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.

Small RNA (sRNA) play pivotal roles in reproductive development, and their biogenesis and action mechanisms are well characterised in angiosperm plants; however, corresponding studies in conifers are very limited. To improve our understanding of the roles of sRNA pathways in the reproductive development of conifers, the genes associated with sRNA biogenesis and action pathways were identified and analysed, and sRNA sequencing and parallel analysis of RNA ends (PARE) were performed in male and female cones of the Chinese pine (Pinus tabuliformis). Based on high-quality reference transcriptomic sequences, 21 high-confidence homologues involved in sRNA biogenesis and action in P. tabuliformis were identified, including two different DCL3 genes and one AGO4 gene. More than 75 % of genes involved in sRNA biogenesis and action have higher expression levels in female than in male cones. Twenty-six microRNA (miRNA) families and 74 targets, including 46 24-nt sRNAs with a 5’ A, which are specifically expressed in male cones or female cones and probably bind to AGO4, were identified. The sRNA pathways have higher activity in female than in male cones, and the miRNA pathways are the main sRNA pathways in P. tabuliformis. The low level of 24-nt short-interfering RNAs in conifers is not caused by the absence of biogenesis-related genes or AGO-binding proteins, but most likely caused by the low accumulation of these key components. The identification of sRNAs and their targets, as well as genes associated with sRNA biogenesis and action, will provide a good starting point for investigations into the roles of sRNA pathways in cone development in conifers.

Sequence-related amplified polymorphism (SRAP) markers were used to investigate the genetic diversity among 30 populations of Toona ciliata Roem. sampled from the species’ distribution area in China. To analyze the polymorphism in the SRAP profiles, 1505 primer pairs were screened and 24 selected. A total of 656 SRAP bands ranging from 100 to 1500 bp were acquired, of these 505 bands (77%) were polymorphic. The polymorphism information content (PIC) values ranged from 0.32 to 0.45, with an average of 0.41. An analysis of molecular variance (AMOVA) indicated that the most significant variation was attributable to differences among the populations and that variation within the populations was small. STRUCTURE analysis divided the 30 populations into two parts. The unweighted pair group method of arithmetic averages (UPGMA) clustering and principal coordinates analysis (PCoA) showed that the 30 populations could be classified into four types. The results demonstrate a clear geographical trend for T. ciliata in China and provide a theoretical basis for future breeding and conservation strategy of T. ciliata.

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 (F_st = 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.