Transcriptional Regulation Associated with Subcutaneous Adipogenesis in Porcine ACSL1 Gene.

Long-chain acyl-CoA synthetase 1 (ACSL1) plays an important role in fatty acid metabolism and fat deposition. The transcription of the ACSL1 gene is regulated specifically among cells and physiological processes, and transcriptional regulation of ACSL1 in adipogenesis remains elusive. Here, we characterize transcription factors (TFs) associated with adipogenesis in the porcine ACSL1 gene. CCAAT-enhancer binding protein (C/EBP)α, a well-known adipogenic marker, was found to enhance the expression of the ACSL1 gene via binding two tandem motifs in the promoter. Further, we demonstrate that ACSL1 mediates C/EBPα effects on adipogenesis in preadipocytes cultured from subcutaneous fat tissue of pigs via gain- and loss-of-function analyses. The cAMP-response element binding protein, another TF involved in adipogenesis, was also identified in the regulation of ACSL1 gene expression. Additionally, single nucleotide polymorphisms (SNPs) were screened in the promoter of ACSL1 among four breeds including the Chinese indigenous Min, and Duroc, Berkshire, and Yorkshire pigs through sequencing of PCR products. Two tightly linked SNPs, -517G>T and -311T>G, were found exclusively in Min pigs. The haplotype mutation decreases promoter activity in PK-15 and ST cells, and in vivo the expression of ACSL1, illustrating a possible role in adipogenesis regulated by C/EBPα/ACSL1 axis. Additionally, a total of 24 alternative splicing transcripts were identified, indicating the complexity of alternative splicing in the ACSL1 gene. The results will contribute to further revealing the regulatory mechanisms of ACSL1 during adipogenesis and to the characterization of molecular markers for selection of fat deposition in pigs.

Enhanced Flame Retardancy of Styrene-Acrylic Emulsion Based Damping Composites Based on an APP/EG Flame-Retardant System.

Developing flame-retarded styrene-acrylic emulsion (SAE) based damping composites is a challenging task because of their very high flammability. A promising approach is the synergistic combination of expandable graphite (EG) and ammonium polyphosphate (APP). In this study, the surface modification of APP was modified by commercial titanate coupling agent ndz-201 through ball milling, and the SAE-based composite material was prepared with SAE and different ratios of modified ammonium polyphosphate (MAPP) and EG. The surface of MAPP was successfully chemically modified by NDZ-201 through scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), Energy Dispersion Spectroscopy (EDS), and contact angle. The effects of different ratios of MAPP and EG on the dynamic and static mechanical properties and flame retardancy of composite materials were explored. The results showed that when MAPP:EG = 1:4, the limiting oxygen index (LOI) of the composite material was 52.5%, and the vertical burning test (UL-94) was at the V0 level. Its LOI increased by 141.9% compared to the composite materials without flame retardant. The optimized formulation of MAPP and EG in SAE-based damping composite materials showed a significant synergistic effect on the flame retardancy of the composite material.

Effects of Maleic Anhydride-Grafted Polyethylene on the Properties of Artificial Marble Waste Powder/Linear Low-Density Polyethylene Composites with Ultra-High Filling Content.

The need to reach carbon neutrality as soon as possible has made the use of recycled materials widespread. However, the treatment of artificial marble waste powder (AMWP) containing unsaturated polyester is a very challenging task. This task can be accomplished by converting AMWP into new plastic composites. Such conversion is a cost-effective and eco-friendly way to recycle industrial waste. However, the lack of mechanical strength in composites and the low filling content of AMWP have been major obstacles to its practical application in structural and technical buildings. In this study, a composite of AMWP/linear low-density polyethylene (LLDPE) filled with a 70 wt% AMWP content was fabricated using maleic anhydride-grafted polyethylene as a compatibilizer (MAPE). The mechanical strength of the prepared composites is excellent (tensile strength ~18.45 MPa, impact strength ~51.6 kJ/m2), making them appropriate as useful building materials. Additionally, laser particle size analysis, Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, and thermogravimetric analysis were used to examine the effects of maleic anhydride-grafted polyethylene on the mechanical properties of AMWP/LLDPE composites and its mechanism of action. Overall, this study offers a practical method for the low-cost recycling of industrial waste into high-performance composites.

Advances and Challenges in Spatial Transcriptomics for Developmental Biology.

