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Article

Oxford Nanopore Full-Length Transcriptome Reveals Alternative Splicing and Its Functional Diversity in Regulating Fruit Ripening in Peach (Prunus persica)

by
Hui Zhou
1,2,†,
Xiao Wang
3,†,
Liuqiong Jiang
1,4,
Pei Shi
1,2,
Yu Sheng
1,2,
Yunyun Wang
1,2,
Qingmei Xie
1,2,
Jinyun Zhang
1,2,* and
Haifa Pan
1,2,*
1
Key Laboratory of Horticultural Crop Germplasm Innovation and Utilization (Co-Construction by Ministry and Province), Institute of Horticulture, Anhui Academy of Agricultural Sciences, Hefei 230031, China
2
Anhui Provincial Key Laboratory for Germplasm Resources Creation and High-Efficiency Cultivation of Horticultural Crops, Hefei 230031, China
3
Soil and Fertilizer Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, China
4
School of Horticulture, Anhui Agricultural University, Hefei 230036, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2026, 16(2), 197; https://doi.org/10.3390/agronomy16020197
Submission received: 9 December 2025 / Revised: 29 December 2025 / Accepted: 12 January 2026 / Published: 13 January 2026
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Abstract

Fruit development and ripening in peach (Prunus persica) involve complex transcriptional and post-transcriptional regulation. While short-read sequencing has advanced transcriptome studies, it often fails to accurately resolve complex transcript isoforms. This study employed Oxford Nanopore Technologies’ (ONT) full-length RNA-Seq to comprehensively characterize the transcriptomic landscape of peach fruits across three key developmental stages: the first exponential stage, the second exponential stage, and the ripening stage. Our analysis identified 44,042 non-redundant isoforms, including 1109 novel genes and 32,289 novel isoforms, significantly expanding the peach genome annotation. We further investigated alternative splicing (AS) events, revealing that intron retention (IR) and alternative 3′ splice site (A3′S) were the most prevalent types, with AS abundance peaking at the S1 stage. A total of 10,236 differentially expressed transcripts (DETs) were identified, highlighting dynamic expression patterns during fruit development. Functional characterization focused on a MADS-box gene, PpMADS6, which produced two isoforms via alternative splicing. Dual luciferase assays in tobacco leaves demonstrated that the full-length isoform, PpMADS6a, specifically activated the promoter of the fruit-softening gene PpPG1, while the truncated isoform, PpMADS6b, lost this transactivation ability. This study provides a valuable resource of full-length transcriptomes for peach and underscores the critical role of alternative splicing in generating functional diversity to fine-tune fruit development and ripening processes.

