Genome-wide identification of tea plant (Camellia sinensis) BAHD acyltransferases reveals their role in response to herbivorous pests.

BACKGROUND: BAHD acyltransferases are among the largest metabolic protein domain families in the genomes of terrestrial plants and play important roles in plant growth and development, aroma formation, and biotic and abiotic stress responses. Little is known about the BAHDs in the tea plant, a cash crop rich in secondary metabolites. RESULTS: In this study, 112 BAHD genes (CsBAHD01-CsBAHD112) were identified from the tea plant genome, with 85% (98/112) unevenly distributed across the 15 chromosomes. The number of BAHD gene family members has significantly expanded from wild tea plants to the assamica type to the sinensis type. Phylogenetic analysis showed that they could be classified into seven subgroups. Promoter cis-acting element analysis revealed that they contain a large number of light, phytohormones, and stress-responsive elements. Many members displayed tissue-specific expression patterns. CsBAHD05 was expressed at more than 500-fold higher levels in purple tea leaves than in green tea leaves. The genes exhibiting the most significant response to MeJA treatment and feeding by herbivorous pests were primarily concentrated in subgroups 5 and 6. The expression of 23 members of these two subgroups at different time points after feeding by tea green leafhoppers and tea geometrids was examined via qPCR, and the results revealed that the expression of CsBAHD93, CsBAHD94 and CsBAHD95 was significantly induced after the tea plants were subjected to feeding by both pricking and chewing pests. Moreover, based on the transcriptome data for tea plants being fed on by these two pests, a transcriptional regulatory network of different transcription factor genes coexpressed with these 23 members was constructed. CONCLUSIONS: Our study provides new insights into the role of BAHDs in the defense response of tea plants, and will facilitate in-depth studies of the molecular function of BAHDs in resistance to herbivorous pests.

Genome-wide association study, population structure, and genetic diversity of the tea plant in Guizhou Plateau.

BACKGROUND: Guizhou Plateau, as one of the original centers of tea plant, has a profound multi-ethnic cultural heritage and abundant tea germplasm resources. However, the impact of indigenous community factors on the genetic diversity, population structure and geographical distribution of tea plant is still unclear. RESULTS: Using the genotyping-by-sequencing (GBS) approach, we collected 415 tea plant accessions from the study sites, estimated genetic diversity, developed a core collection, and conducted a genome-wide association study (GWAS) based on 99,363 high-quality single-nucleotide polymorphisms (SNPs). A total of 415 tea accessions were clustered into six populations (GP01, GP02, GP03, GP04, GP05 and GP06), and the results showed that GP04 and GP05 had the highest and lowest genetic diversity (Pi = 0.214 and Pi = 0.145, respectively). Moreover, 136 tea accessions (33%) were selected to construct the core set that can represent the genetic diversity of the whole collection. By analyzing seven significant SNP markers associated with the traits such as the germination period of one bud and two leaves (OTL) and the germination period of one bud and three leaves (OtL), four candidate genes possibly related to OTL and OtL were identified. CONCLUSIONS: This study revealed the impact of indigenous communities on the population structure of 415 tea accessions, indicating the importance of cultural practices for protection and utilization of tea plant genetic resources. Four potential candidate genes associated with the OTL and OtL of tea plant were also identified, which will facilitate genetic research, germplasm conservation, and breeding.

De novo full length transcriptome analysis of a naturally caffeine-free tea plant reveals specificity in secondary metabolic regulation.

