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Seedling quality is important for subtropical tree species endangered by the degradation of natural habitats in southern China. At present, the cultural regime for raising these seedlings involving fertilizer levels and size of container is not clear. In this study, seedlings of three endangered species, red-seed tree (Ormosia hosiei), Zhejiang phoebe (Phoebe chekiangensis), and Zhejiang camphor (Cinnamomum japonicum) were evaluated along with red-bark oak (Cyclobalanopsis gilva) as a reference, a species which is not endangered. Seedlings were raised in 2.8, 3.6, 5.1, and 6.3 L containers and fertilizer applied at 1.0, 2.0, 3.0 and 4.0 kg m−3. Seedling height and leaf biomass increased in response to higher fertilizer levels while larger containers resulted in greater stem and root biomass. Root biomass of endangered species seedlings did not respond to neither treatments. Zhejiang phoebe seedlings responded to nitrogen and phosphorus uptake but red-seed tree seedlings were unaffected by any nutrient levels. Red-bark oak seedlings had high nitrogen-use efficiency. Based on the results, it is recommended using at least 5.1 L containers to culture Zhejiang phoebe and Zhejiang camphor seedlings with fertilizer at 3.0 kg m−3. Red-bark oak and red-seed tree seedlings should be cultured with 2.0 kg m−3 in smaller containers.

Cypress (Cupressus funebris Endl.) is a major tree species planted for forestland restoration in low-fertility soil and in areas where rocky desertification has occurred. Calcium (Ca) fertilizer can adjust the pH of soil and has an important effect on the growth of cypress. Soil and water losses are serious in Southern China, and soil acidification is increasing, which results in high calcium loss. However, the adaptability of cypress clones to different concentrations of calcium in acidic soils has not been studied. In this investigation, a potted-plant experiment was set up with three concentrations of calcium oxide (CaO) fertilizer (0, 3, and 6 g·kg−1) added under local soil conditions with 0 and 3 g·kg−1 nitrogen (N), phosphorus (P), and potassium (K) fertilizer. The effects of CaO on the growth, root development, and nutrient uptake and utilization efficiency of cypress clones were analyzed. The growth, root development, and nutrient absorption and utilization of cypress differed when calcium fertilizer was applied to acidic soils with different degrees of fertility. In the soil with 0 g·kg−1 NPK fertilizer, the 3 and 6 g·kg−1 CaO treatments significantly increased the clonal growth of cypress seedling height, basal diameter, and dry-matter weight. In addition, the length, surface area, and volume of the roots less than 2.0 mm of root diameter also significantly increased, indicating that the fine cypress roots were somewhat able to adapt to differing Ca levels under lower fertility conditions. Moreover, the efficiency of N, P, and Ca accumulation was highest in the 3 g·kg−1 CaO treatment. After adding 3 g·kg−1 CaO fertilizer to the soil with 3 g·kg−1 NPK fertilizer, only the root dry-matter weight increased significantly, indicating that root development (including root length, surface area, and volume) in the D1–D3 diameter classes (≤1.5 mm in diameter) was significantly elevated. When CaO application reached 6 g·kg−1, the seedling height, basal diameter, and dry-matter weight of each organ decreased, as did the length, surface area, and volume of the roots in the all diameter classes, indicating that the addition of excessive CaO to fertile soil could inhibit the growth and root development of cypress. In Ca-deficient low-quality acidic soils, adding CaO fertilizer can promote the development of fine roots and the uptake and utilization of N, P, and Ca. The results of this study provide a basis for determining the optimal fertilization strategy when growing cypress in acidic soils in Southern China.

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.

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.

Background Schima superba (Theaceae) is a popular woody tree in China. The obscure chromosomal characters of this species are a limitation in the development of high-density genetic linkage maps, which are valuable resources for molecular breeding and functional genomics. Results We determined the chromosome number and the karyotype of S. superba as 2n = 36 = 36 m, which is consistent with the tribe Schimeae (n = 18). A high-density genetic map was constructed using genotyping by sequencing (GBS). A F1 full-sib with 116 individuals and their parents (LC31 × JO32) were sequenced on the Illumina HiSeq™ platform. Overall, 343.3 Gb of raw data containing 1,191,933,474 paired-end reads were generated. Based on this, 99,966 polymorphic SNP markers were developed from the parents, and 2209 markers were mapped onto the integrated genetic linkage map after data filtering and SNP genotyping. The map spanned 2076.24 cM and was distributed among 18 linkage groups. The average marker interval was 0.94 cM. A total of 168 quantitative trait loci (QTLs) for 14 growth traits were identified. Conclusions The chromosome number and karyotype of S. superba was 2n = 36 = 36 m and a linkage map with 2209 SNP markers was constructed to identify QTLs for growth traits. Our study provides a basis for molecular-assisted breeding and genomic studies, which will contribute towards the future research and genetic improvement of S. superba. Electronic supplementary material The online version of this article (10.1186/s12870-019-1655-8) contains supplementary material, which is available to authorized users.