1. Introduction

Most protein-coding genes in eukaryotes contain introns, with approximately 95% of human and 79% of Arabidopsis thaliana genes harboring one or more introns [1,2]. A key consequence of this genetic architecture is alternative splicing (AS), a process through which a single pre-mRNA can generate multiple mRNA isoforms, thereby significantly expanding proteomic diversity. This stands in contrast to constitutive splicing, which yields an invariant mRNA sequence. The prevalence of AS is substantial, occurring in an estimated 95% of human and 78% of Arabidopsis multi-exon genes [3,4]. Therefore, AS is a fundamental mechanism that enables organisms to transcend the limitations of a finite genome, significantly increasing the diversity of transcripts and proteins. AS can be classified into five types, intron retention (IR), alternative 3′ splice site (A3′S), alternative 5′ splice site (A5′S), exon skipping (ES), and mutually exclusive exon (ME). Usually, ES types are the most prevalent type of AS in animals, but IR events are the most prevalent type in plants [5]. Generally, most transcripts generated by AS events are nonsense mRNAs contain in-frame premature termination codons (PTCs) and are degraded by the nonsense-mediated mRNA decay (NMD) pathway [6]. Interestingly, however, splice isoforms containing PTCs generated by IR are often insensitive to NMD and can stably produce truncated proteins [5,7], suggesting that IR may contribute to post-transcriptional regulatory complexity.
AS significantly enhances transcriptomic and proteomic diversity, enabling plants to respond to a wide range of abiotic and biotic stresses. Under abiotic stress conditions, AS participates in key physiological processes by modulating abscisic acid (ABA) homeostasis, circadian rhythm, photosynthesis, sugar metabolism, and antioxidant activity [8,9]. Additionally, AS frequently occurs in plant disease resistance (R) genes, leading to the production of truncated protein isoforms. These truncated variants play significant roles in plant immune responses through multiple mechanisms, such as releasing immune suppression, activating defense signals, or directly engaging in effector-triggered signaling pathways [10].
Beyond stress responses, AS is a critical regulator of plant growth and development. It influences key processes such as vegetative growth, flowering, and aging [11]. The transition to flowering, marked by the shoot apical meristem (SAM) switching from vegetative to reproductive development to produce floral primordia, is tightly controlled by AS. In Arabidopsis, the splicing of central flowering regulators, such as Flowering Locus T (FT), and FLOWERING LOCUS M (FLM), participate in regulation of flowering [12,13,14]. The splicing of key flowering genes in flowering transition is conducted by the U2 auxiliary factors [15]. Prior to the visible switch to flowering, plants undergo earlier, less obvious phase changes during vegetative growth, such as the juvenile-to-adult transition. This phase change is also modulated by age-dependent AS events, as demonstrated in Brachypodium distachyon [16]. Furthermore, AS continues to regulate developmental signals during the final stages of the life cycle. For instance, in Populus, an alternative splicing variant of the NAC transcription factor PtRD26 delays leaf senescence by activating a network of downstream NAC TFs [17]. These examples underscore the pervasive role of AS in coordinating plant development across its entire lifespan.
Increasing evidence also indicates that AS is an important regulator of fruit development and ripening. In fleshy fruits, AS affects diverse processes including cuticle integrity, carbohydrate metabolism, and secondary metabolite accumulation. In tomato, temperature-dependent AS of the cwph gene alters cuticular water permeability and fruit surface integrity [18]. In banana, a truncated AS isoform of MaMYB16 modulates starch degradation by interfering with the activity of its full-length counterpart during ripening [19]. AS has also been linked to pigment accumulation in fruits, as shown by AS variants of MYB transcription factors associated with anthocyanin biosynthesis in grape and tomato [20,21]. Importantly, studies in apple have revealed that truncated AS isoforms do not merely act as inactive byproducts but can function cooperatively with full-length proteins, as exemplified by the Ma1 locus, where isoform interactions fine-tune vacuolar malate transport and fruit acidity [22]. These findings highlight the functional significance of AS-derived isoforms in fruit developmental regulation.
Peach fruit development follows a well-characterized double-sigmoid growth pattern comprising four major stages [23]. Stage 1 (S1, the first exponential growth stage) is characterized by rapid cell division and early cell expansion following fertilization. Stage 2 (S2, pit-hardening stage) corresponds to endocarp lignification and pit hardening, during which mesocarp growth slows markedly. Stage 3 (S3, the second exponential growth stage) involves a second phase of rapid mesocarp cell expansion. Finally, during Stage 4 (S4, the ripening stage), fruits reach their final size and undergo ripening-related physiological and biochemical processes. These developmental transitions are tightly coordinated by hormonal signaling and transcriptional reprogramming, ultimately determining fruit size, texture, flavor, and overall quality. Notably, in many ultra-early-ripening peach cultivars, S2 is absent, and fruit development instead follows a single sigmoidal growth curve [24]. Despite the importance of these stages, the post-transcriptional regulatory mechanisms governing peach fruit development remain incompletely understood. Recent transcriptomic studies have reported widespread AS during peach fruit development and ripening [25]. However, the average number of isoforms per gene identified in peach remains substantially lower than that reported for model species such as Arabidopsis, suggesting that many AS events remain unannotated. Moreover, functional characterization of AS-derived isoforms in peach is still limited, largely due to technical constraints in accurately resolving full-length transcripts using short-read sequencing approaches.
In this study, Oxford Nanopore Technologies (ONT) long-read RNA sequencing was performed on fruit flesh samples of Prunus persica cv. ‘Li Xia Hong’ collected across multiple developmental stages to profile AS and discover previously unannotated isoforms. Newly identified genes and isoforms were examined through functional annotation and expression pattern analysis, with particular attention to AS events in genes implicated in fruit development and ripening. Analysis of the PpMADS6 locus revealed that distinct isoforms generated by AS exhibit divergent roles in regulating these processes. This work broadens the current peach transcriptome landscape and highlights the substantial contribution of AS to fruit development and ripening.

2. Materials and Methods

2.1. Plant Materials

Fruit from the peach cultivar ‘Li Xia Hong’ (LXH) was collected from trees maintained at the Anhui Academy of Agricultural Sciences, Hefei, Anhui Province, China. The peach cultivar ‘Li Xia Hong’ (LXH) is an ultra-early-ripening nectarine derived as a bud sport of ‘Zhong You 4’ and is widely cultivated in the Jianghuai region of China. In Hefei, LXH blooms in late March and ripens in late May, producing round, clingstone fruits with an average weight of ~169 g, soluble solids content of ~13.6%, high yield stability, and a shortened fruit developmental period lacking a distinct pit-hardening (S2) stage. Samples were harvested at three developmental stages: the first exponential expansion (S1, 30 days after full bloom; DAFB), the second exponential expansion (S3, 49 DAFB), and maturity (S4, 65 DAFB). For each stage, three biological replicates were collected, each consisting of at least five fruits. Tissues were peeled, cored, diced, flash-frozen in liquid nitrogen, and stored at −80 °C until analysis.

2.2. Nanopore RNA-Seq cDNA Library Construction

Total RNA was isolated from peach fruit samples using TRIzol reagent (Takara Biotech, Dalian, China) following the manufacturer’s protocol. RNA purity was assessed with a NanoPhotometer spectrophotometer (IMPLEN, Westlake Village, CA, USA). cDNA libraries were prepared from 1 μg of total RNA using the cDNA-PCR Sequencing Kit (SQK-PCS109; Oxford Nanopore Technologies, Oxford, UK). Briefly, full-length cDNAs were synthesized with reverse transcriptase, and defined PCR adapters were ligated to both ends of the first-strand cDNA. This was followed by cDNA amplification via PCR with LongAmp Taq DNA polymerase (NEB, Ipswich, MA, USA) for 14 cycles (8 min elongation per cycle). The amplified products were ligated to ONT adapters using T4 DNA ligase (NEB, Ipswich, MA, USA) and purified with Agencourt XP beads (Beckman Coulter, Brea, CA, USA). The final libraries were sequenced on FLO-MIN109 flow cells using a PromethION platform at Biomarker Technology Company (Beijing, China).