Development from single cells to multicellular tissues and organs involves more than just the exact replication of cells, which is known as differentiation. The primary focus of research into the mechanism of differentiation has been differences in gene expression profiles between individual cells. However, it has predominantly been conducted at low throughput and bulk levels, challenging the efforts to understand molecular mechanisms of differentiation during the developmental process in animals and humans. During the last decades, rapid methodological advancements in genomics facilitated the ability to study developmental processes at a genome-wide level and finer resolution. Particularly, sequencing transcriptomes at single-cell resolution, enabled by single-cell RNA-sequencing (scRNA-seq), was a breath-taking innovation, allowing scientists to gain a better understanding of differentiation and cell lineage during the developmental process. However, single-cell isolation during scRNA-seq results in the loss of the spatial information of individual cells and consequently limits our understanding of the specific functions of the cells performed by different spatial regions of tissues or organs. This greatly encourages the emergence of the spatial transcriptomic discipline and tools. Here, we summarize the recent application of scRNA-seq and spatial transcriptomic tools for developmental biology. We also discuss the limitations of current spatial transcriptomic tools and approaches, as well as possible solutions and future prospects.

Alternative Splicing Isoforms of Porcine CREB Are Differentially Involved in Transcriptional Transactivation.

The cAMP response element-binding protein (CREB), a basic leucine zipper transcription factor, is involved in the activation of numerous genes in a variety of cell types. The CREB gene is rich in alternative splicing (AS) events. However, studies on the AS of CREB genes in pigs are limited, and few reports have compared the roles of isoforms in activating gene expression. Here, five AS transcripts, V1-5, were characterized by RT-PCR and two, V3 and V5, were new identifications. Both V1 and V2 have all the functional domains of the CREB protein, with similar tissue expression profiles and mRNA stability, suggesting that they have similar roles. The transcriptional transactivation activities of four isoforms encoding complete polypeptides were analyzed on the expression of the B-cell CLL/lymphoma 2-like protein 2 and the poly (A)-binding protein, nuclear 1 genes with a dual-luciferase reporter system, and differential activities were observed. Both V1 and V2 have promoting effects, but their roles are gene-specific. V3 has no effect on the promoter of the two genes, while V4 functions as a repressor. The mechanisms underlying the differential roles of V1 and V2 were analyzed with RNA-seq, and the genes specifically regulated by V1 and V2 were identified. These results will contribute to further revealing the role of CREB and to analyzing the significance of AS in genes.

Chimeric RNA TNNI2-ACTA1-V1 Regulates Cell Proliferation by Regulating the Expression of NCOA3.

Chimeric RNA is a crucial target for tumor diagnosis and drug therapy, also having its unique biological role in normal tissues. TNNI2-ACTA1-V1 (TA-V1), a chimeric RNA discovered by our laboratory in porcine muscle tissue, can inhibit the proliferation of Porcine Skeletal Muscle Satellite Cells (PSCs). The regulatory mechanism of TA-V1 in PSCs remains unclear, but we speculate that NCOA3, DDR2 and RDX may be the target genes of TA-V1. In this study, we explored the effects of NCOA3, DDR2 and RDX on cell viability and cell proliferation by CCK-8 assay, EdU staining and flow cytometry. Furthermore, the regulatory pathway of proliferation in PSCs mediated by TA-V1 through NCOA3 or CyclinD1 was elucidated by co-transfection and co-immunoprecipitation (Co-IP). The results revealed that overexpression of NCOA3 significantly increased cell viability and the expression level of CyclinD1, and also promotes cell proliferation by changing cells from the G1 phase to the S phase. In addition, inhibiting the expression of NCOA3 substantially reduced cell viability and inhibited cell proliferation. Overexpression of DDR2 and RDX had no significant effect on cell viability and proliferation. Co-transfection experiments showed that NCOA3 could rescue the proliferation inhibition of PSCs caused by TA-V1. Co-IP assay indicated that TA-V1 directly interacts with NCOA3. Our study explores the hypothesis that TA-V1 directly regulates NCOA3, indirectly regulating CyclinD1, thereby regulating PSCs proliferation. We provide new putative mechanisms of porcine skeletal muscle growth and lay the foundation for the study of chimeric RNA in normal tissues.

