Analysis and evaluation of Camellia oleifera Abel. Germplasm fruit traits from the high-altitude areas of East Guizhou Province, China.

Camellia oleifera, a significant woody edible oil species, was examined using 48 germplasm resources from high-altitude regions in East Guizhou Province, China, to analyze fruit quality. The analysis aimed to identify high-performance germplasm, providing theoretical and research foundations for selecting and cross-breeding superior C. oleifera varieties in these regions. Fifteen primary traits of mature fruits were measured and analyzed, including four phenotypic traits (single fruit weight, transverse diameter, longitudinal diameter, peel thickness) and eleven quality traits (fresh seed yield rate, dry seed yield rate, dry kernel yield rate, seed kernel oil content, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, cis-11-eicosenoic acid). A comprehensive evaluation employing cluster and principal component analyses (PCA) was conducted. The cluster analysis categorized the germplasms into five groups at a squared Euclidean distance of 14, with the first category comprising 17 germplasms, the second 28, and the third, fourth, and fifth each containing one. PCA reduced the 15 traits to five principal components (PCs), with PC1 having the highest eigenvalue of 3.57 and a contribution rate of 23.8%, mainly representing phenotypic traits. PC2, contributing 20.44%, represented linoleic acid, while PC3, PC4, and PC5, with contribution rates of 12.99%, 9.13%, and 7.45% respectively, predominantly represented seed kernel oil content, fresh seed yield, and palmitoleic acid. Employing a weighted sum method, a comprehensive evaluation function was developed to calculate total scores for each superior individual, forming the basis for rankings and selections. Notable variability was detected in single fruit weight, peel thickness, and fresh and dry seed yields, while oleic acid exhibited the lowest coefficient of variation. Dry seed yield showed a robust positive correlation with seed kernel oil content and the concentrations of palmitic and linoleic acids, whereas seed kernel oil content was inversely correlated with cis-11-eicosenoic acid levels. Five PCs with eigenvalues > 1 were identified, highlighting the top ten superior individuals: QD (Qian Dong: the code of eastern Guizhou Province)-33 > QD-34 > QD-48 > QD-38 > QD-27 > QD-15 > QD-35 > QD-5 > QD-14 > QD-36. Thus, the 48 C. oleifera germplasms from East Guizhou's high-altitude areas demonstrate significant potential for enhancing traits such as single fruit weight, peel thickness, and fresh and dry seed yields. Specifically, QD-33, QD-34, and QD-48 exhibited superior comprehensive performance, designating them as prime candidates for variety selection and breeding.

Comparative physiological and transcriptomic analyses provide induction resistance mechanisms of Bacillus tequilensis against Colletotrichum fructicola in Camellia oleifera.

Bacillus tequilensis DZY 6715 was isolated from healthy leaves in Camellia oleifera, and the strain DZY 6715 significantly inhibited anthracnose disease resulting from Colletotrichum fructicola in C. oleifera, besides, its associated mechanism of disease resistance was explored. B. tequilensis DZY 6715 treatment controlled mycelial growth of C. fructicola in C. oleifera, and significantly decreased C. oleifera anthracnose incidence and disease index compared with the control group. B. tequilensis DZY 6715 has strong biofilm forming ability, and also secretes extracellular ß-1, 3-glucanase and chitinase, which could cause cell membranes damage and increased cellular compound leakage. C.oleifera treated with DZY 6715 also effectively enhanced enzyme activities and stimulated the synthesis the substances related to phenylpropane metabolism and reactive oxygen metabolism. Moreover, transcript profiling analysis revealed more differentially expressed genes related to phenylpropanoid pathway metabolism and antioxidant system inducing by DZY 6715 compared with the control in C. oleifera. Thus, it can be concluded that B. tequilensis DZY 6715 is a suitable bio-control agent to control anthracnose disease in C. oleifera.

Comparative Transcriptomic and Lipidomic Analysis of Fatty Acid Accumulation in Three Camellia oleifera Varieties During Seed Maturing.