2.3. Oxford Nanopore Technologies Long Read Processing

Raw reads were filtered to remove sequences with an average Phred quality score < 7 or length < 500 bp. Reads mapping to ribosomal RNA were removed by alignment to an rRNA database. Full-length, non-chimeric (FLNC) transcripts were identified by detecting primer sequences at both ends. FLNC transcripts were mapped to the reference genome using minimap2 v2.16 [26] and clustered to generate consensus sequences, which were polished using pinfish pipeline. Polished consensus sequences were realigned to the genome with minimap2 v2.16 and collapsed using cDNA_Cupcake v5.8 with thresholds of ≥85% coverage and ≥90% identity, ignoring 5′ differences. This produced the final set of nonredundant, polished consensus transcripts.

2.4. Fusion Transcript Identification

To identify candidate fusion transcripts, nonredundant high-quality consensus sequences were screened using the following criteria: alignment to at least two distinct genomic loci; each locus with ≥5% coverage and ≥1 bp aligned; total aligned coverage ≥ 95%; and genomic loci separated by at least 10 kb. Fusion transcripts were identified within each library and subsequently integrated across all libraries.
Fusion transcripts previously identified using PacBio Iso-Seq [25] were compared with ONT-detected fusion events. ONT-derived fusion transcripts were formatted into a BLAST database using BLAST 2.2.31+, and blastn searches were conducted using PacBio-derived fusion transcripts as queries.

2.5. Structure Analysis

The identified transcripts were validated by comparison to known reference annotations using gffcompare (https://github.com/gpertea/gffcompare (accessed on 11 January 2026)). Alternative splicing (AS) events including intron retention (IR), exon skipping (ES), alternative donor (AD), alternative acceptor (AA) and multi-exon exon (MEE) were detected with AStalavista [27].

2.6. Functional Annotation

Functional annotation of transcripts was performed using the following databases: NR (NCBI non-redundant protein sequences) and KOG/COG/eggNOG (Clusters of Orthologous Groups of proteins).

2.7. Expression Quantification and Differential Expression Analysis

Full-length reads were aligned to the reference transcriptome, retaining only reads with mapping quality > 5. Expression levels were quantified as reads per gene/transcript per 10,000 mapped reads. Differential expression analysis was performed using DESeq v1.18.0 [28] with a negative binomial model. p-values were adjusted using the Benjamini–Hochberg method. Transcripts with FDR < 0.01 and ≥2-fold change were considered differentially expressed. Gene clustering was conducted using R packages pheatmap (1.0.13) and Mfuzz (2.58.0).

2.8. RT-qPCR Validation

To validate RNA-Seq expression profiles, total RNA was extracted using the RNAprep Pure Plant Kit (TianGen, Beijing, China) from the same samples used for sequencing, with three biological replicates. First-strand cDNA was synthesized using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Dalian, China). RT-qPCR was performed with the TB Green® Premix Ex Taq™/Tli RNaseH Plus kit (Takara) following the manufacturer’s instructions. Primer sequences are listed in Table S3.

2.9. Construction of Expression Vectors

Coding sequences of the two PpeMADS6 alternative splicing isoforms were amplified from fruit cDNA and cloned into the multiple cloning site (MCS) of pSAK277. Promoter fragments were inserted into pGreenII0800-LUC+. To examine transcription factor–cis-element interactions, a 46 bp minimal CaMV 35S promoter (35Smini) was cloned into pGreenII0800-LUC+ using BamHI and NcoI. Tandem copies of putative MADS-binding cis-elements were inserted upstream of 35Smini via HindIII and BamHI sites. These constructs were used in dual-luciferase assays.

2.10. Dual Luciferase Assays

A dual luciferase reporter assay was performed in Nicotiana benthamiana leaves. Constructs were introduced into Agrobacterium tumefaciens GV3101 and cultured at 28 °C for 2 days. Bacterial suspensions were resuspended in infiltration buffer (10 mM MgCl2, 0.5 μM acetosyringone) and incubated for 2 h. Cultures containing reporter and effector constructs were mixed and infiltrated into leaves. Each assay included at least four independent biological replicates. LUC and Ren activities were measured 3 days post-infiltration using the Luciferase Assay System (Promega, Madison, WI, USA) on an Infinite M200 luminometer (Tecan, Männedorf, Switzerland).

2.11. Assessment of Binding Specificity of MADS-Box Proteins

Putative MADS-binding cis-elements were predicted using the online tool FIMO (https://meme-suite.org/meme/tools/fimo (accessed on 11 January 2026)) from the MEME Suite. Putative MADS-binding cis-elements were predicted using FIMO (MEME Suite). Dual-luciferase assays were employed to evaluate TF–cis-element interactions using constructs containing tandem repeats of predicted cis-elements upstream of the 35Smini promoter in pGreenII0800-LUC+.

