Whole genome sequencing analysis of two sinensis tea (Camellia sinensis var. sinensis) clones: Assessment of molecular variations to search for breeding markers

Freshwater pulmonate snails are prevalent across Kenya and act as intermediate hosts for trematode parasites, some of which are snail vectors for human schistosomiasis. Chemical molluscicides have rarely been used routinely in Kenya to control snails due to high costs of manufacture and the subsequent environmental concerns associated with their use. This study tested extracts from green tea and purple tea plants, Camellia sinensis, which are widely grown in Kenyan highland areas, against Biomphalaria pfeifferi, the obligate intermediate host for Schistosoma mansoni. Snails were exposed to five different concentrations of tea extracts (10, 20, 50, 100, and 150 ppm). All quantitative data analyses were done in R Version 4.4.0. Analysis by LCMS showed that the compounds present in the extracts were epicatechin, epigallocatechin, caffeine (the highest concentration of the compounds), theobromine, and assamsaponin C. The compounds identified by GCMS were hexadecanoic acid, caffeine, octadecenoic acid‐methyl, and octadec‐1‐ene. The crude extracts from the Kenyan tea plant Camellia sinensis (both green and purple tea) induced mortality of the Biomphalaria pfeifferi. Therefore, they can be explored as alternative plant‐based molluscicides against the vector snails of Schistosoma mansoni.

BackgroundCultivated tea is one of the most important economic and ecological trees distributed worldwide. Cultivated tea suffer from long-term targeted selection of traits and overexploitation of habitats by human beings, which may have changed its genetic structure. The chloroplast is an organelle with a conserved cyclic genomic structure, and it can help us better understand the evolutionary relationship of Camellia plants.ResultsWe conducted comparative and evolutionary analyses on cultivated tea and wild tea, and we detected the evolutionary characteristics of cultivated tea. The chloroplast genome sizes of cultivated tea were slightly different, ranging from 157,025 to 157,100 bp. In addition, the cultivated species were more conserved than the wild species, in terms of the genome length, gene number, gene arrangement and GC content. However, comparing Camellia sinensis var. sinensis and Camellia sinensis var. assamica with their cultivars, the IR length variation was approximately 20 bp and 30 bp, respectively. The nucleotide diversity of 14 sequences in cultivated tea was higher than that in wild tea. Detailed analysis on the genomic variation and evolution of Camellia sinensis var. sinensis cultivars revealed 67 single nucleotide polymorphisms (SNPs), 46 insertions/deletions (indels), and 16 protein coding genes with nucleotide substitutions, while Camellia sinensis var. assamica cultivars revealed 4 indels. In cultivated tea, the most variable gene was ycf1. The largest number of nucleotide substitutions, five amino acids exhibited site-specific selection, and a 9 bp sequence insertion were found in the Camellia sinensis var. sinensis cultivars. In addition, phylogenetic relationship in the ycf1 tree suggested that the ycf1 gene has diverged in cultivated tea. Because C. sinensis var. sinensis and its cultivated species were not tightly clustered.ConclusionsThe cultivated species were more conserved than the wild species in terms of architecture and linear sequence order. The variation of the chloroplast genome in cultivated tea was mainly manifested in the nucleotide polymorphisms and sequence insertions. These results provided evidence regarding the influence of human activities on tea.

Chè Shan Tuyet ( Camellia sinensis var. Shan) is one of the precious tea resources of Vietnam; however, there is little research on its chemical composition. The purpose of this study was to characterize the main quality components, such as free amino acids and catechins, in Camellia sinensis var. Shan and Camellia sinensis var. sinensis collected in the high mountain of Ta Xua, Son La, Vietnam by using an amino acid analyzer and liquid chromatography coupled with tandem mass spectrometry, respectively. Principal component analysis (PCA) discrimination analysis of chemical profiles revealed a clear metabolic difference between the young leaves of Shan Tuyet tea and mature leaves of the same variety and of sinensis tea. The amino acids serine, glutamic acid, arginine, ornithine, and aspartic acid contributed mainly to the discrimination and could be considered biomarkers for Shan Tuyet tea. The levels of caffeine and 7 catechins, catechin, catechin 3-gallate, epicatechin, epicatechin-3-gallate, epigallocatechin 3-gallate, gallocatechin, and gallocatechin 3-gallate, in young leaves of Shan Tuyet tea were significantly higher than in the other types. Notably, the pair correlation among catechins revealed strong coefficients of the epistructures and non-epistructures, which suggested that these compounds can be converted naturally to each other. The strong correlation between epicatechin-3-gallate and catechin 3-gallate with antiradical ABTS activity of Shan Tuyet tea leaves indicates that these 2 catechins are mainly responsible for the antioxidant activity. This is the first report on the bioactive compounds of Shan Tuyet tea, as well as its potential for the production of health supplements.

