Hao Cheng

Floral Transcriptome Sequencing for SSR Marker Development and Linkage Map Construction in the Tea Plant (Camellia sinensis)

Despite the worldwide consumption and high economic importance of tea, the plant (Camellia sinensis) is not well studied in molecular biology. Under the few circumstances in which the plant is studied, C. sinensis flowers, which are important for reproduction and cross-breeding, receive less emphasis than investigation of its leaves or roots. Using high-throughput Illumina RNA sequencing, we analyzed a C. sinensis floral transcriptome, and 26.9 million clean reads were assembled into 75,531 unigenes averaging 402 bp. Among them, 50,792 (67.2%) unigenes were annotated with a BLAST search against the NCBI Non-Redundant (NR) database and 10,290 (16.67%) were detected that contained one or more simple sequence repeats (SSRs). From these SSR-containing sequences, 2,439 candidate SSR markers were developed and 720 were experimentally tested, validating 431 (59.9%) novel polymorphic SSR markers for C. sinensis. Then, a consensus SSR-based linkage map was constructed that covered 1,156.9 cM with 237 SSR markers distributed in 15 linkage groups. Both transcriptome information and the genetic map of C. sinensis presented here offer a valuable foundation for molecular biology investigations such as functional gene isolation, quantitative trait loci mapping, and marker-assisted selection breeding in this important species.

Geographical tracing of Xihu Longjing tea using high performance liquid chromatography.

Xihu Longjing tea (XHLJ) is one of the most famous green tea in China. Due to its high price, some inauthentic XHLJ from other tea producing areas appear on the market and hurt the interests of customers and producers. High-performance liquid chromatography (HPLC) combined with the principal component analysis (PCA) and linear discriminant analysis (LDA) methods were applied to the geographical tracing of XHLJ from two other types of Longjing teas and a non-Longjing flatten-shaped tea (non-LJ). The chromatograms of the tea samples from four different regions were highly similar. It was difficult to classify 4 types of teas directly by PCA. However, high total accuracy rates of 94.8% and 87.6% for the training and test set were achieved for distinguishing XHLJ from the other three types of tea by stepwise discriminant analysis. The identification accuracy of XHLJ from non-LJ was the highest, suggesting geographical distance might play an important role in this process. In summary, a combination of chromatographic chemical fingerprints with LDA provides a simple and rapid approach for the identification of XHLJ from other teas.

Identification of genes involved in indole-3-butyric acid-induced adventitious root formation in nodal cuttings of Camellia sinensis (L.) by suppression subtractive hybridization

The plant hormone auxin plays a key role in adventitious rooting. To increase our understanding of genes involved in adventitious root formation, we identified transcripts differentially expressed in single nodal cuttings of Camellia sinensis treated with or without indole-3-butyric acid (IBA) by suppressive subtractive hybridization (SSH). A total of 77 differentially expressed transcripts, including 70 up-regulated and 7 down-regulated sequences, were identified in tea cuttings under IBA treatment. Seven candidate transcripts were selected and analyzed for their response to IBA, and IAA by real time RT-PCR. All these transcripts were up regulated by at least two folds one day after IBA treatment. Meanwhile, IAA showed less positive effects on the expression of candidate transcripts. The full-length cDNA of a F-box/kelch gene was also isolated and found to be similar to a group of At1g23390 like genes. These unigenes provided a new source for mining genes related to adventitious root formation, which facilitate our understanding of relative fundamental metabolism.

Transcriptome Analysis of Indole-3-Butyric Acid-Induced Adventitious Root Formation in Nodal Cuttings of Camellia sinensis (L.)