3. Results

3.1. Peach ONT Transcriptome Sequencing

To obtain complete and previously uncharacterized transcript sequences from peach fruit, nine full-length RNA-Seq libraries were generated using Oxford Nanopore Technologies (ONT). Total RNA was extracted from fruit collected at 30, 49, and 65 days after full bloom (DAFB), representing S1, S3, and S4, respectively (Figure S1). Each developmental stage included three biological replicates. After removal of adapters and low-quality reads, a total of 56.83 Gb of clean data was obtained, with individual libraries ranging from 5.72 to 7.60 Gb (Table 1). The mean read length ranged from 1.05 to 1.30 kb, and maximum read lengths spanned 9100 to 12,746 nt. Following rRNA depletion, clean reads were processed to identify full-length transcripts, resulting in 3,721,799 to 4,525,293 full-length reads per library and full-length read proportions of 74.22–78.48%. The complete set of full-length reads was aligned to the peach reference genome sequences v2.0 [29]. Redundant isoforms with truncated 5′ ends were collapsed, substantially reducing isoform redundancy and yielding a final set of 44,042 non-redundant isoforms.

3.2. Annotation of Novel Genes and Isoforms

All 44,042 non-redundant isoforms were compared with the reference annotation v2.1 [29] using Gffcompare, thereby improving peach genome annotation, particularly with respect to novel gene and isoform discovery. In total, 1109 novel genes and 32,289 novel isoforms were identified. Functional annotation using the eggNOG database showed that most novel genes and isoforms fell into the “function unknown” category (Figure 1A and Figure S2A), followed by categories such as ‘posttranslational modification, protein turnover, chaperones’, ‘transcription’, and ‘signal transduction mechanisms’. To assess sequence similarity, novel gene and isoform sequences were queried against the NCBI Nr database. The majority of novel genes (64.29%) and isoforms (70.42%) showed highest similarity to sequences from Prunus persica (Figure 1B and Figure S2B). A considerable proportion matched genes from other Prunus species, including P. mume and P. avium (23.89% for novel genes and 14.57% for novel isoforms), and a smaller fraction aligned with Rosaceae species outside Prunus (4.45% and 4.80% for novel genes and isoforms, respectively). These findings indicate that some gene and isoform annotations remain incomplete in peach relative to other Prunus and Rosaceae species. Notably, 7.38% of novel genes and 10.29% of novel isoforms lacked detectable similarity to any Rosaceae species, underscoring the extent of missing or incomplete annotation across Rosaceae genomes.

3.3. Discovery of Fusion Transcripts

Fusion transcripts were initially identified within each ONT-derived library. The number of detected fusion transcripts varied considerably among libraries: the 30_DAFB_rep1 library contained the highest number (44), whereas the 65_DAFB_rep1 and 65_DAFB_rep3 libraries each contained the lowest count (20) (Table S1). A subsequent comparative analysis across all libraries identified a total of 66 fusion transcripts, of which only five were consistently detected across biological replicates. Notably, two fusion transcripts, Prupe.6G095300_plus_scaffold_73 and Prupe.4G015900_plus_Prupe.I000100, were present in every library. These transcripts are associated with genes or sequence fragments that remain unanchored to any of the eight chromosomes in the ‘Lovell’ genome v2.0 assembly.
Fusion transcripts are often unstable or expressed at low levels, resulting in substantial variability in their detectability across sequencing platforms, technologies, and tissue types. In our previous SMRT Iso-Seq study involving multiple peach tissues and organs [25], 148 fusion transcripts were identified. A comparison of sequence similarity between fusion transcripts from the earlier study and those identified here showed that 18.2% of previously reported fusion transcripts were also detected in the present dataset, suggesting that a subset of fusion transcripts exhibit greater stability or occur at a higher frequency than others.

3.4. Analysis of as Events

Five major types of alternative splicing (AS) events were examined in this study, including intron retention (IR), exon skipping (ES), alternative 5′ splice site (A5′S), mutually exclusive exons (ME), and alternative 3′ splice site (A3′S), with each ONT library analyzed independently (Figure 2A). Overall, IR and A3′S constituted the predominant AS categories. At the S1 and S3 developmental stages, IR events were significantly more abundant (p < 0.05) than all other AS types (Figure 2B). At the S4 stage, the difference between IR and A3′S decreased, and in some replicates A3′S even exceeded IR (Figure 2B). The total number of AS events of all types peaked during the S1 stage (Figure 2C), indicating that distinct AS patterns may be associated with specific phases of fruit development. Subsequently, a combined analysis of AS events across libraries identified 7943 unique AS events in the ONT dataset. Comparison with our previous PacBio Iso-Seq dataset, which encompassed diverse peach tissues and organs, revealed 2374 AS events shared between the two studies (Figure 3). In addition, 12,113 AS events were unique to the earlier Iso-Seq dataset, while 5569 AS events were detected exclusively in the current ONT dataset.

3.5. Identification of Differentially Expressed Transcripts (DETs)

Gene and transcript expression levels were quantified using counts per million (CPM). We first examined the expression of novel genes, among which 422 showed differential expression between developmental stages (Table S2). These 422 genes were separated into two major groups: one exhibiting relatively small expression changes across stages and the other showing marked stage-specific variation (Figure S3).
Expression levels of all detected transcripts were then compared between each pair of developmental stages (S1 vs. S3, S1 vs. S4, and S3 vs. S4). In total, 10,236 differentially expressed transcripts (DETs) were identified. Among them, 653 DETs were shared across all stage comparisons, whereas 749, 2687, and 1027 transcripts were uniquely differentially expressed in S1 vs. S3, S1 vs. S4, and S3 vs. S4 comparisons, respectively (Figure 4A). Hierarchical clustering categorized the DETs into distinct groups (Figure 4B). Subsequent Mfuzz analysis further partitioned the DETs into six clusters, revealing specific temporal expression patterns (Figure 4C). Transcripts in Clusters 5, 2, and 6 showed peak expression at stages S1, S3, and S4, respectively. In contrast, Clusters 1 and 4 were enriched for transcripts exhibiting either progressive upregulation or downregulation throughout fruit development.
To validate expression patterns observed in ONT RNA-Seq, RT-qPCR was performed on six randomly selected gene loci. For all tested transcripts, expression trends were consistent between nanopore RNA-Seq and RT-qPCR (Figure 5), supporting the reliability of the ONT-based expression analysis.