Phased secondary small interfering RNAs (phasiRNAs) in plants play important roles in regulating genome stability, plant development and stress adaption. Camellia sinensis var. assamica has immense economic, medicinal and cultural significance. However, there are still no studies of phasiRNAs and their putative functions in this valuable plant. We identified 476 and 43 PHAS loci which generated 4290 twenty one nucleotide (nt) and 264 twenty four nt phasiRNAs, respectively. Moreover, the analysis of degradome revealed more than 35000 potential targets for these phasiRNAs. We identified several conserved 21nt phasiRNA generation pathways in tea plant, including miR390 → TAS3, miR482/miR2118 → NB-LRR, miR393 → F-box, miR828 → MYB/TAS4, and miR7122 → PPR in this study. Furthermore, we found that some transposase and plant mobile domain genes could generate phasiRNAs. Our results show that phasiRNAs target genes in the same family in cis- or trans-manners, and different members of the same gene family may generate the same phasiRNAs. The phasiRNAs, generated by transposase and plant mobile domain genes, and their targets, suggest that phasiRNAs may be involved in the inhibition of transposable elements in tea plant. To summarize, these results provide a comprehensive view of phasiRNAs in Camellia sinensis var. assamica.

Camellia sinensis var. assamica ‘Hainan Dayezhong’ (C. sinensis var. assamica ‘Hainan Dayezhong’), an endemic, valuable tea plant from Hainan Province in China, has been utilized as a precious tea resource by local farmers. Nevertheless, no data on the mitochondrial genome of C. sinensis var. assamica ‘Hainan Dayezhong’ is available, which greatly limits our understanding of its phylogenetic classification and population identification. In this study, the Camellia sinensis var. assamica ‘Hainan Dayezhong’ complete mitochondrial genome was successfully assembled using the Nanopore and Illumina sequencing data for the first time. Key findings revealed a complex multi-branched structure comprising 12 contigs, with a total length of 793,531 bp and a GC content of 45.80%. A total of 70 genes were identified, including 38 protein-coding genes (PCGs), 28 tRNA genes, 3 rRNA genes, and one pseudogene (rps19). Out of the 31 PCGs shared by 18 Theaceae species, 28 of the PCGs were undergone purifying selection, and 3 genes (ccmC, rps1, and rps13) showed sign of positive selection. The codon usage analysis demonstrated that Leucine (Leu) and Serine (Ser) were the most preferred amino acids in C. sinensis var. assamica ‘Hainan Dayezhong’, and nearly all of the codons with RSCU > 1 showed the A/U bias at the third position. Tetramer repeats made up 40.71% of the simple sequence repeats (SSRs). 74 RNA editing sites were predicted in 17 PCGs, majority (91.89%) of them were C-to-U conversion pattern. 33 MTPTs were identified between the mitochondrial genome and chloroplast genome, with a combined length of 15,346 bp, accounting for 1.93% of the mitogenome. Moreover, the Maximum Likelihood phylogenetic trees were constructed of 38 shared PCGs in 30 plant species, the results showed that C. sinensis var. assamica ‘Hainan Dayezhong’ was phylogenetically independent of the species of sections Camellia, Chrysantha, and Oleifera. However, it didn’t support the independent subclade of C. sinensis var. assamica ‘Hainan Dayezhong’ due to the low support (BS = 55). Meanwhile, the chloroplast PCG-based phylogenetic analysis revealed that C. sinensis var. assamica ‘Hainan Dayezhong’ clustered together with C. sinensis var. assamica (MH019307.1) into a common subclade with high support (BS = 81). Our work presents the first complete mitogenome of Camellia sinensis var. assamica ‘Hainan Dayezhong’, which could not only provide a reference genome for the comprehensive analysis of Theaceae family, but also contribute to the valuable information for further genomic breeding and evolutionary research of Camellia.

Genetic analyses of ancient tea trees provide insights into the breeding history and dissemination of Chinese Assam tea (Camellia sinensis var. assamica)