Tea plants are crops with economic, health and cultural value. Catechin, caffeine and theanine are the main secondary metabolites of taste. In the process of germplasm collection, we found a resource in the Sandu Aquatic Autonomous County of Guizhou (SDT) that possessed significantly different characteristic metabolites compared with the cultivar 'Qiancha 1'. SDT is rich in theobromine and theophylline, possesses low levels of (-)-epicatechin-3-gallate, (-)-epigallocatechin-3-gallate, and theanine content, and is almost free of caffeine. However, research on this tea resource is limited. Full-length transcriptome analysis was performed to investigate the transcriptome and gene expression of these metabolites. In total, 78,809 unique transcripts were obtained, of which 65,263 were complete coding sequences. RNA-seq revealed 3415 differentially expressed transcripts in the tender leaves of 'Qiancha 1' and 'SDT'. Furthermore, 2665, 6231, and 2687 differentially expressed transcripts were found in different SDT tissues. These differentially expressed transcripts were enriched in flavonoid and amino acid metabolism processes. Co-expression network analysis identified five modules associated with metabolites and found that genes of caffeine synthase (TCS) may be responsible for the low caffeine content in SDT. Phenylalanine ammonia lyase (PAL), glutamine synthetase (GS), glutamate synthase (GOGAT), and arginine decarboxylase (ADC) play important roles in the synthesis of catechin and theanine. In addition, we identified that ethylene resposive factor (ERF) and WRKY transcription factors may be involved in theanine biosynthesis. Overall, our study provides candidate genes to improve understanding of the synthesis mechanisms of these metabolites and provides a basis for molecular breeding of tea plant.

Comparative transcriptomic and proteomic analysis of nutritional quality-related molecular mechanisms of 'Qianmei 419' and 'Qianfu 4' varieties of Camellia sinensis.

In this study, the content of main nutrients in 'QianFu No. 4' were significantly higher than 'QianMei 419.'Transcriptome and proteome were combined to provide new insight of the molecular mechanisms linked to nutritional quality of 'QianFu No. 4' and 'QianMei 419' by leaf function analysis, RNA sequencing and isobaric tags for relative and absolute quantification techniques.A total of 23,813 genes and 361 proteins exhibited differential expression level in 'QianMei 419' when compared with 'QianFu No. 4'. These genes and proteins revealed that the pathway of flavonoids biosynthesis, caffeine metabolism, theanine biosynthesis and amino acid metabolism were linked to nutritional quality of tea. Our results provided transcriptomics and proteomics information with respect to the molecular mechanisms of nutritional changes of tea, identified key genes and proteins that associated with the metabolism and accumulation of nutrients, and helped clarify the molecular mechanisms of nutrient differences.

A Genome-Wide Association Study to Identify Novel Candidate Genes Related to Low-Nitrogen Tolerance in Cucumber (Cucumis sativus L.).

Cucumber is one of the most important vegetables, and nitrogen is essential for the growth and fruit production of cucumbers. It is crucial to develop cultivars with nitrogen limitation tolerance or high nitrogen efficiency for green and efficient development in cucumber industry. To reveal the genetic basis of cucumber response to nitrogen starvation, a genome-wide association study (GWAS) was conducted on a collection of a genetically diverse population of cucumber (Cucumis sativus L.) comprising 88 inbred and DH accessions including the North China type, the Eurasian type, the Japanese and South China type mixed subtype, and the South China type subtype. Phenotypic evaluation of six traits under control (14 mM) and treatment (3.5 mM) N conditions depicted the presence of broad natural variation in the studied population. The GWAS results showed that there were significant differences in the population for nitrogen limitation treatment. Nine significant loci were identified corresponding to six LD blocks, three of which overlapped. Sixteen genes were selected by GO annotation associated with nitrogen. Five low-nitrogen stress tolerance genes were finally identified by gene haplotype analysis: CsaV3_3G003630 (CsNRPD1), CsaV3_3G002970 (CsNRT1.1), CsaV3_4G030260 (CsSnRK2.5), CsaV3_4G026940, and CsaV3_3G011820 (CsNPF5.2). Taken together, the experimental data and identification of candidate genes presented in this study offer valuable insights and serve as a useful reference for the genetic enhancement of nitrogen limitation tolerance in cucumbers.

Study of Camellia sinensis diploid and triploid leaf development mechanism based on transcriptome and leaf characteristics.