3.6. Identification of Fruit Ripening Related as Events in the PpMADS6 Locus

AS serves as a major post-transcriptional regulatory mechanism in fleshy fruit, and isoforms produced from the same transcription factor (TF) locus may cooperate or counteract each other [30]. Members of the MADS-box and NAC families are key regulators of ripening in multiple fleshy fruits [31], including peach [32,33]. In our ONT RNA-Seq dataset, a MADS-box gene locus, a MADS-box gene locus ONT.10265 which was designated as PpMADS5 in a previous study [33] was found to generate two distinct isoforms through AS. The longer isoform, PpMADS5a, contains eight exons and encodes a full-length MADS-box protein with intact MADS, I, K-box, and C-terminal domains (Figure 6A). The shorter isoform, PpMADS5b, is produced through intron retention of the third intron, which introduces a premature stop codon and results in early translational termination. PpMADS5b retains only the MADS and I domains, lacking nearly the entire K-box domain and the full C-terminal region (Figure 6A).
To examine whether these isoforms directly contribute to ripening regulation, dual-luciferase reporter assays were conducted using promoters of four ripening-associated genes: PpACS1, PpACO1, PpPG1, and PpPG2. The first two encode key enzymes in peach ethylene biosynthesis [34], while PpPG1 and PpPG2 encode pectin endo-polygalacturonases essential for fruit softening [35]. Promoter fragments of 2.12, 2.02, 2.17, and 1.73 kb, respectively, were used for these assays. Dual-luciferase results showed that neither PpMADS5a nor PpMADS5b activated the promoters of PpACS1, PpACO1, or PpPG2 in tobacco system. However, PpMADS5a significantly activated the PpPG1 promoter, with a 2.8-fold increase in luciferase/renilla ratio relative to the empty vector, while PpMADS5b showed no detectable activation (Figure 6B). These results indicate that PpPG1 is a downstream target of PpMADS5a but not PpMADS5b. Co-expression of both isoforms revealed that PpMADS5b did not significantly suppress PpMADS5a-mediated activation of the PpPG1 promoter (Figure 7).
To identify cis-elements within the PpPG1 promoter that interact with PpMADS5a, FIMO motif scanning was performed. No canonical CArG motif was detected within the 2.17 kb promoter region, although two sequences showed high similarity to CArG boxes. These elements, located at −1055 bp and −1014 bp relative to the transcription start site and designated proPpPG1_cis1 and proPpPG1_cis2, were fused as tandem repeats to a minimal 35S promoter for dual-luciferase assays (Figure 8A). PpMADS5a strongly activated proPpPG1_cis1, producing a 2.7-fold increase compared with the empty vector, whereas PpMADS5b showed no activation (Figure 8B). Neither isoform strongly activated proPpPG1_cis2, although PpMADS5a showed a modest but statistically significant effect (1.48-fold). These results demonstrate that PpMADS5a activates PpPG1 by binding to the proPpPG1_cis1 element, and that the truncated isoform PpMADS5b lacks this activation capacity. Together, these findings indicate that AS at the PpMADS6 locus generates isoforms with distinct regulatory functions.

4. Discussion

4.1. Comprehensive Characterization of Alternative Splicing Landscapes Using Oxford Nanopore Transcriptome Sequencing in Peach

High-throughput short-read sequencing (SGS) has revolutionized genome-wide transcriptome studies, yet its limited read length often restricts the accurate reconstruction of complex transcript isoforms [36]. This limitation is particularly critical given the high prevalence of alternative splicing in both animals and plants. To overcome these constraints, third-generation sequencing platforms such as PacBio SMRT Iso-Seq and ONT have become indispensable, owing to their ability to generate long reads that frequently span full-length RNA molecules in a single pass [37]. These long reads eliminate the need for fragmentation and in silico assembly, thereby improving isoform resolution. The utility of this approach has been demonstrated in numerous plant species, where long-read sequencing uncovered extensive splicing events and novel full-length transcripts that were undetectable using short-read methods alone [38].
Comparing to PacBio, ONT sequencing offers higher throughput, making it more suitable for quantitative transcript expression analyses and for detecting novel isoforms [39]. However, the two technologies exhibit relatively low overlap in the AS events they identify [40]. For species with transcriptomes sequenced using only one platform, additional sequencing with the alternative technology can therefore markedly improve completeness. In peach, PacBio-based RNA-Seq previously identified 40,477 nonredundant high-quality consensus transcripts [25]. In this study, ONT-based RNA-Seq recovered 44,042 nonredundant consensus transcripts. Notably, PacBio technology played a fundamental role in detecting novel AS events, consistent with findings in Arabidopsis [40]. Nevertheless, only 29.9% of AS events identified by ONT were also detected in the previous PacBio study (Figure 3), indicating that the ONT dataset substantially enriches the peach transcriptome resource, particularly for isoforms abundantly expressed during fruit development and ripening.