Camellia oleifera, a major woody oil crop in China, produces tea oil rich in unsaturated fatty acids, earning it names like liquid gold and eastern olive oil. This study provides an integrated investigation of the transcriptome and lipidome within seeds at the maturing process across three C. oleifera varieties, revealing a significant relationship between fatty acid production and genes involved in lipid synthesis. Through transcriptomic analysis, 26,344 genes with varied expression were found. Functional enrichment analysis highlighted that pathways related to starch and sucrose metabolism, plant hormone signal transduction, and lipid accumulation were highly enriched among the differentially expressed genes. Coordinated high expression of key genes (ACCase, KAS I, KAS II, KAS III, KAR, HAD, EAR, SAD, LPAAT, LACS, DGAT, PDAT) during the late maturation stage contributes largely to high oil content. Additionally, expression variations of SAD and FADs among different varieties were explored. The analysis suggests that high expression of genes such as FAD3, FAD7, and FAD8 notably increased linolenic acid content. This research provides new insights into the molecular mechanisms of oil biosynthesis in C. oleifera, offering valuable references for improving yield and quality.

Genome-wide identification and molecular evolution of Dof gene family in Camellia oleifera.

DNA binding with one finger(Dof) gene family is a class of transcription factors which play an important role on plant growth and development. Genome-wide identification results indicated that there were 45 Dof genes(ColDof) in C.oleifera genome. All 45 ColDof proteins were non-transmembrane and non-secretory proteins. Phosphorylation site analysis showed that biological function of ColDof proteins were mainly realized by phosphorylation at serine (Ser) site. The secondary structure of 44 ColDof proteins was dominated by random coil, and only one ColDof protein was dominated by α-helix. ColDof genes' promoter region contained a variety of cis-acting elements, including light responsive regulators, gibberellin responsive regulators, abscisic acid responsive regulators, auxin responsive regulators and drought induction responsive regulators. The SSR sites analysis showed that the proportion of single nucleotide repeats and the frequency of A/T in ColDof genes were the largest. Non-coding RNA analysis showed that 45 ColDof genes contained 232 miRNAs. Transcription factor binding sites of ColDof genes showed that ColDof genes had 5793 ERF binding sites, 4381 Dof binding sites, 2206 MYB binding sites, 3702 BCR-BPC binding sites. ColDof9, ColDof39 and ColDof44 were expected to have the most TFBSs. The collinearity analysis showed that there were 40 colinear locis between ColDof proteins and AtDof proteins. Phylogenetic analysis showed that ColDof gene family was most closely related to that of Camellia sinensis var. sinensis cv.Biyun and Camellia lanceoleosa. Protein-protein interaction analysis showed that ColDof34, ColDof20, ColDof28, ColDof35, ColDof42 and ColDof26 had the most protein interactions. The transcriptome analysis of C. oleifera seeds showed that 21 ColDof genes were involved in the growth and development process of C. oleifera seeds, and were expressed in 221 C. oleifera varieties. The results of qRT-PCR experiments treated with different concentrations NaCl and PEG6000 solutions indicated that ColDof1, ColDof2, ColDof14 and ColDof36 not only had significant molecular mechanisms for salt stress tolerance, but also significant molecular functions for drought stress tolerance in C. oleifera. The results of this study provide a reference for further understanding of the function of ColDof genes in C.oleifera.

A serine carboxypeptidase-like acyltransferase catalyzes consecutive four-step reactions of hydrolyzable tannin biosynthesis in Camellia oleifera.

Hydrolyzable tannins (HTs), a class of polyphenolic compounds found in dicotyledonous plants, are widely used in food and pharmaceutical industries because of their beneficial effects on human health. Although the biosynthesis of simple HTs has been verified at the enzymatic level, relevant genes have not yet been identified. Here, based on the parent ion-fragment ion pairs in the feature fragment data obtained using UPLC-Q-TOF-/MS/MS, galloyl phenolic compounds in the leaves of Camellia sinensis and C. oleifera were analyzed qualitatively and quantitatively. Correlation analysis between the transcript abundance of serine carboxypeptidase-like acyltransferases (SCPL-ATs) and the peak area of galloyl products in Camellia species showed that SCPL3 expression was highly correlated with HT biosynthesis. Enzymatic verification of the recombinant protein showed that CoSCPL3 from C. oleifera catalyzed the four consecutive steps involved in the conversion of digalloylglucose to pentagalloylglucose. We also identified the residues affecting the enzymatic activity of CoSCPL3 and determined that SCPL-AT catalyzes the synthesis of galloyl glycosides. The findings of this study provide a target gene for germplasm innovation of important cash crops that are rich in HTs, such as C. oleifera, strawberry, and walnut.

