Improving Sedum plumbizincicola genetic transformation with the SpGRF4-SpGIF1 gene and the self-excision CRE/LoxP system.

MAIN CONCLUSION: S. plumbizincicola genetic transformation was optimized using a self-excision molecular-assisted transformation system by integrating the SpGRF4/SpGIF1 gene with XVE and Cre/loxP. Sedum plumbizincicola, despite being an excellent hyperaccumulator of cadmium and zinc with significant potential for soil pollution phytoremediation on farmland, has nonetheless trailed behind other major model plants in genetic transformation technology. In this study, different explants and SpGRF4-SpGIF1 genes were used to optimize the genetic transformation of S. plumbizincicola. We found that petiole and stem segments had higher genetic transformation efficiency than cluster buds. Overexpression of SpGRF4-SpGIF1 could significantly improve the genetic transformation efficiency and shorten the period of obtaining regenerated buds. However, molecular assistance with overexpression of SpGRF4-SpGIF1 leads to abnormal morphology, resulting in plant tissue enlargement and abnormal growth. Therefore, we combined SpGRF4-SpGIF1 with XVE and Cre/loxP to obtain DNA autocleavage transgenic plants induced by estradiol, thereby ensuring normal growth in transgenic plants. This study optimized the S. plumbizincicola genetic transformation system, improved the efficiency of genetic transformation, and established a self-excision molecular-assisted transformation system. This work also established the basis for studying S. plumbizincicola gene function, and for S. plumbizincicola breeding and germplasm innovation.

Phosphorus deficiency-induced cell wall pectin demethylesterification enhances cadmium accumulation in roots of Salix caprea.

Regions affected by heavy metal contamination frequently encounter phosphorus (P) deficiency. Numerous studies highlight crucial role of P in facilitating cadmium (Cd) accumulation in woody plants. However, the regulatory mechanism by which P affects Cd accumulation in roots remains ambiguous. This study aims to investigate the effects of phosphorus (P) deficiency on Cd accumulation, Cd subcellular distribution, and cell wall components in the roots of Salix caprea under Cd stress. The results revealed that under P deficiency conditions, there was a 35.4% elevation in Cd content in roots, coupled with a 60.1% reduction in Cd content in shoots, compared to the P sufficiency conditions. Under deficient P conditions, the predominant response of roots to Cd exposure was the increased sequestration of Cd in root cell walls. The sequestration of Cd in root cell walls increased from 37.1% under sufficient P conditions to 66.7% under P deficiency, with pectin identified as the primary Cd binding site under both P conditions. Among cell wall components, P deficiency led to a significant 31.7% increase in Cd content within pectin compared to P sufficiency conditions, but did not change the pectin content. Notably, P deficiency significantly increased pectin methylesterase (PME) activity by regulating the expression of PME and PMEI genes, leading to a 10.4% reduction in the degree of pectin methylesterification. This may elucidate the absence of significant changes in pectin content under P deficiency conditions and the concurrent increase in Cd accumulation in pectin. Fourier transform infrared spectroscopy (FTIR) results indicated an increase in carboxyl groups in the root cell walls under P deficiency compared to sufficient P treatment. The results provide deep insights into the mechanisms of higher Cd accumulation in root mediated by P deficiency.

VirGrapher: a graph-based viral identifier for long sequences from metagenomes.

Viruses are the most abundant biological entities on earth and are important components of microbial communities. A metagenome contains all microorganisms from an environmental sample. Correctly identifying viruses from these mixed sequences is critical in viral analyses. It is common to identify long viral sequences, which has already been passed thought pipelines of assembly and binning. Existing deep learning-based methods divide these long sequences into short subsequences and identify them separately. This makes the relationships between them be omitted, leading to poor performance on identifying long viral sequences. In this paper, VirGrapher is proposed to improve the identification performance of long viral sequences by constructing relationships among short subsequences from long ones. VirGrapher see a long sequence as a graph and uses a Graph Convolutional Network (GCN) model to learn multilayer connections between nodes from sequences after a GCN-based node embedding model. VirGrapher achieves a better AUC value and accuracy on validation set, which is better than three benchmark methods.