4.2. As Orchestrates Transcriptional Diversity in Fruit

AS is dynamically modulated during fruit development and ripening and serves as a key post-transcriptional mechanism that adjusts gene expression to support major physiological transitions [41,42]. Comparative studies across species reveal distinct patterns of AS regulation. In kiwifruit, the total number of AS events increases during fruit maturation [43]. In contrast, species such as papaya and peach exhibit a decrease, while cucumber and melon show an increase [41]. These divergent patterns suggest that AS dynamics may be lineage-specific. Consistent with the pattern reported for peach, the total number of AS events in our study decreased from S1 to S3 (Figure 2C). The clustering of samples by species rather than by ripening stage underscores the idea that AS profiles are species-specific traits that likely contribute to distinct developmental and quality attributes among fleshy fruits. This divergence further implies that regulatory networks governing splice-site selection have evolved independently to fine-tune gene expression in ways that align with species-specific developmental requirements.
Among plant AS types, intron retention is generally the most prevalent, accounting for approximately 56% and 53.5% of all AS events in Arabidopsis and rice, respectively [5,44]. Although an earlier Illumina-based analysis in peach suggested that A3′ splice site usage is the most abundant type [41], both PacBio [25] and ONT long-read datasets, including the present study, consistently identify intron retention as the predominant AS category, with A3′ splice sites being the second most frequent (Figure 2C). This concordance across long-read platforms highlights the improved accuracy with which third-generation sequencing resolves splicing complexity.
IR type AS is one of the best-studied AS types in plants. Retention of introns often introduces premature stop codons, leading to the production of truncated transcripts that may be retained in the nucleus to escape NMD, exported to the cytoplasm and degraded, or translated into truncated proteins [45]. The functional consequences of AS greatly depend on the relative positions of introns and protein domains. When an intron is retained within a region encoding a critical domain, the resulting isoform typically lacks function [46]. Evolutionary conservation can provide strong clues to AS function, as conserved AS patterns are more likely to yield adaptive isoforms [47]. MADS-box genes in several plant species generate alternative isoforms through intron retention within the 5′ region, disrupting canonical domains [48,49,50]. In this study, we identified an intron retention event in the peach PpMADS6 gene that produced an isoform lacking the K-box domain. This coordinated alteration between protein-domain architecture and AS pattern suggests that domain-separating intron retention may represent a conserved mechanism within certain gene families.

4.3. MADS-Box TFs Participate Contribute to Fruit Ripening

MADS-box and NAC transcription factors are central regulatory hubs governing the ripening of fleshy fruits. Based on their predominant regulatory modules, fruits can be broadly classified into MADS-type, NAC-type, and dual-type categories [31]. Peach has been classified as an NAC-type fruit due to coordinated expression and methylation changes of PpNAC1, a regulator of ethylene biosynthesis, anthocyanin accumulation, sugar and aroma compound production, and ripening time PpNAC1 [32,51,52,53]. This classification is mainly due to the relatively stable expression of many MADS-box genes throughout development, including early stages. However, this does not imply that MADS genes are uninvolved in peach fruit ripening. Several MADS-box genes, including PpMADS2, PpMADS6, and PpMADS7, have documented roles in modulating ripening processes [33,54,55,56,57]. Among these, PpMADS6 (also referred to as PrupeFUL4) has been proposed to promote ripening, potentially through activation of the ethylene biosynthesis genes PpACS1 and PpACO1 [57]. In our study, results of dual luciferase assays in tobacco leaves showed that PpACS1 and PpACO1 were not significantly activated by PpMADS6a, but the softening-related gene PpPG1 was identified as a downstream target (Figure 6B). Considering that MADS-box genes are also highly expressed in non-climacteric fruits such as watermelon [31], we propose that these genes may perform a conserved function related to fruit softening across species.
MADS proteins typically function as dimers, with the I and K domains contributing to dimer formation [58]. In this study, the PpMADS6b isoform lacking the complete K-box domain did not exhibit a dominant-negative effect on PpMADS6a activity in tobacco leaves based dual luciferase assays, suggesting that PpMADS6b may still participate in dimerization, potentially through its intact I domain (Figure 6A). Future work is needed to clarify whether PpMADS6b can physically interact with PpMADS6a and how such interactions contribute to developmental regulation.

