Metabolomic Study of Flavonoids in Camellia drupifera under Aluminum Stress by UPLC-MS/MS.

Aluminum (Al) affects the yield of forest trees in acidic soils. The oil tea plant (Camellia drupifera Lour.) has high Al tolerance, with abundant phenolic compounds in its leaves, especially flavonoid compounds. The role of these flavonoids in the Al resistance of oil tea plants is unclear. In this metabolomic study of C. drupifera under Al stress, ultra-pressure liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was utilized to identify metabolites, while principal component analysis, cluster analysis, and orthogonal partial least squares discriminant analysis were applied to analyze the data on the flavonoid metabolites. The leaf morphology of C. drupifera revealed significant damage by excess aluminum ions under each treatment compared with the control group. Under Al stress at 2 mmol/L (GZ2) and 4 mmol/L (GZ4), the total flavonoid content in C. drupifera leaves reached 24.37 and 35.64 mg/g, respectively, which are significantly higher than the levels measured in the control group (CK) (p < 0.01). In addition, we identified 25 upregulated and 5 downregulated metabolites in the GZ2 vs. CK comparison and 31 upregulated and 7 downregulated flavonoid metabolites in GZ4 vs. CK. The results demonstrate that different levels of Al stress had a significant influence on the metabolite profile of C. drupifera. It was found that the abundance of the 24 differential flavonoid metabolites was gradually elevated with increasing concentrations of Al stress, including catechin, epicatechin, naringenin-7-glucoside, astilbin, taxifolin, miquelianin, quercitrin, and quercimeritrin. Moreover, the most significant increase in antioxidant activity (about 30%) was observed in C. drupifera precultured in leaf extracts containing 7.5 and 15 µg/mL of active flavonoids. The qRT-PCR results showed that the expression levels of key genes involved in the synthesis of flavonoids were consistent with the accumulation trends of flavonoids under different concentrations of Al. Therefore, our results demonstrate the key role of flavonoid compounds in the oil tea plant C. drupifera in response to Al stress, which suggests that flavonoid metabolites in C. drupifera, as well as other aluminum-tolerant plants, may help with detoxifying aluminum.

Transcriptome and metabolome analyses provide insights into the relevance of pericarp thickness variations in Camellia drupifera and Camellia oleifera.

Camellia fruit is a woody edible oil source with a recalcitrant pericarp, which increases processing costs. However, the relevance of pericarp thickness variations in Camellia species remains unclear. Therefore, this study aimed to identify pericarp differences at the metabolic and transcription levels between thick-pericarp Camellia drupifera BG and thin-pericarp Camellia oleifera SG. Forty differentially accumulated metabolites were screened through non-targeted UHPLC-Q-TOF MS-based metabolite profiling. S-lignin was prominently upregulated in BG compared with SG, contributing to the thick pericarp of BG. KEGG enrichment and coexpression network analysis showed 29 differentially expressed genes associated with the lignin biosynthetic pathway, including 21 genes encoding catalysts and 8 encoding transcription factors. Nine upregulated genes encoding catalysts potentially led to S-lignin accumulation in BG pericarp, and transcription factors NAC and MYB were possibly involved in major transcriptional regulatory mechanisms. Conventional growth-related factors WRKYs and AP2/ERFs were positively associated while pathogenesis-related proteins MLP328 and NCS2 were negatively associated with S-lignin content. Thus, Camellia balances growth and defense possibly by altering lignin biosynthesis. The results of this study may guide the genetic modifications of C. drupifera to optimize its growth-defense balance and improve seed accessibility.

Climate-Driven Adaptive Differentiation in Melia azedarach: Evidence from a Common Garden Experiment.

