Gene mining and genomics-assisted breeding empowered by the pangenome of tea plant Camellia sinensis.

Tea is one of the world's oldest crops and is cultivated to produce beverages with various flavours. Despite advances in sequencing technologies, the genetic mechanisms underlying key agronomic traits of tea remain unclear. In this study, we present a high-quality pangenome of 22 elite cultivars, representing broad genetic diversity in the species. Our analysis reveals that a recent long terminal repeat burst contributed nearly 20% of gene copies, introducing functional genetic variants that affect phenotypes such as leaf colour. Our graphical pangenome improves the efficiency of genome-wide association studies and allows the identification of key genes controlling bud flush timing. We also identified strong correlations between allelic variants and flavour-related chemistries. These findings deepen our understanding of the genetic basis of tea quality and provide valuable genomic resources to facilitate its genomics-assisted breeding.

Allele-specific expression and chromatin accessibility contribute to heterosis in tea plants (Camellia sinensis).

Heterosis is extensively used to improve crop productivity, yet its allelic and chromatin regulation remains unclear. Based on our resolved genomes of the maternal TGY and paternal HD, we analyzed the contribution of allele-specific expression (ASE) and chromatin accessibility of JGY and HGY, the artificial hybrids of oolong tea with the largest cultivated area in China. The ASE genes (ASEGs) of tea hybrids with maternal-biased were mainly related to the energy and terpenoid metabolism pathways, whereas the ASEGs with paternal-biased tend to be enriched in glutathione metabolism, and these parental bias of hybrids may coordinate and lead to the acquisition of heterosis in more biological pathways. ATAC-seq results showed that hybrids have significantly higher accessible chromatin regions (ACRs) compared with their parents, which may confer broader and stronger transcriptional activity of genes in hybrids. The number of ACRs with significantly increased accessibility in hybrids was much greater than decreased, and the associated alleles were also affected by differential ACRs across different parents, suggesting enhanced positive chromatin regulation and potential genetic effects in hybrids. Core ASEGs of terpene and purine alkaloid metabolism pathways with significant positive heterosis have greater chromatin accessibility in hybrids, and were potentially regulated by several members of the MYB, DOF and TRB families. The binding motif of CsMYB85 in the promoter ACR of the rate-limiting enzyme CsDXS was verified by DAP-seq. These results suggest that higher numbers and more accessible ACRs in hybrids contribute to the regulation of ASEGs, thereby affecting the formation of heterotic metabolites.

Changes in Non-Volatile and Volatile Metabolites Associated with Heterosis in Tea Plants (Camellia sinensis).

Heterosis or hybrid vigor is extensively used in plant breeding. However, the contribution of metabolites to heterosis is still elusive. Here, we systematically identified the non-volatile and volatile metabolites of two hybrids and their parents in Camellia sinensis. The metabolomics analysis showed prevalent non-additive accumulation in hybrids, among which the non-additive nucleotides, alkaloids, organic acids, and tannins contribute to the positive heterosis of hybrids, including typical inosine, guanosine, adenosine, caffeine, succinic acid, adipic acid, xylonic acid, and gallic acid. The catechins and free amino acids in hybrids showed negative heterosis compared to its maternal cultivar TGY. Furthermore, the significant accumulation of non-additive terpenes combined with the mild heterosis of other types of volatiles contributes to the aroma of tea plant hybrids. The genetics of volatiles from different parents affect the aroma of hybrids processed into oolong tea. The comprehensive heterosis of these non-additive metabolites may play an important role in the formation of desirable breeding traits for hybrids. Our results provide insights into the utilization of heterosis breeding and the regulation of heterosis metabolites in tea plants.

Lipidomics analysis unravels changes from flavor precursors in different processing treatments of purple-leaf tea.