Polyploidization results in significant changes in the morphology and physiology of plants, with increased growth rate and genetic gains as the number of chromosomes increases. In this study, the leaf functional traits, photosynthetic characteristics, leaf cell structure and transcriptome of Camellia sinensis were analyzed. The results showed that triploid tea had a significant growth advantage over diploid tea, the leaf area was 59.81% larger, and the photosynthetic capacity was greater. The morphological structure of triploid leaves was significantly different, the xylem of the veins was more developed, the cell gap between the palisade tissue and the sponge tissue was larger and the stomata of the triploid leaves were also larger. Transcriptome sequencing analysis revealed that in triploid tea, the changes in leaf morphology and physiological characteristics were affected by the expression of certain key regulatory genes. We identified a large number of genes that may play important roles in leaf development, especially genes involved in photosynthesis, cell division, hormone synthesis and stomata development. This research will enhance our understanding of the molecular mechanism underlying tea and stomata development and provide a basis for molecular breeding of high-quality and high-yield tea varieties.

The complete chloroplast genome sequence of Camellia sinensis cultivar 'Qiancha1' from Guizhou Province, China.

The complete chloroplast genome sequence of Camellia sinensis cultivar 'Qiancha 1' (QC1), an excellent tea plant cultivar was determined in this study. The cp genome of QC1 is 157,024 bp in length and includes a large single copy (LSC, 86,585 bp), a small single copy (SSC, 18,277 bp) and a pair of inverted repeats (IRa and IRb, 26,081 bp). The overall GC content is 37.3%. A total of 137 genes were annotated, including 92 protein coding, 37 tRNA, and eight rRNA genes. Phylogenetic analysis showed that QC1 has the closest evolutionary relationship with C. sinensis cultivar 'Anhua' from Hunan, China. The complete cp genome of QC1 provides a resource for further research on the phylogeny and taxonomy of Sect. Thea (L.) Dyer.

Population structure analysis to explore genetic diversity and geographical distribution characteristics of cultivated-type tea plant in Guizhou Plateau.

BACKGROUND: Tea plants originated in southwestern China. Guizhou Plateau is an original center of tea plants, and is rich in germplasm resources. However, the genetic diversity, population structure and distribution characteristics of cultivated-type tea plants in the region are unknown. In this study, we explored the genetic diversity and geographical distribution of cultivated-type tea accessions in Guizhou Plateau. RESULTS: We used 112,072 high-quality genotyping-by-sequencing to analyze the genetic diversity, principal components, phylogeny, population structure, and linkage disequilibrium, and develop a core collection of 253 cultivated-type tea plant accessions from Guizhou Plateau. The results showed Genetic diversity of the cultivated-type tea accessions of the Pearl River Basin was significantly higher than that of the cultivated-type tea accessions of the Yangtze River Basin. Three inferred pure groups (CG-1, CG-2 and CG-3) and one inferred admixture group (CG-4), were identified by a population structure analysis, and verified by principal component and phylogenetic analyses. The highest genetic distance and differentiation coefficients were determined for CG-2 vs CG-3. The lower genetic distance and differentiation coefficients were determined for CG-4 vs CG-2 and CG-4 vs CG-3, respectively. We developed a core set and a primary set. The primary and core sets contained 77.0 and 33.6% of all individuals in the initial set, respectively. The primary set may serve as the primary population in genome-wide association studies, while the core collection may serve as the core population in multiple treatment setting studies. CONCLUSIONS: The present study demonstrated the genetic diversity and geographical distribution characteristics of cultivated-type tea plants in Guizhou Plateau. Significant differences in genetic diversity and evolutionary direction were detected between the ancient landraces of the Pearl River Basin and the those of the Yangtze River Basin. Major rivers and ancient hubs were largely responsible for the genetic exchange between the Pearl River Basin and the Yangtze River Basin ancient landraces as well as the formation of the ancient hubs evolutionary group. Genetic diversity, population structure and core collection elucidated by this study will facilitate further genetic studies, germplasm protection, and breeding of tea plants.

Genome-wide identification and expression analysis of SABATH methyltransferases in tea plant (Camellia sinensis): insights into their roles in plant defense responses.

