Chun-Lei Ma

Global transcriptome profiles of Camellia sinensis during cold acclimation

BackgroundTea is the most popular non-alcoholic health beverage in the world. The tea plant (Camellia sinensis (L.) O. Kuntze) needs to undergo a cold acclimation process to enhance its freezing tolerance in winter. Changes that occur at the molecular level in response to low temperatures are poorly understood in tea plants. To elucidate the molecular mechanisms of cold acclimation, we employed RNA-Seq and digital gene expression (DGE) technologies to the study of genome-wide expression profiles during cold acclimation in tea plants.ResultsUsing the Illumina sequencing platform, we obtained approximately 57.35 million RNA-Seq reads. These reads were assembled into 216,831 transcripts, with an average length of 356 bp and an N50 of 529 bp. In total, 1,770 differentially expressed transcripts were identified, of which 1,168 were up-regulated and 602 down-regulated. These include a group of cold sensor or signal transduction genes, cold-responsive transcription factor genes, plasma membrane stabilization related genes, osmosensing-responsive genes, and detoxification enzyme genes. DGE and quantitative RT-PCR analysis further confirmed the results from RNA-Seq analysis. Pathway analysis indicated that the “carbohydrate metabolism pathway” and the “calcium signaling pathway” might play a vital role in tea plants’ responses to cold stress.ConclusionsOur study presents a global survey of transcriptome profiles of tea plants in response to low, non-freezing temperatures and yields insights into the molecular mechanisms of tea plants during the cold acclimation process. It could also serve as a valuable resource for relevant research on cold-tolerance and help to explore the cold-related genes in improving the understanding of low-temperature tolerance and plant-environment interactions.

Identification and characterization of 74 novel polymorphic EST-SSR markers in the tea plant, Camellia sinensis (Theaceae)

UNLABELLED PREMISE OF THE STUDY New simple sequence repeat (SSR) markers were developed in the tea plant (Camellia sinensis) using published Expressed Sequence Tag (EST) sequences for further genetic studies and breeding programs. • METHODS AND RESULTS A total of 74 EST-based SSR markers were generated. Polymorphism and transferability validation in 45 individuals of seven species and varieties of Camellia L. sect. Thea (L.) Dyer revealed that the number of alleles (N(A)) per locus varied between one and four or five in each species or variety. The observed heterozygosity (H(O)) and expected heterozygosity (H(E)) ranged from 0.000 to 1.000 and 0.893, respectively. • CONCLUSIONS These new polymorphic and transferable EST-SSR markers will have potential for applications in genetic diversity evaluation, molecular fingerprinting identification, comparative genomics analysis, and genetic mapping in the tea plant.

Diversity distribution and population structure of tea germplasms in China revealed by EST-SSR markers

Tea plant (Camellia sinensis (L.) O. Kuntze) originated from China, where distributed abundant genetic resources. It is of critical importance to well understanding of genetic diversity and population structure for effective collection, conservation, and utilization of tea germplasms. In this study, 96 new polymorphic EST-SSR markers were developed and used to analyze 450 tea accessions collected from 14 tea-producing regions across China. A total of 409 alleles were observed, and the gene diversity (H) and polymorphic information content (PIC) were estimated to be averagely 0.64 and 0.61, respectively, across all the tested samples. The higher level of genetic diversity was observed in original regions like Guangxi, Yunnan, and Guizhou provinces. The allele number, H, and PIC showed decreasing trend when the region was more and more away from origin center of tea plant, which gave us implications on the spreading route of tea plant in China. The clustering of 450 samples both showed a clear separation according to their geographic origin based on either model simulation or genetic distance. The genetic differentiation was further analyzed among five inferred populations represented different eco-geographic regions. The lowest Fst and the closest relationship were revealed between proximal populations, which indicated that gene exchanges occurred frequently between nearby regions than distance ones. The majority of genetic variation resulted from differentiation within population (81.36%) rather than among inferred (13.6%) and regional (5.04%) populations based on analysis of molecular variance. Our study also revealed that the lower diversity and simpler population structure were found in improved cultivars than wild teas and landraces, which indicated that genetic base of developed cultivars became narrow because of long-standing domestication and artificial selection. So more attentions should be focused to conserve and utilize the beneficial genes in wild teas and landraces to broaden genetic variation of new cultivars in future breeding of the tea plant.