5. Conclusions

This study employs ONT to generate a comprehensive transcriptomic profile of peach fruit across three major developmental stages. The long-read sequencing dataset substantially enriches the current genome annotation, revealing 44,042 nonredundant isoforms, including 1109 novel genes and 32,289 novel isoforms, and provides an important resource for functional genomics in Prunus persica. Our analysis shows that alternative splicing is both pervasive and dynamically regulated during fruit development, with intron retention and alternative 3′ splice site events being the most abundant types. The highest abundance of AS events occurs at the early developmental stage (S1), highlighting the particular importance of AS in orchestrating early fruit growth.
A central finding of this work is the functional characterization of alternative splicing at the PpMADS6 locus, which produces two isoforms: the full-length PpMADS6a and a truncated PpMADS6b generated through intron retention. We demonstrate that PpMADS6a specifically activates the promoter of the fruit-softening gene PpPG1 by recognizing a key cis-element (proPpPG1_cis1), whereas PpMADS6b lacks this regulatory activity. This illustrates how alternative splicing fine-tunes transcription factor function by generating isoforms with distinct regulatory capacities, thereby adding complexity to the control of fruit ripening. Although peach has been characterized as an NAC-type fruit with respect to ripening regulation, our findings underscore the significant contribution of MADS-box transcription factors, particularly through alternative splicing.
In conclusion, our ONT-based transcriptome analysis refines the genomic annotation of peach and highlights alternative splicing as a critical post-transcriptional mechanism that enhances transcriptional diversity during fruit development. The functional divergence of PpMADS6 isoforms further demonstrates the role of splicing variation in modulating key ripening-related processes. These results deepen our understanding of the molecular basis of peach fruit development and provide a framework for future efforts to improve fruit quality through the targeted manipulation of splicing events.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy16020197/s1, Figure S1: Different fruit developmental stages of peach ‘Li Xia Hong’; Figure S2: Annotation of the novel isoforms. Annotation by eggNOG database (A) and Nr database (B); Figure S3: Heatmap analysis of all novel genes; Table S1: Distribution of identified fusion genes in different libraries; Table S2: Expression levels of novel genes in different libraries; Table S3: Primers used in this study.

Author Contributions

Conceptualization, H.Z.; methodology, H.Z. and X.W.; software, H.Z. and Q.X.; validation, L.J., P.S., Y.S. and Y.W.; formal analysis, X.W., L.J., P.S., Y.S., Y.W. and Q.X.; investigation, H.Z. and X.W.; resources, H.P. and J.Z.; data curation, H.Z., X.W., L.J., P.S., Y.S. and Y.W.; writing—original draft preparation, H.Z.; writing—review and editing, H.Z., X.W., L.J., P.S., Y.S., Y.W., Q.X., H.P. and J.Z.; visualization, H.Z. and Q.X.; supervision, J.Z.; project administration, H.P. and J.Z.; funding acquisition, H.Z., H.P. and J.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This project was supported by funds received from Anhui Province’s Key Research and Development Project (2023n06020053), Natural Science Foundation of Anhui Province (2508085MC070), The Agriculture Research System of Anhui Province (AHNYCYTX-10), and Agriculture Research System of China (CARS-30-Z-18).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw RNA-seq data reported in this study were deposited in NCBI BioProject PRJNA939397.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviation

Oxford Nanopore Technologies, ONT; alternative splicing, AS; intron retention, IR; alternative 3′ splice site, A3′S; alternative 5′ splice site, A5′S; exon skipping, ES; mutually exclusive exon, ME; premature termination codons, PTCs; nonsense-mediated mRNA decay, NMD; abscisic acid, ABA; shoot apical meristem, SAM; minimal CaMV 35S promoter,35Smini; days after full bloom (DAFB); transcription factor, TF; differentially expressed transcripts, DETs; counts per million, CPM.

