Transcriptome of lncRNAs and mRNAs and Their Network Profile in Relation to Phenotypic Variation in Grafted Peach–Apricot Chimeras

Abstract

Grafted plants carrying DNA from both species are prone to new phenotypes. Specific long non-coding RNA (lncRNA) sequences are known to play roles in the formation and development of grafted plants. However, the roles of lncRNAs in phenotypic variation in grafts between peach and apricot remain unexplored. In this study, mixed tissues (leaves, buds and fully bloomed flowers) of peach branches from heterografts between apricot/peach (A/P) and peach/apricot (P/A) and homografted peach (SP) were collected for transcriptome sequencing. The differentially expressed genes (DEGs) and lncRNAs (DElncRNAs) between A/P and P/A were identified as candidates mediating the formation of divergent traits. Compared with SP, 1115 and 624 DEGs were detected in A/P and P/A, respectively. There were 173 DEGs shared between A/P and P/A, whereas the transcripts of 942 genes were specifically altered in A/P and 451 DEGs were specific to P/A. There were 29 DElncRNAs in A/P and 26 DElncRNAs in P/A, of which, 21 DElncRNAs were specific to A/P and 18 were specific to P/A. The biological functions of the DEGs and DElncRNAs were predicted via GO and KEGG enrichment analyses. A total of 24 co-expressed ‘lncRNA-mRNA’ pairs were identified, including 14 ‘lncRNA-mRNA’ pairs in A/P and 10 ‘lncRNA-mRNA’ pairs in P/A. The ‘MSTRG.17020.2-XM_007210198-2’ pair potentially participates in aminoacyl biosynthesis, and the ‘MSTRG.8395.1-XM_007217967.2’ pair may regulate galactose metabolism. The lncRNA MSTRG.6365.3 may regulate defense response through altering the levels of XM_020556240.1 and XM_020556234.1. These findings provide valuable insights into the molecular mechanisms underlying grafting-induced differential trait formation and establish a foundation for further research on the functional roles of ‘lncRNA-mRNA’ pairs in fruit tree grafting systems.

1. Introduction

Grafting is a traditional technique that joins the shoot system (scion) of one plant to the root system (rootstock) of another plant. Grafting has been used for more than 2000 years and continues to be widely used in the propagation of agricultural and forestry crops [1,2,3]. Grafting between plants of distinct genotypes can induce the exchange of genetic materials between the rootstock and scion and even an interstock between the two, leading to significant changes in the traits of the scion and the emergence of new phenotypes [4,5]. This phenomenon of rootstock–scion interaction is termed ‘graft hybridization’, and the resulting novel plants are referred to as ‘grafted hybrids’ or ‘grafted chimeras’. Chimeras derived from heterografting are more prone to developing new, stably heritable traits, which provides a novel approach for creating a new germplasm [6,7,8].

Grafting exerts significant effects on the chimeric plants, altering traits related to height [9,10], growth vigor [11], flowering period [12,13], fruit size and shape [14], yield and quality [15,16,17], stress tolerance [18,19], and physiological metabolism [20]. For instance, the addition of an interstock between persimmon scions and rootstock induces dwarfing of the scion [9]; soybeans grafted onto early-flowering rootstocks bloom 27–30 days earlier [12]; and grafting of ‘Sahahmiveh’ pear on six different rootstocks significantly increased both fruit weight and yield [20,21]. Grafting European grape varieties onto pest-resistant wild American grape reduces phylloxera damage [22], while pumpkin rootstocks can protect scion cucumbers from soil-borne diseases [23]. In tomato, using msh1 mutants as rootstocks boosted the yield in the progeny by 35%, and this effect can persist for up to five generations [11]. Grafting can be reliably used to improve various plant traits.

