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Pinus massoniana Lamb. is a crucial timber and resin conifer in China, but its plantation industry is threatened by outbreaks of pine wilt disease (PWD) caused by Bursaphelenchus xylophilus (pinewood nematode; PWN). However, as of yet, there is no comprehensive analysis of NBS-LRR genes in P. massoniana involved in its defense against PWN. In this study, 507 NBS genes were identified in the transcriptome of resistant and susceptible P. masoniana inoculated with the PWN. The phylogenetic analysis and expression profiles of resistant and susceptible P. massoniana revealed that the up-regulated PmNBS-LRR97 gene was involved in conferring resistance to PWN. The results of real-time quantitative PCR (qRT-PCR) showed that PmNBS-LRR97 was significantly up-regulated after PWN infection, especially in the stems. Subcellular localization indicated that PmNBS-LRR97 located to the cell membrane. PmNBS-LRR97 significantly activated the expression of reactive oxygen species (ROS)-related genes in P. massoniana. In addition, the overexpression of PmNBS-LRR97 was capable of promoting the production of ROS, aiding in plant growth and development. In summary, PmNBS-LRR97 participates in the defense response to PWN and plays an active role in conferring resistance in P. massoniana. This finding provides new insight into the regulatory mechanism of the R gene in P. massoniana.

Taxus mairei (Lemee & Lev.) S.Y. Hu ex T. S. Liu is a vulnerable tree species, and it is also a precious timber species in China. We used 13 microsatellites to assess the genetic diversity and differentiation of 665 T. mairei samples from 18 natural populations. A total of 291 alleles were detected. The average number of alleles (Na = 22.39), expected heterozygosity (He = 0.74), polymorphic information content (PIC = 0.86) and Shannon diversity index (I = 1.66) of the loci indicated a high level of genetic diversity in natural T. mairei populations. Moreover, gene flow was more active among populations (Nm = 1.62) than within populations. Among the 18 populations, the Xinfeng population in Jiangxi Province has the highest genetic diversity. Although each of the studied populations should be protected from further deforestation and agricultural expansion, the Xinfeng population deserves the highest conservation priority. The results based on analysis of molecular variance showed that genetic variation occurred mainly within populations (84.90%; P

Ormosia hosiei is a tree species native to China that has been extensively used for ornamental and furniture purposes due to its valued timber. The mating system has substantial impact on genetic diversity and structure of plant natural population. Such information should be considered when planning tree planting for forest restoration. Here we used 12 microsatellite markers and described the mating system of single families and the population genetic diversity of O. hosiei. A high level of genetic diversity was observed in both adults and progenies, although slight differences existed among populations and their progenies, with the expected heterozygosity ranging from 0.763 to 0.794. Overall, O. hosiei displayed a predominantly outcrossed mating. The estimate of multi-locus outcrossing rate (tm) was high with low variations among families, ranged from 0.997 to 1.000. The value of tm-ts, ranged from 0.000 to 0.139, indicated that biparental inbreeding occurred in progenies. Therefore, to obtain a reasonable genetic representation of native tree species and prevent problems associated with inbreeding depression, we suggested effective in situ conservation by replanting seedlings, but seedling production for restoration purposes may require a much larger sampling effort than is currently used. Moreover, it is necessary to conduct further multiple population and multi-year experiments to verify our conclusions.

Twenty SSR markers were used to evaluate the genetic diversity of 304 s-generation parentals of masson pine (Pinus massoniana Lamb.), which was collected from eight locations in China. The average ...

