harvested in 2021.A total of about 50 fruits at each stage were collected, and RNA was isolated using an OminiPlant RNA

Figure 1 .
Figure 1.Phylogenetic tree of expansins from Rubus chingii, Fragaria vesca, Rubus idaeus, and R. occidentalis.EXPA, EXPB, EXPLA, and EXPLB subfamilies were presented in light orange, yellow, blue, and green."⋆"indicates the same expression tendency during fruit development stages which was highest at the red stage, while "▲" indicates that these gene expressions were initiated in earlier stages of fruit.

Figure 2 .
Figure 2. Phylogenetic relationships, gene structures of exon/intron, and motif compositions of expansins in Rubus chingii.Figure 2. Phylogenetic relationships, gene structures of exon/intron, and motif compositions of expansins in Rubus chingii.

Figure 2 .
Figure 2. Phylogenetic relationships, gene structures of exon/intron, and motif compositions of expansins in Rubus chingii.Figure 2. Phylogenetic relationships, gene structures of exon/intron, and motif compositions of expansins in Rubus chingii.

Figure 4 .
Figure 4. Synteny analyses of R. chingii and F. vesa genomes.(a) Synteny analysis of R. chingii genome.The gene density was displayed in the form of heat maps and lines.Syntenic blocks were linked by gray lines, and syntenic relationships of EXP members were highlighted by red color.(b) Syntenic relationships of R. chingii and F. vesa genomes.Syntenic EXP gene pairs between R. chingii and F. vesa were highlighted by red color.

Figure 4 .
Figure 4. Synteny analyses of R. chingii and F. vesa genomes.(a) Synteny analysis of R. chingii genome.The gene density was displayed in the form of heat maps and lines.Syntenic blocks were linked by gray lines, and syntenic relationships of EXP members were highlighted by red color.(b) Syntenic relationships of R. chingii and F. vesa genomes.Syntenic EXP gene pairs between R. chingii and F. vesa were highlighted by red color.

Figure 5 .
Figure 5. Predicted cis-acting elements and protein-protein interaction networks of expansins in R. chingii.(a) Predicted cis-acting elements and their numbers in the promoters of expansin genes in R. chingii.(b) Construction of protein-protein interaction networks of expansins and other proteins in R. chingii.

Figure 5 .
Figure 5. Predicted cis-acting elements and protein-protein interaction networks of expansins in R. chingii.(a) Predicted cis-acting elements and their numbers in the promoters of expansin genes in R. chingii.(b) Construction of protein-protein interaction networks of expansins and other proteins in R. chingii.

Plants 2024 , 20 Figure 6 .
Figure 6.Expression profile of RchEXPs during fruit ripening and softening.(a) RchEXPs expression levels at the four representative stages, BGI, GY, YO, and Re, by RNA-seq.(b) RchEXPs expression levels at the red stages of the different degrees of ripeness by RNA-seq.(c-f) qPCR of RchEXPA6, RchEXPA26, RchEXPA5, and RchEXPA15.Different lower cases mean the significant difference at p < 0.05 by a one-way analysis of variance with the LSD method.

Figure 6 .
Figure 6.Expression profile of RchEXPs during fruit ripening and softening.(a) RchEXPs expression levels at the four representative stages, BGI, GY, YO, and Re, by RNA-seq.(b) RchEXPs expression levels at the red stages of the different degrees of ripeness by RNA-seq.(c-f) qPCR of RchEXPA6, RchEXPA26, RchEXPA5, and RchEXPA15.Different lower cases mean the significant difference at p < 0.05 by a one-way analysis of variance with the LSD method.

Figure 7 .
Figure 7. Biomass and real-time quantitative PCR of RchEXPs in red fruit of different R. chingii lineages.(a) Fresh weight (Fw); (b) fruit firmness; (c-f) qPCR of RchEXPA6, RchEXPA26, RchEXPA7, and RchEXPA15.Different lower cases mean a significant difference at p < 0.05 by a one-way analysis of variance with the LSD method.(g) Pearson correlation analysis of RchEXPs and fruit fresh weight and firmness based on the qPCR data of red fruits from different lineages."*" means a significant difference at p ≤ 0.05.

Table 1 .
Characteristics of RchEXP family members in Rubus chingii Hu.

Figure 1 .
Phylogenetic tree of expansins from Rubus chingii, Fragaria vesca, Rubus idaeus, and R. dentalis.EXPA, EXPB, EXPLA, and EXPLB subfamilies were presented in light orange, yellow, and green."★"indicates the same expression tendency during fruit development stages which highest at the red stage, while "▲" indicates that these gene expressions were initiated in ea stages of fruit.