The success of grafting relies on a successful interaction between the rootstock and the scion that enables the long-distance transport and exchange of endogenous substances [24,25,26], then the two separate plants appear to grow as a single unit. A vascular connection between the scion and the rootstock is an essential prerequisite in grafted plants [8]. When successful, the communication of endogenous various molecules, including DNA, RNA, protein, and hormone via graft union, might play key roles in the new traits’ formation and influence the development of graft hybrids [7,27,28]. The plastid DNA can move across cellular barriers adjacent to the graft junction [6], and nuclear genomes can be transferred between plant-fused grafting cells [29], while heritable changes induced by epigenetic modifications of genomic DNA may occur as a result of the movement [27]. Long-distance translocation of mRNA between rootstocks and scions has also been documented in several diverse grafting systems such as cucumber/pumpkin (Cucurbita maxima) [30,31], melon/squash [32], and Vitis vinifera/Schisandra chinensis [33]. mRNA can form mRNA–protein complexes by binding to specific ribonucleoproteins, which not only protects them from degradation but also facilitates their translocation [34]. In addition, small RNAs and other epigenetic-related factors have been demonstrated to modulate the phenotypic traits of grafted plants [25,35,36]. For instance, various non-coding small RNAs, such as microRNAs [37,38] and small interfering RNAs [39,40], can undergo both short-distance cell-to-cell transport and long-distance movement across graft junctions, where they act as signaling factors, regulating distal cell or tissue development or stress responses, ultimately leading to significant phenotypic changes in grafted plants [41]. Thus, successful interaction between the rootstock and the scion is crucial for the generation of new phenotypes in grafted plants.

Non-coding RNAs vary in length, with lncRNAs categorized as longer than 200 nucleotides, which exert their cis or trans regulatory roles in the nucleus to modulate the gene expression. An lncRNA transcript may function in cis via interaction with proteins and RNAs [42]. Meanwhile, some trans regulatory mechanisms of lncRNA can alter the transcriptional output of a cell [43], mRNA translation [44] or mRNA degradation [45].

In A. thaliana, lncRNAs (COOLAIR and COLDAIR) can inhibit the FLC (FLOWERING LOCUS C) gene expression [46]. In Gossypium hirsutum, lnc883 acts as a cis regulator to modulate GhD03G0339 expression under salt stress [47]. In apple (Malus spp.), the lncRNA (MSTRG.85814.11) can promote the expression of the SAUR32 gene, thereby activating proton transport under iron-deficient conditions [48]. These studies indicate that lncRNAs play crucial regulatory roles in plant growth, development, and stress acclimation [46,47,48]. However, the function of lncRNAs in regulating trait differences in heterografting remains unreported.

‘Ziye’ peach (Prunus persica), with purple leaves and double-petaled red flowers, offers excellent ornamental value. Its fruits are large and edible. Kernel apricot cultivar ‘Youyi’, with green leaves and a single-petaled flower, small fruit and kernels, has its flowering period earlier than that of peaches. In our previous study, heterografting of ‘Ziye’ peach and ‘Youyi’ kernel apricot was performed using different scion/rootstock combinations, including peach/apricot (P/A), apricot/peach (A/P), and self-rooted peach (SP). The resulting grafted chimeras exhibited notable variations in leaf and flower morphology (Figure 1), flowering period and the size of pits and kernels [49,50]. However, the mRNA and lncRNA molecules involved in regulating the formation of differential traits in peach chimeras remain unclear. In this study, the key lncRNAs and mRNAs in peach chimeras were identified by transcriptome sequencing, and further identify co-expressed ‘lncRNA-mRNA’ pairs. These results establish a theoretical foundation for investigating the molecular mechanisms underlying the formation of differential traits in peach chimeras and provide new insights into creating an improved peach or kernel apricot germplasm through the heterografting of genetically distant plants.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Trees of peach cultivar ‘Ziye’ and kernel apricot cultivar ‘Youyi’ were maintained in an open field with sandy soil under normal cultivation and management conditions at the Mengzhou County Experimental Base (112°42′51″ E, 34°51′35″ N), affiliated with the Research Institute of Non-timber Forestry, Chinese Academy of Forestry, Zhengzhou, China. This region is a warm temperate monsoon climate. Reciprocal peach–apricot heterografted chimeras, namely peach/apricot (P/A) and apricot/peach (A/P), were obtained by grafting the peach cultivar ‘Ziye’ as the scion onto the apricot cultivar ‘Youyi’ as the rootstock, and vice versa, on 15 June 2014 [49,50], with the homografted peach cultivar ‘Ziye’ (SP) used as a control (Figure 1a). After successful grafting, chimeric branches of P/A and A/P above the graft union were retained, and began to blossom and bear fruit in 2016. For each combination (P/A, A/P and SP), more than 10 chimeric trees were preserved. Phenotypic analyses were conducted on the leaves, flower buds and fully opened flowers of each scion from the peach (P) portion of each graft. These tissues were harvested, immediately immersed in liquid nitrogen for flash freezing, and subsequently stored in –80 °C until further use. The samples for RNA-seq were carried out with three biological replicates, and the samples for qRT-PCR were carried out with three technical replications.

