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As the core of heterosis utilization, cytoplasmic male sterility (CMS) has been widely used in hybrid seed production. Previous studies have shown that CMS is always closely related to the altered programming of mitochondrial genes. To explore candidate CMS genes in cotton (Gossypium hirsutum), sequencing and de novo assembly were performed on the mitochondrial genome of the G. hirsutum CMS line SI3A, with G. harknessii CMS-D2 cytoplasm, and the corresponding G. hirsutum restorer line 0-613-2R. Remarkable variations in genome structure and gene transcripts were detected. The mitochondrial genome of SI3A has three circle molecules, including one main circle and two sub-circles, while 0-613-2R only has one. RNA-seq and RT-qPCR analysis proved that orf606a and orf109a, which have a chimeric structure and transmembrane domain, were highly expressed in abortive anthers of SI3A. In addition, comparative analysis of RNA-seq and full-length transcripts revealed the complex I gene nad4 to be expressed at a lower level in SI3A than in its restorer and that it featured an intron retention splicing pattern. These two novel chimeric ORFs and nad4 are potential candidates that confer CMS character in SI3A. This study provides new insight into the molecular basis of the nuclear–cytoplasmic interaction mechanism, and that putative CMS genes might be important sources for future precise design cross-breeding of cotton.

The technological advancements in single‐cell transcriptome analysis make significant progress in both depth and breadth. However, balancing the cell analysis throughput with full‐length transcript coverage remains a persistent challenge. Here, CBTi‐seq (Combinational Barcoded Tn5 Transposon Insertion sequencing) is reported, leveraging Tn5 transposase‐mediated molecular assembly of combinatorial barcodes and unique molecular identifiers (UMIs) to enable high‐resolution multiplexed sequencing of the full‐length transcriptome in single cells. This approach achieves molecular resolution by end‐to‐end sequencing, enabling unambiguous reconstruction of splice variants and structural variations with base‐pair precision. The design of orthogonal combination barcode Tn5 reduces DNA barcode diversity while enhancing multiplexing flexibility, and Tn5‐delivered UMIs insertion eliminates read bias, providing accurately quantifies transcript abundance through the tagging of each fragment. The method is compatible with both single‐cell and spatially resolved tissue microenvironment. Compared with commercial terminal library and other full‐length sequencing methods, CBTi‐seq achieves superior sensitivity and resolution while significantly reducing costs and work time (≈5 h). Moreover, cell‐type‐specific alternative splicing patterns are robustly identified in both gene‐edited cells and human testicular cells, leveraging this high‐resolution capability to further reveal modality dynamic events and isoform switching independent of gene expression changes during spermatogenesis with the potential to reproductive development and diagnostic treatment. CBTi‐seq is reported, leveraging Tn5 transposase‐mediated molecular assembly of combinatorial barcodes and UMIs for high‐resolution multiplexed sequencing of the full‐length transcriptome in single cells. This approach achieves molecular resolution by end‐to‐end sequencing, enabling reconstruction of splice variants and accurate quantification of transcript abundance. CBTi‐seq offers superior sensitivity and resolution, significantly reducing costs (≈0.7 USD cell−1) and fast work time (≈5 h).

Simple sequence repeats (SSRs) are found in nonrandom distributions in genomes and are thought to impact gene expression. The distribution patterns of 48 295 SSRs of Paphiopedilum malipoense are mined and characterized based on the first full‐length transcriptome and comprehensive transcriptome dataset from 12 organs. Statistical genomics analyses are used to investigate how SSRs in transcripts affect gene expression. The results demonstrate the correlations between SSR distributions, characteristics, and expression level. Nine expression‐modulating motifs (expMotifs) are identified and a model is proposed to explain the effect of their key features, potency, and gene function on an intra‐transcribed region scale. The expMotif‐transcribed region combination is the most predominant contributor to the expression‐modulating effect of SSRs, and some intra‐transcribed regions are critical for this effect. Genes containing the same type of expMotif‐SSR elements in the same transcribed region are likely linked in function, regulation, or evolution aspects. This study offers novel evidence to understand how SSRs regulate gene expression and provides potential regulatory elements for plant genetic engineering. Statistical genomics analyses reveal a high‐density SSR landscape of slipper orchid P. malipoense transcriptome. Transcribed SSRs can alter the gene expression potential and tissue‐specificity where they are located. The expression regulation effects of expMotifs are finely delineated, hotspots of expMotifs exist within the transcribed regions, and the mechanisms are illustrated by a model.