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Figure 1. Annotation of the novel genes. Annotation by eggNOG database (A) and Nr database (B).
Figure 1. Annotation of the novel genes. Annotation by eggNOG database (A) and Nr database (B).
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Figure 2. Overview and proportion of alternative splicing (AS) events. (A) Schematic illustration of the five main types of AS events analyzed: IR, intron retention, A3′S, alternative 3′ splice site, A5′S, alternative 5′ splice site, ES, exon skipping, ME, mutually exclusive exon. (B) The relative abundance (percentage) of each AS event type. (C) Changes in the number of each AS event type during fruit development and ripening.
Figure 2. Overview and proportion of alternative splicing (AS) events. (A) Schematic illustration of the five main types of AS events analyzed: IR, intron retention, A3′S, alternative 3′ splice site, A5′S, alternative 5′ splice site, ES, exon skipping, ME, mutually exclusive exon. (B) The relative abundance (percentage) of each AS event type. (C) Changes in the number of each AS event type during fruit development and ripening.
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Figure 3. Venn diagram of the AS events identified in this study and the previous Nanopore RNA-Seq.
Figure 3. Venn diagram of the AS events identified in this study and the previous Nanopore RNA-Seq.
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Figure 4. Overall view of differently expressed transcripts (DETs). (A) Venn diagram of DETs in different developmental stages. (B) Heatmap analysis of all transcripts. (C) Clustering of DETs by Fitted curve analysis.
Figure 4. Overall view of differently expressed transcripts (DETs). (A) Venn diagram of DETs in different developmental stages. (B) Heatmap analysis of all transcripts. (C) Clustering of DETs by Fitted curve analysis.
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Figure 5. Comparison of expression profiles for randomly selected transcripts measured by RT–qPCR and RNA-Seq. Data are presented as the mean ± SE from three biological replicates.
Figure 5. Comparison of expression profiles for randomly selected transcripts measured by RT–qPCR and RNA-Seq. Data are presented as the mean ± SE from three biological replicates.
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Figure 6. Activation test of fruit ripening-related gene promoters by PpMADS6a and PpMADS6b. (A) Coding sequence (CDS) and protein domain structures of PpMADS6a and PpMADS6b. Blue, green, and brown boxes represent the MADS, I, and K-box domains, respectively. (B) Activation test of the PpACS1, PpACO1, PpPG1, and PpPG2 promoters by PpMADS6a and PpMADS6b, as determined by dual-luciferase reporter assays in young Nicotiana benthamiana leaves. EV, empty vector. Data are presented as the mean ± standard error (SE) of four biological replicates. Statistical significance was determined by one-way ANOVA. Different letters above the bars indicate statistically significant differences (p < 0.05).
Figure 6. Activation test of fruit ripening-related gene promoters by PpMADS6a and PpMADS6b. (A) Coding sequence (CDS) and protein domain structures of PpMADS6a and PpMADS6b. Blue, green, and brown boxes represent the MADS, I, and K-box domains, respectively. (B) Activation test of the PpACS1, PpACO1, PpPG1, and PpPG2 promoters by PpMADS6a and PpMADS6b, as determined by dual-luciferase reporter assays in young Nicotiana benthamiana leaves. EV, empty vector. Data are presented as the mean ± standard error (SE) of four biological replicates. Statistical significance was determined by one-way ANOVA. Different letters above the bars indicate statistically significant differences (p < 0.05).
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Figure 7. Transcriptional activation of the PpPG1 promoter by PpMADS6a and PpMADS6b. Transcriptional activity was measured using a dual-luciferase reporter assay. Data are presented as the mean ± standard error (SE) of four biological replicates. Statistical significance was determined by one-way ANOVA. Different letters above the bars indicate statistically significant differences (p < 0.05).
Figure 7. Transcriptional activation of the PpPG1 promoter by PpMADS6a and PpMADS6b. Transcriptional activity was measured using a dual-luciferase reporter assay. Data are presented as the mean ± standard error (SE) of four biological replicates. Statistical significance was determined by one-way ANOVA. Different letters above the bars indicate statistically significant differences (p < 0.05).
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Figure 8. Identification of cis-elements within the PpPG1 promoter mediating PpMADS6a interaction. (A) Schematic diagrams of the effector and reporter constructs used in the dual-luciferase assay. (B) Transcriptional activation analysis of tandem repeats of putative MADS-domain binding cis-elements by PpMADS6a, measured by dual-luciferase reporter assays. Data are presented as the mean ± standard error (SE) from four biological replicates. Statistical significance was determined by one-way ANOVA. Different letters above the bars indicate statistically significant differences (p < 0.05).
Figure 8. Identification of cis-elements within the PpPG1 promoter mediating PpMADS6a interaction. (A) Schematic diagrams of the effector and reporter constructs used in the dual-luciferase assay. (B) Transcriptional activation analysis of tandem repeats of putative MADS-domain binding cis-elements by PpMADS6a, measured by dual-luciferase reporter assays. Data are presented as the mean ± standard error (SE) from four biological replicates. Statistical significance was determined by one-way ANOVA. Different letters above the bars indicate statistically significant differences (p < 0.05).
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Table 1. Summary of the ONT full-length RNA-Seq data of the peach fruits.
Table 1. Summary of the ONT full-length RNA-Seq data of the peach fruits.
SampleIDReadNumMeanLengthMaxLengthClean ReadsFull-Length Percentage
S1_rep15,841,309130112,5305,765,8790.7848
S1_rep25,575,180119510,7455,509,7250.7333
S1_rep35,102,038116110,0875,043,3010.738
S3_rep15,519,868109898815,465,8330.7514
S3_rep25,353,968112799275,299,4820.7479
S3_rep35,286,179118591005,224,8140.7516
S4_rep15,205,734112495995,152,4390.7627
S4_rep25,628,959118912,7465,565,7270.7449
S4_rep35,458,163104897475,402,1630.7422
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Zhou, H.; Wang, X.; Jiang, L.; Shi, P.; Sheng, Y.; Wang, Y.; Xie, Q.; Zhang, J.; Pan, H. Oxford Nanopore Full-Length Transcriptome Reveals Alternative Splicing and Its Functional Diversity in Regulating Fruit Ripening in Peach (Prunus persica). Agronomy 2026, 16, 197. https://doi.org/10.3390/agronomy16020197

AMA Style

Zhou H, Wang X, Jiang L, Shi P, Sheng Y, Wang Y, Xie Q, Zhang J, Pan H. Oxford Nanopore Full-Length Transcriptome Reveals Alternative Splicing and Its Functional Diversity in Regulating Fruit Ripening in Peach (Prunus persica). Agronomy. 2026; 16(2):197. https://doi.org/10.3390/agronomy16020197

Chicago/Turabian Style

Zhou, Hui, Xiao Wang, Liuqiong Jiang, Pei Shi, Yu Sheng, Yunyun Wang, Qingmei Xie, Jinyun Zhang, and Haifa Pan. 2026. "Oxford Nanopore Full-Length Transcriptome Reveals Alternative Splicing and Its Functional Diversity in Regulating Fruit Ripening in Peach (Prunus persica)" Agronomy 16, no. 2: 197. https://doi.org/10.3390/agronomy16020197

APA Style

Zhou, H., Wang, X., Jiang, L., Shi, P., Sheng, Y., Wang, Y., Xie, Q., Zhang, J., & Pan, H. (2026). Oxford Nanopore Full-Length Transcriptome Reveals Alternative Splicing and Its Functional Diversity in Regulating Fruit Ripening in Peach (Prunus persica). Agronomy, 16(2), 197. https://doi.org/10.3390/agronomy16020197

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