Integrating histology and phytohormone/metabolite profiling to understand rooting in yellow camellia cuttings.

Vegetative propagation through cutting is a widely used clonal approach for maintaining desired genotypes. However, some woody species have difficulty forming adventitious roots (ARs) with this approach, including yellow camellia (YC) C. nitidissima. Yellow camellias, prized for their ornamental value and potential health benefits in tea, remain difficult to propagate clonally due to this rooting recalcitrance. As part of the efforts to understand YC cuttings' recalcitrance, we conducted a detailed investigation into AR formation in yellow camellia cuttings via histology and endogenous phytohormone dynamics during this process. We also compared YC endogenous phytohormone and metabolite phytohormone profiles with those of easy-to-root poplar and willow cuttings. Our results indicate that the induction of ARs in YC cuttings is achievable through auxin treatment, and YC ARs are initiated from cambial derivatives and develop a vascular system connected with that of the stem. During AR induction, endogenous hormones showed a dynamic profile, with IAA continuing to increase starting 9 days after auxin induction. JA, JA-Ile, and OPDA showed a similar trend as IAA but decreased by the 45th day. Cytokinin first decreased to its lowest level by the 18th day and then increased. SA largely exhibited an increasing trend with a drop on the 36th day, while ABA first increased to its peak level by the 18th day and then decreased. Compared to poplar, YC cuttings had a low level of IAA, IAA-Asp, and OPDA, and a high level of cytokinin and SA. Metabolite profiling highlighted significant down-accumulation of compounds associated with AR formation in yellow camellias, such as citric and ascorbic acid, fructose, sucrose, flavonoids, and phenolic acid derivatives. Our study reveals the unfavorable endogenous hormone and metabolite profiles underlying the rooting recalcitrance of YC cuttings, providing valuable knowledge for addressing this challenge in clonal propagation.

Analysis of population structure and genetic diversity of Camellia tachangensis in Guizhou based on SNP markers.

BACKGROUND: Camellia tachangensis F. C. Zhang is a five-compartment species in the ovary of tea group plants, which represents the original germline of early differentiation of some tea group plants. METHODS AND RESULTS: In this study, we analyzed single-nucleotide polymorphisms (SNPs) at the genome level, constructed a phylogenetic tree, analyzed the genetic diversity, and further investigated the population structure of 100 C. tachangensis accessions using the genotyping-by-sequencing (GBS) method. A total of 91,959 high-quality SNPs were obtained. Population structure analysis showed that the 100 C. tachangensis accessions clustered into three groups: YQ-1 (Village Group), YQ-2 (Forest Group) and YQ-3 (Transition Group), which was further consistent with the results of phylogenetic analysis and principal component analyses (PCA). In addition, a comparative analysis of the genetic diversity among the three populations (Forest, Village, and Transition Groups) detected the highest genetic diversity in the Transition Group and the highest differentiation between Forest and Village Groups. CONCLUSIONS: C. tachangensis plants growing in the forest had different genetic backgrounds from those growing in villages. This study provides a basis for the effective protection and utilization of C. tachangensis populations and lays a foundation for future C. tachangensis breeding.

Chromosome-scale genome assembly of oil-tea tree Camellia crapnelliana.

Camellia crapnelliana Tutch., belonging to the Theaceae family, is an excellent landscape tree species with high ornamental values. It is particularly an important woody oil-bearing plant species with high ecological, economic, and medicinal values. Here, we first report the chromosome-scale reference genome of C. crapnelliana with integrated technologies of SMRT, Hi-C and Illumina sequencing platforms. The genome assembly had a total length of ~2.94 Gb with contig N50 of ~67.5 Mb, and ~96.34% of contigs were assigned to 15 chromosomes. In total, we predicted 37,390 protein-coding genes, ~99.00% of which could be functionally annotated. The chromosome-scale genome of C. crapnelliana will become valuable resources for understanding the genetic basis of the fatty acid biosynthesis, and greatly facilitate the exploration and conservation of C. crapnelliana.

