Pine wilt disease caused by pine wood nematode (Bursaphelenchus xylophilus, PWN) is a severe forest disease of the genus Pinus. Masson pine as an important timber and oleoresin resource in South China, is the major species infected by pine wilt disease. However, the underlying mechanism of pine resistance is still unclear. Here, we performed a transcriptomics analysis to identify differentially expressed genes associated with resistance to PWN infection. By comparing the expression profiles of resistant and susceptible trees inoculated with PWN at 1, 15, or 30 days post-inoculation (dpi), 260, 371 and 152 differentially expressed genes (DEGs) in resistant trees and 756, 2179 and 398 DEGs in susceptible trees were obtained. Gene Ontology enrichment analysis of DEGs revealed that the most significant biological processes were “syncytium formation” in the resistant phenotype and “response to stress” and “terpenoid biosynthesis” in the susceptible phenotype at 1 and 15 dpi, respectively. Furthermore, some key DEGs with potential regulatory roles to PWN infection, including expansins, pinene synthases and reactive oxidation species (ROS)-related genes were evaluated in detail. Finally, we propose that the biosynthesis of oleoresin and capability of ROS scavenging are pivotal to the high resistance of PWN.

Masson pine is an important timber and resource for oleoresin in South China. Increasing yield of oleoresin in stems can raise economic benefits and enhance the resistance to bark beetles. However, the genetic mechanisms for regulating the yield of oleoresin were still unknown. Here, high-throughput sequencing technology was used to investigate the transcriptome and compare the gene expression profiles of high and low oleoresin-yielding genotypes. A total of 40,690,540 reads were obtained and assembled into 137,499 transcripts from the secondary xylem tissues. We identified 84,842 candidate unigenes based on sequence annotation using various databases and 96 unigenes were candidates for terpenoid backbone biosynthesis in pine. By comparing the expression profiles of high and low oleoresin-yielding genotypes, 649 differentially expressed genes (DEGs) were identified. GO enrichment analysis of DEGs revealed that multiple pathways were related to high yield of oleoresin. Nine candidate genes were validated by QPCR analysis. Among them, the candidate genes encoding geranylgeranyl diphosphate synthase (GGPS) and (-)-alpha/beta-pinene synthase were up-regulated in the high oleoresin-yielding genotype, while tricyclene synthase revealed lower expression level, which was in good agreement with the GC/MS result. In addition, DEG encoding ABC transporters, pathogenesis-related proteins (PR5 and PR9), phosphomethylpyrimidine synthase, non-specific lipid-transfer protein-like protein and ethylene responsive transcription factors (ERFs) were also confirmed to be critical for the biosynthesis of oleoresin. The next-generation sequencing strategy used in this study has proven to be a powerful means for analyzing transcriptome variation related to the yield of oleoresin in masson pine. The candidate genes encoding GGPS, (-)-alpha/beta-pinene, tricyclene synthase, ABC transporters, non-specific lipid-transfer protein-like protein, phosphomethylpyrimidine synthase, ERFs and pathogen responses may play important roles in regulating the yield of oleoresin. These DEGs are worthy of special attention in future studies.

Real-time quantitative PCR (RT-qPCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. Schima superba is a fast-growing timber species with strong resistance. However, thus far, reliable reference gene identifications have not been reported in S. superba. In this study, 19 candidate reference genes were selected and evaluated for their expression stability in different tissues of S. superba. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the reference gene transcript stabilities, and comprehensive stability ranking was generated by the geometric mean method. Our results show that SsuACT was the most stable reference gene and that SsuACT + SsuRIB was the best reference gene combination for different tissues. Finally, the stable and less stable reference genes were verified using SsuSND1 expression in different tissues. To our knowledge, this is the first report to verify appropriate reference genes for normalizing gene expression in S. superba for different tissues, which will facilitate the future elucidation of gene regulations in this species and useful references for relative species.