Outbreaks of pine wood nematode (PWN; Bursaphelenchus xylophilus) represent a severe biotic epidemic for the Pinus massoniana in China. When invaded by the PWN, the resistant P. massoniana might secret abundant oleoresin terpenoid to form certain defensive fronts for survival. However, the regulatory mechanisms of this process remain unclear. Here, the geranyl diphosphate synthase (PmGPPS1) gene was identified from resistant P. massoniana. Tissue-specific expression patterns of PmGPPS1 at transcript and protein level in resistant P. massoniana were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. Functional characteristics analysis of PmGPPS1 was performed on transgenic Nicotiana benthamiana by overexpression, as genetic transformation of P. massoniana is, so far, not possible. In summary, we identified and functionally characterized PmGPPS1 from the resistant P. massoniana following PWN inoculation. Tissue-specific expression patterns and localization of PmGPPS1 indicated that it may play a positive role involved in the metabolic and defensive processes of oleoresin terpenes production in response to PWN attack. Furthermore, overexpression of PmGPPS1 may enhance the production of monoterpene, among which limonene reduced the survival of PWN in vitro. In addition, PmGPPS1 upregulated the expression level of key genes involved in mevalonic acid (MVA) pathway, the methylerythritol phosphate (MEP) pathway and gibberellins (GAs) biosynthesis to boost the growth and development of tobacco through a feedback regulation mechanism. Our results offered new insights into the pivotal role of the PmGPPS1 involved in terpene-based defense mechanisms responding to the PWN invasion in resistant P. massoniana and provided a new metabolic engineering scenario to improve monoterpene production in tobacco.

Clonal forestry offers the opportunity to increase yields, enhance uniformity and improve wood characteristics. Evaluation of growth differences between clones and families has rarely been studied in slow-growing Cupressus funebris species, and whether such growth differences are related to biomass allocation patterns remains to be revealed and is essential for assessing the genetic selection potential and carbon sink capacity of superior species. We studied the genetic variation, heritability (replication power) and gain levels of 36 clones and 33 families of superior trees and analysed their biomass allocation patterns. The results showed that the early growth of Cupressus funebris was dominated by high growth, and the clones grew rapidly, with 253.36% and 51.77% higher diameter at breast height and height at 8 years old than the family lines, respectively. However, the genetic variation of growth traits in the families was higher than that of the clones. The clone repeatability and family heritability levels were evaluated. According to the 10% selection rate, the genetic gains of DBH and tree height of clones were 39.53% and 24.23%, respectively, 5.22 times and 2.05 times the genetic gains of families. The ratio of narrow and broad heritability of each trait was estimated to be 0.55–0.68, with an average value of 0.63, indicating that clones obtained higher additional genetic gains through nonadditive effects. The growth advantage of the clones was to increase the biomass of the aboveground part (86.03%) at the expense of the belowground biomass allocation, which was mainly reflected by the proportion of branch and leaf biomass allocation. The branches and leaves of the clones were dominated by the middle and upper layers, which accounted for 180.64% and 60.14%, respectively, while the middle and lower layers of the branches and leaves of the family lines accounted for a higher percentage. The model ln W=k0+k1 lnD + k2 lnH +k3 has the best prediction of biomass and could be used for early genetic evaluation of Cupressus funebris. In general, Cupressus funebris clones are fast growing and will help to improve the productivity and carbon storage of stands through genetic selection and utilization.

Climate change is causing changes in tree species performance and distribution, impacting breeding programme effectiveness. Our aim was to analyse the effects of provenance and climatic factorson the annual ring density of Masson pine (Pinus massoniana Lamb.) at different experimental sites and potential breeding strategies that may be developed in response to future climate change. The study trees represented provenances originating from the western, east-central, northern, and southern regions of P. massoniana distribution in China. The wood density differed significantly among provenances. A multisite variance analysis test showed that the type B correlation coefficients for ring density at the two sites studied were less than 0.8, indicating an interaction effect of genotype by environment (G×E) on tree ring density. Climatic factors directly affected the wood density properties. At Chun’an (CA), the maximum latewood density (MXD) and minimum earlywood density (MND) were positively correlated with absolute maximum temperatures in August and May of the current growing season, respectively. At Taizi Mountain (TZS), MXD was significantly positively correlated with absolute maximum temperature in September ofthe current year and significantly negatively correlated with precipitation in June. MND was significantly positively correlated with absolute maximum temperature in May of the current year and significantly negatively correlated with precipitation in April. The climatic effects on P. massoniana wood density differed among seed-source origins. This study showed that ring density characteristics differed significantly among provenances, and provenance selection could promote wood density. MXD and MND exhibited significant genotype-by-environment interaction effects, and significant correlations were found between ring density and temperature, and precipitation conditions. These findings suggest that climatic factors and site conditionsin addition to genetics could be strong drivers of wood density variation, and/or that wood density is a highly plastic trait.

Cholecystokinin (CCK) is widely distributed in the gastrointestinal tract and central nervous system, regulating a range of physiological functions by activating its receptors (CCK1R and CCK2R). Compared to those in mammals, the CCK gene and its receptors have already been cloned in various birds, such as chickens. However, knowledge regarding their functionality and tissue expression is limited. In this study, we examined the expression of CCK and its 2 receptors in chicken tissues. In addition, the functionality of the 2 receptors was investigated. Using 3 cell-based luciferase reporter systems and western blots, we demonstrated that chicken (c-) CCK1R could be potently activated by cCCK-8S but not cCCK-4, whereas cCCK2R could be activated by cCCK-8S and cCCK-4 with similar efficiency. Using RNA-sequencing, we revealed that cCCK is abundantly expressed in the testis, ileum, and several brain regions (cerebrum, midbrain, cerebellum, hindbrain, and hypothalamus). The abundant expression of CCK in the hypothalamus was further supported by immunofluorescence. In addition, cCCK1R is highly expressed in the pancreas and moderately expressed in various intestinal regions (ileum, cecum, and rectum) and the pituitary gland, whereas cCCK2R expression is primarily restricted to the brain. Our data reveal the differential specificities of CCK receptors for various CCK peptides. In combination with the differential tissue distribution of CCK and its receptors, the present study helps to understanding the physiological functions of CCK/CCKRs in birds.

Xyloglucan endotransglucosylase/hydrolase (XTH), belonging to glycoside hydrolase family 16, is one of the key enzymes in plant cell wall remodeling.

Background In angiosperm, Self-incompatibility (SI) is a common and widespread mechanism for plant preventing inbreeding risk and late-acting self-incompatibility (LSI) is even more ancestral conserved. In this work, we worked on Schima superba, one species in Theaceae, commercially important timer and fire-resistant tree species and revealed its LSI mechanism. Results The self-pollen tubes grew to the bottom of the stylus and enter ovary until 48h, but failed to penetrate the ovule. Meanwhile, the hormone and peroxidase levels were dramatically changed. Transcriptome and proteome analysis explored the molecular mechanisms of LSI and candidate genes related to LSI in S.superba. Overall 586.71 million reads were obtained and 79,642 (39.08%) unigenes were annotated. KEGG and GO showed that 4531 differentially expressed genes (DEGs) and 82 differentially expressed proteins (DEPs) were at 48h in self- (SP) vs outcross-pollination (OP). Among these, 160 DEGs and 33 DEPs involved in pollen-pistil interaction. The “pollen-pistil interaction”, “signal recognition” and “component of membrane” were downregulated in SP whereas the “cell wall and membrane biosynthetic process”, “oxidoreductase activity” were upregulated. The DEGs involved with S-RNases and SCF during SP suggested that the LSI was happened at 48h ovary, and the LSI in S.superba was under gametophyte control. The calcium ion increase-releasement and F-actin depolymerization initiated metacaspases-1 activity and lead to PCD progress. Also, mitochondrial function loss and ROS disruption further aggravated the PCD progress and cell death. Cell wall biosynthetic progress upregulated and cellulose synthase downregulated could lead to ovary xylification and withering. Conclusions The LSI of S.superba which happened at 48h after pollination was key time point. The incompatibility PT ceased growth in ovary because of the S-RNase recognition and PCD happened in this organ. This study highlight the LSI molecular mechanism in S.superba and also gave a reference to other species in Theaceae.

Adrenoceptors are suggested to mediate the functions of norepinephrine (NE) and epinephrine (EPI) in the central nervous system (CNS) and peripheral tissues in vertebrates. Compared to mammals, the functionality and expression of adrenoceptors have not been well characterized in birds. Here, we reported the structure, expression, and functionality of chicken functional α

Currently, there are no published studies that have used the concurrent operant preference assessment procedure to identify functions of challenging behaviors displayed by individuals with comorbid diagnoses. Four participants (aged 11–16 years) with comorbid diagnoses who displayed multiple challenging behaviors were referred to this study. We modified the standard concurrent operant preference assessment and used the new modified version, the pictorial concurrent operant preference assessment, to identify the functions of the challenging behaviors. Utilizing the triangulation mixed-methods design, we compared the indirect functional behavioral assessment (FBA) and the direct FBA with the pictorial concurrent operant preference assessment. The results obtained successfully demonstrated the concordance among these assessments in identifying the behavioral function for each participant. The results further showed that (1) the preferences served the same functional effects on both the challenging behaviors and the adaptive behaviors and (2) the pictorial concurrent operant preference assessment can be used independently to identify potential behavioral function and to specify the reinforcing potency of each behavioral function. The significance of the study results, limitations of this study, and directions for future research and clinical practice are discussed.

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.

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.

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.

Excessive nutrient inputs imperil the stability of forest ecosystems via modifying the interactions among soil properties, microbes, and plants, particularly in forests composed of cash crops that are under intensive disturbances of agricultural activities, such as Torreya grandis. Understanding the potential drivers of soil microbial community helps scientists develop effective strategies for balancing the protection and productivity of the ancient Torreya forest. Here, we assayed the link between plant and soil parameters and prokaryote communities in bulk soil and T. grandis rhizosphere in 900-year-old stands by detecting plant and soil properties in two independent sites in southeastern China. Our results showed no apparent influence of stand age on the compositions of prokaryote communities in bulk soil and T. grandis rhizosphere. In contrast, soil abiotic factors (i.e., soil pH) overwhelm plant characteristics (i.e., height, plant tissue carbon, nitrogen, and phosphorus content) and contribute most to the shift in prokaryote communities in bulk soil and T. grandis rhizosphere. Soil pH leads to an increase in microbiota alpha diversity in both compartments. With the help of a random forest, we found a critical transition point of pH (pH = 4.9) for the dominance of acidic and near-neutral bacterial groups. Co-occurrence network analysis further revealed a substantially simplified network in plots with a pH of4.9 versus samples with a pH of ≥4.9, indicating that soil acidification induces biodiversity loss and disrupts potential interactions among soil microbes. Our findings provide empirical evidence that soil abiotic properties nearly completely offset the roles of host plants in the assembly and potential interactions of rhizosphere microorganisms. Hence, reduction in inorganic fertilization and proper liming protocols should be seriously considered by local farmers to protect ancient Torreya forests.

Maire yew (Taxus wallichiana var. mairei (Lemée H. Léveillé.) L. K. Fu et Nan Li) is a rare and endangered tree species, and it is also a precious timber species in China. We used 13 microsatellites to assess the genetic diversity and differentiation of 665 Maire yew samples from 18 natural populations. A total of 291 alleles were detected. The average number of alleles (Na=22.39), expected heterozygosity (He=0.74), polymorphic information content (PIC = 0.86) and Shannon diversity index (I = 1.66) of the loci indicated a high level of genetic diversity in natural Maire yew populations. Moreover, gene flow was more active among populations (Nm=1.62) than within populations. Among the 18 populations, the Xinfeng population in Jiangxi Province has the highest genetic diversity. Although each of the studied populations should be protected from further deforestation and agricultural expansion, the Xinfeng population deserves the highest conservation priority. The results based on analysis of molecular variance showed that genetic variation occurred mainly within populations (84.90%; P

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: 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 strong resistance and fast-growing timber specie. However, so far, reliable reference gene identifications have not been reported in S. superba. In this study, we screened and verified the stably expressed reference genes in different tissues of S. superba.Results: Nineteen candidate reference genes were selected and evaluated for their expression stability in different tissues. 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 identified that SsuACT was the most stable reference gene, SsuACT + SsuRIB was the best reference genes combination for different tissues. Finally, the stable and less stable reference genes were verified using the SsuSND1 expression in different tissues.Conclusions: This is the first report to verify the appropriate reference genes for normalizing gene expression in S. superba, which will facilitate future elucidation of gene regulations in this species, and useful references for relative species.

Background Quantitative real-time PCR (qRT-PCR) 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 strong resistance and fast-growing timber specie. However, so far, reliable reference gene identifications have not been reported in S. superba. In this study, we screened and verified the stably expressed reference genes in different tissues of S. superba.Results Nineteen candidate reference genes were selected and evaluated for their expression stability in different tissues. 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 identified that SsuACT was the most stable reference gene, SsuACT + SsuRIB was the best reference genes combination for different tissues. Finally, the stable and less stable reference genes were verified using the SsuSND1 expression in different tissues.Conclusions This is the first report to verify the appropriate reference genes for normalizing gene expression in S. superba for different tissues, which will facilitate future elucidation of gene regulations in this species, and useful references for relative species.

Background: Both NH4+ and NO3– are capable of greatly influencing plants’ growth and biomass. However, the belowground responses of subtropical trees to either NH4+ or NO3– deposition remain poorly understood. Here, we discuss how these two forms of N deposition can affect root development, and experimentally analyzed how they could impact nitrogen and phosphorus absorption in two types (broadleaved with a fibrous root system vs. conifer with a tap root system) of subtropical tree species. Results: In a greenhouse in southern China, 1-year-old S. superba and P. massoniana seedlings grown on P-limited and P-normal soil were treated with NaNO3 and NH4Cl solutions of 0, 80, and 200 kg N ha–1 year–1, corresponding to the control, N80, and N200 groups, respectively. Root phenotype characteristics and metabolism ability were measured after 8 months of growth. The results showed that the root morphology and physiology variables differed significantly between the two species under different N and P treatments. Although S. superba had a larger quantity of roots than P. massoniana, both its root growth rate and root absorption were respectively lower and weaker. N addition differentially affected root growth and activity as follows: (1) NO3–-N80 and NH4+-N80 increased root growth and activity of the two species, but NH4+-N80 led to thicker roots in S. superba; (2) NO3–-N200 and NH4+-N200 had inhibitory effects on the roots of P. massoniana, for which NH4+-N200 led to thinner and longer roots and even the death of some roots; and (3) NH4+-N could promote metabolic activity in thicker roots (> 1.5 mm) and the NO3–-N was found to stimulate activity in thinner roots (0.5–1.5 mm) in the fibrous root system having a larger quantity of roots, namely S. superba. By contrast, NO3–-N and NH4+-N had an opposite influence upon functioning in the tap root system with a slender root, namely P. massoniana. Conclusion: We conclude P. massoniana has a much higher root absorption efficiency; however, nitrogen deposition is more beneficial to the root growth of S. superba.

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: Masson pine (Pinus massoniana Lamb.), the dominant native coniferous species in South China, is commercially important for timber and oleoresin. However, knowledge of the genetic variability of the masson pine germplasm is still scarce. Here, the genetic diversity and population structure of masson pine germplasm were assessed using 204 wild accessions from 10 main distribution regions by 94,194 core 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% variation sourced from the genetic differentiation. Structure analysis identified two geographically distinct groups. Discriminant analysis of principal components (DAPC) showed one geographically distinct group was divided two clusters furtherly. Sichuan and Chongqing provenance is the geographical origin, and diffuses outward along two lines. Oleoresin yield might be reflected by the evolution, and exhibits two different changing trends between two diffusing line. It may be associated with the genes of chitinase, CYP720B, Cytochrome P450, ABC transporter and AP2/ERF based on SNPs and expression. Conclusions: SNP markers by transcriptome sequencing have a strong power in evaluating genetic diversity within species and the genetic control of objective trait. The function of these genes will be verified and the strong associated genes with oleoresin yield will be used in the improvement of oleoresin yield by early genotype selection or genetic engineering.

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.

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.

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.