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.

Development of core-collections for Guizhou tea genetic resources and GWAS of leaf size using SNP developed by genotyping-by-sequencing.

An accurate depiction of the genetic relationship, the development of core collection, and genome-wide association analysis (GWAS) are key for the effective exploitation and utilization of genetic resources. Here, genotyping-by-sequencing (GBS) was used to characterize 415 tea accessions mostly collected from the Guizhou region in China. A total of 30,282 high-quality SNPs was used to estimate the genetic relationships, develop core collections, and perform GWAS. We suggest 198 and 148 accessions to represent the core set and mini-core set, which consist of 47% and 37% of the whole collection, respectively, and contain 93-95% of the total SNPs. Furthermore, the frequencies of all alleles and genotypes in the whole set were very well retained in the core set and mini-core set. The 415 accessions were clustered into 14 groups and the core and the mini-core collections contain accessions from each group, species, cultivation status and growth habit. By analyzing the significant SNP markers associated with multiple traits, nine SNPs were found to be significantly associated with four leaf size traits, namely MLL, MLW, MLA and MLSI (P < 1.655E-06). This study characterized the genetic distance and relationship of tea collections, suggested the core collections, and established an efficient GWAS analysis of GBS result.

Genetic diversity, linkage disequilibrium, and population structure analysis of the tea plant (Camellia sinensis) from an origin center, Guizhou plateau, using genome-wide SNPs developed by genotyping-by-sequencing.

BACKGROUND: To efficiently protect and exploit germplasm resources for marker development and breeding purposes, we must accurately depict the features of the tea populations. This study focuses on the Camellia sinensis (C. sinensis) population and aims to (i) identify single nucleotide polymorphisms (SNPs) on the genome level, (ii) investigate the genetic diversity and population structure, and (iii) characterize the linkage disequilibrium (LD) pattern to facilitate next genome-wide association mapping and marker-assisted selection. RESULTS: We collected 415 tea accessions from the Origin Center and analyzed the genetic diversity, population structure and LD pattern using the genotyping-by-sequencing (GBS) approach. A total of 79,016 high-quality SNPs were identified; the polymorphism information content (PIC) and genetic diversity (GD) based on these SNPs showed a higher level of genetic diversity in cultivated type than in wild type. The 415 accessions were clustered into three groups by STRUCTURE software and confirmed using principal component analyses (PCA)-wild type, cultivated type, and admixed wild type. However, unweighted pair group method with arithmetic mean (UPGMA) trees indicated the accessions should be grouped into more clusters. Further analyses identified four groups, the Pure Wild Type, Admixed Wild Type, ancient landraces and modern landraces using STRUCTURE, and the results were confirmed by PCA and UPGMA tree method. A higher level of genetic diversity was detected in ancient landraces and Admixed Wild Type than that in the Pure Wild Type and modern landraces. The highest differentiation was between the Pure Wild Type and modern landraces. A relatively fast LD decay with a short range (kb) was observed, and the LD decays of four inferred populations were different. CONCLUSIONS: This study is, to our knowledge, the first population genetic analysis of tea germplasm from the Origin Center, Guizhou Plateau, using GBS. The LD pattern, population structure and genetic differentiation of the tea population revealed by our study will benefit further genetic studies, germplasm protection, and breeding.

Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis).

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant (Camellia sinensis), which account for the tea's unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762 bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.

Surface Passivation of Bismuth-Based Perovskite Variant Quantum Dots To Achieve Efficient Blue Emission.

Metal halide perovskite quantum dots (QDs) recently have attracted great research attentions. However, blue-emitting perovskite QDs generally suffer from low photoluminescence quantum yield (PLQY) because of easily formed defects and insufficient surface passivation. Replacement of lead with low toxicity elements is also preferred toward potential commercial applications. Here, we apply Cl-passivation to boost the PLQY of MA3Bi2Br9 QDs to 54.1% at the wavelength of 422 nm, a new PLQY record for blue emissive, lead-free perovskite QDs. Because of the incompatible crystal structures between MA3Bi2Br9 and MA3Bi2Cl9 and the careful kinetic control during the synthesis, Cl- anions are engineered to mainly locate on the surface of QDs acting as passivating ligands, which effectively suppress surface defects and enhance the PLQY. Our results highlight the potential of MA3Bi2Br9 QDs for applications of phosphors, scintillators, and light-emitting diodes.