Schima superba is a precious broadleaf tree species that produces excellent timber. Analyzing the radial variation of wood anatomical properties within stems and determining the boundary between the juvenile and mature wood can provide a theoretical basis for the breeding and cultivation of S. superba. Herein, 30 54-year-old trees were used to analyze the radial variation of wood anatomical properties qualitatively. The correlation among wood properties was analyzed. On this basis, four types of model were used to predict the wood properties quantitatively, and the optimal segmentation model was used to determine the boundary between juvenile and mature wood. The results showed that the variation of the early stage (before the 20th year) was larger than that of the later stage (after the 20th year); moreover, the variation of the vessel features (9.56%) was greater than that of the fiber features (7.42%), the vessel lumen diameter (11.94%) and the fiber length (8.00%) had the most variation. There were three radial variation patterns in the wood properties. In the early stage, there was a significant positive correlation between the ring width and wood air-dry density (0.78) and wood basic density (0.89), and a significant negative correlation between the ring width and the fiber characters (−0.79~−0.93) and vessel characters (−0.64~−0.90). The growth models of Logistic and Richards were more suitable (R² = 84.2%~95.2%) for the radial variation of wood anatomical properties than the nonlinear least square model (R² = 67.7%~90.9%). The transition wood between juvenile and mature wood of S. superba was from the 11th to the 16th year based on aggregative indicators from the pith to bark at breast height. Finally, when taken together, the wood anatomical properties of S. superba had regular radial variation. Breeding programs need to consider both growth and wood anatomical properties in the early stage, but they can be neglected in the mature stage. The formation of mature wood of S. superba occurred after the 16th year.

Xyloglucan endotransglucosylase/hydrolase (XTH), belonging to glycoside hydrolase family 16, is one of the key enzymes in plant cell wall remodeling. Schima superba is an important timber and fireproof tree species in southern China. However, little is known about XTHs in S. superba. In the present study, a total of 34 SsuXTHs were obtained, which were classified into three subfamilies based on the phylogenetic relationship and unevenly distributed on 18 chromosomes. Furthermore, the intron–exon structure and conserved motif composition of them supported the classification and the members belonging to the same subfamily shared similar gene structures. Segmental and tandem duplication events did not lead to SsuXTH gene family expansion, and strong purifying selection pressures during evolution led to similar structure and function of SsuXTH gene family. The interaction network and cis-acting regulatory elements analysis revealed the SsuXTH expression might be regulated by multiple hormones, abiotic stresses and transcription factors. Finally, expression profiles and GO enrichment analysis showed most of the tandem repeat genes were mainly expressed in the phloem and xylem and they mainly participated in glycoside metabolic processes through the transfer and hydrolysis of xyloglucan in the cell wall and then regulated fiber elongation.

Background: Masson pine (Pinus massoniana Lamb.), the dominant native coniferous species in southern China, is commercially important for supplying timber and oleoresin. However, knowledge of the genetic variability of masson pine germplasm is still limited. In this study, the genetic diversity and population structure of masson pine germplasm were assessed using 204 wild accessions from 10 main distribution regions using 94,194 core single-nucleotide polymorphisms (SNPs) obtained from transcriptome sequencing data. Results: The average expected heterozygosity was 0.2724, implying abundant genetic diversity within masson pine germplasm. Analysis of molecular variance (AMOVA) revealed that 3.29% of the variation was sourced from genetic differentiation. Structure analysis identified two geographically distinct groups. Discriminant analysis of principal components (DAPC) showed that one of those groups was further divided into two clusters. Sichuan and Chongqing provenance is the geographical origin, which diffused outward along two different lines. Oleoresin yield is reflected in the evolution of the two groups, and exhibits two different trends along the two lines of diffusion. The oleoresin yield may be associated with the genes of chitinase, CYP720B, cytochrome P450, ABC transporter, and AP2/ethylene-responsive transcription factor (ERF) based on SNPs and expression. Conclusions: SNP markers from transcriptome sequencing are highly capable of evaluating genetic diversity within different species, as well as the genetic control of objective traits. The functions of these genes will be verified in future studies, and those genes strongly associated with oleoresin yield will be used to improve yields by means of early genotype selection and genetic engineering.