Deposition rates of NH4+ and NO3−, two naturally occurring forms of nitrogen (N), vary worldwide. Here, we discuss the combined effect of deposition of N forms on plant growth in low-phosphorus (P) soil and analyze the patterns that trees utilize to adapt to specific N forms in low-P and high-P soils. One-year-old Schima superba and Pinus massoniana seedlings grown in soil with 1.1 mg kg−1 available P (low-P) and 25 mg kg−1 available P (high-P) were treated with NaNO3 or NH4Cl solutions: N0 = 0 (control), N80 = 80 kg N ha−1 year−1, and N200 = 200 kg N ha−1 year−1. Soil N, P, seedling traits,and leaf N and P contents were measured. Leaf N/P ratios were calculated to determine plant nutrient status. Pinus massoniana had a larger suitable P range and thus showed better growth in low-P soil than S. superba. Although S. superba preferred NH4+ compared with P. massoniana, the addition of NH4+ aggravated the plant growth disadvantage in low-P soil, and high concentrations resulted in the death of P. massoniana plants. The moderate addition of NO3− promoted the growth of the two species. However, the addition of a high dose of NO3− inhibited P. massoniana growth in both low-P and high-P soil. The addition of NH4+-N and NO3−-N resulted in an increase and decrease in the leaf N/P ratios and made plants prone to P and N limitation, respectively. The addition of NH4+ and NO3−-N resulted in different species responses. The responses of tree species to N deposition may vary depending on the forms and rates of N deposition, and species-dependent N-requirements, which in turn are based on the species-specific P-requirements and soil P content. We conclude that plant biomass is related to soil NH4+/NO3− ratios in high-P soil.

In this study, leaf nitrogen (N) and phosphorus (P) stoichiometry were used as indicators of nitrogen saturation and to assess ecosystem nutrient limitations. Schima superba, a representative and widely distributed dominant evergreen broadleaf tree species of the subtropical forests in southern China, was used for this purpose. A nutrient-addition experiment and a field survey were conducted to test the responses of trees from different provenances to N deposition. The relationships between leaf N and P stoichiometry and biomass, nutrient limitation, and soil N:P were analyzed. There was a relationship between leaf N, P, N:P, soil N:P and plant dry biomass. A threshold leaf N:P ratio (16.3) divided the five provenances into different nutrient-limitation classes that were related to the soil N:P ratio or N deposition. The leaf N:P ratio provided an indication of P limitation. A higher soil P level reduced the N deposition effect on plant growth. The leaf N:P ratio of individuals from different provenances can be used as a predictor of nutrient limitation, and this was related to the soil N:P ratio.

Adaptive of trees and its correlation with the climatic are causing changes in tree species performance and distribution, which will change breeding programs and influence forest productivity. To further evaluate the joint influence of climatic factors and provenance on the ring width (RW) and ring density (RD) of Masson pine. We selected 18 provenances at Chun’an (CA) and Taizi Mountain (TZS) test site, which representing four different breeding regions, including the south, west, north and east-central regions. The results showed that the provenance effects were significantly for the RW and RD. The provenances from high temperature and low latitude regions had greater mean RW compared to species from local and cold sources. The geographical genetic variation in wood traits is generally weak. The correlation between RW of Masson pine and precipitation was stronger in the relatively arid TZS site compared with that in relatively wet CA site, as well as the effect of temperature and precipitation on RD was earlier than that in CA test site. The response relationship between establishing the width of tree rings and the environmental variables of provenance indicated that during the transition from the northern and western breeding regions to the eastern and southern breeding regions, the response of RW to climate factors changed from being temperature-based to being precipitation-based. In addition, the response of provenance to the climate of seed sources origin showed their own variation characteristics in each breeding area. Therefore, genetic improvement of big diameter wood and wood density can be gain through selection of provenance and analysis of adaptability.