BACKGROUND: Lipids are one of the most important bioactive compounds, affecting the character and quality of tea. However, the contribution of lipids to tea productions is still elusive. Here, we systematically identified the lipid profiles of green, oolong, and black teas in purple-leaf tea (Jinmingzao, JMZ) and green-leaf tea (Huangdan, HD), respectively. RESULTS: The lipids analysis showed regular accumulation in tea products with different manufacturing processes, among which the fatty acids, glycerolipids, glycerophospholipids, and sphingolipids contribute to the quality characteristics of tea products, including typical fatty acyl (FA), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerols (DGDG), and phosphatidylcholine (PC). Compared tea materials with products, levels of fatty acids were up-regulated, while glycerolipids and glycerophospholipids were down-regulated in tea products. FA 18:3, FA 16:0, MGDG 36:6, DGDG 36:6, PC 34:3, and PC 36:6 were the negative contributors to green tea flavor formation of purple-leaf tea. The pathway analysis of significant lipids in materials and products of purple-leaf tea were enriched linolenic acid metabolism pathway and glycerolipid metabolism. CONCLUSION: This study provides insights into the lipid metabolism profiles of different tea leaf colors, and found that fatty acids are essential precursors of black tea flavor formation. © 2021 Society of Chemical Industry.

Identification of Co-Expressed Genes Related to Theacrine Synthesis in Tea Flowers at Different Developmental Stages.

Jiaocheng kucha is the first reported tea germplasm resource which contains theacrine founded in Fujian Province. Currently, the anabolic mechanism of theacrine within tea leaves is clear, but there are few studies focused on its flowers. In order to further explore the mechanism of theacrine synthesis and related genes in flowers, current study applied Jiaocheng kucha flowers (JC) as test materials and Fuding Dabaicha flowers (FD) as control materials to make transcriptome sequencing, and determination of purine alkaloid content in three different developmental periods (flower bud stage, whitening stage and full opening stage). The results showed that the flower in all stages of JC contained theacrine. The theacrine in the flower bud stage was significantly higher than in the other stages. The differentially expressed genes (DEGs) at three different developmental stages were screened from the transcriptome data, and were in a total of 5642, 8640 and 8465. These DEGs related to the synthesis of theacrine were primarily annotated to the pathways of purine alkaloids. Among them, the number of DEGs in xanthine synthesis pathway was the largest and upregulated in JC, while it was the smallest in caffeine synthesis pathway and downregulated in JC. Further weighted gene co-expression network (WGCNA) indicated that ADSL (CsTGY03G0002327), ADSL (CsTGY09G0001824) and UAZ (CsTGY06G0002694) may be a hub gene for the regulation of theacrine metabolism in JC. Our results will contribute to the identification of candidate genes related to the synthesis of theacrine in tea flowers, and explore the molecular mechanism of theacrine synthesis in JC at different developmental stages.

Genetic basis of high aroma and stress tolerance in the oolong tea cultivar genome.

Tea plants (Camellia sinensis) are commercially cultivated in >60 countries, and their fresh leaves are processed into tea, which is the most widely consumed beverage in the world. Although several chromosome-level tea plant genomes have been published, they collapsed the two haplotypes and ignored a large number of allelic variations that may underlie important biological functions in this species. Here, we present a phased chromosome-scale assembly for an elite oolong tea cultivar, "Huangdan", that is well known for its high levels of aroma. Based on the two sets of haplotype genome data, we identified numerous genetic variations and a substantial proportion of allelic imbalance related to important traits, including aroma- and stress-related alleles. Comparative genomics revealed extensive structural variations as well as expansion of some gene families, such as terpene synthases (TPSs), that likely contribute to the high-aroma characteristics of the backbone parent, underlying the molecular basis for the biosynthesis of aroma-related chemicals in oolong tea. Our results uncovered the genetic basis of special features of this oolong tea cultivar, providing fundamental genomic resources to study evolution and domestication for the economically important tea crop.

R2R3-MYB transcription factor family in tea plant (Camellia sinensis): Genome-wide characterization, phylogeny, chromosome location, structure and expression patterns.