Large-Scale SNP Discovery and Genotyping for Constructing a High-Density Genetic Map of Tea Plant Using Specific-Locus Amplified Fragment Sequencing (SLAF-seq).

Genetic maps are important tools in plant genomics and breeding. The present study reports the large-scale discovery of single nucleotide polymorphisms (SNPs) for genetic map construction in tea plant. We developed a total of 6,042 valid SNP markers using specific-locus amplified fragment sequencing (SLAF-seq), and subsequently mapped them into the previous framework map. The final map contained 6,448 molecular markers, distributing on fifteen linkage groups corresponding to the number of tea plant chromosomes. The total map length was 3,965 cM, with an average inter-locus distance of 1.0 cM. This map is the first SNP-based reference map of tea plant, as well as the most saturated one developed to date. The SNP markers and map resources generated in this study provide a wealth of genetic information that can serve as a foundation for downstream genetic analyses, such as the fine mapping of quantitative trait loci (QTL), map-based cloning, marker-assisted selection, and anchoring of scaffolds to facilitate the process of whole genome sequencing projects for tea plant.

Construction of a SSR-based genetic map and identification of QTLs for catechins content in tea plant (Camellia sinensis).

Catechins are the most important bioactive compounds in tea, and have been demonstrated to possess a wide variety of pharmacological activities. To characterize quantitative trait loci (QTLs) for catechins content in the tender shoots of tea plant, we constructed a moderately saturated genetic map using 406 simple sequence repeat (SSR) markers, based on a pseudo-testcross population of 183 individuals derived from an intraspecific cross of two Camellia sinensis varieties with diverse catechins composition. The map consisted of fifteen linkage groups (LGs), corresponding to the haploid chromosome number of tea plant (2n = 2x = 30). The total map length was 1,143.5 cM, with an average locus spacing of 2.9 cM. A total of 25 QTLs associated with catechins content were identified over two measurement years. Of these, nine stable QTLs were validated across years, and clustered into four main chromosome regions on LG03, LG11, LG12 and LG15. The population variability explained by each QTL was predominantly at moderate-to-high levels and ranged from 2.4% to 71.0%, with an average of 17.7%. The total number of QTL for each trait varied from four to eight, while the total population variability explained by all QTLs for a trait ranged between 38.4% and 79.7%. This is the first report on the identification of QTL for catechins content in tea plant. The results of this study provide a foundation for further cloning and functional characterization of catechin QTLs for utilization in improvement of tea plant.

Determination of catechin content in representative Chinese tea germplasms.

To understand tea germplasms better and to use them effectively for production and breeding, the catechin content of 403 accessions of representative tea germplasms collected from various locations in China were studied using HPLC. The catechin content of these tea germplasms varied from 56.6 to 231.9 mg/g and averaged 154.5 ± 18.1 mg/g. One germplasm with low total catechin (TC) content (<60 mg/g) and three with high TC (>200 mg/g) contents were found. Averages of the TC content of the three varieties of Camellia sinensis (L.) O. Kuntze, namely, sinensis, assamica, and pubilimba, were 152.9 ± 16.2 mg/g, 162.8 ± 22.3 mg/g, and 165.1 ± 21.3 mg/g, respectively. The TC content of the sinensis variety was significantly lower (P < 0.05) than that of the other two varieties. The assamica variety had the highest levels of (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC), whereas the pubilimba variety had the highest levels of (-)-epigallocatechin gallate (EGCG), (+)-gallocatechin (GC), (+)-catechin (C), and (-)-gallocatechin gallate (GCG). Factor analysis indicated that GC, C, GCG, catechin index, and ECG greatly influenced the classification. The TC content of germplasms collected from the various provinces showed significant differences (P < 0.05). Tea germplasms of the southern provinces had higher degrees of variation in TC.