Transcriptomic and Metabolomic Analyses Reveal Differences in Flavonoid Pathway Gene Expression Profiles between Two Dendrobium Varieties during Vernalization.

Dendrobium (Orchidaceae, Epidendoideae) plants have flowers with a wide variety of colors that persist for a long period throughout the year. The yellow coloration of Dendrobium flowers is mainly determined by the flavonol pathway and the flavone pathway, but the relevant biosynthesis mechanisms during vernalization remain unclear. To explore the similarities and differences in flavonoid biosynthesis in different tissues during vernalization, we selected two species of Dendrobium for a flower color study: Dendrobium capillipes Rchb (which has yellow flowers) and Dendrobium nobile Lindl (which has white flowers). We collected a total of 36 samples from six tissue types and both Dendrobium species during vernalization and subjected the samples to metabolic profiling and transcriptome sequencing. A total of 31,504 differentially expressed genes (DEGs) were identified between different tissues of the two Dendrobium species by transcriptomic analysis. However, many differentially accumulated metabolites (DAMs) and DEGs were enriched not only in the general pathway of "flavonoid biosynthesis" but also in multiple subpathways of "flavone and flavonol biosynthesis". According to a combined transcriptome and metabolome analysis, Putrescine hydroxycinnamoyl transferase 1 (LOC110093422) may be the main gene responsible for the differences in flavonoid accumulation during vernalization, which is closely associated with yellow flowers. Taken together, the results of our study preliminarily revealed the metabolites responsible for and the key genes regulating flavonoid biosynthesis during vernalization. These results provide a basis for the further study of the molecular mechanism of flavonoid synthesis during vernalization.

Exploring Transcriptional Regulation of Hyperaccumulation in Sedum plumbizincicola through Integrated Transcriptome Analysis and CRISPR/Cas9 Technology.

The cadmium hyperaccumulator Sedum plumbizincicola has remarkable abilities for cadmium (Cd) transport, accumulation and detoxification, but the transcriptional regulation mechanisms responsible for its Cd hyperaccumulation remain unknown. To address this knowledge gap, we conducted a comparative transcriptome study between S. plumbizincicola and the non-hyperaccumulating ecotype (NHE) of Sedum alfredii with or without Cd treatment. Our results revealed many differentially expressed genes involved in heavy metal transport and detoxification that were abundantly expressed in S. plumbizincicola. Additionally, we identified a large number of differentially expressed transcription factor genes, highlighting the complexity of transcriptional regulatory networks. We further screened four transcription factor genes that were highly expressed in the roots of S. plumbizincicola as candidate genes for creating CRISPR/Cas9 knockout mutations. Among these, the SpARR11 and SpMYB84 mutant lines exhibited decreased Cd accumulation in their aboveground parts, suggesting that these two transcription factors may play a role in the regulation of the Cd hyperaccumulation in S. plumbizincicola. Although further research will be required to determine the precise targeted genes of these transcription factors, combined transcriptome analysis and CRISPR/Cas9 technology provides unprecedented opportunities for identifying transcription factors related to Cd hyperaccumulation and contributes to the understanding of the transcriptional regulation mechanism of hyperaccumulation in S. plumbizincicola.

Factor structure of the patient health questionnaire-9 and measurement invariance across gender and age among Chinese university students.

