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.

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.

Exogenous salicylic acid induces defense responses in tea plants against Toxoptera aurantii (Hemiptera: Aphididae).

Toxoptera aurantii is one of the most destructive pests, threatening the yield and quality of tea plantations. The salicylic acid (SA)-mediated signaling pathway is vital for the induction of plant defense responses; however, its role in tea plant resistance to T. aurantii remains unclear. Thus, this study used and electrical penetration graph and monitoring of population dynamics to evaluate the effects of exogenous SA application on T. aurantii feeding behavior and population growth in tea seedlings. Moreover, the effects of SA treatment on the activities of defense-related enzymes were analyzed. Probe counts and the duration of xylem sap ingestion were significantly higher in SA-treated plants than those in the control group. The total duration of passive phloem ingestion was significantly decreased in 0.5 mmol/l SA-treated plants, and the application of 0.5, 1, and 4 mmol/l SA significantly inhibited T. aurantii population growth. In addition, the activities of polyphenol oxidase, peroxidase, and superoxide dismutase were significantly increased in the 0.5 mmol/l SA-treated plants. Overall, this study demonstrates the capacity of exogenous SA to activate defense responses against T. aurantii. These results have crucial implications for understanding the mechanisms of enhanced resistance, thereby providing a sustainable approach for managing T. aurantii.

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

BACKGROUND: Tea, the second largest consumer beverage in the world after water, is widely cultivated in tropical and subtropical areas. However, the effect of environmental factors on the distribution of wild tea plants is unclear. RESULTS: A total of 159 wild tea plants were collected from different altitudes and geological types of the Guizhou Plateau. Using the genotyping-by-sequencing method, 98,241 high-quality single nucleotide polymorphisms were identified. Genetic diversity, population structure analysis, principal component analysis, phylogenetic analysis, and linkage disequilibrium were performed. The genetic diversity of the wild tea plant population from the Silicate Rock Classes of Camellia gymnogyna was higher than that from the Carbonate Rock Classes of Camellia tachangensis. In addition, the genetic diversity of wild tea plants from the second altitude gradient was significantly higher than that of wild tea plants from the third and first altitude gradients. Two inferred pure groups (GP01 and GP02) and one inferred admixture group (GP03) were identified by population structure analysis and were verified by principal component and phylogenetic analyses. The highest differentiation coefficients were determined for GP01 vs. GP02, while the lowest differentiation coefficients were determined for GP01 vs. GP03. CONCLUSIONS: This study revealed the genetic diversity and geographical distribution characteristics of wild tea plants in the Guizhou Plateau. There are significant differences in genetic diversity and evolutionary direction between Camellia tachangensis with Carbonate Rock Classes at the first altitude gradient and Camellia gymnogyna with Silicate Rock Classes at the third altitude gradient. Geological environment, soil mineral element content, soil pH, and altitude markedly contributed to the genetic differentiation between Camellia tachangensis and Camellia gymnogyna.

Genome-wide association study of leaf-related traits in tea plant in Guizhou based on genotyping-by-sequencing.

BACKGROUND: Studying the genetic characteristics of tea plant (Camellia spp.) leaf traits is essential for improving yield and quality through breeding and selection. Guizhou Plateau, an important part of the original center of tea plants, has rich genetic resources. However, few studies have explored the associations between tea plant leaf traits and single nucleotide polymorphism (SNP) markers in Guizhou. RESULTS: In this study, we used the genotyping-by-sequencing (GBS) method to identify 100,829 SNP markers from 338 accessions of tea germplasm in Guizhou Plateau, a region with rich genetic resources. We assessed population structure based on high-quality SNPs, constructed phylogenetic relationships, and performed genome-wide association studies (GWASs). Four inferred pure groups (G-I, G-II, G-III, and G-IV) and one inferred admixture group (G-V), were identified by a population structure analysis, and verified by principal component analyses and phylogenetic analyses. Through GWAS, we identified six candidate genes associated with four leaf traits, including mature leaf size, texture, color and shape. Specifically, two candidate genes, located on chromosomes 1 and 9, were significantly associated with mature leaf size, while two genes, located on chromosomes 8 and 11, were significantly associated with mature leaf texture. Additionally, two candidate genes, located on chromosomes 1 and 2 were identified as being associated with mature leaf color and mature leaf shape, respectively. We verified the expression level of two candidate genes was verified using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and designed a derived cleaved amplified polymorphism (dCAPS) marker that co-segregated with mature leaf size, which could be used for marker-assisted selection (MAS) breeding in Camellia sinensis. CONCLUSIONS: In the present study, by using GWAS approaches with the 338 tea accessions population in Guizhou, we revealed a list of SNPs markers and candidate genes that were significantly associated with four leaf traits. This work provides theoretical and practical basis for the genetic breeding of related traits in tea plant leaves.

