Population Structure and Genetic Diversity of the Toona ciliata (Meliaceae) Complex Assayed with Chloroplast DNA Markers.

Toona ciliata is a deciduous or semi-deciduous tree species and belongs to the Toona genus of the Meliaceae family. Owing to low natural regeneration and over-exploitation, the species is listed as an endangered species at level II in China and its conservation has received increasing concern. Here, we sampled 447 individuals from 29 populations across the range-wide distribution of the T. ciliata complex in China and assessed their genetic variation using two chloroplast DNA markers. The results showed that the overall haplotype diversity and nucleotide diversity per site were high at h = 0.9767 and π = 0.0303 for the psbA-trnH fragment and h= 0.8999 and π = 0.0189 for the trnL-trnL fragment. Phylogenetic analysis supported the division of the natural distribution of T. ciliata complex into western and eastern regions. The genetic diversity was higher in the western region than in the eastern region, showing significant phylogeographic structure. Genetic differentiation among populations was moderate (Φst=42.87%), and the effects of isolation by distance (IBD) were significant. A neutrality test and mismatch distribution analysis indicated that the distribution of the T. ciliata complex generally did not expand, although a few local populations could likely expand after bottleneck effects. The overall results were complementary to and consolidated previous studies using mitochondrial and nuclear DNA markers. We finally discussed strategies for the genetic conservation of the T. ciliata complex.

Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves.

Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. Its tender young buds exhibit a diverse range of colors, primarily determined by the presence and composition of anthocyanins and flavonoids. However, the underlying mechanisms of anthocyanin biosynthesis in Toona sinensis have been rarely reported. To explore the related genes and metabolites associated with composition of leaf color, we conducted an analysis of the transcriptome and metabolome of five distinct Toona clones. The results showed that differentially expressed genes and metabolites involved in anthocyanin biosynthesis pathway were mainly enriched. A conjoint analysis of transcripts and metabolites was carried out in JFC (red) and LFC (green), resulting in the identification of 510 genes and 23 anthocyanin-related metabolites with a positive correlation coefficient greater than 0.8. Among these genes and metabolites, 23 transcription factors and phytohormone-related genes showed strong coefficients with 13 anthocyanin derivates, which mainly belonged to the stable types of delphinidin, cyanidin, peonidin. The core derivative was found to be Cyanidin-3-O-arabinoside, which was present in JFC at 520.93 times the abundance compared to LFC. Additionally, the regulatory network and relative expression levels of genes revealed that the structural genes DFR, ANS, and UFGT1 might be directly or indirectly regulated by the transcription factors SOC1 (MADS-box), CPC (MYB), and bHLH162 (bHLH) to control the accumulation of anthocyanin. The expression of these genes was significantly higher in red clones compared to green clones. Furthermore, RNA-seq results accurately reflected the true expression levels of genes. Overall, this study provides a foundation for future research aimed at manipulating anthocyanin biosynthesis to improve plant coloration or to derive human health benefits.

Sequencing and analysis of the complete mitochondrial genomes of Toona sinensis and Toona ciliata reveal evolutionary features of Toona.

BACKGROUND: Toona is a critical genus in the Meliaceae, and the plants of this group are an asset for both restorative and restorative purposes, the most flexible of which are Toona sinensis and Toona ciliata. To concentrate on the advancement of mitochondrial(Mt) genome variety in T.sinensis and T.ciliata, the Mt genomes of the two species were sequenced in high throughput independently, after de novo assembly and annotation to construct a Mt genome map for comparison in genome structure. Find their repetitive sequences and analyze them in comparison with the chloroplast genome, along with Maximum-likelihood(ML) phylogenetic analysis with 16 other relatives. RESULTS: (1) T. sinensis and T.ciliata are both circular structures with lengths of 683482 bp and 68300 bp, respectively. They share a high degree of similarity in encoding genes and have AT preferences. All of them have the largest Phe concentration and are the most frequently used codons. (2) Both of their Mt genome are highly preserved in terms of structural and functional genes, while the main variability is reflected in the length of tRNA, the number of genes, and the value of RSCU. (3) T. siniensis and T. ciliata were detected to have 94 and 87 SSRs, respectively, of which mononucleotides accounted for the absolute proportion. Besides, the vast majority of their SSRs were found to be poly-A or poly-T. (4)10 and 11 migrating fragments were identified in the comparison with the chloroplast genome, respectively. (5) In the ML evolutionary tree, T.sinensis and T.ciliata clustered individually into a small branch with 100% support, reflecting two species of Toona are very similarly related to each other. CONCLUSIONS: This research provides a basis for the exploitation of T.sinensis and T.ciliata in terms of medicinal, edible, and timber resources to avoid confusion; at the same time, it can explore the evolutionary relationship between the Toona and related species, which does not only have an important practical value, but also provides a theoretical basis for future hybrid breeding of forest trees, molecular markers, and evolutionary aspects of plants, which has great scientific significance.