The Patient Health Questionnaire-9 (PHQ-9) has been widely used to screen depression symptoms. The present research aimed to assess the reliability and validity of PHQ-9, besides measurement invariance of the PHQ-9 across gender and age among Chinese university students. A total of 12,957 Chinese college students from 2 universities in Henan and Hainan provinces (China) completed the questionnaires via WeChat. This research reported the psychometric properties of PHQ-9 and measurement invariance of the PHQ-9 across gender and age among Chinese university students. Compared with 1-factor model, the 2-factor (affective factor and somatic factor) model of PHQ-9 showed a better fit index in Chinese university students. Without the last 2 items, the 2-factor model of the PHQ-9 showed satisfactory reliability, validity, and good fit index (e.g., Root mean square error of approximation = 0.060, Goodness-of-fit index = 0.982, Comparative fit index = 0.986, and Tucker-Lewis index = 0.974). The Cronbach's alpha of PHQ-9 was 0.874. Multi-group analysis across gender and age demonstrated that measurement equivalency for the 2-factor model of the PHQ-9 was established (e.g., Root mean square error of approximation < 0.08, Comparative fit index > 0.90 and Tucker-Lewis index > 0.90). The 2-factor model of the PHQ-9 without the items of "movement" and "desire to die" showed a better fit index in Chinese university students. The measurement equivalence across gender and age for the 2-factor model of the PHQ-9 can be established among Chinese university students.

Sexual differences in root growth and antioxidant characteristics in Salix viminalis exposed to cadmium stress.

NOVELTY STATEMENT: Salix viminalis, a dioecious shrub willow, has been widely used in phytoremediation, yet sexually differences in tolerance to cadmium of which remained unclear. This study focused on different responses to cadmium stress between roots of male and female S. viminalis. Results show that male plants of S. viminalis have stronger cadmium tolerance than female plants, which indicates male S. viminalis should be more considered to be applied for phytoremediation and ecological restoration of cadmium-accumulated soil considering cadmium tolerance characteristics. The findings can provide valuable evidence and insights for researches focused on phytoremediation with dioecious woody plants and sexual dimorphism under abiotic stress.

Proteomic insights into the photosynthetic divergence between bark and leaf chloroplasts in Salix matsudana.

Bark chloroplasts play important roles in carbon balancing by recycling internal stem CO2 into assimilated carbon. The photosynthetic response of bark chloroplasts to interior stem environments has been studied recently in woody plants. However, the molecular regulatory mechanisms underlying specific characteristics of bark photosynthesis remain unclear. To address this knowledge gap, differences in the structure, photosynthetic activity and protein expression profiles between bark and leaf chloroplasts were investigated in Salix matsudana in this study. Bark chloroplasts exhibited broader and lower grana stacks and higher levels of starch relative to leaf chloroplasts. Concomitantly, decreased oxygen evolution rates and decreased saturated radiation point were observed in bark chloroplasts. Furthermore, a total of 293 differentially expressed proteins (DEPs) were identified in bark and leaf chloroplast profile comparisons. These DEPs were significantly enriched in photosynthesis-related biological processes or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with photosynthesis. All 116 DEPs within the KEGG pathways associated with photosynthesis light reactions were downregulated in bark chloroplasts, including key proteins responsible for chlorophyll synthesis, light energy harvesting, nonphotochemical quenching, linear electron transport and photophosphorylation. Interestingly, seven upregulated proteins involved in dark reactions were identified in bark chloroplasts that comprised two kinds of malic enzymes typical of C4-type photosynthesis. These results provide comprehensive proteomic evidence to understand the low photochemical capability of bark chloroplasts and suggest that bark chloroplasts might fix CO2 derived from malate decarboxylation.

Soil microbial community succession and interactions during combined plant/white-rot fungus remediation of polycyclic aromatic hydrocarbons.