2.2. Total RNA Extraction and Transcriptome Sequencing

The collected leaf, flower bud and fully opened flower samples from the peach branches of P/A, A/P, and SP grafts were taken out of the freezer and quickly ground into powder with liquid nitrogen. For each sample, 100 mg of powder was used for total RNA extraction with the Plant RNA Extraction Kit (Vazyme, Nanjing, China). RNA purity and concentration were evaluated in a NanoDrop 2000c spectrophotometer (Termo Scientifc, Waltham, MA, USA), and RNA integrity was assessed using electrophoresis on a 1.0% agarose gel. For each combination, the high-quality (A_260/280 and A_260/230 ratios between 1.8 and 2.2) RNA isolated from the three different tissues were selected and mixed, respectively. cDNA for library construction was synthesized using PrimeScript™ RT-PCR Kit (Takara, Dalian, China) with 2 μg of total RNA. Each library was constructed three independent times using mixed peach samples of P/A, A/P and SP, for a total of nine cDNA libraries. After a library quality test, transcriptome sequencing was performed via the Illumina Novaseq 4000 platform at the LC-BIO (Hangzhou, China), using the method of 2 × 150 bp paired-end sequencing.

2.3. Screening and Analysis of Differentially Expressed Genes (DEGs)

The raw RNA-Seq data were uploaded to the National Biotechnology Data Center database after sequencing was completed. Adapter sequences, empty reads, and low-quality reads were removed from the raw RNA-Seq data using Trimmomatic v.0.36 to generate high-quality reads. The GC content and the quality scores (Q20/Q30) were also calculated. The clean reads were subsequently aligned to the peach reference genome (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Ppersica, accessed on 5 March 2023) using Tophat (http://tophat.cbcb.umd.edu) [51] and mapped to gene IDs.

To evaluate the expression levels of DEGs, the fragments per kilobase of exon model per million mapped 23 fragments (FPKM) value for each transcript was calculated using the R package DESeq. The obtained p-value was adjusted using the Benjamini–Hochberg program to determine the False Discovery Rate (FDR), and then the expression of each transcript was quantified using RSEM. Finally, DEGs were isolated using DESeq2 and a relatively strict criterion, with |log₂^{(fold change)}| > 1 and p-value/FDR < 0.05 as the thresholds [52]. To further elucidate the biological processes in which these DEGs may be involved, enrichment analysis was performed on the identified DEGs using GO seq R (https://bioconductor.org/packages/2.6/bioc/html/GOSemSim.html, accessed on 5 March 2023) and the KEGG Orthology database (https://www.kegg.jp/kegg/ko.html, accessed on 5 March 2023).

2.4. Identification and Analysis of lncRNA

To identify the lncRNAs in peach chimeras, comprehensive analysis of lncRNAs in the peach chimeras was performed via BLAST (version 2.12.0+) alignment combined with predictions using Crawford Network Consulting Inc (CNCI), Coding Potential Calculator (CPC), and PfamScan. Firstly, transcripts overlapping with annotated protein-coding genes were discarded. In addition, transcripts shorter than 200 bp were removed. The remaining transcripts were evaluated for coding potential using the CPC and the CNCI. Transcripts with CPC score ≥ 1 or CNCI score ≥ 0 were considered to have coding potential and were excluded. Only transcripts with a CPC score < 1 and CNCI score < 0 were retained as non-coding candidates. Finally, transcripts classified with class codes “i”, “j”, “o”, “u”, or “x” by gff compare were identified as putative lncRNAs.

2.5. Screening of DElncRNA and the Analysis of Co-Expressed lncRNAs and mRNAs

The differentially expressed lncRNAs (DElncRNAs) in peach chimeras of A/P and P/A were identified using DESeq2 (version1.38.3) with |log₂^{(fold change)}| > 1 and FDR < 0.05 as screening criteria. To predict potential ‘lncRNA-mRNA’ networks associated with the growth and development of peach chimeras, Pearson’s correlation coefficients (r) were calculated using an R package (version 3.2.5), with |r| values ≥ 0.6 identifying co-expressed ‘lncRNA-mRNA’ pairs. GO and KEGG enrichment analyses were performed for these co-expressed mRNAs to predict possible biological functions based on the peach reference genome. Finally, the ‘lncRNA-mRNA’ co-expression networks were constructed and visualized using Cytoscape software (v.3.7.1).