Characterization of a Read-through Fusion Transcript, BCL2L2-PABPN1, Involved in Porcine Adipogenesis.

cis-Splicing of adjacent genes (cis-SAGe) has been involved in multiple physiological and pathological processes in humans. However, to the best of our knowledge, there is no report of cis-SAGe in adipogenic regulation. In this study, a cis-SAGe product, BCL2L2-PABPN1 (BP), was characterized in fat tissue of pigs with RT-PCR and RACE method. BP is an in-frame fusion product composed of 333 aa and all the functional domains of both parents. BP is highly conserved among species and rich in splicing variants. BP was found to promote proliferation and inhibit differentiation of primary porcine preadipocytes. A total of 3074/44 differentially expressed mRNAs (DEmRs)/known miRNAs (DEmiRs) were identified in porcine preadipocytes overexpressing BP through RNA-Seq analysis. Both DEmRs and target genes of DEmiRs were involved in various fat-related pathways with MAPK and PI3K-Akt being the top enriched. PPP2CB, EGFR, Wnt5A and EHHADH were hub genes among the fat-related pathways identified. Moreover, ssc-miR-339-3p was found to be critical for BP regulating adipogenesis through integrated analysis of mRNA and miRNA data. The results highlight the role of cis-SAGe in adipogenesis and contribute to further revealing the mechanisms underlying fat deposition, which will be conductive to human obesity control.

Characterization of Alternative Splicing Events in Porcine Skeletal Muscles with Different Intramuscular Fat Contents.

Meat quality is one of the most important economic traits in pig breeding and production. Intramuscular fat (IMF) is a major factor that improves meat quality. To better understand the alternative splicing (AS) events underlying meat quality, long-read isoform sequencing (Iso-seq) was used to identify differential (D)AS events between the longissimus thoracis (LT) and semitendinosus (ST), which differ in IMF content, together with short-read RNA-seq. Through Iso-seq analysis, we identified a total of 56,789 novel transcripts covering protein-coding genes, lncRNA, and fusion transcripts that were not previously annotated in pigs. We also identified 456,965 AS events, among which 3930 were DAS events, corresponding to 2364 unique genes. Through integrative analysis of Iso-seq and RNA-seq, we identified 1174 differentially expressed genes (DEGs), among which 122 were DAS genes, i.e., DE-DAS genes. There are 12 overlapped pathways between the top 20 DEGs and DE-DAS genes, as revealed by KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, indicating that DE-DAS genes play important roles in the differential phenotype of LT and ST. Further analysis showed that upregulated DE-DAS genes are more important than downregulated ones in IMF deposition. Fatty acid degradation and the PPAR (peroxisome proliferator-activated receptor) signaling pathway were found to be the most important pathways regulating the differential fat deposition of the two muscles. The results update the existing porcine genome annotations and provide data for the in-depth exploration of the mechanisms underlying meat quality and IMF deposition.

Identification of Chimeric RNAs in Pig Skeletal Muscle and Transcriptomic Analysis of Chimeric RNA TNNI2-ACTA1 V1.

Chimeric RNA was considered a special marker of cancer. However, recent studies have demonstrated that chimeric RNAs also exist in non-cancerous cells and tissues. Here, we analyzed and predicted jointly 49 chimeric RNAs by Star-Fusion and FusionMap. One chimeric RNA, we named TNNI2-ACTA1, and its eight transcript variants were identified by reverse transcriptase-polymerase chain reaction. The overexpression of TNNI2-ACTA1 V1 inhibited the proliferation of porcine skeletal muscle satellite cells through down-regulating the mRNA expression levels of cell cycle-related genes cyclinD1. However, as parental genes, there is no such effect in the TNNI2 and ACTA1. To explore the underlying mechanism for this phenomenon, we used RNA-seq to profile the transcriptomes of PSCs with overexpression. Compared with the negative control group, 1,592 differentially expressed genes (DEGs) were upregulated and 1,077 DEGs downregulated in TNNI2 group; 1,226 DEGs were upregulated and 902 DEGs downregulated in ACTA1 group; and 13 DEGs were upregulated and 16 DEGs downregulated in TNNI2-ACTA1 V1 group, respectively. Compared with the parental gene groups, three specific genes were enriched in the TNNI2-ACTA1 V1 group (NCOA3, Radixin, and DDR2). These three genes may be the key to TNNI2-ACTA1 V1 regulating cell proliferation. Taken together, our study explores the role of chimeric RNAs in normal tissues. In addition, our study as the first research provides the foundation for the mechanism of chimeric RNAs regulating porcine skeletal muscle growth.