Phylogeography of Toona ciliata (Meliaceae) Complex in China Inferred from Cytonuclear Markers.

Toona ciliata is an important timber species but is recognized as an endangered species at level II in China. Its genetic conservation is of increasing concern. Provenance trials and other breeding programs were conducted to develop seed transfer rules and multiplications. Here, we investigated twenty-nine populations sampled across the natural distribution of the T. ciliata complex using mtDNA and nrDNA ITS (ribosomal internal transcribed spacer) markers. Haplotype diversity was h = 0.190 ± 0.202 and nucleotide diversity was π = 0.000383 ± 0.000536 for mtDNA marker. Nucleotide diversity for ITS sequences was 0.00837 ± 0.000783. Haplotypes exhibited phylogeographic structure in spatial distribution. The extent of genetic differentiation was significant (Fst = 0.6994 ± 0.0079 for ITS and 0.8870 ± 0.0077 for mtDNA marker). Isolation by distance (IBD) and by elevation (IBE) occurred among populations. Phylogenetic relationships from mtDNA marker indicated three genetically distinct regions, each without IBD effects. Compared with pollen flow, seed flow was strongly impeded in the western region, but extensive in the central region, and less impeded in the eastern region. Most populations did not exhibit expansion, with only a few populations showing expansion after bottleneck effects. We discussed a strategy of region-based genetic conservation and proposed to conserve multiple populations in the western and eastern regions and a few populations in the central region.

Long read sequencing of Toona sinensis (A. Juss) Roem: A chromosome-level reference genome for the family Meliaceae.

Chinese mahogany (Toona sinensis) is a woody plant that is widely cultivated in China and Malaysia. Toona sinensis is important economically, including as a nutritious food source, as material for traditional Chinese medicine and as a high-quality hardwood. However, the absence of a reference genome has hindered in-depth molecular and evolutionary studies of this plant. In this study, we report a high-quality T. sinensis genome assembly, with scaffolds anchored to 28 chromosomes and a total assembled length of 596 Mb (contig N50 = 1.5 Mb and scaffold N50 = 21.5 Mb). A total of 34,345 genes were predicted in the genome after homology-based and de novo annotation analyses. Evolutionary analysis showed that the genomes of T. sinensis and Populus trichocarpa diverged ~99.1-103.1 million years ago, and the T. sinensis genome underwent a recent genome-wide duplication event at ~7.8 million years and one more ancient whole genome duplication event at ~71.5 million years. These results provide a high-quality chromosome-level reference genome for T. sinensis and confirm its evolutionary position at the genomic level. Such information will offer genomic resources to study the molecular mechanism of terpenoid biosynthesis and the formation of flavour compounds, which will further facilitate its molecular breeding. As the first chromosome-level genome assembled in the family Meliaceae, it will provide unique insights into the evolution of members of the Meliaceae.

Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis.

BACKGROUND: Before studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. Toona ciliata Roem (T. ciliata). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions. RESULTS: The experimental results showed that TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta (H. robusta) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 (TcMYB3) gene. CONCLUSIONS: This is the first report to verify the best reference genes for normalizing gene expression in T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.