Genome-Wide Identification of 2-Oxoglutarate and Fe (II)-Dependent Dioxygenase (2ODD-C) Family Genes and Expression Profiles under Different Abiotic Stresses in Camellia sinensis (L.).

The 2-oxoglutarate and Fe (II)-dependent dioxygenase (2ODD-C) family of 2-oxoglutarate-dependent dioxygenases potentially participates in the biosynthesis of various metabolites under various abiotic stresses. However, there is scarce information on the expression profiles and roles of 2ODD-C genes in Camellia sinensis. We identified 153 Cs2ODD-C genes from C. sinensis, and they were distributed unevenly on 15 chromosomes. According to the phylogenetic tree topology, these genes were divided into 21 groups distinguished by conserved motifs and an intron/exon structure. Gene-duplication analyses revealed that 75 Cs2ODD-C genes were expanded and retained after WGD/segmental and tandem duplications. The expression profiles of Cs2ODD-C genes were explored under methyl jasmonate (MeJA), polyethylene glycol (PEG), and salt (NaCl) stress treatments. The expression analysis showed that 14, 13, and 49 Cs2ODD-C genes displayed the same expression pattern under MeJA and PEG treatments, MeJA and NaCl treatments, and PEG and NaCl treatments, respectively. A further analysis showed that two genes, Cs2ODD-C36 and Cs2ODD-C21, were significantly upregulated and downregulated after MeJA, PEG, and NaCl treatments, indicating that these two genes played positive and negative roles in enhancing the multi-stress tolerance. These results provide candidate genes for the use of genetic engineering technology to modify plants by enhancing multi-stress tolerance to promote phytoremediation efficiency.

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.

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.

The Zea mays mutants opaque2 and opaque16 disclose lysine change in waxy maize as revealed by RNA-Seq.

In maize, opaque2 (o2) and opaque16 (o16) alleles can increase lysine content, while the waxy (wx) gene can enhance the amylopectin content of grains. In our study, o2 and o16 alleles were backcrossed into waxy maize line (wxwx). The o2o2o16o16wxwx lines had amylopectin contents similar to those of waxy line. Their nutritional value was better than waxy line, but the mechanism by which the o2 and o16 alleles increased the lysine content of waxy maize remained unclear. The o2o2o16o16wxwx lines and their parents on kernels (18th day after pollination) were subjected to RNA sequencing (RNA-Seq). The RNA-Seq analysis revealed 272 differentially expressed genes (DEGs). Functional analyses revealed that these DEGs were mainly related to biomass metabolism. Among them, in o2o2o16o16wxwx lines, 15 genes encoding α-zein were down-regulated, which resulted in the reduction of α-zein synthesis and increased lysine content; lkr/sdh1 and Zm00001d020984.1 genes involved in the lysine degradation pathway were down-regulated, thereby inhibited lysine degradation; sh2, bt2 and ae1 genes involved in starch metabolism were upregulated, leaded to wrinkling kernel and farinaceous endosperm. Our transcriptional-level identification of key genes responsible for increased grain lysine content and farinaceous endosperm formation following introgression of o2 and o16 alleles should promote molecular breeding for maize quality.

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.

Molecular Mechanisms Underlying Increase in Lysine Content of Waxy Maize through the Introgression of the opaque2 Allele.

The opaque2 (o2) mutation in maize is associated with high lysine content in endosperm and good nutritional value. To improve the nutritional quality of waxy maize, the o2 allele was introgressed into the wxwx line using marker-assisted backcrossing selection technology. The lysine content of o2o2wxwx lines was higher than that of the wxwx line. To reveal the mechanism of increasing lysine content through introgression of the o2 in waxy maize, the transcriptome on kernels (18th day after pollination) of the o2o2wxwx and parent lines was analyzed using RNA-sequencing (RNA-Seq). The RNA-Seq analysis revealed 49 differentially expressed genes (DEGs). Functional analysis showed that these DEGs were mostly related to the catalytic activity and metabolic processes. The O2 gene regulated multiple metabolic pathways related to biological processes (BP) and molecular function (MP) during waxy maize endosperm development. In particular, in the o2o2wxwx lines, the two genes that encode the EF-1α and LHT1 were up-regulated, but the gene that encodes sulfur-rich proteins was down-regulated, raising the grain lysine content. These findings are of great importance for understanding the molecular mechanism underlying the lysine content increase due to o2 allele introgression into waxy maize.