High-yield hybrid breeding of Camellia oleifolia based on ISSR molecular markers.

BACKGROUND: C. Oleifera is among the world's largest four woody plants known for their edible oil production, yet the contribution rate of improved varieties is less than 20%. The species traditional breeding is lengthy cycle (20-30 years), occupation of land resources, high labor cost, and low accuracy and efficiency, which can be enhanced by molecular marker-assisted selection. However, the lack of high-quality molecular markers hinders the species genetic analysis and molecular breeding. RESULTS: Through quantitative traits characterization, genetic diversity assessment, and association studies, we generated a selection population with wide genetic diversity, and identified five excellent high-yield parental combinations associated with four reliable high-yield ISSR markers. Early selection criteria were determined based on kernel fresh weight and cultivated 1-year seedling height, aided by the identification of these 4 ISSR markers. Specific assignment of selected individuals as paternal and maternal parents was made to capitalize on their unique attributes. CONCLUSIONS: Our results indicated that molecular markers-assisted breeding can effectively shorten, enhance selection accuracy and efficiency and facilitate the development of a new breeding system for C. oleifera.

Genome-Wide Analysis of MADS-Box Gene Family Reveals CjSTK as a Key Regulator of Seed Abortion in Camellia japonica.

The plant MADS-box transcription factor family is a major regulator of plant flower development and reproduction, and the AGAMOUS-LIKE11/SEEDSTICK (AGL11/STK) subfamily plays conserved functions in the seed development of flowering plants. Camellia japonica is a world-famous ornamental flower, and its seed kernels are rich in highly valuable fatty acids. Seed abortion has been found to be common in C. japonica, but little is known about how it is regulated during seed development. In this study, we performed a genome-wide analysis of the MADS-box gene the in C. japonica genome and identified 126 MADS-box genes. Through gene expression profiling in various tissue types, we revealed the C/D-class MADS-box genes were preferentially expressed in seed-related tissues. We identified the AGL11/STK-like gene, CjSTK, and showed that it contained a typical STK motif and exclusively expressed during seed development. We found a significant increase in the CjSTK expression level in aborted seeds compared with normally developing seeds. Furthermore, overexpression of CjSTK in Arabidopsis thaliana caused shorter pods and smaller seeds. Taken together, we concluded that the fine regulation of the CjSTK expression at different stages of seed development is critical for ovule formation and seed abortion in C. japonica. The present study provides evidence revealing the regulation of seed development in Camellia.

Identification and analysis of MAPK cascade gene families of Camellia oleifera and their roles in response to cold stress.

BACKGROUND: Low-temperature severely limits the growth and development of Camellia oleifera (C. oleifera). The mitogen-activated protein kinase (MAPK) cascade plays a key role in the response to cold stress. METHODS AND RESULTS: Our study aims to identify MAPK cascade genes in C. oleifera and reveal their roles in response to cold stress. In our study, we systematically identified and analyzed the MAPK cascade gene families of C. oleifera, including their physical and chemical properties, conserved motifs, and multiple sequence alignments. In addition, we characterized the interacting networks of MAPKK kinase (MAPKKK)-MAPK kinase (MAPKK)-MAPK in C. oleifera. The molecular mechanism of cold stress resistance of MAPK cascade genes in wild C. oleifera was analyzed by differential gene expression and real-time quantitative reverse transcription-PCR (qRT-PCR). CONCLUSION: In this study, 21 MAPKs, 4 MAPKKs and 55 MAPKKKs genes were identified in the leaf transcriptome of C. oleifera. According to the phylogenetic results, MAPKs were divided into 4 groups (A, B, C and D), MAPKKs were divided into 3 groups (A, B and D), and MAPKKKs were divided into 2 groups (MEKK and Raf). Motif analysis showed that the motifs in each subfamily were conserved, and most of the motifs in the same subfamily were basically the same. The protein interaction network based on Arabidopsis thaliana (A. thaliana) homologs revealed that MAPK, MAPKK, and MAPKKK genes were widely involved in C. oleifera growth and development and in responses to biotic and abiotic stresses. Gene expression analysis revealed that the CoMAPKKK5/CoMAPKKK43/CoMAPKKK49-CoMAPKK4-CoMAPK8 module may play a key role in the cold stress resistance of wild C. oleifera at a high-elevation site in Lu Mountain (LSG). This study can facilitate the mining and utilization of genetic resources of C. oleifera with low-temperature tolerance.