2.6. qRT-PCR Analysis of DEmRNAs and DElncRNAs

To verify the reliability of the RNA-Seq data and the identified ‘lncRNA-mRNA’ co-expression pairs, quantitative real-time PCR (qRT-PCR) was used to analyze the transcriptional levels of 10 DEmRNAs and 8 DElncRNAs derived from the predicted ‘lncRNA-mRNA’ pairs. The target molecules were randomly selected and included 5 DEmRNAs and 4 DElncRNAs from the A/P vs. SP comparison, and 5 DEmRNAs and 4 DElncRNAs from P/A vs. SP. The peach 18s rRNA and RPL13 genes were used as internal references to normalize the relative expression levels of mRNAs and lncRNAs, respectively. The primer sequences were designed by Primer Premier 5.0 (Tables S1 and S2). The qRT-PCR assay followed the manufacturer’s protocol for the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) using an ABI PRISM 7500 FAST Sequence Detection System (Applied Biosystems, USA) with 20 μL reaction mixtures. Each experiment was repeated three times with three biological replicates. The relative expression levels of the target genes were normalized using the 2^−ΔΔCT method against the reference gene, and then the multiples of changes in expression levels in A/P vs. SP and P/A vs. SP were analyzed.

3. Results

3.1. Identification of DEGs in Peach Chimeras

For each sample used for RNA-seq in this study, 7.65–11.69 Gb clean bases were generated using the Illumina Novaseq 4000 platform (Illumina, Inc., San Diego, CA, USA). The sequencing quality parameters were calculated to be Q20 ≥ 99.97%, Q30 ≥ 97.62%, and an average GC content ≥ 43% (Table S3). Principal component analysis (PCA) confirmed that the profiles of differed significantly between the A/P, P/A, and SP transcriptomes (Figure 2a). Furthermore, Spearman correlation values between the nine transcriptomes were calculated. The correlation coefficient (r) values were high between the three biological replicates, with an average of 0.9 for the A/P samples, 0.94 for P/A and 0.95 for SP (Figure 2b). These results indicated that the sequencing quality was relatively high and that the RNA-seq data of the biological replicates were suitable for further analysis.

To identify the DEGs related to the growth and development of peach–apricot graft chimeras, the transcriptome data of peach samples from A/P, P/A and SP were analyzed using the DESeq2 R software (version1.38.3) package to identify DEGs with a relatively strict criterion [False Discovery Rate (FDR) ≤ 0.05, fold change ≥ 2]. Compared to the SP transcriptomes, 1739 DEGs were detected in A/P and P/A (all DEGs are listed in Table S4). Specifically, 1115 DEGs were identified in A/P vs. SP, among which 734 genes were up-regulated and 381 genes were down-regulated in A/P compared to SP. There were 624 DEGs between P/A vs. SP, comprising 415 up-regulated and 209 down-regulated DEGs in P/A compared to SP (Table S4, Figure 2c). A Venn diagram revealed that 173 DEGs were shared between P/A vs. SP and A/P vs. SP. There were relatively more DEGs (942) specific to A/P vs. SP, with only 451 DEGs unique to P/A vs. SP (Figure 2d). These unique DEGs in P/A vs. SP and A/P vs. SP may be related to the differential phenotypes in the peach–apricot grafted chimeras.

3.2. Functional Analysis of DEGs in Peach Chimeras

To investigate the potential biological functions of DEGs in the peach–apricot grafted chimeras, the 1739 DEGs in A/P and P/A were classified by GO enrichment analysis (Figure 3). The detected DEGs were mainly enriched in 25 functional terms within the biological processes (BPs) category, 15 functional terms within the cellular components (CCs) category, and 10 functional terms within the molecular functions (MFs) category (Figure 3a,b). In the comparison between A/P vs. SP, a total of 833 DEGs may be involved in regulating CCs, mainly related to the nucleus, plasma membrane, cytoplasm, integral component of membranes and chloroplast. The nucleus category was the most enriched. Additionally, 401 DEGs were assigned to BPs, predominantly involving biological processes, regulation of transcription, D-templated, oxidation reduction process, and defense responses. Within the MFs category, the DEGs were mainly concentrated in molecular function, protein binding and D binding transcription factor activity, accounting for 52.37%, with fewer genes related to metal ion binding, ATP binding, and sequence-specific D binding (Figure 3a). In the P/A vs. SP group, 537 DEGs were associated with regulating CCs, including the nucleus, plasma membrane, integral component of membrane, cytoplasm, extracellular region, chloroplast and cytoplasm, with the nucleus again being the most enriched category. Within BPs, DEGs were mainly involved in biological processes, regulation of transcription, D-templated, transcription, D-templated defense regulation, and oxidation reduction processes. Among the DEGs annotated to the MF category, protein binding and ATP binding exceeded 50% (Figure 3b).