BackgroundChloroplast genome resources can provide useful information for the evolution of plant species. Tea plant (Camellia sinensis) is among the most economically valuable member of Camellia. Here, we determined the chloroplast genome of the first natural triploid Chinary type tea (‘Wuyi narcissus’ cultivar of Camellia sinensis var. sinensis, CWN) and conducted the genome comparison with the diploid Chinary type tea (Camellia sinensis var. sinensis, CSS) and two types of diploid Assamica type teas (Camellia sinensis var. assamica: Chinese Assamica type tea, CSA and Indian Assamica type tea, CIA). Further, the evolutionary mechanism of the chloroplast genome of Camellia sinensis and the relationships of Camellia species based on chloroplast genome were discussed.ResultsComparative analysis showed the evolutionary dynamics of chloroplast genome of Camellia sinensis were the repeats and insertion-deletions (indels), and distribution of the repeats, indels and substitutions were significantly correlated. Chinese tea and Indian tea had significant differences in the structural characteristic and the codon usage of the chloroplast genome. Analysis of sequence characterized amplified region (SCAR) using sequences of the intergenic spacers (trnE/trnT) showed none of 292 different Camellia sinensis cultivars had similar sequence characteristic to triploid CWN, but the other four Camellia species did. Estimations of the divergence time showed that CIA diverged from the common ancestor of two Assamica type teas about 6.2 Mya (CI: 4.4–8.1 Mya). CSS and CSA diverged to each other about 0.8 Mya (CI: 0.4–1.5 Mya). Moreover, phylogenetic clustering was not exactly consistent with the current taxonomy of Camellia.ConclusionsThe repeat-induced and indel-induced mutations were two important dynamics contributed to the diversification of the chloroplast genome in Camellia sinensis, which were not mutually exclusive. Chinese tea and Indian tea might have undergone different selection pressures. Chloroplast transfer occurred during the polyploid evolution in Camellia sinensis. In addition, our results supported the three different domestication origins of Chinary type tea, Chinese Assamica type tea and Indian Assamica type tea. And, the current classification of some Camellia species might need to be further discussed.

Effect of shading on trichome formation and CAPRICE-like gene expression in tea (Camellia sinensis var. sinensis) leaves

SABATH methyltransferases convent plant small-molecule metabolites into volatile methyl esters, which play important roles in many biological processes and defense reactions in plants. In this study, a total of 32 SABATH genes were identified in the Camellia sinensis var. sinensis (CSS) genome, which were renamed CsSABATH1 to CsSABATH32. Genome location annotation suggested that tandem duplication was responsible for the expansion of SABATH genes in tea plant. Multiple sequence alignment and phylogenetic analysis showed that the CsSABATHs could be classified into three groups (I, II and III), which were also supported by gene structures and conserved motifs analysis. Group II contained only two CsSABATH proteins, which were closely related to PtIAMT, AtIAMT and OsIAMT. The group III SABATH genes of tea plant exhibited expansion on the CSS genome compared with Camellia sinensis var. assamica (CSA) genome. Based on RNA-seq data, the CsSABATHs exhibited tissue-specific expression patterns, and the members with high expression in buds and young leaves were also obviously upregulated after MeJA treatment. The expression of many transcription factors was significantly correlated with that of different members of the CsSABATH gene family, suggesting a potential regulatory relationship between them. Quantitative real-time PCR (qPCR) expression analysis showed that CsSABATHs could respond to exogenous JA, SA and MeSA treatments in tea plants. RNA-seq data analysis and qPCR validation suggested that CsSABATH8, 11, 16, 25, 29 and 32 might play a special role in plant defense against insect herbivory. These results provide references for evolutionary studies of the plant SABATH family and the exploration of the potential roles of CsSABATHs in tea plant defense responses.

BackgroundSingle nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) are the major genetic variations and are distributed extensively across the whole plant genome. However, few studies of these variations have been conducted in the long-lived perennial tea plant.ResultsIn this study, we investigated the genome-wide genetic variations between Camellia sinensis var. sinensis ‘Shuchazao’ and Camellia sinensis var. assamica ‘Yunkang 10’, identified 7,511,731 SNPs and 255,218 InDels based on their whole genome sequences, and we subsequently analyzed their distinct types and distribution patterns. A total of 48 InDel markers that yielded polymorphic and unambiguous fragments were developed when screening six tea cultivars. These markers were further deployed on 46 tea cultivars for transferability and genetic diversity analysis, exhibiting information with an average 4.02 of the number of alleles (Na) and 0.457 of polymorphism information content (PIC). The dendrogram showed that the phylogenetic relationships among these tea cultivars are highly consistent with their genetic backgrounds or original places. Interestingly, we observed that the catechin/caffeine contents between ‘Shuchazao’ and ‘Yunkang 10’ were significantly different, and a large number of SNPs/InDels were identified within catechin/caffeine biosynthesis-related genes.ConclusionThe identified genome-wide genetic variations and newly-developed InDel markers will provide a valuable resource for tea plant genetic and genomic studies, especially the SNPs/InDels within catechin/caffeine biosynthesis-related genes, which may serve as pivotal candidates for elucidating the molecular mechanism governing catechin/caffeine biosynthesis.

The distribution of catechins and their derivatives among 114 Camellia plants and their correlation in different species and tea-processing suitability.