Despite combined plant/white-rot fungus remediation being effective for remediating polycyclic aromatic hydrocarbon (PAH)-contaminated soil, the complex organismal interactions and their effects on soil PAH degradation remain unclear. Here, we used quantitative PCR, analysis of soil enzyme activities, and sequencing of representative genes to characterize the ecological dynamics of natural attenuation, mycoremediation (MR, using Crucibulum laeve), phytoremediation (PR, using Salix viminalis), and plant-microbial remediation (PMR, using both species) for PAHs in soil for 60 days. On day 60, PMR achieved the highest removal efficiency of all three representative PAHs (65.5%, 47.5%, and 62.4% for phenanthrene, pyrene, and benzo(a)pyrene, respectively) when compared with the other treatments. MR significantly increased the relative abundance of Rhizobium and Bacillus but antagonized the other putative indigenous PAH-degrading bacteria, which were enriched by PR. PR significantly reduced soil nutrients, such as NO3- and NH4+, and available potassium (AK), thereby changing the microbial community composition as reflected by redundancy analysis, significantly reducing the soil bacterial biomass relative to that in other treatments. These disadvantages hampered phenanthrene and pyrene removal. MR provided additional nutrients, which counteracted the nutrient consumption associated with PR, thereby maintaining the microbial community diversity and bacterial biomass of PMR at a level achieved in the NA treatment. Combination remediation therefore overcame the disadvantages of using PR alone. These results indicated that inoculation with the combination of S. viminalis and C. laeve synergistically stimulated the growth of indigenous PAH-degrading microorganisms and maintained bacterial biomass, thus accelerating the dissipation of soil PAHs.

Mid-wave infrared polarization imaging system for detecting moving scene.

In this Letter, a polarimetric analyzer is designed for a mid-wave infrared camera. This kind of infrared camera transforms into the mid-wave infrared polarization imaging system to measure the infrared polarization characteristics of the object in the moving scene. The polarimetric analyzer is designed by using the ultra-high-speed and high-position method to drive the polarizer to rotate uniformly at the speed of 900 rpm. The polarization state of the object scene is changed, and the mid-wave infrared camera synchronously acquires the infrared intensity image in different polarized directions, those of 0°, 120°, and 240°. Then, a Stokes vector model is established with the basic rotation angles, and a sort-iteration method is proposed to process the original infrared intensity image. Three continuously neighboring infrared intensity images are used to calculate the degree of linear polarization (DoLP) and the angle of polarization (AoP), which make the infrared polarization image the same imaging frame as the infrared intensity image. Test results show that the mid-wave infrared polarization imaging system can complete the acquisition of the DoLP and the AoP images well with the frame frequency of 45 fps, which is suitable for the infrared polarization detection of the moving scenes. The study has great potential for polarization remote sensing and marine object detection.

Enhancing Salix viminalis L.-mediated phytoremediation of polycyclic aromatic hydrocarbon-contaminated soil by inoculation with Crucibulum laeve (white-rot fungus).

Although plant-white-rot fungi (WRF) remediation is considered efficient in improving polycyclic aromatic hydrocarbon (PAH)-contaminated soil, the prospects for using it remain poorly known. Therefore, we evaluated whether the WRF Crucibulum laeve could improve the phytoremediation of PAH-contaminated soil by Salix viminalis L. A 60-day pot experiment was conducted to investigate the effects of C. laeve inoculation (using two inoculation treatments and a non-inoculated control) on the phytoremediation potential, growth, and antioxidant metabolism of S. viminalis cultivated in PAH-contaminated soil. The S. viminalis-C. laeve association synergistically caused the highest PAH removal rate. Under the S. viminalis-C. laeve treatment, 80% of the biological concentration and translocation factors for all tissues of S. viminalis were > 1, whereas only 20% of these factors were > 1 when S. viminalis was used alone. C. laeve inoculation remarkably enhanced phytoremediation by promoting S. viminalis-based phytoextraction of PAHs from soils. Furthermore, although C. laeve inoculation altered the antioxidant metabolism of S. viminalis by inducing oxidative stress, thereby inhibiting plant growth, the plant's hardiness enabled it to survive and grow normally for 60 days after treatment. Therefore, phytoremediation using S. viminalis inoculated with C. laeve can be considered a feasible approach for the phytoremediation of PAH-contaminated soil.

Responses of Manila Grass (Zoysia matrella) to chilling stress: From transcriptomics to physiology.