KEGG pathway enrichment analysis was also performed for the 1739 DEGs identified in the A/P vs. SP and P/A vs. SP. Among the 1115 DEGs in A/P vs. SP, the most DEGs were enriched in the plant–pathogen interaction (ko04626) pathway, followed by plant hormone signal transduction (ko04075), starch and sucrose metabolism (ko00500), MAPK signaling pathway–plant (ko04016), and phenylpropanoid biosynthesis (ko00940) (Figure 3c). Among the 624 DEGs in P/A vs. SP, the majority of DEGs were enriched in pentose and glucuronate interconversions (ko00040), followed by phenylpropanoid biosynthesis (ko00940) and starch and sucrose metabolism (ko00500) pathways (Figure 3d).

3.3. Analysis of lncRNA and mRNA in Peach Chimeras

In addition to identifying the mRNAs in the transcriptomes of the peach chimeras, comprehensive analysis of lncRNAs in the peach genome was performed via BLAST alignment combined with predictions using CNCI and CPC, 4445 novel lncRNAs were identified from the RNA-seq data, and these lncRNAs were classified into five categories based on their class codes (x, u, o, j and i), with their proportion distribution shown in Figure 4a. Among these categories, the u-type lncRNAs (usually containing one or more U-rich regions) constituted the largest proportion (45.08%). Comparative analysis revealed distinct distributions based on transcript length between the lncRNAs and mRNAs: over 80% of mRNAs were longer than 1000 bp, whereas more than 60% of lncRNAs were shorter than 500 bp (Figure 4b). A higher proportion of lncRNAs contained 1–3 exons, with single-exon lncRNAs being the most prevalent (Figure 4c). Furthermore, the number of lncRNAs and mRNA with different lengths of ORFs were analyzed based on their amino acid (aa), and lncRNAs exhibited significantly shorter predicted amino acid sequences from their ORFs compared to the mRNAs (Figure 4d,e). Most lncRNAs contain short ORF-encoding peptides less than 100 aa (Figure 4d), while most mRNAs containing ORF-encoding peptides are between 100 aa and 700 aa (Figure 4e). Finally, the lncRNAs had lower FPKM values compared to mRNAs (Figure 4f), and the number of lncRNAs was fewer than the number of mRNAs (Figure 4g). These findings collectively demonstrated that the lncRNAs generally possessed simpler structural features than mRNAs.

3.4. Identification of DElncRNAs in Peach Chimeras

The differentially expressed lncRNAs (DElncRNAs) in A/P and P/A were further analyzed using DEseq2 R software package, with a relatively strict criterion [False Discovery Rate (FDR) ≤ 0.05, fold change ≥ 2]. A total of 55 lncRNAs showing significant differences in expression were detected in A/P and P/A, with 29 and 26 DElncRNAs identified in A/P vs. SP and P/A vs. SP, respectively (Figure 5a). The detailed information for all significant DElncRNAs is listed in Table S5. In the group of A/P vs. SP, the transcriptional levels of 16 significant DElncRNAs were up-regulated and 13 significant lncRNAs were down-regulated in A/P compared to SP. Among the 26 DElncRNAs in P/A vs. SP, 17 were up-regulated and nine were down-regulated (Figure 5a). A Venn diagram of the 55 DElncRNAs revealed that eight DElncRNAs were present in both the P/A vs. SP and A/P vs. SP comparisons. On the other hand, 21 DElncRNAs were specific to the A/P graft and 18 DElncRNAs were specific to the P/A graft (Figure 5b). These specific DElncRNAs may be related to the differential phenotypes of the peach chimeras.

Heatmap analysis was performed to visualize the expression levels of the 29 DElncRNAs detected from the A/P vs. SP comparison and the 26 DElncRNAs from the P/A vs. SP comparison. The result showed that MSTRG.18445.1 exhibited the highest expression level in SP, and its transcript levels were significantly down-regulated in both A/P and P/A compared to SP (Figure 5c,d). In contrast, MSTRG.23668.1 in SP had relatively low expression, but its transcript level was significantly up-regulated in both A/P and P/A compared to SP (Figure 5c,d).