Glutathione-mediated alleviation of chromium toxicity in rice plants.

A hydroponic experiment was conducted to determine the possible effect of exogenous glutathione (GSH) in alleviating chromium (Cr) stress through examining plant growth, chlorophyll contents, antioxidant enzyme activity, and lipid peroxidation in rice seedlings exposed to Cr toxicity. The results showed that plant growth and chlorophyll content were dramatically reduced when rice plants were exposed to 100 µM Cr. Addition of GSH in the culture solution obviously alleviated the reduction of plant growth and chlorophyll content. The activities of some antioxidant enzymes, including superoxide dismutase, catalase (CAT) and glutathione reductase in leaves, and CAT and glutathione peroxidase in roots showed obvious increase under Cr stress. Addition of GSH reduced malondialdehyde accumulation and increased the activities of these antioxidant enzymes in both leaves and roots, suggesting that GSH may enhance antioxidant capacity in Cr-stressed plants. Furthermore, exogenous GSH caused significant decrease of Cr uptake and root-to-shoot transport in the Cr-stressed rice plants. It can be assumed that GSH is involved in Cr compartmentalization in root cells.

[QTL analysis of starch content in maize kernels using the trisomic inheritance of the endosperm model].

Two hundred and fifty-nine F2:3 families, developed from the cross between a dent corn inbred line Dan232 and a popcorn inbred line N04, were evaluated for its starch content in the replicated experiments under two environments. One hundred and eighty three pairs of SSR markers were selected to construct a genetic linkage map, covering 1,762.2 cM (centiMorgan) and on the average distance of 9.63 cM between the markers. Quantative trait loci (QTL) were identified and its effects were analyzed, using the triploid endosperm genetic model and the interval mapping method. Ten QTL, detected on chromosome 1, 3, 4, 5 and 7 respectively, were mapped in spring and in summer. All detected QTL in spring were also identified in summer. The phenotypic variation of a single QTL varied from 4.74% to 11.26%, and the total contributions of all QTL under two environments were 36.84% and 72.65%, respectively. Two QTL of all identified QTL in spring showed over-dominant effects, but additive and partially dominant in summer respectively, the amounts of other QTL showing Additive, partially dominant and over-dominant effects were 2, 1, 5 respectively. The positive alleles of three QTL were contributed by the normal corn parent Dan232, and the other positive alleles were contributed by the popcorn parent N04.

QTL analysis of popping fold and the consistency of QTLs under two environments in popcorn.

Popping fold (PF) is the most important quality trait in popcorn. In this study, a total of 259 F2:3 families, derived from the cross between a dent corn inbred Dan232 and a popcorn inbred N04, were evaluated for their popping folds in replicated experiments under two environments. Of 613 simple sequence repeat (SSR) primer pairs screened, 183 pairs were selected to construct a genetic linkage map with the genetic distance of 1,762.2 cM (centimorgan) and on average 9.63 cM every marker. Quantative trait loci (QTL) were identified, and their genetic effects were estimated using CIM (composite interval mapping) method. The interactions among QTLs detected were calculated using MIM (multiple interval mapping) method. In all, 22 QTLs were detected, and only 5 of them were common under two environments. Contribution to phenotypic variation of a single QTL varied from 3.07% to 12.84%, and total contributions of all QTLs under two environments were 66.46% and 51.90%, respectively. Three QTLs (qPF-6-1, qPF-8-1 and qPF-1-3) with more than 10% contributions were observed. The additive effects were larger than dominant effects for most QTLs. The amount of QTLs showing additive, partially dominant, dominant and over-dominant effects were 4, 5, 0, 2 in spring sowing and 2, 5, 2, 2 in summer sowing, respectively. There were only 2.60% pairs of QTLs or maker intervals expressing AA, DA or DD interactions.