Transcriptomic analysis of differentially expressed genes in flower-buds of genetic male sterile and wild type cucumber by RNA sequencing.

Cucumber (Cucumis sativus L.) pollen development involves a diverse range of gene interactions between sporophytic and gametophytic tissues. Previous studies in our laboratory showed that male sterility was controlled by a single recessive nuclear gene, and occurred in pollen mother cell meiophase. To fully explore the global gene expression and identify genes related to male sterility, a RNA-seq analysis was adopted in this study. Young male flower-buds (1-2 mm in length) from genetic male sterility (GMS) mutant and homozygous fertile cucumber (WT) were collected for two sequencing libraries. Total 545 differentially expressed genes (DEGs), including 142 up-regulated DEGs and 403 down-regulated DEGs, were detected in two libraries (Fold Change ≥ 2, FDR < 0.01). These genes were involved in a variety of metabolic pathways, like ethylene-activated signaling pathway, sporopollenin biosynthetic pathway, cell cycle and DNA damage repair pathway. qRT-PCR analysis was performed and showed that the correlation between RNA-Seq and qRT-PCR was 0.876. These findings contribute to a better understanding of the mechanism that leads to GMS in cucumber.

Fine mapping of a male sterility gene ms-3 in a novel cucumber (Cucumis sativus L.) mutant.

KEY MESSAGE: The cucumber male sterility gene ms - 3 was fine mapped in a 76 kb region harboring an MMD1 -like gene Csa3M006660 that may be responsible for the male sterile in cucumber. A cucumber (Cucumis sativus L.) male sterile mutant (ms-3) in an advanced-generation inbred line was identified, and genetic analysis revealed that the male sterility trait was controlled by a recessive nuclear gene, ms-3, which was stably inherited. Histological studies suggested that the main cause of the male sterility was defective microsporogenesis, resulting in no tetrad or microspores being formed. Bulked segregant analysis (BSA) and genotyping of an F2 population of 2553 individuals were employed used to fine map ms-3, which was delimited to a 76 Kb region. In this region, a single non-synonymous SNP was found in the Csa3M006660 gene locus, which was predicted to result in an amino acid change. Quantitative RT-PCR analysis of Csa3M006660 was consistent with the fact that it plays a role in the early development of cucumber pollen. The protein encoded by Csa3M006660 is predicted to be homeodomain (PHD) finger protein, and the high degree of sequence conservation with homologs from a range of plant species further suggested the importance of the ms-3 non-synonymous mutation. The data presented here provide support for Csa3M006660 as the most likely candidate gene for Ms-3.

Lead-Free, Blue Emitting Bismuth Halide Perovskite Quantum Dots.

Lead halide perovskite quantum dots (QDs) are promising candidates for future lighting applications, due to their high quantum yield, narrow full width at half maximum (FWHM), and wide color gamut. However, the toxicity of lead represents a potential obstacle to their utilization. Although tin(II) has been used to replace lead in films and QDs, the high intrinsic defect density and oxidation vulnerability typically leads to unsatisfactory material properties. Bismuth, with much lower toxicity than lead, is promising to constitute lead-free perovskite materials because Bi3+ is isoelectronic to Pb2+ and more stable than Sn2+ . Herein we report, for the first time, the synthesis and optical characterization of MA3 Bi2 Br9 perovskite QDs with photoluminescence quantum yield (PLQY) up to 12 %, which is much higher than Sn-based perovskite nanocrystals. Furthermore, the photoluminescence (PL) peaks of MA3 Bi2 X9 QDs could be easily tuned from 360 to 540 nm through anion exchange.