Platycodon grandiflorus (Jacq.) A. DC. (P. grandiflorus), a traditional Chinese medicine, contains considerable triterpenoid saponins with broad pharmacological activities. Triterpenoid saponins are the major components of P. grandiflorus. Here, single-molecule real-time and next-generation sequencing technologies were combined to comprehensively analyse the transcriptome and identify genes involved in triterpenoid saponin biosynthesis in P. grandiflorus. We quantified four saponins in P. grandiflorus and found that their total content was highest in the roots and lowest in the stems and leaves. A total of 173,354 non-redundant transcripts were generated from the PacBio platform, and three full-length transcripts of β-amyrin synthase, the key synthase of β-amyrin, were identified. A total of 132,610 clean reads obtained from the DNBSEQ platform were utilised to explore key genes related to the triterpenoid saponin biosynthetic pathway in P. grandiflorus, and 96 differentially expressed genes were selected as candidates. The expression levels of these genes were verified by quantitative real-time PCR. Our reliable transcriptome data provide valuable information on the related biosynthesis pathway and may provide insights into the molecular mechanisms of triterpenoid saponin biosynthesis in P. grandiflorus.

Background The flower of the safflower ( Carthamus tinctorius L.) has been widely used in traditional Chinese medicine for the ability to improve cerebral blood flow. Flavonoids are the primary bioactive components in safflower, and their biosynthesis has attracted widespread interest. Previous studies mostly used second-generation sequencing platforms to survey the putative flavonoid biosynthesis genes. For a better understanding of transcription data and the putative genes involved in flavonoid biosynthesis in safflower, we carry our study. Results High-quality RNA was extracted from six types of safflower tissue. The RNAs of different tissues were mixed equally and used for multiple size-fractionated libraries (1–2, 2–3 and 3-6 k) library construction. Five cells were carried (2 cells for 1–2 and for 2-3 k libraries and 1 cell for 3-6 k libraries). 10.43Gb clean data and 38,302 de-redundant sequences were captured. 44 unique isoforms were annotated as encoding enzymes involved in flavonoid biosynthesis. The full length flavonoid genes were characterized and their evolutional relationship and expressional pattern were analyzed. They can be divided into eight families, with a large differences in the tissue expression. The temporal expressions under MeJA treatment were also measured, 9 genes are significantly up-regulated and 2 genes are significantly down-regulated. The genes involved in flavonoid synthesis in safflower were predicted in our study. Besides, the SSR and lncRNA are also analyzed in our study. Conclusions Full-length transcriptome sequences were used in our study. The genes involved in flavonoid synthesis in safflower were predicted in our study. Combined the determination of flavonoids, CtC4H2 , CtCHS3 , CtCHI3 , CtF3H3 , CtF3H1 are mainly participated in MeJA promoting the synthesis of flavonoids. Our results also provide a valuable resource for further study on safflower.

Actinidia latifolia is one of the very few kiwifruit genotypes with extremely high ascorbic acid (AsA) content. However, a transcriptome atlas of this species is lacking. The accumulation of AsA during fruit development and ripening and the associated molecular mechanisms are still poorly understood. Herein, dynamic changes in AsA content at six different stages of A. latifolia fruit development and ripening were determined. AsA content of A. latifolia fruit reached 1108.76 ± 35.26 mg 100 g−1 FW at full maturity. A high-quality, full-length (FL) transcriptome of A. latifolia was successfully constructed for the first time using third-generation sequencing technology. The transcriptome comprises 326,926 FL non-chimeric reads, 15,505 coding sequences, 2882 transcription factors, 18,797 simple sequence repeats, 3328 long noncoding RNAs, and 231 alternative splicing events. The genes involved in AsA biosynthesis and recycling pathways were identified and compared with those in different kiwifruit genotypes. The correlation between the AsA content and expression levels of key genes in AsA biosynthesis and recycling pathways was revealed. LncRNAs that participate in AsA-related gene expression regulation were also identified. Gene expression patterns in AsA biosynthesis and metabolism exhibited a trend similar to that of AsA accumulation. Overall, this study paves the way for genetic engineering to develop kiwifruits with super-high AsA content.

Spinibarbus hollandi is an economically significant subtropical fish in China, valued for both ornamental and aquaculture purposes. However, the full-length transcriptomic resources for this species remain scarce. To address this gap, we constructed the first multi-tissue, full-length transcriptome of S. hollandi using PacBio single-molecule real-time sequencing. We assembled 23,403 non-redundant transcripts, including 15,197 unigenes, with a mean length of 2,147 bp and an N50 of 2,868 bp. Among these, 14,567 unigenes (95.85%) were annotated in public databases. Our analysis identified 373 alternative splicing events, with retained intron being the most common type. We also predicted 2,397 long non-coding RNAs. Furthermore, a comprehensive screening revealed 7,449 simple sequence repeat (SSR) loci, comprising 1,198 compound and 6,251 perfect SSRs, with an occurrence frequency of 30.96%. Di- and tri-nucleotide repeats were the predominant types. Thirteen highly polymorphic SSR loci showed robust polymorphism across four geographical populations. This study provides a crucial data foundation and genetic resource for functional gene research, molecular marker-assisted breeding, and germplasm conservation in S. hollandi .