Transcriptomic Analysis of Tea Plant Responding to Drought Stress and Recovery.

Tea plant (Camellia sinensis) is an economically important beverage crop. Drought stress (DS) seriously limits the growth and development of tea plant, thus affecting crop yield and quality. To elucidate the molecular mechanisms of tea plant responding to DS, we performed transcriptomic analysis of tea plant during the three stages [control (CK) and during DS, and recovery (RC) after DS] using RNA sequencing (RNA-Seq). Totally 378.08 million high-quality trimmed reads were obtained and assembled into 59,674 unigenes, which were extensively annotated. There were 5,955 differentially expressed genes (DEGs) among the three stages. Among them, 3,948 and 1,673 DEGs were up-regulated under DS and RC, respectively. RNA-Seq data were further confirmed by qRT-PCR analysis. Genes involved in abscisic acid (ABA), ethylene, and jasmonic acid biosynthesis and signaling were generally up-regulated under DS and down-regulated during RC. Tea plant potentially used an exchange pathway for biosynthesis of indole-3-acetic acid (IAA) and salicylic acid under DS. IAA signaling was possibly decreased under DS but increased after RC. Genes encoding enzymes involved in cytokinin synthesis were up-regulated under DS, but down-regulated during RC. It seemed probable that cytokinin signaling was slightly enhanced under DS. In total, 762 and 950 protein kinases belonging to 26 families were differentially expressed during DS and RC, respectively. Overall, 547 and 604 transcription factor (TF) genes belonging to 58 families were induced in the DS vs. CK and RC vs. DS libraries, respectively. Most members of the 12 TF families were up-regulated under DS. Under DS, genes related to starch synthesis were down-regulated, while those related to starch decomposition were up-regulated. Mannitol, trehalose and sucrose synthesis-related genes were up-regulated under DS. Proline was probably mainly biosynthesized from glutamate under DS and RC. The mechanism by which ABA regulated stomatal movement under DS and RC was partly clarified. These results document the global and novel responses of tea plant during DS and RC. These data will serve as a valuable resource for drought-tolerance research and will be useful for breeding drought-resistant tea cultivars.

Biochemical and transcriptomic analyses reveal different metabolite biosynthesis profiles among three color and developmental stages in ‘Anji Baicha’ (Camellia sinensis)

BackgroundThe new shoots of the albino tea cultivar ‘Anji Baicha’ are yellow or white at low temperatures and turn green as the environmental temperatures increase during the early spring. ‘Anji Baicha’ metabolite profiles exhibit considerable variability over three color and developmental stages, especially regarding the carotenoid, chlorophyll, and theanine concentrations. Previous studies focused on physiological characteristics, gene expression differences, and variations in metabolite abundances in albino tea plant leaves at specific growth stages. However, the molecular mechanisms regulating metabolite biosynthesis in various color and developmental stages in albino tea leaves have not been fully characterized.ResultsWe used RNA-sequencing to analyze ‘Anji Baicha’ leaves at the yellow-green, albescent, and re-greening stages. The leaf transcriptomes differed considerably among the three stages. Functional classifications based on Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that differentially expressed unigenes were mainly related to metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and carbon fixation in photosynthetic organisms. Chemical analyses revealed higher β-carotene and theanine levels, but lower chlorophyll a levels, in the albescent stage than in the green stage. Furthermore, unigenes involved in carotenoid, chlorophyll, and theanine biosyntheses were identified, and the expression patterns of the differentially expressed unigenes in these biosynthesis pathways were characterized. Through co-expression analyses, we identified the key genes in these pathways. These genes may be responsible for the metabolite biosynthesis differences among the different leaf color and developmental stages of ‘Anji Baicha’ tea plants.ConclusionsOur study presents the results of transcriptomic and biochemical analyses of ‘Anji Baicha’ tea plants at various stages. The distinct transcriptome profiles for each color and developmental stage enabled us to identify changes to biosynthesis pathways and revealed the contributions of such variations to the albino phenotype of tea plants. Furthermore, comparisons of the transcriptomes and related metabolites helped clarify the molecular regulatory mechanisms underlying the secondary metabolic pathways in different stages.