MYB transcription factors play essential roles in many biological processes and environmental stimuli. However, the functions of the MYB transcription factor family in tea plants have not been elucidated. Here, a total of 122 CsR2R3-MYB genes were identified from the chromosome level genome of tea plant (Camellia sinensis). The CsR2R3-MYB genes were phylogenetically classified into 25 groups. Results from the structure analysis of the gene, conserved motifs, and chromosomal distribution supported the relative conservation of the R2R3-MYB genes family in the tea plant. Synteny analysis indicated that 122, 34, and 112 CsR2R3-MYB genes were orthologous to Arabidopsis thaliana, Oryza sativa and C. sinensis var. 'huangdan' (HD), respectively. Tissue-specific expression showed that all CsR2R3-MYB genes had different expression patterns in the tea plant tissues, indicating that these genes may perform diverse functions. The expression patterns of representative R2R3-MYB genes and the regulatory network of the main anthocyanin components were analyzed, which suggested that CsMYB17 may played a key role in the regulation of cya-3-O-gal, del-3-O-gal, cya-3-O-glu and pel-3-O-glu. Results from the qRT-PCR validation of selected genes suggested that CsR2R3-MYB genes were induced in response to drought, cold, GA, and ABA treatments. Overall, this study provides comprehensive and systematic information for research on the function of R2R3-MYB genes in tea plants.

Exploration of the Effects of Different Blue LED Light Intensities on Flavonoid and Lipid Metabolism in Tea Plants via Transcriptomics and Metabolomics.

Blue light extensively regulates multiple physiological processes and secondary metabolism of plants. Although blue light quantity (fluence rate) is important for plant life, few studies have focused on the effects of different blue light intensity on plant secondary metabolism regulation, including tea plants. Here, we performed transcriptomic and metabolomic analyses of young tea shoots (one bud and two leaves) under three levels of supplemental blue light, including low-intensity blue light (LBL, 50 µmol m-2 s-1), medium-intensity blue light (MBL, 100 µmol m-2 s-1), and high-intensity blue light (HBL, 200 µmol m-2 s-1). The total number of differentially expressed genes (DEGs) in LBL, MBL and HBL was 1, 7 and 1097, respectively, indicating that high-intensity blue light comprehensively affects the transcription of tea plants. These DEGs were primarily annotated to the pathways of photosynthesis, lipid metabolism and flavonoid synthesis. In addition, the most abundant transcription factor (TF) families in DEGs were bHLH and MYB, which have been shown to be widely involved in the regulation of plant flavonoids. The significantly changed metabolites that we detected contained 15 lipids and 6 flavonoid components. Further weighted gene co-expression network analysis (WGCNA) indicated that CsMYB (TEA001045) may be a hub gene for the regulation of lipid and flavonoid metabolism by blue light. Our results may help to establish a foundation for future research investigating the regulation of woody plants by blue light.

Comparison of Metabolome and Transcriptome of Flavonoid Biosynthesis Pathway in a Purple-Leaf Tea Germplasm Jinmingzao and a Green-Leaf Tea Germplasm Huangdan reveals Their Relationship with Genetic Mechanisms of Color Formation.

Purple-leaf tea is a phenotype with unique color because of its high anthocyanin content. The special flavor of purple-leaf tea is highly different from that of green-leaf tea, and its main ingredient is also of economic value. To probe the genetic mechanism of the phenotypic characteristics of tea leaf color, we conducted widely targeted metabolic and transcriptomic profiling. The metabolites in the flavonoid biosynthetic pathway of purple- and green-leaf tea were compared, and results showed that phenolic compounds, including phenolic acids, flavonoids, and tannins, accumulated in purple-leaf tea. The high expression of genes related to flavonoid biosynthesis (e.g., PAL and LAR) exhibits the specific expression of biosynthesis and the accumulation of these metabolites. Our result also shows that two CsUFGTs were positively related to the accumulation of anthocyanin. Moreover, genes encoding transcription factors that regulate flavonoids were identified by coexpression analysis. These results may help to identify the metabolic factors that influence leaf color differentiation and provide reference for future research on leaf color biology and the genetic improvement of tea.