BackgroundLow temperature restricts the planting range of all crops, but cold acclimation induces adaption to cold stress in many plants. Camellia sinensis, a perennial evergreen tree that is the source of tea, is mainly grown in warm areas. Camellia sinensis var. sinensis (CSS) has greater cold tolerance than Camellia sinensis var. assamica (CSA). To gain deep insight into the molecular mechanisms underlying cold adaptation, we investigated the physiological responses and transcriptome profiles by RNA-Seq in two tea varieties, cold resistant SCZ (classified as CSS) and cold susceptible YH9 (classified as CSA), during cold acclimation.ResultsUnder freezing stress, lower relative electrical conductivity and higher chlorophyll fluorescence (Fv/Fm) values were detected in SCZ than in YH9 when subjected to freezing acclimation. During cold treatment, 6072 and 7749 DEGs were observed for SCZ and YH9, respectively. A total of 978 DEGs were common for both SCZ and YH9 during the entire cold acclimation process. DEGs were enriched in pathways of photosynthesis, hormone signal transduction, and transcriptional regulation of plant-pathogen interactions. Further analyses indicated that decreased expression of Lhca2 and higher expression of SnRK2.8 are correlated with cold tolerance in SCZ.ConclusionsCompared with CSA, CSS was significantly more resistant to freezing after cold acclimation, and this increased resistance was associated with an earlier expression of cold-induced genes. Because the greater transcriptional differentiation during cold acclimation in SCZ may contribute to its greater cold tolerance, our studies identify specific genes involved in photoinhibition, ABA signal conduction, and plant immunity that should be studied for understanding the processes involved in cold tolerance. Marker-assisted breeding focused on the allelic variation at these loci provides an avenue for the possible generation of CSA cultivars that have CSS-level cold tolerance.

Endophytic actinobacteria exist widely in plant tissues and are considered as a potential bioresource library of natural products. Tea plants play important roles in human health and in the lifestyles of Asians, especially the Chinese. However, little is known about the endophytic actinobacteria of tea plants. In this study, 16 actinobacteria of 7 different genera and 28 actinobacteria of 8 genera were isolated and analyzed by 16S rRNA gene sequencing from tea cultivars of Zijuan and Yunkang-10 (Camellia sinensis var. assamica), respectively. The diversity of actinobacteria species from Zijuan were higher in July than December (6 vs. 3 genera), but the diversity of species from Yunkang-10 were higher in December than July (7 vs. 3 genera). No actinobacteria isolates were obtained from any tea cultivar in September. Ten isolates from Yunkang-10 exhibited antimicrobial activity against at least one human pathogenic microorganism (Staphylococcus epidermidis, Shigella flexneri, and Escherichia coli), but none of the isolates from Zijuan exhibited antimicrobial activities. Fourteen strains were further exammined the genes of polyketide synthetase (PKS)-I and PKS-II and non-ribosomal peptide synthetase (NRPS). Brevibacterium sp. YXT131 from Yunkang-10 showed strong inhibitory activity against S. epidermidis, Sh. flexneri, and E. coli, and PKS-I and PKS-II and NRPS genes were obtained from the strain. In in vitro assays, extracts from 14 actinobacteria that were tested for antibiotic biosynthetic genes showed no inhibition of concanavalin A (ConA)-induced murine splenocyte proliferation. In in vivo assays, the crude extract of YXT131 modulated the immune response by decreasing the proinflammatory cytokines interleukin (IL)-12/IL-23 p40 and tumor necrosis factor (TNF)-α in the serum of mice. These results confirm that endophytic actinobacteria from tea plants might be an undeveloped bioresource library for active compounds.

Genetic diversity and genetic variation of 240 adult plants of four tea populations in Hunan – Camellia sinensis var. sinensis, C. sinensis var. assamica cv. Duntsa, C. ptilophylla and C. sinensis var. assamica cv. Jianghua – were studied by rapid amplification of polymorphic DNA (RAPD) markers. The results showed 226 loci using 21 random primers (10 bp), of which 201 (88.9%) were polymorphic. The genetic diversity analysis indicated that Shannon's index was 0.43; 74.7% of which was within-population genetic diversity while 25.3% was among-population variation. The gene diversity indexes of total populations (HT), within populations (HS) and among populations (HST) were, respectively, 0.37, 0.28 and 0.09. The coefficient of gene differentiation (GST) among populations was 0.23, indicating a 76.7% variation within populations and 23.3% among populations. These results displayed a rich within-population genetic variation, as in Shannon's diversity index. Interpopulation gene flow (Nm) was 0.74, which indicates the limited genetic exchange between populations.

Deciphering codon usage patterns and evolutionary forces in chloroplast genes of Camellia sinensis var. assamica and Camellia sinensis var. sinensis in comparison to Camellia pubicosta