Tea (Camellia sinensis L.) is a popular world beverage, and propagation of tea plants chiefly depends on the formation of adventitious roots in cuttings. To better understand potential mechanisms involved in adventitious root formation, we performed transcriptome analysis of single nodal cuttings of C. sinensis treated with or without indole-3-butyric acid (IBA) using the Illumina sequencing method. Totally 42.5 million RNA-Seq reads were obtained and these were assembled into 59,931 unigenes, with an average length of 732 bp and an N50 of 1292 bp. In addition, 1091 differentially expressed unigenes were identified in the tea cuttings treated with IBA compared to controls, including 656 up- and 435 down-regulated genes. Further real time RT-PCR analysis confirmed RNA-Seq data. Functional annotation analysis showed that many genes were involved in plant hormone signal transduction, secondary metabolism, cell wall organization and glutathione metabolism, indicating potential contributions to adventitious rooting. Our study presents a global view of transcriptome profiles of tea cuttings in response to IBA treatment and provides new insights into the fundamental mechanisms associated with auxin-induced adventitious rooting. Our data will be a valuable resource for genomic research about adventitious root formation in tea cuttings, which can be used to improve rooting for difficult-to-root varieties.

Fingerprinting 128 Chinese clonal tea cultivars using SSR markers provides new insights into their pedigree relationships

The improved clonal tea cultivars play a crucial role in the modern tea industry. In present study, we analyzed 128 elite clonal tea cultivars in China with 30 well-chosen simple sequence repeat (SSR) markers, aiming at (1) characterizing a set of DNA markers for unambiguously fingerprinting clonal tea cultivars and (2) confirming or identifying the parent-offspring (PO) relationships among them. The results showed that the markers are highly polymorphic among the tested cultivars with an average allele number of 10.4 per locus and an average polymorphic information content of 0.704. Robust fingerprinting power was demonstrated: It was possible to fully discriminate all 128 cultivars by a combination of four markers, and the overall possibility of finding two random individuals having the same genotypes across the 30 loci was estimated to be 4.8 × 10−33. Eight SSR loci were further recommended as a core marker set for fingerprinting the tea plant. Meanwhile, parentage analysis based on the fingerprint data revealed 47 pairs of putative PO relationships, among which 33 were in agreement with the known pedigree information, whereas the other 14 were newly identified in this study. The SSR markers and pedigree relationships reported here are valuable for tea cultivar identification and new breeding programs.

Short communication: Identification of geographical indication tea with Fisher’s discriminant classification and principal components analysis

This study attempted to identify authentic geographical indication tea using near infrared (NIR) spectroscopy with a combination of Fishers discriminant classification and principal components analysis (PCA). This rapid and accurate NIR-based approach has shown an accuracy rate for identifying the geographical indication tea equal to 96.7% in a training set, 95.3% using cross-validation and 96.7% in a test set. The overall results suggest that the combination of NIR spectroscopy with Fishers discriminant classification with PCA could be successfully applied as a rapid and reliable way to identify geographical indication tea.

Characteristics of NH4+ and NO3- fluxes in tea (Camellia sinensis) roots measured by scanning ion-selective electrode technique

As a vital beverage crop, tea has been extensively planted in tropical and subtropical regions. Nitrogen (N) levels and forms are closely related to tea quality. Based on different N levels and forms, we studied changes in NO3− and NH4+ fluxes in tea roots utilizing scanning ion-selective electrode technique. Our results showed that under both single and mixed N forms, influx rates of NO3− were much lower than those of NH4+, suggesting a preference for NH4+ in tea. With the increase in N concentration, the influx rate of NO3− increased more than that of NH4+. The NH4+ influx rates in a solution without NO3− were much higher than those in a solution with NO3−, while the NO3− influx rates in a solution without NH4+ were much lower than those in a solution with NH4+. We concluded that (1) tea roots showed a preference for NH4+, (2) presence of NO3− had a negative effect on NH4+ influx, and (3) NH4+ had a positive effect on NO3− influx. Our findings not only may help advance hydroponic tea experiments but also may be used to develop efficient fertilization protocols for soil-grown tea in the future.