Pine wood nematode (PWN; Bursaphelenchus xylophilus), a destructive pest of Pinus massoniana, is causing a severe epidemic of pine wilt disease in China. When invaded by PWN, resistant P. massoniana secrete an abundance of oleoresin terpenoids as a defensive strategy. However, regulatory mechanisms of this defense in resistant P. massoniana have yet to be elucidated. Here, we characterized two terpene synthase genes, α-pinene synthase (PmTPS4) and longifolene synthase (PmTPS21) identified in resistant P. massoniana and investigate the contribution of these genes to the oleoresin defense strategy in resistant masson pines. Upregulation of these two genes in the stem supported their involvement in terpene biosynthesis as part of the defense against PWN. Recombinant protein expression revealed catalytic activity for the two PmTPSs, with PmTPS4 primarily producing α-pinene, while PmTPS21 produced α-pinene and longifolene simultaneously. The major enzymatic products of the two terpene synthases had inhibitory effects on PWN in vitro. We demonstrated that PmTPS4 and PmTPS21 played positive roles in terpene-defense mechanisms against PWN infestation. The major products of these terpene synthases could directly inhibit the survival rate of PWN in vitro. We revealed that PmTPS21 was a novel bifunctional enzyme capable of simultaneous production of both monoterpene and sesquiterpene. This article is protected by copyright. All rights reserved.

The Masson pine breeding programme has advanced to the third cycle. However, breeding and deployment are based largely on plantation inventory zones rather than biological patterns of genotype by environment interaction (G × E) and consequently cannot deliver optimal genetic gains across the whole plantation estate. The present study analysed G × E in family tests of Masson pine planted in the eastern and central regions of China. Twenty-six controlled pollination families were selected at three experimental sites. Measurements were performed at 8 years of age for diameter at breast height, total height, and volume. The genotype and G × E (GGE) biplot was used to study the effects of G × E, and the type B correlation coefficients was used to evaluate the extent of G × E. Significant site, family and family × site effects on growth traits were found (p h f 2 ) and individual heritability ( h s 2 ) ranged from 0.59 to 0.84 and 0.16 to 0.38. The type B correlation refers to genetic correlation between different environments. In this study, type B correlation coefficients between tested sites were all less than 0.8, indicating significant G × E among families at different sites. The volume of family 6 was highest at the TZS and XJ sites, and that of family 4 was highest at the SW site. 12 superior families and 49 superior individual trees were selected, and, as determined by comparison with the controls, the real gain (G) of volume was 23.70% and 279.53%, respectively. At the three sites, the individual volumes of families 6, and 15 were larger and more stable than those of other families, while family 4 was large and unstable. The analyses revealed that those families that perform best in all the tests can be planted in areas with similar environmental traits to the sites tested here. Excellent genotypes with stable and high yield can provide a basis for follow-up breeding work.

Exponential fertilization (EF) can feed seedlings with more nutrients than needed for growth and continuous lighting is expected to reduce the risk of loading at high application rate. In this study, red-seed tree (Ormosia hosiei Hemsley & E. H. Wilson) seedlings were raised by EF at a low rate of 160 mg nitrogen (N) plant-1 (conventional) (N-phosphorus [P]-potassium [K], 12-9-12) and a high rate of 600 mg N plant-1 (intensive), which was adapted from previous studies with large-pot-seedlings. Both fertilizer regimes were nested to either of two light spectra from high-pressure sodium (HPS) and light-emitting diode (LED) sources with three replicates for each combined treatment. Seedlings subjected to the conventional regime in the LED spectrum showed better growth and greater biomass accumulation with higher leaf N and P contents than other ones. In the conventional fertilizer regime, the LED spectrum also resulted in higher photosynthesis shown by more pigments and higher N synthesis than the HPS spectrum. The HPS spectrum strengthened P synthesis in the intensive regime. The antioxidative activity was stimulated by a high dose of EF, hence excessive toxicity was likely induced. We recommend using the normal rate of 160 mg N plant-1 for the culture of red-seed tree seedlings with LED lighting to promote seedling quality without causing excessive nutrient toxicity.

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.

Pollen flow and mating patterns are the most important factors influencing the genetic structure of insect-pollinated forest trees and are essential parts of genetic management in seed orchards. We investigated pollen flow, the mating system and the level of pollen contamination in a clonal seed orchard of Schima superba Gardn. et Champ. In total, 328 open-pollination progenies coming from 11 mother trees were identified using 13 polymorphic microsatellite loci. A total of 203 full-sib families were identified and were nested among the 11 studied seed donors. The male reproductive success rate from 0.49 to 7.77% for most male parents, with an average of 2.44%. More than 80% of the crosses were found within a distance of 60 m, and the most frequent pollination distance between female parent and male parents was approximately 20 m. Mating system analysis showed that the outcrossing level was very high (t m = 1.000, outcrossing rate = 98.5%) in the seed orchard and that there was an average of 2.3 effective pollen donors (N ep ) per female parent. In addition, the mating success of individual males within neighborhoods was moderately influenced by their fecundity and the direction of their location relative to mother trees. The pollen contamination from outside the seed orchard was high (7.01%). Our findings are valuable for the assessment of seed orchards, and it may be worthwhile to use pollen management strategies to decrease pollen contamination and increase the genetic quality of the seeds produced.