Identification of differential gene expression profiles between winter dormant and sprouting axillary buds in tea plant (Camellia sinensis) by suppression subtractive hybridization

Tea plant (Camellia sinensis (L.) O. Kuntze) is an important cash crop. In temperate regions, bud dormancy in winter and budbreak in spring are important biological phenomena for the tea plant life cycle. To understand the molecular mechanism of dormancy maintenance and release in tea plant, the differentially expressed genes in dormant and sprouting axillary buds were investigated in two cultivars (special early-sprouting tea cultivar ‘Camellia sinensis cv. Longjing 43’ (‘LJ’) and late-sprouting tea cultivar ‘C. sinensis cv. Zhenghe Dabaicha’ (‘ZD’)), using suppression subtractive hybridization (SSH) approach. Four high performance complementary DNA (cDNA)-SSH libraries (‘LJ’ sprouting bud library, ‘LJ’ dormant bud library, ‘ZD’ sprouting bud library and ‘ZD’ dormant bud library) were constructed, and 1,736 valid ESTs were obtained, in which 1,242 ESTs were unique to the sprouting bud libraries and 494 ESTs were unique to the dormant bud libraries. Based on sequence matching and gene ontology analysis, 1,287 unigenes consisting of 208 contigs and 1,079 singletons were identified, in which 995 had Blast hits. The putative functions of differentially regulated sequences were involved in most aspects of plant biological processes. The quality and expression patterns of partial ESTs from these four libraries were validated by qRT-PCR. We identified numerous differentially expressed genes mainly involved in stress response, water metabolism, cell division regulation, energy metabolism and hormone regulation from dormant and sprouting bud libraries. This study provides general information on tea plant axillary bud dormancy and release at the transcriptional level and provides some hypotheses for further exploration on the mechanism of bud dormancy and budbreak in tea plant.

Proteome and Acetyl-Proteome Profiling of Camellia sinensis cv. ‘Anji Baicha’ during Periodic Albinism Reveals Alterations in Photosynthetic and Secondary Metabolite Biosynthetic Pathways

Tea leaf color is not only important from an aesthetics standpoint but is also related to tea quality. To investigate the molecular mechanisms that determine tea leaf color, we examined Camellia sinensis cv. ‘Anji Baicha’ (an albino tea cultivar) by tandem mass tag isobaric labeling to generate a high-resolution proteome and acetyl-proteome atlas of three leaf developmental stages. We identified a total of 7,637 proteins and quantified 6,256; of these, 3,232 were classified as differentially accumulated proteins (DAPs). We also identified 3,161 lysine acetylation sites in 1,752 proteins and quantified 2,869 in 1,612 proteins. The acetylation levels at 468 sites were significantly altered across the three developmental stages during periodic albinism; the corresponding proteins were associated with a variety of biological processes. Interestingly, a large number of DAPs and acetylated proteins with increased/decreased acetylation were related to photosynthesis and secondary metabolite biosynthetic pathways, suggesting that the accumulation or acetylation level of these proteins regulates periodic albinism in ‘Anji Baicha.’ Additionally, overlap between succinylome and acetylome among three ‘Anji Baicha’ developmental stages were found. These data provide important insight into the mechanisms of leaf coloration in the tea plant. The mass spectrometry data have been deposited to Proteome X change via the PRIDE partner repository with the data set identifier PXD008134.