A new nuclear phylogeny of the tea family (Theaceae) unravels rapid radiations in genus Camellia.

Molecular analyses of rapidly radiating groups often reveal incongruence between gene trees. This mainly results from incomplete lineage sorting, introgression, and gene tree estimation error, which complicate the estimation of phylogenetic relationships. In this study, we reconstruct the phylogeny of Theaceae using 348 nuclear loci from 68 individuals and two outgroup taxa. Sequence data were obtained by target enrichment using the recently released Angiosperm 353 universal probe set applied to herbarium specimens. The robustness of the topologies to variation in data quality was established under a range of different filtering schemes, using both coalescent and concatenation approaches. Our results confirmed most of the previously hypothesized relationships among tribes and genera, while clarifying additional interspecific relationships within the rapidly radiating genus Camellia. We recovered a remarkably high degree of gene tree heterogeneity indicative of rapid radiation in the group and observed cytonuclear conflicts, especially within Camellia. This was especially pronounced around short branches, which we primarily associate with gene tree estimation error. Our analysis also indicates that incomplete lineage sorting (ILS) contributed to gene-tree conflicts and accounted for approximately 14 % of the explained variation, whereas inferred introgression levels were low. Our study advances the understanding of the evolution of this important plant family and provides guidance on the application of target capture methods and the evaluation of key processes that influence phylogenetic discordances.

Metabolome and transcriptome integration reveals insights into petals coloration mechanism of three species in Sect. Chrysantha chang.

Background: Sect. Chrysantha Chang, belonging to the Camellia genus, is one of the rare and precious ornamental plants distinguished by a distinctive array of yellow-toned petals. However, the variation mechanisms of petal color in Sect. Chrysantha Chang remains largely unclear. Methods: We conducted an integrated analysis of metabolome and transcriptome to reveal petal coloration mechanism in three species, which have different yellow tones petals, including C. chuongtsoensis (CZ, golden yellow), C. achrysantha (ZD, light yellow), and C. parvipetala (XB, milk white). Results: A total of 356 flavonoid metabolites were detected, and 295 differential metabolites were screened. The contents of 74 differential metabolites showed an upward trend and 19 metabolites showed a downward trend, among which 11 metabolites were annotated to the KEGG pathway database. We speculated that 10 metabolites were closely related to the deepening of the yellowness. Transcriptome analysis indicated that there were 2,948, 14,018 and 13,366 differentially expressed genes (DEGs) between CZ vs. ZD, CZ vs. XB and ZD vs. XB, respectively. Six key structural genes (CcCHI, CcFLS, CcDFR1, CcDFR2, CcDFR3, and CcCYP75B1) and five candidate transcription factors (MYB22, MYB28, MYB17, EREBP9, and EREBP13) were involved in the regulation of flavonoid metabolites. The findings indicate that flavonoid compounds influence the color intensity of yellow-toned petals in Sect. Chrysantha Chang. Our results provide a new perspective on the molecular mechanisms underlying flower color variation and present potential candidate genes for Camellia breeding.

Genome-Wide Identification and Expression Analysis of YTH Gene Family for Abiotic Stress Regulation in Camellia chekiangoleosa.