Manila grass (Zoysia matrella), a warm-season turfgrass, usually wilts and browns by late autumn because of low temperature. To elucidate the molecular mechanisms regarding Manila grass responses to cold stress, we performed transcriptome sequencing of leaves exposed to 4°C for 0 (CK), 2h (2h_CT) and 72h (72h_CT) by Illumina technology. Approximately 250 million paired-end reads were obtained and de novo assembled into 82,605 unigenes. A total of 34,879 unigenes were annotated by comparing their sequence to public protein databases. At the 2h- and 72h-cold time points, 324 and 5,851 differentially expressed genes (DEGs) were identified, respectively. Gene ontology (GO) and metabolism pathway (KEGG) enrichment analyses of DEGs indicated that auxin, gibberellins, ethylene and calcium took part in the cold signal transduction in the early period. And in the late cold period, electron transport activities, photosynthetic machinery and activity, carbohydrate and nitrogen metabolism, redox equilibrium and hormone metabolism were disturbed. Low temperature stress triggered high light, drought and oxidative stress. At the physiological level, cold stress induced a decrease in water content, an increase in levels of total soluble sugar, free proline and MDA, and changes in bioactive gibberellins levels, which supported the changes in gene expression. The results provided a large set of sequence data of Manila grass as well as molecular mechanisms of the grass in response to cold stress. This information will be helpful for future study of molecular breeding and turf management.

Heterologous Expression of SvMBD5 from Salix viminalis L. Promotes Flowering in Arabidopsis thaliana L.

Methyl-CpG-binding domain (MBD) proteins have diverse molecular and biological functions in plants. Most studies of MBD proteins in plants have focused on the model plant Arabidopsis thaliana L. Here we cloned SvMBD5 from the willow Salix viminalis L. by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed the structure of SvMBD5 and its evolutionary relationships with proteins in other species. The coding sequence of SvMBD5 is 645 bp long, encoding a 214 amino acid protein with a methyl-CpG-binding domain. SvMBD5 belongs to the same subfamily as AtMBD5 and AtMBD6 from Arabidopsis. Subcellular localization analysis showed that SvMBD5 is only expressed in the nucleus. We transformed Arabidopsis plants with a 35S::SvMBD5 expression construct to examine SvMBD5 function. The Arabidopsis SvMBD5-expressing line flowered earlier than the wild type. In the transgenic plants, the expression of FLOWERING LOCUS T and CONSTANS significantly increased, while the expression of FLOWERING LOCUS C greatly decreased. In addition, heterologously expressing SvMBD5 in Arabidopsis significantly inhibited the establishment and maintenance of methylation of CHROMOMETHYLASE 3 and METHYLTRANSFERASE 1, as well as their expression, and significantly increased the expression of the demethylation-related genes REPRESSOR OF SILENCING1 and DEMETER-LIKE PROTEIN3. Our findings suggest that SvMBD5 participates in the flowering process by regulating the methylation levels of flowering genes, laying the foundation for further studying the role of SvMBD5 in regulating DNA demethylation.

Corticular photosynthesis drives bark water uptake to refill embolized vessels in dehydrated branches of Salix matsudana.

It is well known that xylem embolism can be repaired by bark water uptake and that the sugar required for embolism refilling can be provided by corticular photosynthesis. However, the relationship between corticular photosynthesis and embolism repair by bark water uptake is still poorly understood. In this study, the role of corticular photosynthesis in embolism repair was assessed using Salix matsudana branch segments dehydrated to -1.9 MPa (P50 , water potential at 50% loss of conductivity). The results indicated that corticular photosynthesis significantly promoted water uptake and nonstructural carbohydrate (NSC) accumulation in the bark and xylem during soaking, thereby effectively enhancing the refilling of the embolized vessels and the recovery of hydraulic conductivity. Furthermore, the influence of the extent of dehydration on the embolism refilling enhanced by corticular photosynthesis was investigated. The enhanced refilling effects were much higher in the mildly dehydrated (-1.5 MPa) and moderately dehydrated (-1.9 MPa) branch segments than in the severely dehydrated (-2.2 MPa) branch segments. This study provides evidence that corticular photosynthesis plays a crucial role in xylem embolism repair by bark water uptake for mildly and moderately dehydrated branches.