Studies of local adaptation in populations of chinaberry (Melia azedarach L.) are important for clarifying patterns in the population differentiation of this species across its natural range. M. azedarach is an economically important timber species, and its phenotype is highly variable across its range in China. Here, we collected M. azedarach seeds from 31 populations across its range and conducted a common garden experiment. We studied patterns of genetic differentiation among populations using molecular markers (simple sequence repeats) and data on phenotypic variation in six traits collected over five years. Our sampled populations could be subdivided into two groups based on genetic analyses, as well as patterns of isolation by distance and isolation by environment. Significant differentiation in growth traits was observed among provenances and families within provenances. Geographic distance was significantly correlated with the quantitative genetic differentiation (QST) in height (HEIT) and crown breadth. Climate factors were significantly correlated with the QST for each trait. A total of 23 climatic factors were examined. There was a significant effect of temperature on all traits, and minimum relative humidity had a significant effect on the survival rate over four years. By comparing the neutral genetic differentiation (FST) with the QST, the mode of selection acting on survival rate varied, whereas HEIT and the straightness of the main trunk were subject to the same mode of selection. The variation in survival rate was consistent with the variation in genetic differentiation among populations, which was indicative of local adaptation. Overall, our findings provide new insights into the responses of the phenological traits of M. azedarach to changes in the climate conditions of China.

The complete genome sequence of Melia azedarach Linn. (Meliaceae): a multi-purpose pesticide species.

The plant genus Melia has two or four species in the modern world, and is a natural source of a traditional pesticide, Toosendanin. In this study, we report the complete chloroplast genome of Melia azedarach, assembled from whole-genome high-throughput sequencing data, as a resource for future studies on the taxonomy and evolution of Melia. The chloroplast genome was 160,393 bp in length, with a large single-copy region of 87,598 bp, a small single-copy region of 18,709 bp, separated by two inverted repeat regions of 27,043 bp each. It was predicted to contain a total of 133 genes, with an overall GC content of 37.37%. Phylogenetic analysis placed M. azedarach closest to Azadirachta sp. in Meliaceae.

The complete genome sequence of Sorbus insignis (Rosaceae: Amygdaloideae), an epiphytic shrub in this genus.

Sorbus insignis (Hook. f.) Hedl., belonging to S. sect. Multijugae Yu is an epiphytic shrub. Its phylogenetic position is still poorly understood. In this study, we assembled its complete chloroplast genome from whole-genome high-throughput sequencing data. The chloroplast genome was 159, 993 bp in length, with a large single-copy (LSC) region of 87, 932 bp, a small single-copy (SSC) region of 19, 255 bp, separated by two inverted repeat (IR) regions of 26, 403 bp each. It was predicted to contain a total of 132 genes, with an overall GC content of 36.56%. Phylogenetic analysis suggested S. insignis belongs to S. L. sensu stricto and closest to S. prattii Koehne among the published chloroplast genome.

Development and characterization of genomic microsatellite markers in the tree species, Rhodoleia championii, R. parvipetala, and R. forrestii (Hamamelidaceae).

Rhodoleia Champion ex Hooker is one of the most primitive relict genera of Hamamelidaceae, a key family exploited to understand the origin and early evolution of flowering plants. Genomic simple sequence repeats (SSRs) were developed for R. championii to perform genetic diversity, phylogeographical structure or even systematic evolution studies of the genus. Among the 278,743 contigs (105,758,242 bps) de novo assembled from the low-coverage whole genome sequencing of R. championii, a total of 9106 SSRs were detected in 8370 contigs, and SSR primer pairs were successfully designed for 6677 SSRs. Among the 110 selected primer pairs, 41 were amplified successfully in the preliminary test of SSR screening. Further amplification of these 41 primer pairs across the 122 individuals collected from six populations of the three Rhodoleia species showed that 32 and 40 SSR markers can be amplified in Vietnam and Jinping populations of R. parvipetala, 41, 33, and 41 SSR markers in Boluo, Hongkong and Xinyi populations of R. championii, 25 SSR markers in Fugong population of R. forrestii, and 20 SSR markers demonstrated to be polymorphic across the three species. Genetic analysis for these 20 polymorphic SSRs showed that Allele number (A) ranged from four to 13 and polymorphic information content (PIC) ranged from 0.479 to 0.876 across the three species. At the population level, observed heterozygosity (HO) ranged from 0.000 to 1.000, and expected heterozygosity (HE) ranged from 0.091 to 0.851. In the present study, we provided the first whole-genome sequencing database for the species R. championii, identified ample SSR loci with designed primers, and revealed that 20 of the 110 selected SSRs were polymorphic across three Rhodoleia species. These provide valuable resources for future studies on genetic study, species delimitation, phylogeography, and conservation of this genus.