N6-methyladenosine (m6A) is essential for RNA metabolism in cells. The YTH domain, conserved in the kingdom of Eukaryotes, acts as an m6A reader that binds m6A-containing RNA. In plants, the YTH domain is involved in plant hormone signaling, stress response regulation, RNA stability, translation, and differentiation. However, little is known about the YTH genes in tea-oil tree, which can produce edible oil with high nutritional value. This study aims to identify and characterize the YTH domains within the tea-oil tree (Camellia chekiangoleosa Hu) genome to predict their potential role in development and stress regulation. In this study, 10 members of the YTH family containing the YTH domain named CchYTH1-10 were identified from C. chekiangoleosa. Through analysis of their physical and chemical properties and prediction of subcellular localization, it is known that most family members are located in the nucleus and may have liquid-liquid phase separation. Analysis of cis-acting elements in the CchYTH promoter region revealed that these genes could be closely related to abiotic stress and hormones. The results of expression profiling show that the CchYTH genes were differentially expressed in different tissues, and their expression levels change under drought stress. Overall, these findings could provide a foundation for future research regarding CchYTHs in C. chekiangoleosa and enrich the world in terms of epigenetic mark m6A in forest trees.

Selection and Validation of qRT-PCR Internal Reference Genes to Study Flower Color Formation in Camellia impressinervis.

Real-time quantitative PCR (qRT-PCR) is a pivotal technique for gene expression analysis. To ensure reliable and accurate results, the internal reference genes must exhibit stable expression across varied experimental conditions. Currently, no internal reference genes for Camellia impressinervis have been established. This study aimed to identify stable internal reference genes from eight candidates derived from different developmental stages of C. impressinervis flowers. We employed geNorm, NormFinder, and BestKeeper to evaluate the expression stability of these candidates, which was followed by a comprehensive stability analysis. The results indicated that CiTUB, a tubulin gene, exhibited the most stable expression among the eight reference gene candidates in the petals. Subsequently, CiTUB was utilized as an internal reference for the qRT-PCR analysis of six genes implicated in the petal pigment synthesis pathway of C. impressinervis. The qRT-PCR results were corroborated by transcriptome sequencing data, affirming the stability and suitability of CiTUB as a reference gene. This study marks the first identification of stable internal reference genes within the entire genome of C. impressinervis, establishing a foundation for future gene expression and functional studies. Identifying such stable reference genes is crucial for advancing molecular research on C. impressinervis.

Genomics insights into flowering and floral pattern formation: regional duplication and seasonal pattern of gene expression in Camellia.

BACKGROUND: The formation and domestication of ornamental traits are influenced by various aspects, such as the recognition of esthetic values and cultural traditions. Camellia japonica is widely appreciated and domesticated around the world mainly due to its rich variations in ornamental traits. Ornamental camellias have a diverse range of resources, including different bud variations from Camellia spp. as well as inter- and intra- specific hybridization. Despite research on the formation of ornamental traits, a basic understanding of their genetics and genomics is still lacking. RESULTS: Here, we report the chromosomal-level reference genome of C. japonica through combining multiple DNA-sequencing technologies and obtain a high-density genetic linkage map of 4255 markers by sequencing 98 interspecific F1 hybrids between C. japonica and C. chekiangoleosa. We identify two whole-genome duplication events in C. japonica: one is a shared ancient γ event, and the other is revealed to be specific to genus Camellia. Based on the micro-collinearity analysis, we find large-scale segmental duplication of chromosome 8, resulting to two copies of the AGAMOUS loci, which may play a key role in the domestication of floral shapes. To explore the regulatory mechanisms of seasonal flowering, we have analyzed year-round gene expression patterns of C. japonica and C. azalea-a sister plant of continuous flowering that has been widely used for cross breeding. Through comparative analyses of gene co-expression networks and annual gene expression patterns, we show that annual expression rhythms of some important regulators of seasonal growth and development, including GIGANTEA and CONSTANS of the photoperiod pathway, have been disrupted in C. azalea. Furthermore, we reveal that the distinctive expression patterns of FLOWERING LOCUS T can be correlated with the seasonal activities of flowering and flushing. We demonstrate that the regulatory module involved in GIGANTEA, CONSTANS, and FLOWERING LOCUS T is central to achieve seasonality. CONCLUSIONS: Through the genomic and comparative genomics characterizations of ornamental Camellia spp., we propose that duplication of chromosomal segments as well as the establishment of gene expression patterns has played a key role in the formation of ornamental traits (e.g., flower shape, flowering time). This work provides a valuable genomic platform for understanding the molecular basis of ornamental traits.