A Monolithic Electrochemical Micro Seismic Sensor Capable of Monitoring Three-Dimensional Vibrations.

A monolithic electrochemical micro seismic sensor capable of monitoring three-axial vibrations was proposed in this paper. The proposed micro sensor mainly consisted of four sensing units interconnected within flow channels and by interpreting the voltage outputs of the sensing units, vibrations with arbitrary directions can be quantified. The proposed seismic sensors are fabricated based on MEMS technologies and characterized, which produced sensitivities along x, y, and z axes as 2473.2 ± 184.5 V/(m/s), 2261.7 ± 119.6 V/(m/s), and 3480.7 ± 417.2 V/(m/s) at 30 Hz. In addition, the vibrations in x-y, x-z, and y-z planes were applied to the developed seismic sensors, leading to comparable monitoring results after decoupling calculations with the input velocities. Furthermore, the results have shown its feasibilities for seismic data recording.

A Flexible Sensing Unit Manufacturing Method of Electrochemical Seismic Sensor.

This paper presents an electrochemical seismic sensor in which paraylene was used as a substrate and insulating layer of micro-fabricated electrodes, enabling the detection of seismic signals with enhanced sensitivities in comparison to silicon-based counterparts. Based on microfabrication, paralene-based electrochemical seismic sensors were fabricated in which the thickness of the insulating spacer was 6.7 µm. Compared to silicon-based counterparts with ~100 µm insulating layers, the parylene-based devices produced higher sensitivities of 490.3 ± 6.1 V/(m/s) vs. 192.2 ± 1.9 V/(m/s) at 0.1 Hz, 4764.4 ± 18 V/(m/s) vs. 318.9 ± 6.5 V/(m/s) at 1 Hz, and 4128.1 ± 38.3 V/(m/s) vs. 254.5 ± 4.2 V/(m/s) at 10 Hz. In addition, the outputs of the parylene vs. silicon devices in response to two transit inputs were compared, producing peak responses of 2.97 V vs. 0.22 V and 2.41 V vs. 0.19 V, respectively. Furthermore, the self-noises of parylene vs. silicon-based devices were compared as follows: -82.3 ± 3.9 dB vs. -90.4 ± 9.4 dB at 0.1 Hz, -75.7 ± 7.3 dB vs. -98.2 ± 9.9 dB at 1 Hz, and -62.4 ± 7.7 dB vs. -91.1 ± 8.1 dB at 10 Hz. The developed parylene-based electrochemical seismic sensors may function as an enabling technique for further detection of seismic motions in various applications.

Nondestructive Estimation of Muscle Contributions to STS Training with Different Loadings Based on Wearable Sensor System.

Partial body weight support or loading sit-to-stand (STS) rehabilitation can be useful for persons with lower limb dysfunction to achieve movement again based on the internal residual muscle force and external assistance. To explicate how the muscles contribute to the kinetics and kinematics of STS performance by non-invasive in vitro detection and to nondestructively estimate the muscle contributions to STS training with different loadings, a wearable sensor system was developed with ground reaction force (GRF) platforms, motion capture inertial sensors and electromyography (EMG) sensors. To estimate the internal moments of hip, knee and ankle joints and quantify the contributions of individual muscle and gravity to STS movement, the inverse dynamics analysis on a simplified STS biomechanical model with external loading is proposed. The functional roles of the lower limb individual muscles (rectus femoris (RF), gluteus maximus (GM), vastus lateralis (VL), tibialis anterior (TA) and gastrocnemius (GAST)) during STS motion and the mechanism of the muscles' synergies to perform STS-specific subtasks were analyzed. The muscle contributions to the biomechanical STS subtasks of vertical propulsion, anteroposterior (AP) braking and propulsion for body balance in the sagittal plane were quantified by experimental studies with EMG, kinematic and kinetic data.