Aerial bulbils are important vegetative reproductive organs in Lilium . They are often perpetually dormant in most Lilium species, and little is known about the induction of these vegetative structures. The world-famous Oriental hybrid lily cultivar ‘Sorbonne’, which blooms naturally devoid of aerial bulbils, is known for its lovely appearance and sweet fragrance. We found that decapitation stimulated the outgrowth of aerial bulbils at lower stems (LSs) and then application of low and high concentrations of IAA promoted aerial bulbils emergence around the wound at upper stems (USs) of ‘Sorbonne’. However, the genetic basis of aerial bulbil induction is still unclear. Herein, ‘Sorbonne’ transcriptome has been sequenced for the first time using the combination of third-generation long-read and next-generation short-read technology. A total of 46,557 high-quality non-redundant full-length transcripts were generated. Transcriptomic profiling was performed on seven tissues and stems with treatments of decapitation and application of low and high concentrations of IAA, respectively. Functional annotation of 1918 DEGs within stem samples of different treatments showed that hormone signaling, sugar metabolism and wound-induced genes were crucial to bulbils outgrowth. The expression pattern of auxin-, shoot branching hormone-, plant defense hormone- and wound-inducing-related genes indicated their crucial roles in bulbil induction. Then we established five hormone- and wounding-regulated co-expression modules and identified some candidate transcriptional factors, such as MYB, bZIP, and bHLH, that may function in inducing bulbils. High connectivity was observed among hormone signaling genes, wound-induced genes, and some transcriptional factors, suggesting wound- and hormone-invoked signals exhibit extensive cross-talk and regulate bulbil initiation-associated genes via multilayered regulatory cascades. We propose that the induction of aerial bulbils at LSs after decapitation can be explained as the release of apical dominance. In contrast, the induction of aerial bulbils at the cut surface of USs after IAA application occurs via a process similar to callus formation. This study provides abundant candidate genes that will deepen our understanding of the regulation of bulbil outgrowth, paving the way for further molecular breeding of lily.

Genes with similar or related functions in chloroplasts are often arranged in close proximity, forming clusters on chromosomes. These clusters are transcribed coordinated to facilitate the expression of genes with specific function. Our previous study revealed a significant negative correlation between the chloroplast gene expression level of the rare medicinal fern Ophioglossum vulgatum and its evolutionary rates as well as selection pressure. Therefore, in this study, we employed a combination of SMRT and Illumina sequencing technology to analyze the full-length transcriptome sequencing of O. vulgatum for the first time. In particular, we experimentally identified gene clusters based on transcriptome data and investigated the effects of chloroplast gene clustering on expression and evolutionary patterns. The results revealed that the total sequenced data volume of the full-length transcriptome of O. vulgatum amounted to 71,950,652,163 bp, and 110 chloroplast genes received transcript coverage. Nine different types of gene clusters were experimentally identified in their transcripts. The chloroplast cluster genes may cause a decrease in non-synonymous substitution rate and selection pressure, as well as a reduction in transversion rate, transition rate, and their ratio. While expression levels of chloroplast cluster genes in leaf, sporangium, and stem would be relatively elevated. The Mann–Whitney U test indicated statistically significant in the selection pressure, sporangia and leaves groups (P < 0.05). We have contributed novel full-length transcriptome data resources for ferns, presenting new evidence on the effects of chloroplast gene clustering on expression land evolutionary patterns, and offering new theoretical support for transgenic research through gene clustering.Key messageThe clustering of chloroplast genes in Ophioglossum vulgatum demonstrates a tendency towards elevated expression levels in sporangium, leaf, and stem, while exhibiting a decline in evolutionary rates and selection pressure.

Light, as an energy source for plant photosynthesis, can not only affect the growth and development of plants, but also affect their leaf color. This study used white (WL), red (RL), and blue light (BL) to treat tea cuttings, aiming to investigate the effect of light quality on the color of tea leaves. The results showed that tea leaves turned yellow under red light, the SPAD and Fv/Fm values were significantly lower than WL and BL. Full-length transcriptome was analyzed, photosynthesis and chlorophyll biosynthesis related genes such as PsbS , Psb28 , HemL , and POR had the lowest expression levels under RLCarotenoid biosynthesis related genes ZEP , ABA2 , and CRTISO had the higher expression levels under RL. This study revealed the molecular mechanism of RL induced leaf yellowing in tea plants, providing new insights for the application of light quality in tea plants.