3.5. Functional Analysis of Co-Expressed mRNA with DElncRNAs in Peach Chimeras

Potential functions of lncRNAs can be inferred based on the functional annotation of their target mRNAs. To further dissect the potential functions of DElncRNAs, the target mRNAs of DElncRNAs in peach–apricot graft chimeras were identified by finding co-expressed genes [Pearson’s correlation coefficients (r) between the transcripts of mRNAs and DElncRNAs were calculated, with |r| values ≥ 0.6 identifying co-expressed genes]. Specifically, 67 mRNAs were found to be co-expressed with the 29 DElncRNAs in the A/P vs. SP (Table S6), and 52 mRNAs were targeted by the 26 DElncRNAs from the P/A vs. SP (Table S7), resulting in a total of 119 potential target mRNAs corresponding to the 55 DElncRNAs.

For these 119 mRNAs co-expressed with the DElncRNAs, GO and KEGG enrichment analyses were performed (Figure 6). The 67 potential mRNAs targeted by the 29 DElncRNAs in A/P vs. SP were significantly enriched in the cellular component category, including the nucleus, integral component of membrane, plasma membrane, chloroplast, and cytoplasm, as well as biochemical process and molecular function categories, with a particular focus on ATP binding (Figure 6a). The 52 mRNAs targeted by the 26 DElncRNAs in P/A vs. SP were significantly enriched in cellular component categories such as the nucleus, cytoplasm, and integral component of membrane, followed by molecular functions such as metal ion binding, protein binding, and DNA-binding transcription factor activity (Figure 6b).

The results of the KEGG analysis indicated that the mRNAs targeted by DElncRNAs in A/P vs. SP were mainly enriched in pathways such as RNA degradation, protein processing in endoplasmic reticulum, aminoacyl–tRNA biosynthesis, glycolysis/gluconeogenesis, and spliceosome (Figure 6c). The mRNAs targeted by DElncRNAs in P/A vs. SP were mainly enriched in key pathways, namely protein processing in endoplasmic reticulum, aminoacyl–tRNA biosynthesis, starch and sucrose metabolism, spliceosome, pyrimidine metabolism, and amino sugar and nucleotide sugar metabolism (Figure 6d).

3.6. Analysis of Co-Expression Regulatory Patterns of ‘lncRNA-mRNA’

To identify the ‘lncRNA-mRNA’ pairs related to growth and development in peach and apricot grafts, we constructed a visual co-expression network of DElncRNAs and DEmRNAs using Cytoscape software. The results showed that DElncRNAs and mRNAs exhibited both one-to-one and one-to-many co-expression patterns (Figure 7). A total of 24 ‘lncRNA-mRNA’ pairs were discovered, including 14 ‘lncRNA-mRNA’ pairs in A/P vs. SP (Figure 8a) and 10 ‘lncRNA-mRNA’ pairs in P/A vs. SP (Figure 7b). Among these pairs, 13 DElncRNAs were positively correlated with their target DEmRNAs, while 11 pairs were negatively correlated (Figure 7a,b). For example, MSTRG.18445.1 was down-regulated and its target XM_007207084.2 was up-regulated in A/P compared with SP, indicating that MSTRG.18445.1 may negatively regulate the transcription of XM_007207084.2 in ungrafted peach. MSTRG.17020.2 was down-regulated in A/P, whereas XM_007210198.2 and XM_00721476.2 were both up-regulated in A/P. MSTRG.8395.1 and XM_007217967.2 were both up-regulated in A/P (Figure 7a). In the P/A vs. SP comparison, MSTRG.6365.3 was down-regulated in the P/A graft, while XM_020556240.1 and XM_020556234.1 were both up-regulated in the P/A graft (Figure 7b). Based on the functional annotations of the DEmRNAs, it can be extrapolated that MSTRG.17020.2, found in the A/P vs. SP comparison, may participate in aminoacyl–tRNA biosynthesis by negatively regulating XM_007210198.2, which encodes a phenylalanine–tRNA ligase beta subunit, cytoplasmic protein. MSTRG.8395.1 may be involved in galactose metabolism by positively regulating the expression of XM_007217967.2, which encodes an alpha-galactosidase 3. MSTRG.6365.3, found in the P/A vs. SP comparison, may be involved in plant defense response by negatively regulating the expression of two putative disease resistance protein-encoding genes XM_020556240.1 and XM_020556234.1.

