The tea plant reference genome and improved gene annotation using long-read and paired-end sequencing data

Tea is a globally consumed non-alcohol beverage with great economic importance. Lacking of the reference genome has largely hampered the utilization of the precious tea plant genetic resources towards breeding. To address this, we generated a high-quality reference genome of tea plant using Illumina and PacBio sequencing technology that totally produced 2,124 Gb (~700-fold coverage) short and 125 Gb (~41-fold) long read data, respectively. We employed a hybrid strategy to assemble the genome that has been publicly released. We here described the data framework used to generate, annotate and validate the genome assembly. Besides, we re-predicted the protein-coding genes and annotated their putative functions using more complete omics datasets and improved training models. We reassessed the assembly and annotation quality using the latest version of BUSCO. These data can be used to develop new methodologies/tools for better assembly of complex genomes, aid in finding of novel genes, variations and evolutionary clues associated with tea quality, and thus help to breed new varieties with high yields and quality in the future.</p

Data_Sheet_1_Antimicrobial Metabolites Produced by Penicillium mallochii CCH01 Isolated From the Gut of Ectropis oblique, Cultivated in the Presence of a Histone Deacetylase Inhibitor.pdf

Three chemical epigenetic modifiers [5-azacytidine, nicotinamide, and suberoylanilide hydroxamic acid (SAHA)] were applied to induce the metabolites of Penicillium mallochii CCH01, a fungus isolated from the gut of Ectropis oblique. Metabolite profiles of P. mallochii CCH01 were obviously changed by SAHA treatment. Four metabolites (1–4), including two new natural sclerotioramine derivatives, isochromophilone XIV (1) and isochromophilone XV (2), and two known compounds, sclerotioramine (3) and (+)-sclerotiorin (4), were isolated and purified from P. mallochii CCH01 treated with SAHA. Their structures were determined by spectroscopic analyzes. Anti-phytopathogenic activities of the isolated compounds 1–4 were investigated under laboratory conditions, and compound 4 showed broad and important inhibition activities against Curvularia lunata (IC50 = 2.1 μg/mL), Curvularia clavata (IC50 = 21.0 μg/mL), Fusarium oxysporum f. sp. Mornordica (IC50 = 40.4 μg/mL), and Botryosphaeria dothidea (IC50 = 27.8 μg/mL), which were comparable to those of referenced cycloheximide, with IC50 values of 0.3, 5.0, 12.4, and 15.3 μg/mL, respectively. Ingredients 2 and 3 showed selective and potent activities against Colletotrichum graminicola with IC50 values of 29.9 and 9.7 μg/mL, respectively. Furthermore, the antibacterial bioassays showed that compounds 3 and 4 exhibited strong inhibition activities against Bacillus subtilis, with disc diameters of zone of inhibition (ZOI) of 9.1 mm for both compounds, which were a bit weaker than that of referenced gentamycin with a ZOI of 10.8 mm. Additionally, the new metabolite 1 showed a promising activity against Candida albicans (ZOI = 10.5 mm), comparable to that of positive amphotericin B with a ZOI of 23.2 mm. The present results suggest that chemical epigenetic modifier induction was a promising approach to obtaining antimicrobial metabolites encoded by silent biosynthetic genes of P. mallochii.</p

Table_4_Identification of Regulatory Networks of MicroRNAs and Their Targets in Response to Colletotrichum gloeosporioides in Tea Plant (Camellia sinensis L.).xls

Anthracnose disease is caused by Colletotrichum gloeosporioides, and is common in leaves of the tea plant (Camellia sinensis). MicroRNAs (miRNAs) have been known as key modulators of gene expression in response to environmental stresses, disease resistance, defense responses, and plant immunity. However, the role of miRNAs in responses to C. gloeosporioides remains unexplored in tea plant. Therefore, in the present study, six miRNA sequencing data sets and two degradome data sets were generated from C. gloeosporioides-inoculated and control tea leaves. A total of 485 conserved and 761 novel miRNAs were identified. Of those, 239 known and 369 novel miRNAs exhibited significantly differential expression under C. gloeosporioides stress. One thousand one hundred thirty-four and 596 mRNAs were identified as targets of 389 conserved and 299 novel miRNAs by degradome analysis, respectively. Based on degradome analysis, most of the predicted targets are negatively correlated with their corresponding conserved and novel miRNAs. The expression levels of 12 miRNAs and their targets were validated by quantitative real-time PCR. A negative correlation between expression profiles of five miRNAs (PC-5p-80764_22, csn-miR160c, csn-miR828a, csn-miR164a, and csn-miR169e) and their targets (WRKY, ARF, MYB75, NAC, and NFY transcription factor) was observed. The predicted targets of five interesting miRNAs were further validated through 5’RLM-RACE. Furthermore, Gene Ontology and metabolism pathway analysis revealed that most of the target genes were involved in the regulation of auxin pathway, ROS scavenging pathway, salicylic acid mediated pathway, receptor kinases, and transcription factors for plant growth and development as well as stress responses in tea plant against C. gloeosporioides stress. This study enriches the resources of stress-responsive miRNAs and their targets in C. sinensis and thus provides novel insights into the miRNA-mediated regulatory mechanisms, which could contribute to the enhanced susceptibility of C. gloeosporioides in tea plant.</p