A Flexible Multimodal Sole Sensor for Legged Robot Sensing Complex Ground Information during Locomotion.

Recent achievements in the field of computer vision, reinforcement learning, and locomotion control have largely extended legged robots' maneuverability in complex natural environments. However, little research focuses on sensing and analyzing the physical properties of the ground, which is crucial to robots' locomotion during their interaction with highly irregular profiles, deformable terrains, and slippery surfaces. A biomimetic, flexible, multimodal sole sensor (FMSS) designed for legged robots to identify the ontological status and ground information, such as reaction force mapping, contact situation, terrain, and texture information, to achieve agile maneuvers was innovatively presented in this paper. The FMSS is flexible and large-loaded (20 Pa-800 kPa), designed by integrating a triboelectric sensing coat, embedded piezoelectric sensor, and piezoresistive sensor array. To evaluate the effectiveness and adaptability in different environments, the multimodal sensor was mounted on one of the quadruped robot's feet and one of the human feet then traversed through different environments in real-world tests. The experiment's results demonstrated that the FMSS could recognize terrain, texture, hardness, and contact conditions during locomotion effectively and retrain its sensitivity (0.66 kPa-1), robustness, and compliance. The presented work indicates the FMSS's potential to extend the feasibility and dexterity of tactile perception for state estimation and complex scenario detection.

Enhancing the performance of photoelectrochemical glucose sensor via the electron cloud bridge of Au in SrTiO3/PDA electrodes.

Developing photoelectrochemical biosensors via efficient photogenerated-charge separation remains a challenging task in biomolecular detection. In this study, we utilised a simple approach for constructing an efficient photoactive organic-inorganic heterojunction interface composed of SrTiO3 with high photocatalytic activity and polydopamine (PDA) with high biocompatibility and electrical conductivity. Gold nanoparticles with dense electron cloud properties were introduced as a bridge between SrTiO3 and PDA (SrTiO3/Au/PDA). The Au bridge allowed the PDA to uniformly and tightly attach on the surface of SrTiO3 electrodes and also provided a separate transmission channel for electrons from PDA to SrTiO3. The rapidly transmitted electrons were captured by a signal-acquisition system, thereby improving the photocurrent signal output. The 3D hollowed out SrTiO3/Au/PDA biosensor manufactured herein was used for glucose detection. The biosensor achieved ultrahigh sensitivities reaching 23.7 µA mM-1 cm-2, an extended linear range (1-20 mM), and a low detection limit (0.012 mM). The excellent results of glucose analysis in serum samples further confirmed the feasibility of the biosensor in clinical applications. In summary, the proposed strategy allowed for the use of an electronic cloud bridge in the construction of glucose biosensors with satisfactory performances, which is promising for the future fabrication of high-performance biosensors.

Bioinspired Hydrogel-Polymer Hybrids with a Tough and Antifatigue Interface via One-Step Polymerization.

Hydrogel hybrids are one of the key factors in life activities and biomimetic science; however, their development and utilization are critically impeded by their inadequate adhesive strength and intricate process. In nature, barnacles can stick to a variety of solid surfaces firmly (adhesive strength above 300 kPa) using a hydrophobic interface, which inspires us to firmly combine hydrogels and polymers through introducing an adhesive layer. By spreading a hydrophobic liquid membrane directly, tough combination of a hydrogel and a polymer substrate could be achieved after one-step polymerization. The fracture energy of the hydrogel attached to the surface of polyvinyl chloride was up to 1200 J m-2 and the tensile strength reached 1.21 MPa. Furthermore, the adhesion samples with this method exhibit an antifatigue performance, having withstood large bends and twists. It should be pointed out that this approach can also be applied to a variety of complicated surfaces. This work may expand the application range of hydrogels and provides an inspiration for hydrogel adhesion.

3D hollow-out TiO2 nanowire cluster/GOx as an ultrasensitive photoelectrochemical glucose biosensor.