Development and characterization of EST-SSR markers via transcriptome sequencing in Brainea insignis (Aspleniaceae s.l.).

PREMISE OF THE STUDY: Brainea insignis (Aspleniaceae) is an endangered tree fern in China whose wild populations have been seriously damaged due to overexploitation. Expressed sequence tag-simple sequence repeat (EST-SSR) primers were developed to investigate its genetic diversity and provide resources for future conservation studies. METHODS AND RESULTS: We obtained 72,897 unigenes of B. insignis using transcriptome sequencing and detected 15,006 SSRs in 12,058 unigenes. Based on these results, we designed 100 EST-SSR primer pairs and successfully amplified 52 of them in six individuals; 27 demonstrated polymorphisms after amplification against 72 individuals across three populations. Allele numbers ranged from three to 10, and the observed and expected heterozygosities ranged from 0.105 to 1.000 and from 0.523 to 0.865, respectively, in the tested populations. Most of these primers could be successfully amplified in two other fern species (Blechnumorientale and Chieniopteris harlandii). CONCLUSIONS: These selected EST-SSRs are valuable for genetic diversity and conservation studies in B. insignis and other related fern species.

Development and characterization of genomic SSR markers for Anneslea fragrans (Pentaphylacaceae).

PREMISE OF THE STUDY: The genus Anneslea (Pentaphylacaceae) contains four species and six varieties, most of which are locally endemic. Here, simple sequence repeat (SSR) markers were developed for the conservation of these species. METHODS AND RESULTS: The genome of A. fragrans was sequenced and de novo assembled into 445,162 contigs, of which 30,409 SSR loci were detected. Primers for 100 SSR loci were validated with PCR amplification in three populations of A. fragrans. Seventy-nine loci successfully amplified, and 30 were polymorphic. The mean number of alleles, observed heterozygosity, and expected heterozygosity were 7.01 ± 1.60, 0.817 ± 0.241, and 0.796 ± 0.145, respectively. Most primers could be amplified in Ternstroemia gymnanthera, T. kwangtungensis, and Cleyerapachyphylla. CONCLUSIONS: Our study demonstrated that shotgun genome sequencing is an efficient way to develop genomic SSR markers for nonmodel species. These genomic SSR loci will be valuable in population genetic studies in Anneslea and its relatives.

Phylogeographic pattern suggests a general northeastward dispersal in the distribution of Machilus pauhoi in South China.

Machilus pauhoi Kanehira is an important timber species in China. A provenance trial was recently set up to evaluate the growth performance of trees from different localities, with the aim of designing seed transfer guidelines. Here, we tested twelve nuclear microsatellite markers derived from other species of the Lauraceae family and investigated population genetic structure in M. pauhoi. Both the number of observed alleles per locus (Na) and the polymorphic information content (PIC) significantly decreased against the latitude, but showed an insignificant decrease against the longitude. Heterozygosity (Ho) and gene diversity (h) exhibited a weak correlation with geographic location. Private alleles were present in multiple populations, and a moderate level of population genetic differentiation was detected (Gst = 0.1691). The joint pattern of genetic diversity (Na, PIC, Ho, and h) suggests that general northeastward dispersal led to the current distribution of M. pauhoi. Significant but weak effects of isolation-by-distance (IBD) occurred, implicating the mountain ranges as the major barrier to gene flow. Both STRUCTURE and hierarchical clustering analyses showed three distinct groups of populations related to the physical connectivity among mountain ranges. A priority in designing genetic conservation should be given to the populations at the southwest side of the species' distribution. This conservation strategy can also be combined with the pattern of adaptive genetic variation from the provenance trial for comprehensive genetic resource management of native M. pauhoi.