Transcriptome analysis reveals the potential mechanism of the response to scale insects in Camellia sasanqua Thunb.

BACKGROUND: Camellia sasanqua Thunb. is an essential woody ornamental plant. Our continuous observation found that scale insects often infest C. sasanqua all year round in Kunming, China, resulting in poor growth. Scientifically preventing and controlling the infestation of scale insects should be paid attention to, and the mechanism of scale insects influencing C. sasanqua should be used as the research basis. RESULTS: The scale insect was identified as Pseudaulacaspis sasakawai Takagi. We analyzed transcriptome sequencing data from leaves of C. sasanqua infested with scale insects. A total of 1320 genes were either up-regulated or down-regulated and differed significantly in response to scale insects. GO (Gene Ontology) annotation analysis showed that the pathway of catalytic activity, binding, membrane part, cell part, and cellular process were affected. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed that most DEGs (differentially expressed genes) involved in plant hormone signal transduction, MAPK signaling pathway, flavonoid biosynthesis, tropane, piperidine and pyridine alkaloid biosynthesis. We also observed that the expression of galactose metabolism and carotenoid biosynthesis were significantly influenced. In addition, qRT-PCR (quantitative real-time PCR) validated the expression patterns of DEGs, which showed an excellent agreement with the transcriptome sequencing. CONCLUSIONS: Our transcriptomic analysis revealed that the C. sasanqua had an intricate resistance strategy to cope with scale insect attacks. After sensing the attack signal of scale insects, C. sasanqua activated the early signal MAPK (mitogen-activated protein kinase) to activate further transcription factors and Auxin, ET, JA, ABA, and other plant hormone signaling pathways, ultimately leading to the accumulation of lignin, scopolin, flavonoids and other secondary metabolites, produces direct and indirect resistance to scale insects. Our results suggested that it provided some potential resources of defense genes that would benefit the following resistance breeding in C. sasanqua to scale insects.

Complete chloroplast genomes of 13 species of sect. Tuberculata Chang (Camellia L.): genomic features, comparative analysis, and phylogenetic relationships.

BACKGROUND: Sect. Tuberculata belongs to Camellia, and its members are characterized by a wrinkled pericarp and united filaments. All the plants in this group, which are endemic to China, are highly valuable for exploring the evolution of Camellia and have great potential for use as an oil source. However, due to the complex and diverse phenotypes of these species and the difficulty of investigating them in the field, their complex evolutionary history and interspecific definitions have remained largely unelucidated. RESULTS: Therefore, we newly sequenced and annotated 12 chloroplast (cp) genomes and retrieved the published cp genome of Camellia anlungensis Chang in sect. Tuberculata. In this study, comparative analysis of the cp genomes of the thirteen sect. Tuberculata species revealed a typical quadripartite structure characterized by a total sequence length ranging from 156,587 bp to 157,068 bp. The cp.genome arrangement is highly conserved and moderately differentiated. A total of 130 to 136 genes specific to the three types were identified by annotation, including protein-coding genes (coding sequences (CDSs)) (87-91), tRNA genes (35-37), and rRNA genes (8). The total observed frequency ranged from 23,045 (C. lipingensis) to 26,557 (C. anlungensis). IR region boundaries were analyzed to show that the ycf1 gene of C. anlungensis is located in the IRb region, while the remaining species are present only in the IRa region. Sequence variation in the SSC region is greater than that in the IR region, and most protein-coding genes have high codon preferences. Comparative analyses revealed six hotspot regions (tRNA-Thr(GGT)-psbD, psbE-petL, ycf15-tRNA-Leu(CAA), ndhF-rpl32, ndhD, and trnL(CAA)-ycf15) in the cp genomes that could serve as potential molecular markers. In addition, the results of phylogenetic tree construction based on the cp genomes showed that the thirteen sect. Tuberculata species formed a monophyletic group and were divided into two evolutionarily independent clades, confirming the independence of the section. CONCLUSIONS: In summary, we obtained the cp genomes of thirteen sect. Tuberculata plants and performed the first comparative analysis of this group. These results will help us better characterize the plants in this section, deepen our understanding of their genetic characteristics and phylogenetic relationships, and lay the theoretical foundation for their accurate classification, elucidation of their evolutionary changes, and rational development and utilization of this section in the future.