Significant genetic variations in N and P nutritional traits exist under combined conditions of soil P deficiency and N deposition and interactively contribute to growth benefits. Forest ecosystems encounter combined conditions of soil phosphorus (P) deficiency and atmospheric nitrogen (N) deposition. As such, determining genetic variations and interactions of N and P nutritional traits is important for silviculture. Low N (LN) and high N (HN) were combined with three P treatments in a pot experiment with five full-sib families of Masson pine (Pinus massoniana). The three P treatments are: low P in soil profile (low-P condition, LP), enriched P in topsoil and poor P in subsoil (medium–low-P condition, MLP), and high P in soil profile (high-P condition, HP). P acquisition was more significant for growth benefits than P utilization under LP. Growth response to N was higher under MLP and HP than that under LP because of more balanced plant N:P ratios in the two former conditions. Under HN, there were significant correlation between nitrogen acquisition efficiency (NAE) and phosphorus use efficiency (PUE) and they interactively contributed to growth promotion under N deposition. Significant genetic variations, as well as large heritability of growth and N and P nutritional traits, were detected under all N–P conditions. This finding indicated the feasibility of breeding families with improved nutrient efficiency under N–P conditions.

Schima superba Gardn. et Champ. is a perennial, evergreen tree valued for its eco-protection and commercial values in China. In this study, we investigate the breeding system, reproductive ecology and pollination biology of S. superba in a seed orchard. The flowers are hermaphrodite and protogynous. The viability of the pollen is inactivated rapidly, and the stigma maintains a high receptivity within the flower lifespan. Flowers typically offer pollen and nectar to visitors. The flowers possess a typical insect pollination syndrome, and three visitors (Apis cerana cerana Fabricius, Protaetia brevitarsis Lewis, and Popillia mutans Newman) are observed on flowers during the study period. The visitation frequency per minute and capability of pollen removal and deposition of A. cerana are significantly higher than P. brevitarsis and P. mutans, although the pollinator efficiency is lower than those shown by the two beetles. Fruit set (28.27%) and seed set (6.57%) percentages resulting from open-pollination are significantly lower than those resulting from cross-pollination (fruit/seed set, 43.73%/11.66%), and the pollen limitation index (L) was 0.34, suggesting that seed production is pollen-limited in the seed orchard. The pollen/ovule ratio (P/O) and outcrossing index (OCI) values are 6686.67 and 4, respectively. The self-incompatibility index (ISI) was estimated to be 0.95. Results from hand-pollination, pollen tube growth experiments and the ISI value show that S. superba is late-acting self-incompatible. The synthetic results indicate that A. cerana is the most efficient pollinator of S. superba, and seed production is frequently limited by pollinators, fruit abortion, and pollen quality.

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.

To identify genetic diversity, genetic structure and the relationship among accessions, and further establish a core collection for the long-term breeding of cypress (Cupressus funebris Endl.), the genealogy of breeding parents was reconstructed using simple sequence repeat (SSR) molecular markers. Seventeen SSR markers were used to detect molecular polymorphisms among 290 cypress accessions from five provinces and 53 accessions with unknown origin in China. A total of 92 alleles (Na) were detected with 5.412 alleles per locus and an average polymorphism information content (PIC) of 0.593. The haplotype diversity (H) ranged from 0.021 to 0.832, with an average of 0.406. The number of alleles (Na) and the effective number of alleles (Ne) ranged from 4.294 to 5.176 and from 2.488 to 2.817 among five populations, respectively. The pairwise population matrix of Nei’s genetic distance ranged from 0.008 to 0.023. Based on the results of unweighted pair group method average (UPGMA) cluster and population structure analyses, 343 breeding parents were divided into two major groups. Lower genetic differentiation coefficients and closer genetic relationships were observed among cypress breeding parents, suggesting that the genetic basis was narrow, and the genetic relationship was confused by frequent introduction and wide cultivation. Moreover, we reconstructed the genealogy between breeding parents and 30 accessions of breeding parents from an identified core collection. According to the present study, not only geographic origin but also the relationship of the individuals should be considered in future crossbreeding work.

Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana.