PmHs1pro-1 monitors Bsursaphelenchus xylophilus infection and activates defensive response in resistant Pinus massoniana.

Plant resistance (R) genes play a crucial role in the detection of effector proteins secreted by pathogens, either directly or indirectly, as well as in the subsequent activation of downstream defence mechanisms. However, little is known about how R genes regulate the defence responses of conifers, particularly Pinus massoniana, against the destructive pine wood nematode (PWN; Bursaphelenchus xylophilus). Here, we isolated and characterised PmHs1pro-1, a nematode-resistance gene of P. massoniana, using bioinformatics, molecular biology, histochemistry and transgenesis. Tissue-specific expressional pattern and localisation of PmHs1pro-1 suggested that it was a crucial positive regulator in response to PWN attack in resistant P. massoniana. Meanwhile, overexpression of PmHs1pro-1 was found to activate reactive oxygen species (ROS) metabolism-related enzymes and the expressional level of their key genes, including superoxide dismutase, peroxidase and catalase. In addition, we showed that PmHs1pro-1 directly recognised the effector protein BxSCD1of PWN, and induced the ROS burst responding to PWN invasion in resistant P. massoniana. Our findings illustrated the molecular framework of R genes directly recognising the effector protein of pathology in pine, which offered a novel insight into the plant-pathogen arms race.

Comprehensive Analysis and Functional Verification of the Pinus massoniana NBS-LRR Gene Family Involved in the Resistance to Bursaphelenchus xylophilus.

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.

Identification and Defensive Characterization of PmCYP720B11v2 from Pinus massoniana.

Pinus massoniana is a pioneer species for afforestation timber and oleoresin, while epidemics of pinewood nematode (PWN; Bursaphelenchus xylophilus) are causing a serious biotic disaster for P. massoniana in China. Importantly, resistant P. massoniana could leak copious oleoresin terpenoids to build particular defense fronts for survival when attacked by PWN. However, the defense mechanisms regulating this process remain unknown. Here, PmCYP720B11v2, a cytochrome P450 monooxygenase gene, was first identified and functionally characterized from resistant P. massoniana following PWN inoculation. The tissue-specific expression pattern and localization of PmCYP720B11v2 at the transcript and protein levels in resistant P. massoniana indicated that its upregulation in the stem supported its involvement in the metabolic processes of diterpene biosynthesis as a positive part of the defense against PWN attack. Furthermore, overexpression of PmCYP720B11v2 may enhance the growth and development of plants. In addition, PmCYP720B11v2 activated the metabolic flux of antioxidases and stress-responsive proteins under drought conditions and improved drought stress tolerance. Our results provide new insights into the favorable role of PmCYP720B11v2 in diterpene defense mechanisms in response to PWN attack in resistant P. massoniana and provide a novel metabolic engineering scenario to reform the stress tolerance potential of tobacco.

Two terpene synthases in resistant Pinus massoniana contribute to defence against Bursaphelenchus xylophilus.

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 secretes an abundance of oleoresin terpenoids as a defensive strategy. However, regulatory mechanisms of this defence 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 defence strategy in resistant masson pines. Up-regulation of these two genes in the stem supported their involvement in terpene biosynthesis as part of the defence 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-defence 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.

A transcriptomic variation map provides insights into the genetic basis of Pinus massoniana Lamb. evolution and the association with oleoresin yield.

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.

A high-density genetic map of Schima superba based on its chromosomal characteristics.

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.

The Application of the Acellular Dermal Matrix in the Correction of the Tear Trough Deformity.

OBJECTIVE: The acellular dermal matrix (ADM) used in correcting the tear trough deformity has been reported, but there were only a few cases. The long-term effectiveness of ADM was not clear. We aim to discuss the technique and the effect of using ADM to correct the tear trough deformity through more cases. METHODS: A retrospective study was conducted from January 2012 to January 2017. Twenty-six patients who showed obvious tear trough deformity with moderate or severe orbital fat bulging and excess of lower eyelid skin were treated with ADM to improve the appearance of the midface. Follow-up was performed for 2-12 months in 26 cases. The level of postoperative satisfaction was assessed by interview during the follow-up and rated as very satisfied, satisfied, acceptable, or unacceptable. RESULT: Twenty patients were very satisfied for having achieved complete correction. Three patients were satisfied for having achieved obvious improvement. Three patients felt the results were just acceptable and were refilled because of the insufficiency of the filler. No one was unacceptable. There were no complications such as rapid resorption, rejection, or inflammation. CONCLUSION: The method of using ADM for the correction of tear trough deformity has the advantages of low absorption rate, good appearance, and high security. It provides a new choice for the treatment of tear trough deformity. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

A fast phenotype approach of 3D point clouds of Pinus massoniana seedlings.

The phenotyping of Pinus massoniana seedlings is essential for breeding, vegetation protection, resource investigation, and so on. Few reports regarding estimating phenotypic parameters accurately in the seeding stage of Pinus massoniana plants using 3D point clouds exist. In this study, seedlings with heights of approximately 15-30 cm were taken as the research object, and an improved approach was proposed to automatically calculate five key parameters. The key procedure of our proposed method includes point cloud preprocessing, stem and leaf segmentation, and morphological trait extraction steps. In the skeletonization step, the cloud points were sliced in vertical and horizontal directions, gray value clustering was performed, the centroid of the slice was regarded as the skeleton point, and the alternative skeleton point of the main stem was determined by the DAG single source shortest path algorithm. Then, the skeleton points of the canopy in the alternative skeleton point were removed, and the skeleton point of the main stem was obtained. Last, the main stem skeleton point after linear interpolation was restored, while stem and leaf segmentation was achieved. Because of the leaf morphological characteristics of Pinus massoniana, its leaves are large and dense. Even using a high-precision industrial digital readout, it is impossible to obtain a 3D model of Pinus massoniana leaves. In this study, an improved algorithm based on density and projection is proposed to estimate the relevant parameters of Pinus massoniana leaves. Finally, five important phenotypic parameters, namely plant height, stem diameter, main stem length, regional leaf length, and total leaf number, are obtained from the skeleton and the point cloud after separation and reconstruction. The experimental results showed that there was a high correlation between the actual value from manual measurement and the predicted value from the algorithm output. The accuracies of the main stem diameter, main stem length, and leaf length were 93.5%, 95.7%, and 83.8%, respectively, which meet the requirements of real applications.

Cholecystokinin (CCK) and its receptors (CCK1R and CCK2R) in chickens: functional analysis and tissue expression.

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.

Geographical provenance variation of growth and wood properties of 18-year-old Schima superba.

We analyzed the variation patterns of growth and wood properties of 24 different provenances of 18-year-old Schima superba in Jian'ou, Fujian Province. A total of 11 growth and wood indices were measured, including tree height, diameter at breast height, wood basic density and anatomical structure. We analyzed the geographical variation patterns of growth and wood properties, and the provenance areas were divided. Further, the excellent timber provenances were selected according to different uses. The results showed that the variation of growth traits, which was 17.6%-27.3% with mean value of 22.4%, was larger than that of wood properties (7.0%-21.0%, mean 12.7%). Growth properties and some wood properties (fiber length, fiber lumen diameter and fiber cell wall thickness) had significant differences among provenances. Growth traits were not correlated with fiber traits, and they could be selected independently without emphasis on other traits. There was significant correlation between the longitudinal and radial growth indicators of wood properties, but they were not correlated with the wood basic density, which could be selected independently. In addition, the growth and wood properties were significantly influenced by temperature and precipitation, which showed a latitudinal variation pattern. According to Q-type clustering analysis, 24 provenances could be divided into four categories, of which southern provenances from distribution area of S. superba had vigorous growth and supper wood properties. They had smaller microfibril angle, higher maturity, longer fiber length, and thicker fiber cell wall. Finally, five excellent provenances were selected according to pulpwood and building use.

The XTH Gene Family in Schima superba: Genome-Wide Identification, Expression Profiles, and Functional Interaction Network Analysis.

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.

Incompatible pollen abortion and late-acting self-incompatibility in Schima superba.

In angiosperms, self-incompatibility (SI) is a common and widespread mechanism for plant prevention of inbreeding, and late-acting self-incompatibility (LSI) may be ancestral in the group. In this work, we studied Schima superba, a species in Theaceae that is a commercially important timer and fire-resistant tree, and revealed its LSI mechanism. Hormones, enzymes, transcriptomes, and proteins were compared between self-pollination (SP) and outcross pollination (OP) in the styles and ovaries from 0 to 120 h after pollination. The self-pollen tubes grew to the bottom of the style and entered the ovary within 48 h but failed to penetrate the ovule. Meanwhile, the hormone and peroxidase levels dramatically changed. Transcriptome and proteome analyses 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 analysis showed that there were 4531 differentially expressed genes (DEGs) and 82 differentially expressed proteins (DEPs) at 48 h in self- (SP) versus outcross pollination (OP). Among these, 160 DEGs and 33 DEPs were involved in pollen-pistil interactions. "Pollen-pistil interaction," "signal recognition," and "component of membrane" were downregulated in SP, whereas "cell wall and membrane biosynthetic process," and "oxidoreductase activity" were upregulated. The DEGs involved with S-RNases and SCF during SP suggested that the LSI occurred at 48 h in the ovary and that the LSI in S. superba was under gametophyte control. Calcium ion increase and release, mitochondrial function loss, and ROS disruption further aggravated PCD progress and cell death. The LSI of S. superba, which happened 48 h after pollination, was a key time point. The incompatibility PT ceased growth in the ovary because of S-RNase recognition and PCD in this organ. This study highlights the LSI molecular mechanism in S. superba and provides a reference to other species in Theaceae.

Mating Systems of Single Families and Population Genetic Diversity of Endangered Ormosia hosiei in South China.

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.

Overexpression of geranyl diphosphate synthase (PmGPPS1) boosts monoterpene and diterpene production involved in the response to pine wood nematode invasion.

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.

Comprehensive analysis of LRR-RLKs and key gene identification in Pinus massoniana resistant to pine wood nematode.

Pinus massoniana is a pioneer tree widely planted for afforestation on barren hills in southern China where the total planted area is 8.04 million ha. The invasive pine wood nematode (Bursaphelenchus xylophilus) poses a serious threat to the survival of P. massoniana. Plant resistance genes encoded by leucine-rich repeat-containing transmembrane-receptor proteins play important roles in plant defense. Leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest subfamily of the RLK protein family, play an important role in sensing stress signals in plants. However, the LRR-RLKs of P. massoniana have not been characterized previously, and their role in resistance to B. xylophilus is unknown. In this study, 185 members of the LRR-RLK subfamily were identified in P. massoniana and were categorized into 14 subgroups. Transcriptomic and quantitative real-time RT-PCR analyses showed that PmRLKs32 was highly expressed in the stem tissue after inoculation with B. xylophilus. The gene exhibited high homology with AtFLS2 of Arabidopsis thaliana. PmRLKs32 was localized to the plasma membrane and was significantly upregulated in nematode-resistant and nematode-susceptible individuals. The transient expression of PmRLKs32 resulted in a burst of reactive oxygen species production in P. massoniana and Nicotiana benthamiana seedlings. These results lay a foundation for further exploration of the regulatory mechanism of LRR-RLKs in response to biotic stress in P. massoniana.

Factors driving the assembly of prokaryotic communities in bulk soil and rhizosphere of Torreya grandis along a 900-year age gradient.

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 of <4.9 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.

Characterization of Chicken α2A-Adrenoceptor: Molecular Cloning, Functional Analysis, and Its Involvement in Ovarian Follicular Development.

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 α2A-adrenoceptor, named ADRA2A. The cloned chicken ADRA2A cDNA is 1335 bp in length, encoding the receptor with 444 amino acids (a.a.), which shows high amino acid sequence identity (63.4%) with its corresponding ortholog in humans. Using cell-based luciferase reporter assays and Western blot, we demonstrated that the ADRA2A could be activated by both NE and EPI through multiple signaling pathways, including MAPK/ERK signaling cascade. In addition, the mRNA expression of ADRA2A is found to be expressed abundantly in adult chicken tissues including thyroid, lung, ovary and adipose from the reported RNA-Seq data sets. Moreover, the mRNA expression of ADRA2A is also found to be highly expressed in the granulosa cells of 6-8 mm and F5 chicken ovarian follicles, which thus supports that ADRA2A signaling may play a role in ovarian follicular growth and differentiation. Taken together, our data provide the first proof that the α2A-adrenoceptor is functional in birds involving avian ovarian follicular development.

Identification and Validation of Reference Genes for Gene Expression Analysis in Schima superba.

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.

Shining a Light on the Challenging Behaviors of Adolescents with Comorbid Diagnoses: Use of Pictorial Concurrent Operant Preference Assessment.

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.

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In order to realize precise fertilization and high yield management of Pinus massoniana clonal seed orchard, clones with different fruiting abilities were used as the materials. Four P fertilization levels were at 0, 400, 800 and 1600 g per plant (P0, P4, P8 and P16 respectively). Fertilization was applied before floral primordia formation and after cone picked, respectively. The effects of P fertilizer on the female strobilus of P. massoniana clones and the changes of N, P contents in needles of different positions during floral primordia formation stage and early stage of flower bud differentiation were investigated. The results showed that compared with P0, the female strobilus of P8 and P16 were significantly increased by 67.4% and 61.2% in 2018 and 28.9% and 14.1% in 2019, respectively. There was a significant negative correlation between the female strobilus with the N content and N/P, a significant positive correlation between the female strobilus and the P content in needles. The responses of N and P contents in needles to P fertilization differed in clones with different fruiting abilities. In floral primordia formation stage, the N content of clones with weak fruiting ability was high, and the N/P was 11.5-12.5, while the P content of clones with strong fruiting abilities was high, and the N/P was 9.5-10.5. During this period, the P content of most clones under P8 treatment was the highest, while the N/P was lowest. In the early stage of flower bud differentiation, the N/P of two fruiting clones was 15.3-17.0 and 13.2-15.1, respectively. The P content in upside layer was significantly higher while N/P was significantly lower than that in middle and lower layers. In conclusion, the 800 g P fertilization per plant could increase the P content and reduce the N/P of needles during the floral primordia formation stage of clones with diffe-rent fruiting abilities, which was beneficial to the formation of female strobilus and promote the yield of clone seed orchards.