Heterologous overexpression of heat shock protein 20 genes of different species of yellow Camellia in Arabidopsis thaliana reveals their roles in high calcium resistance.

Yellow Camellia (Camellia sect. chrysantha) is a rare ornamental plant and an important germplasm resource globally. Camellia nitidissima thrives in normal acidic soils, while Camellia limonia can adapt to the calcareous soils found in karst areas. Our previous study on the karst adaptation of yellow camellias revealed that the expression levels of heat shock protein 20(HSP20) were higher in Camellia limonia than in Camellia nitidissima. However, the functions of the HSP20 gene of Camellia limonia remain unclear to data. In this study, the HSP20 genes of Camellia limonia (ClHSP20-OE lines) and Camellia. nitidissima (CnHSP20-OE lines) were cloned and overexpressed heterologously in Arabidopsis thaliana. Additionally, we overexpressed the HSP20 gene of Arabidopsis (AtHSP20-OE lines) was also overexpressed, and the T-DNA inserted mutants (athspmutant lines) were also used to determine the functions of HSP20 genes. Under high calcium stress, the chlorophyll, nitrogen, water content and humidity of leaves were increased in ClHSP20-OE lines, while those of other lines were declined. The size of the stomatal apertures, stomatal conductance, and the photosynthetic efficiency of ClHSP20-OE lines were higher than those of the other lines. However, the accumulation of H2O2 and O2- in the leaves of ClHSP20-OE lines was the lowest among all the lines. Energy spectrum scanning revealed that the percentage of calcium on the surfaces of the leaves of ClHSP20-OE lines was relatively low, while that of athspmutant lines was the highest. The ClHSP20 gene can also affected soil humidity and the contents of soil nitrogen, phosphorus, and potassium. Transcriptome analysis revealed that the expressions of FBA5 and AT5G10770 in ClHSP20-OE lines was significantly up-regulated compared to that of CnHSP20-OE lines. Compared to that of athspmutant lines, the expressions of DREB1A and AT3G30460 was significantly upregulated in AtHSP20-OE lines, and the expression of POL was down-regulated. Our findings suggest that the HSP20 gene plays a crucial role in maintained photosynthetic rate and normal metabolism by regulating the expression of key genes under high-calcium stress. This study elucidates the mechanisms underlying the karst adaptation in Camellia. limonia and provides novel insights for future research on karst plants.

Integrative metabolome and transcriptome analyses reveal the coloration mechanism in Camellia oleifera petals with different color.

BACKGROUND: Camellia olelfera petals are colorful, and have high ornamental value. However, the color formation mechanism of C. olelfera petals with different color is still unclear. In our study, WGCNA method was applied to integrate metabolites and transcriptomes to investigate the coloration mechanism of four C. olelfera cultivars with different petal colors. RESULTS: Here, a total of 372 flavonoids were identified (including 27 anthocyanins), and 13 anthocyanins were significantly differentially accumulated in C. olelfera petals. Among them, cyanidin-3-O-(6''-O-p-Coumaroyl) glucoside was the main color constituent in pink petals, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-(6''-O-malonyl) glucoside were the main contributors to candy pink petals, and peonidin-3-O-glucoside was the important color substance responsible for the red petals of C. oleifera. Furthermore, six structural genes (Co4CL1, CoF3H1, CoF3'H, CoANS, CoUGT75C1-4, and CoUGT75C1-5), three MYBs (CoMYB1, CoMYB4, and CoMYB44-3), three bHLHs (CobHLH30, CobHLH 77, and CobHLH 79-1), and two WRKYs (CoWRKY7 and CoWRKY22) could be identified candidate genes related to anthocyanins biosynthesis and accumulation, and lead to the pink and red phenotypes. The regulatory network of differentially accumulated anthocyanins and the anthocyanins related genes in C. olelfera petals were established. CONCLUSIONS: These findings elucidate the molecular basis of the coloration mechanisms of pink and red color in C. olelfera petals, and provided valuable target genes for future improvement of petals color in C. olelfera.