3.7. qRT-PCR Validation of DElncRNAs and DEmRNAs

To verify the accuracy and reliability of the RNA-Seq results and the ‘lncRNA-mRNA’ co-expression network construction, 10 DEmRNAs and eight DElncRNAs were randomly selected from the co-expressed ‘lncRNA-mRNA’ pairs for qRT-PCR verification. The fold change trends of the DEmRNAs and DElncRNAs as detected by qRT-PCR were generally consistent with those from RNA-Seq (Figure 8), indicating that the RNA sequencing results are reliable. Compared with SP, the expression levels in P/A of XM_020558689.1, XM_007222168.2 and XM_020557431.1 were up-regulated (Figure 8a) as were the expression levels of MSTRG.5074.2, MSTRG.8229.3, MSTRG.5610.4 and MSTRG.8131.3 (Figure 8c). In the P/A vs. SP comparison, the expression of XM_020558689.1 was positively correlated with MSTRG.8229.3, XM_007222168.2 was positively correlated with MSTRG.5610.4, and XM_020557431.1 was positively correlated with MSTRG.8131.3. These results were consistent with the analysis of the ‘lncRNA-mRNA’ co-expression network (Figure 8b). In addition, compared with SP, the expressions of XM_007207084.2, XM_020566895.1, XM_007210198.2, XM_020556234.1 and XM_007217967.2 were all up-regulated in A/P (Figure 8b). The expression of MSTRG.18445.1 was down-regulated in A/P, while MSTRG.19893.1, MSTRG.5074.2 and MSTRG.17005.1 were all up-regulated in A/P compared to SP (Figure 8d). And in the A/P vs. SP comparison, the expression trend of XM_007207084.2 was negatively correlated with that of MSTRG.18445.1, and the expression trend of XM_020566895.1 was positively correlated with that of MSTRG.19893.1. These results were also consistent with the analysis of the co-expressed ‘lncRNA-mRNA’ pairs (Figure 7a). Collectively, these results indicated that the transcriptome and ‘lncRNA-mRNA’ co-expression network are reliable, and thus can be used to further explore and characterize key genes involved in the growth and development of peach and apricot grafts.

4. Discussion

LncRNAs are a class of non-coding RNAs widely found in eukaryotes that lack protein-coding capability [53]. Although their processing mechanisms may resemble those of mRNAs, lncRNAs exhibit higher cell-type specificity, lower abundance, and a degree of sequence conservation. Nevertheless, lcnRNAs play crucial regulatory roles in biological processes such as gene silencing, mRNA splicing, translation, genomic rearrangement, and chromatin modification [43,46,54]. To date, numerous lncRNAs have been identified in diverse species, including Arabidopsis thaliana [53], tomato (Solanum lycopersicum) [55,56], strawberry (Fragaria spp.) [57], and melon (Cucumis melo) [58], and so on. lncRNAs have emerged as important regulators of gene expression across diverse organisms, functioning through epigenetic modification, transcriptional regulation, and post-transcriptional regulation [59,60]. However, research on the roles of lcnRNAs in mediating differential phenotypic traits in plant grafts remains relatively limited.

Our previous study revealed significant differences in important agronomic traits, such as leaf shape, flower morphology, flowering period, and kernel size, between peach–apricot grafts (A/P and P/A) and a peach homograft (SP) (Figure 1) [50]. Additionally, we identified several key mRNAs and miRNAs that may be involved in regulating the growth and development of peach chimeras [49]. In this study, a total of 1739 DEGs were detected in A/P and P/A compared to SP. Specifically, 1115 DEGs were identified in A/P vs. SP, and 624 DEGs were found in P/A vs. SP (Figure 2). GO enrichment analysis showed that these DEGs may participate in a series of highly coordinated biological processes during graft growth and development. Notably, the significant changes in the expression levels of genes related to cytoplasm, membrane proteins, membrane channels, and ion pumps (Figure 3a,b) may play important roles in the growth and development of peach chimeras [61,62]. Subsequent KEGG enrichment analysis of DEGs demonstrated that in A/P vs. SP, DEGs were significantly enriched in pathways including “Plant hormone signal transduction”, “Plant MAPK signaling pathway”, “Phenylpropanoid biosynthesis”, and “Amino sugar and nucleotide sugar metabolism”. In contrast, in P/A vs. SP, DEGs were mainly involved in pathways such as “Starch and sucrose metabolism”, “Plant–pathogen interaction”, and “Plant hormone signal transduction” (Figure 3c,d). These findings indicated that these DEGs may be involved in regulating biological processes, substance transport, signal transmission, and graft junction healing [33,63,64].