Transcriptome Analysis Reveals Key Flavonoid 3′-Hydroxylase and Flavonoid 3′,5′-Hydroxylase Genes in Affecting the Ratio of Dihydroxylated to Trihydroxylated Catechins in Camellia sinensis

The ratio of dihydroxylated to trihydroxylated catechins (RDTC) is an important indicator of tea quality and biochemical marker for the study of genetic diversity. It is reported to be under genetic control but the underlying mechanism is not well understood. Flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) are key enzymes involved in the formation of dihydroxylated and trihydroxylated catechins. The transcriptome and HPLC analysis of tea samples from Longjing43 and Zhonghuang2 under control and shading treatment were performed to assess the F3′H and F3′5′H genes that might affect RDTC. A total of 74.7 million reads of mRNA seq (2×101bp) data were generated. After de novo assembly, 109,909 unigenes were obtained, and 39,982 of them were annotated using 7 public databases. Four key F3′H and F3′5′H genes (including CsF3′5′H1, CsF3′H1, CsF3′H2 and CsF3′H3) were identified to be closely correlated with RDTC. Shading treatment had little effect on RDTC, which was attributed to the stable expression of these key F3′H and F3′5′H genes. The correlation of the coexpression of four key genes and RDTC was further confirmed among 13 tea varieties by real time PCR and HPLC analysis. The coexpression of three F3′H genes and a F3′5′H gene may play a key role in affecting RDTC in Camellia sinensis. The current results may establish valuable foundation for further research about the mechanism controlling catechin composition in tea.

Accumulation of catechins and expression of catechin synthetic genes in Camellia sinensis at different developmental stages

BackgroundCatechins are the main polyphenol compounds in tea (Camellia sinensis). To understand the relationship between gene expression and product accumulation, the levels of catechins and relative expressions of key genes in tea leaves of different developmental stages were analyzed.ResultsThe amounts of catechins differed significantly in leaves of different stages, except for gallocatechin gallate. Close correlations between the expression of synthesis genes and the accumulation of catechins were identified. Correlation analysis showed that the expressions of chalcone synthase 1, chalcone synthase 3, anthocyanidin reductase 1, anthocyanidin reductase 2 and leucoanthocyanidin reductase genes were significantly and positively correlated with total catechin contents, suggesting their expression may largely affect total catechin accumulation. Anthocyanidin synthase was significantly correlated with catechin. While both ANRs and LAR were significantly and positively correlated with the contents of (−)-epigallocatechin gallate and (−)-epicatechin gallate.ConclusionOur results suggest synergistic changes between the expression of synthetic genes and the accumulation of catechins. Based on our findings, anthocyanidin synthase may regulate earlier steps in the conversion of catechin, while the anthocyanidin reductase and leucoanthocyanidin reductase genes may both play important roles in the biosynthesis of galloylated catechins.

Expression of Key Structural Genes of the Phenylpropanoid Pathway Associated with Catechin Epimerization in Tea Cultivars

Catechin epimerization is an important factor affecting tea catechin compositions and thereby tea quality. However, a lack of tea germplasms with high non-epicatechins limits relative research. Here, a tea cultivar Y510 with high non-epicatechins was firstly reported and used for catechin and RNA sequencing (RNA-Seq) analysis. Results showed that the (-)-gallocatechin gallate and (+)-catechin (C) contents in Y510 were at least 136 and 6 times higher than those in Fudingdabaicha and 0306I, but the epicatechins (-)-epigallocatechin and (-)-epicatechin (EC) were significantly lower. Eleven unigenes potentially involved in catechin epimerization were identified by RNA-Seq analysis. Based on a combination of catechin and gene expression analysis, it was hypothesized that two anthocyanidin reductase genes (CsANR1, CsANR2) and an anthocyanidin synthase gene (CsANS) are the key genes affecting catechin epimerization in tea. Non-epicatechin formations were hypothesized to be mainly influenced by the expression ratio of CsANR2 to CsANR1 and the expression of CsANS. Overexpression of CsANS in an Arabidopsis mutant tds4-2 led to a significant increase of EC accumulation in seeds, revealing CsANS is important for catechin epimerization. These results shed new light on breeding tea cultivars with special catechin compositions.