High diversity of the Ganzhou Oviraptorid Fauna increased by a new "cassowary-like" crested species.

A new oviraptorid dinosaur from the Late Cretaceous of Ganzhou, bringing oviraptrotid diversity of this region to seven taxa, is described. It is characterized by a distinct cassowary-like crest on the skull, no pleurocoels on the centra from the second through fourth cervical vertebrae, a neck twice as long as the dorsal vertebral column and slightly longer than the forelimb (including the manus). Phylogenetic analysis recovers the new oviraptorid taxon, Corythoraptor jacobsi, as closely related to Huanansaurus from Ganzhou. Osteochronology suggests that the type specimen of Corythoraptor had not reached stationary growth stage but died while decreasing growth rates. The histology implies that it would correspond to an immature individual approximately eight years old. We hypothesize, based on the inner structure compared to that in modern cassowaries, that the prominent casque of Corythoraptor was a multifunction-structure utilized in display, communication and probably expression of the fitness during mating seasons.

Male and Female Subpopulations of Salix viminalis Present High Genetic Diversity and High Long-Term Migration Rates between Them.

Dioecy distributed in 157 flowering plant families and 959 flowering plant genera. Morphological and physiological differences between male and female plants have been studied extensively, but studies of sex-specific genetic diversity are relatively scarce in dioecious plants. In this study, 20 SSR loci were employed to examine the genetic variance of male subpopulations and female subpopulations in Salix viminalis. The results showed that all of the markers were polymorphic (Na = 14.15, He = 0.7566) and workable to reveal the genetic diversity of S. viminalis. No statistically significant difference was detected between male and female subpopulations, but the average genetic diversity of male subpopulations (Na = 7.12, He = 0.7071) and female subpopulations (Na = 7.31, He = 0.7226) were high. Under unfavorable environments (West Liao basin), the genetic diversity between male and female subpopulations was still not significantly different, but the genetic diversity of sexual subpopulations were lower. The differentiation of the ten subpopulations in S. viminalis was moderate (FST = 0.0858), which was conformed by AMOVA that most of genetic variance (94%) existed within subpopulations. Pairwise FST indicated no differentiation between sexual subpopulations, which was accompanied by high long-term migrate between them (M = 0.73~1.26). However, little recent migration was found between sexual subpopulations. Therefore, artificial crossing or/and transplantation by cutting propagation should be carried out so as to increase the migration during the process of ex situ conservation.

Identification of suitable reference genes in buffalo grass for accurate transcript normalization under various abiotic stress conditions.

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a sensitive technique for normalization of the gene expression level of target genes. Buffalograss (Buchloe dactyloides), a warm-season turfgrass with strong abiotic stress resistance, is widely used in North China. Up to now, no work was performed to evaluate the reference genes in buffalograss. In this study, the expression profiles of ten potential reference genes were examined by qRT-PCR in 24 buffalograss samples, which were subjected to a different treatment (salt, osmotic, cold and heat). Three qRT-PCR analysis methods (GeNorm, NormFinder, and Bestkeeper) were used to evaluate the stability of gene expression. The results indicated that DNAJ and ß-ACTIN were the optimal reference genes for salt-treated leaves, and the combination of PP2A and GAPDH was better reference genes for PEG-treated leaves. Under cold stress, DNAJ and ß-ACTIN showed less variety of expression level in leaves. DNAJ and GAPDH exhibited the most stable expression in heat-treated samples. To sum up, glyceral-dehyde-3-phosphate dehydrogenase (GAPDH), ß-ACTIN, DNAJ-like protein (DNAJ) and protein phosphatase 2A (PP2A) were selected as the most stable reference gene among all tested samples. To further validate the suitability of these reference genes, the expression levels of DREB2 (homologs of AtDREB2) were analyzed in parallel. Our results show that the best reference genes differed across different experimental conditions, and these results should enable better normalization and quantification of transcript levels in buffalograss in the future.