Additional file 9: of Comparative transcriptomic analysis reveals gene expression associated with cold adaptation in the tea plant Camellia sinensis

Table S8. Differentially expressed genes related to osmoprotectans in response to cold treatment of SCZ and YH9 leaves. Significant (P < 0.05) decrease of transcript abundance is highlighted in blue and bold, significant transcript increase is highlighted in red and bold. (XLSX 15 kb

Additional file 1: of Global dissection of alternative splicing uncovers transcriptional diversity in tissues and associates with the flavonoid pathway in tea plant (Camellia sinensis)

Figure S1. Hierarchical cluster tree showing co-expression modules identified by WGCNA. Figure S2. The proposed pathway related to flavonoid biosynthesis. Black and red numbers in brackets following each gene indicate the number of expressed full-length and AS genes identified in eight tissues, respectively. Figure S3. Alternative spliced isoforms of theanine-related genes in tea plant. The orange solid dot indicate the targeted AS transcripts identified in specific tissues. Figure S4. Alternatively spliced isoforms of caffeine-related genes in tea plant. Figure S5. An unrooted neighbor joining phylogenetic tree constructed from 12 amino acid sequences of MYBs identified in C. sinensis and other species. The CsMYB genes are highlighted by a solid purple circle. The bold font indicates the CsMYBs which were identified as alternatively spliced genes. Figure S6. Validation of AS isoforms using independent samples by RT-PCR. The AS isoforms were verified by RT-PCR in four tissues (bud, young leaf, root and flower) and each tissue was performed with three biological replicates. Lane 1, 500 bp DNA maker. Lane 2, 6, 10 and 14, full-length transcripts of corresponding genes. Lane 3, 4 and 5, AS isforms in bud. Lane 7, 8 and 9, AS isoforms in young leaf. Lane 11, 12 and 13, AS isoforms in root. Lane 15, 16 and 17, AS isoforms in flower. Table S1. Statistics of different AS events obtained from the Illumina libraries generated in this study. Table S2. Statistic analysis of characterization of identified AS genes in different tissues. Table S3. Statistics of different AS events obtained from the specific modules based on WGCNA analysis. Table S4. Statistical analysis of characterized AS genes identified in different tissues. The solid dot indicated the targeted AS transcripts identified in specific tissues. Table S5. Primers used in this study (application details of primers are described in the materials and method. (PDF 1036 kb

Table_3_Identification of Regulatory Networks of MicroRNAs and Their Targets in Response to Colletotrichum gloeosporioides in Tea Plant (Camellia sinensis L.).xls

Anthracnose disease is caused by Colletotrichum gloeosporioides, and is common in leaves of the tea plant (Camellia sinensis). MicroRNAs (miRNAs) have been known as key modulators of gene expression in response to environmental stresses, disease resistance, defense responses, and plant immunity. However, the role of miRNAs in responses to C. gloeosporioides remains unexplored in tea plant. Therefore, in the present study, six miRNA sequencing data sets and two degradome data sets were generated from C. gloeosporioides-inoculated and control tea leaves. A total of 485 conserved and 761 novel miRNAs were identified. Of those, 239 known and 369 novel miRNAs exhibited significantly differential expression under C. gloeosporioides stress. One thousand one hundred thirty-four and 596 mRNAs were identified as targets of 389 conserved and 299 novel miRNAs by degradome analysis, respectively. Based on degradome analysis, most of the predicted targets are negatively correlated with their corresponding conserved and novel miRNAs. The expression levels of 12 miRNAs and their targets were validated by quantitative real-time PCR. A negative correlation between expression profiles of five miRNAs (PC-5p-80764_22, csn-miR160c, csn-miR828a, csn-miR164a, and csn-miR169e) and their targets (WRKY, ARF, MYB75, NAC, and NFY transcription factor) was observed. The predicted targets of five interesting miRNAs were further validated through 5’RLM-RACE. Furthermore, Gene Ontology and metabolism pathway analysis revealed that most of the target genes were involved in the regulation of auxin pathway, ROS scavenging pathway, salicylic acid mediated pathway, receptor kinases, and transcription factors for plant growth and development as well as stress responses in tea plant against C. gloeosporioides stress. This study enriches the resources of stress-responsive miRNAs and their targets in C. sinensis and thus provides novel insights into the miRNA-mediated regulatory mechanisms, which could contribute to the enhanced susceptibility of C. gloeosporioides in tea plant.</p