Ultra-high sensitivity is difficult to achieve using conventional enzymatic glucose biosensors due to the lack of exposed active sites and steric-hinderance effects. Thus, in the present study, we report a photoelectrochemical (PEC) enzymatic glucose biosensor based on 3-dimensional (3D) hollow-out titanium dioxide (TiO2) nanowire cluster (NWc)/glucose oxidase (GOx), providing more number of exposed active sites, thus constructing a sensor with a higher affinity toward glucose reaction. Excellent performance with an ultra-high sensitivity of 58.9 µA mM-1 cm-2 and 0-2 mM linear range with a determination limit of 8.7 µM was obtained for the detection of glucose. This study might provide a new approach to expose active sites efficiently for remarkable photoelectrochemical performances.

Porcine reproductive and respiratory syndrome virus infection promotes C1QBP secretion to enhance inflammatory responses.

Complement component 1, q subcomponent binding protein (C1QBP) is a receptor for the globular heads of C1q and modulates various biological processes including infection, inflammation, autoimmunity, and cancer. In our previous study to identify differentially expressed secretory proteins in Marc-145 cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), mass spectrum data showed that C1QBP was secreted after PRRSV infection. However, the biological significance of secreted C1QBP remains unclear. In this study, we confirmed that PRRSV infection promoted C1QBP secretion in Marc-145 cells and porcine alveolar macrophages (PAMs), the target cells of PRRSV in vivo. Knockdown of endogenous C1QBP decreased PRRSV-induced inflammatory responses. The purified recombinant porcine C1QBP (poC1QBP) had proinflammatory effects. The exogenous addition of poC1QBP significantly enhanced PRRSV-induced inflammatory responses and abolished the inhibitory effects mediated by poC1QBP-knockdown. Taken together, these results demonstrate that PRRSV infection promotes poC1QBP secretion that enhances inflammatory responses.

Fatty Acids Regulate Porcine Reproductive and Respiratory Syndrome Virus Infection via the AMPK-ACC1 Signaling Pathway.

Lipids play a crucial role in the replication of porcine reproductive and respiratory syndrome virus (PRRSV), a porcine virus that is endemic throughout the world. However, little is known about the effect of fatty acids (FAs), a type of vital lipid, on PRRSV infection. In this study, we found that treatment with a FA biosynthetic inhibitor significantly inhibited PRRSV propagation, indicating the necessity of FAs for optimal replication of PRRSV. Further study revealed that 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), a key kinase antagonizing FA biosynthesis, was strongly activated by PRRSV and the pharmacological activator of AMPK exhibited anti-PRRSV activity. Additionally, we found that acetyl-CoA carboxylase 1 (ACC1), the first rate-limiting enzyme in the FA biosynthesis pathway, was phosphorylated (inactive form) by PRRSV-activated AMPK, and active ACC1 was required for PRRSV proliferation, suggesting that the PRRSV infection induced the activation of the AMPK-ACC1 pathway, which was not conducive to PRRSV replication. This work provides new evidence about the mechanisms involved in host lipid metabolism during PRRSV infection and identifies novel potential antiviral targets for PRRSV.

Isolation and Characterization of CsWRKY7, a Subgroup IId WRKY Transcription Factor from Camellia sinensis, Linked to Development in Arabidopsis.

WRKY transcription factors (TFs) containing one or two WRKY domains are a class of plant TFs that respond to diverse abiotic stresses and are associated with developmental processes. However, little has been known about the function of WRKY gene in tea plant. In this study, a subgroup IId WRKY gene CsWRKY7 was isolated from Camellia sinensis, which displayed amino acid sequence homology with Arabidopsis AtWRKY7 and AtWRKY15. Subcellular localization prediction indicated that CsWRKY7 localized to nucleus. Cis-acting elements detected in the promotor region of CsWRKY7 are mainly involved in plant response to environmental stress and growth. Consistently, expression analysis showed that CsWRKY7 transcripts responded to NaCl, mannitol, PEG, and diverse hormones treatments. Additionally, CsWRKY7 exhibited a higher accumulation both in old leaves and roots compared to bud. Seed germination and root growth assay indicated that overexpressed CsWRKY7 in transgenic Arabidopsis was not sensitive to NaCl, mannitol, PEG, and low concentration of ABA treatments. CsWRKY7 overexpressing Arabidopsis showed a late-flowering phenotype under normal conditions compared to wild type. Furthermore, gene expression analysis showed that the transcription levels of the flowering time integrator gene FLOWERING LOCUS T (FT) and the floral meristem identity genes APETALA1 (AP1) and LEAFY (LFY) were lower in WRKY7-OE than in the WT. Taken together, these findings indicate that CsWRKY7 TF may participate in plant growth. This study provides a potential strategy to breed late-blooming tea cultivar.