The identification of lncRNAs and their target mRNAs, along with the analysis of their interactions, can help elucidate the biological processes regulated by ‘lncRNA-mRNA’ pairs. In this study, the characteristics of lncRNA transcripts, including transcript length, exon number, ORF length, and FPKM values, were identified in peach chimeras (Figure 4). These characteristics are similar to those reported for lncRNAs in tea sugar beet [65] and Chinese cabbage (Brassica campestris L. ssp. pekinensis) [66]. A total of 55 DElncRNAs in A/P and P/A were further analyzed, with 29 DElncRNAs in A/P vs. SP and 26 identified in P/A vs. SP, respectively (Figure 5a). Compared with SP, 21 DElncRNAs were specific to A/P, while 18 were only in P/A, respectively (Figure 5b). These specific DElncRNAs may be related to the formation of differential phenotypes of the peach grafts. Since many lncRNAs regulate mRNAs, the biological functions of the lncRNAs could be predicted based on their positional relationships and expression correlations with protein-coding genes. GO and KEGG enrichment analyses were performed on the 119 mRNAs potentially targeted by the 55 DElncRNAs (Figure 6).

The identification of lncRNAs, their target genes, and their interactions provides a valuable resource for future research. To further explore the regulatory roles of the lncRNAs, we used Cytoscape to construct a co-expression network of ‘lncRNA-mRNA’, identifying 24 co-expressed ‘lncRNA-mRNA’ pairs (Figure 7). Subsequently, functional annotation of the mRNAs targeted by the DElncRNAs was performed to explore potential regulatory roles. The results revealed that in A/P vs. SP, the pair ‘MSTGR.17020.2-XM_007210198.2’ may be involved in aminoacyl–tRNA biosynthesis, while ‘MSTRG.8395.1-XM_007217967.2’ may participate in galactose metabolism. In P/A vs. SP, and ‘MSTRG.6365.3-XM_020556240.1/XM_020556234.1’ may play a role in plant defense responses. These findings suggest that the identified ‘lncRNA-mRNA’ pairs are potentially involved in metabolic and defense processes in peach chimeras, thereby contributing to the formation of differential phenotypes.

LncRNA act in both cis or trans regulatory roles to modulate the expression levels of protein-coding genes [48]. So, the ‘lncRNA–mRNA’ regulatory pairs include both sense-oriented and antisense-oriented relationships with respect to the target gene. In Arabidopsis thaliana [67], HDA6, a homolog of the gene XM_007210198.2, functions as a histone deacetylase. It integrates into the SANT complex and promotes flowering by repressing floral repressors such as FLC, MAF4, and MAF5. In this study, peach chimeras contain ‘lncRNA-mRNA’ pairs differentially expressed in the A/P vs. SP and P/A vs. SP comparisons. And the lncRNA MSTRG.17020.2, which shows differential expression in A/P compared to SP, was found to negatively regulate XM_007210198.2, suggesting that the ‘MSTRG.17020.2-XM_007210198.2’ pair may influence the floral development and flowering time in peach chimeras. In tomato, the lncRNA16397-SIGRX22 module has been shown to reduce membrane damage by suppressing reactive oxygen species (ROS) accumulation [58]. In Arabidopsis, HPU26, a homolog of XM_007328760.2, modulates hypoxic and biotic stress responses by regulating PR1 and ROS levels, thereby increasing resistance to bacterial pathogens [67]. We observed that MSTRG.7244.1—a DElncRNA in A/P vs. SP—negatively regulates XM_007218760.2, including its potential role in the ROS pathway. This leads us to hypothesize that apricot–peach grafting may also alter the biotic stress response in peach chimeras. Overall, this study establishes a theoretical foundation for understanding how ‘lncRNA-mRNA’ pairs contribute to trait variation in grafting chimeras and offers insights for germplasm innovation through cross-species grafting.

5. Conclusions

We identified 1739 DEGs and 55 DElncRNAs between peach chimeras of A/P, P/A and SP. Compared with SP, 1115 and 624 DEGs were detected in A/P and P/A, and 942 and 451 DEGs were specifically altered in A/P and P/A, respectively. A total of 29 and 26 DElncRNAs were found in A/P and P/A, of which, 21 DElncRNAs were specific to A/P and 18 were specific to P/A. The biological functions of the 1739 DEGs and 55 DElncRNAs were predicted via GO and KEGG enrichment analyses. And 24 co-expressed ‘lncRNA-mRNA’ pairs were identified, including 14 and 10 ‘lncRNA-mRNA’ pairs in A/P and P/A, respectively. Altogether, these findings contribute to our understanding of molecular mechanisms underlying grafting-induced differential trait formation and establish a foundation for further research on the functional roles of ‘lncRNA-mRNA’ pairs in grafted peach chimeras.