Additional file 7: of Comparative transcriptomic analysis reveals gene expression associated with cold adaptation in the tea plant Camellia sinensis

Table S6. Differentially expressed genes related to free radical scavengers and flavonoid in response to cold treatment of SCZ and YH9 leaves. Significant (P < 0.05) decrease of transcript abundance is highlighted in blue and bold, significant transcript increase is highlighted in red and bold. (XLSX 19 kb

Table_5_Identification of Regulatory Networks of MicroRNAs and Their Targets in Response to Colletotrichum gloeosporioides in Tea Plant (Camellia sinensis L.).xls

Anthracnose disease is caused by Colletotrichum gloeosporioides, and is common in leaves of the tea plant (Camellia sinensis). MicroRNAs (miRNAs) have been known as key modulators of gene expression in response to environmental stresses, disease resistance, defense responses, and plant immunity. However, the role of miRNAs in responses to C. gloeosporioides remains unexplored in tea plant. Therefore, in the present study, six miRNA sequencing data sets and two degradome data sets were generated from C. gloeosporioides-inoculated and control tea leaves. A total of 485 conserved and 761 novel miRNAs were identified. Of those, 239 known and 369 novel miRNAs exhibited significantly differential expression under C. gloeosporioides stress. One thousand one hundred thirty-four and 596 mRNAs were identified as targets of 389 conserved and 299 novel miRNAs by degradome analysis, respectively. Based on degradome analysis, most of the predicted targets are negatively correlated with their corresponding conserved and novel miRNAs. The expression levels of 12 miRNAs and their targets were validated by quantitative real-time PCR. A negative correlation between expression profiles of five miRNAs (PC-5p-80764_22, csn-miR160c, csn-miR828a, csn-miR164a, and csn-miR169e) and their targets (WRKY, ARF, MYB75, NAC, and NFY transcription factor) was observed. The predicted targets of five interesting miRNAs were further validated through 5’RLM-RACE. Furthermore, Gene Ontology and metabolism pathway analysis revealed that most of the target genes were involved in the regulation of auxin pathway, ROS scavenging pathway, salicylic acid mediated pathway, receptor kinases, and transcription factors for plant growth and development as well as stress responses in tea plant against C. gloeosporioides stress. This study enriches the resources of stress-responsive miRNAs and their targets in C. sinensis and thus provides novel insights into the miRNA-mediated regulatory mechanisms, which could contribute to the enhanced susceptibility of C. gloeosporioides in tea plant.</p

MOESM1 of Characterization of genome-wide genetic variations between two varieties of tea plant (Camellia sinensis) and development of InDel markers for genetic research

Additional file 1: Figure S1. Flowchart diagram for identifying genome-wide genetic variations between ‘Shuchazao’ and ‘Yunkang 10’ and functional annotation

Table_2_Identification of Regulatory Networks of MicroRNAs and Their Targets in Response to Colletotrichum gloeosporioides in Tea Plant (Camellia sinensis L.).xls

Anthracnose disease is caused by Colletotrichum gloeosporioides, and is common in leaves of the tea plant (Camellia sinensis). MicroRNAs (miRNAs) have been known as key modulators of gene expression in response to environmental stresses, disease resistance, defense responses, and plant immunity. However, the role of miRNAs in responses to C. gloeosporioides remains unexplored in tea plant. Therefore, in the present study, six miRNA sequencing data sets and two degradome data sets were generated from C. gloeosporioides-inoculated and control tea leaves. A total of 485 conserved and 761 novel miRNAs were identified. Of those, 239 known and 369 novel miRNAs exhibited significantly differential expression under C. gloeosporioides stress. One thousand one hundred thirty-four and 596 mRNAs were identified as targets of 389 conserved and 299 novel miRNAs by degradome analysis, respectively. Based on degradome analysis, most of the predicted targets are negatively correlated with their corresponding conserved and novel miRNAs. The expression levels of 12 miRNAs and their targets were validated by quantitative real-time PCR. A negative correlation between expression profiles of five miRNAs (PC-5p-80764_22, csn-miR160c, csn-miR828a, csn-miR164a, and csn-miR169e) and their targets (WRKY, ARF, MYB75, NAC, and NFY transcription factor) was observed. The predicted targets of five interesting miRNAs were further validated through 5’RLM-RACE. Furthermore, Gene Ontology and metabolism pathway analysis revealed that most of the target genes were involved in the regulation of auxin pathway, ROS scavenging pathway, salicylic acid mediated pathway, receptor kinases, and transcription factors for plant growth and development as well as stress responses in tea plant against C. gloeosporioides stress. This study enriches the resources of stress-responsive miRNAs and their targets in C. sinensis and thus provides novel insights into the miRNA-mediated regulatory mechanisms, which could contribute to the enhanced susceptibility of C. gloeosporioides in tea plant.</p