A Novel Chimeric Mitochondrial Gene Confers Cytoplasmic Effects on Seed Oil Content in Polyploid Rapeseed (Brassica napus).

Cytoplasmic effects (CEs) have been discovered to influence a diverse array of agronomic traits in crops, and understanding the underlying mechanisms can help accelerate breeding programs. Seed oil content (SOC) is of great agricultural, nutritional, and economic importance. However, the genetic basis of CEs on SOC (CE-SOC) remains enigmatic. In this study, we use an optimized approach to sequence the cytoplasmic (plastid and mitochondrial) genomes of allotetraploid oilseed rape (Brassica napus) cultivars, 51218 and 56366, that bear contrasting CE-SOC. By combining comparative genomics and genome-wide transcriptome analysis, we identify mitochondria-encoded orf188 as a potential CE-SOC determinant gene. Functional analyses in the model system Arabidopsis thaliana and rapeseed demonstrated that orf188 governs CE-SOC and could significantly increase SOC, strikingly, through promoting the yield of ATP. Consistent with this finding, transcriptional profiling with microarray and RNA sequencing revealed that orf188 affects transcriptional reprogramming of mitochondrial energy metabolism to facilitate ATP production. Intriguingly, orf188 is a previously uncharacterized chimeric gene, and the presence of this genetic novelty endows rapeseed with positive CE-SOC. Our results shed light on the molecular basis of CEs on a key quantitative trait in polyploid crops and enrich the theory of maternal control of oil content, providing new scientific guidance for breeding high-oil rapeseed germplasms.

The complete chloroplast genome of an albino tea, Camellia sinensis cultivar 'Baiye 1'.

For obtaining the sequence and phylogenetic position of Camellia sinensis cultivar 'Baiye1', the complete chloroplast genome was determined. This chloroplast genome is 156,691 bp in length with overall GC content of 37.3%. It was comprised of a large single-copy (LSC) region of 86,585bp, a small single-copy (SSC) region of 18,276bp, and two inverted repeat (IR) regions of 26,083 bp. It contains 87 protein-coding, 8 rRNA, and 35 tRNA genes. Phylogenetic analysis showed 'Baiye1' and C. sinensis cv. 'Longjing43' were clustered into a group. These results may contribute to the further understanding of the albino phenotype and genetic evolution.

The complete chloroplast genome sequence of Camellia tachangensis F. C. Zhang (Theaceae).

To understand genetic background and phylogenetic position of Camellia tachangensis, we determined its complete chloroplast genome sequence which is 157,026 bp in length with overall GC content of 36.7%. It has four sub regions: a large single-copy (LSC) region (86,669 bp) and a small single-copy (SSC) region (18,253 bp) are separated by two inverted repeat (IR) regions (26,052 bp each). A total of 129 genes were annotated, containing 86 protein-coding genes, 35 tRNA genes, and 8 rRNA genes. Phylogenetic trees showed C. tachangensis clustered with Camellia gymnogyna and Camellia taliensis and separated from Camellia sinensis and its two varieties, Camellia sinensis var. assamica and Camellia sinensis var. pubilimba.

Quantitative Trait Loci Mapping for Theobromine and Caffeine Contents in Tea Plant ( Camellia sinensis).

Understanding the genetic basis of theobromine and caffeine accumulation in the tea plant is important due to their contribution to tea flavor. Quantitative trait loci (QTL) analyses were carried out to identify genetic variants associated with theobromine and caffeine contents and ratio using a pseudo-testcross population derived from an intervarietal cross between two varieties of Camellia sinensis. A total of 10 QTL controlling caffeine content (CAF), theobromine content (TBR), sum of caffeine and theobromine (SCT), and caffeine-to-theobromine ratio (CTR) were identified over four measurement years. The major QTL controlling CAF, qCAF1, was mapped onto LG01 and validated across years, explaining an average of 20.1% of the phenotypic variance. The other QTL were detected in 1 or 2 years, and of them there were four, two, and three for TBR, SCT, and CTR, respectively. The present results provide valuable information for further fine mapping and cloning functional genes and for genetic improvement in tea plant.