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Rapeseed (Brassica napus L.) seedlings are rich in vitamin C (Vc), which is beneficial for humans. Understanding the genetic variance in Vc content has practical significance for the breeding of “oil–vegetable dual-purpose” rapeseed. In this study, the joint segregation analysis of a mixed genetic model of the major gene plus polygene was conducted on the Vc content in rapeseed seedlings. Six generations, including two parents, P1 (high Vc content) and P2 (low Vc content), F1, and the populations of F2, BC1P1, and BC1P2 from two crosses were investigated. Genetic analysis revealed that the genetic model MX2-A-AD was the most fitting genetic model, which indicates that Vc content is controlled by two additive major genes plus additive and dominance polygenes. In addition, the whole heritability in F2 and BC1P1 was higher than that in BC1P2. The largest coefficient of variation for Vc content appeared in the F2 generation. Therefore, for Vc content, the method of single cross recross or single backcross are suggested to transfer major genes, and the selection in F2 would be more efficient than that in other generations. Our findings provide a theoretical basis for the quantitative trait locus (QTL) mapping and breeding of Vc content in rapeseed seedlings.

Abstract Background Rapeseed (Brassica napus L.) is an essential source of edible oil and livestock feed, as well as a promising source of biofuel. Breeding crops with an ideal root system architecture (RSA) for high phosphorus use efficiency (PUE) is an effective way to reduce the use of phosphate fertilizers. However, the genetic mechanisms that underpin PUE in rapeseed remain elusive. To address this, we conducted a genome-wide association study (GWAS) in 327 rapeseed accessions to elucidate the genetic variability of 13 root and biomass traits under low phosphorus (LP; 0.01 mM P +). Furthermore, RNA-sequencing was performed in root among high/low phosphorus efficient groups (HP1/LP1) and high/low phosphorus stress tolerance groups (HP2/LP2) at two-time points under control and P-stress conditions. Results Significant variations were observed in all measured traits, with heritabilities ranging from 0.47 to 0.72, and significant correlations were found between most of the traits. There were 39 significant trait–SNP associations and 31 suggestive associations, which integrated into 11 valid quantitative trait loci (QTL) clusters, explaining 4.24–24.43% of the phenotypic variance observed. In total, RNA-seq identified 692, 1076, 648, and 934 differentially expressed genes (DEGs) specific to HP1/LP1 and HP2/LP2 under P-stress and control conditions, respectively, while 761 and 860 DEGs common for HP1/LP1 and HP2/LP2 under both conditions. An integrated approach of GWAS, weighted co-expression network, and differential expression analysis identified 12 genes associated with root growth and development under LP stress. In this study, six genes (BnaA04g23490D, BnaA09g08440D, BnaA09g04320D, BnaA09g04350D, BnaA09g04930D, BnaA09g09290D) that showed differential expression were identified as promising candidate genes for the target traits. Conclusion 11 QTL clusters and 12 candidate genes associated with root and development under LP stress were identified in this study. Our study's phenotypic and genetic information may be exploited for genetic improvement of root traits to increase PUE in rapeseed.

ABSTRACTZika virus (ZIKV) infections are typically asymptomatic but cause severe neurological complications (e.g. Guillain–Barré syndrome in adults, and microcephaly in newborns). There are currently no specific therapy or vaccine options available to prevent ZIKV infections. Temporal gene expression profiles of ZIKV-infected human brain microvascular endothelial cells (HBMECs) were used in this study to identify genes essential for viral replication. These genes were then used to identify novel anti-ZIKV agents and validated in publicly available data and functional wet-lab experiments. Here, we found that ZIKV effectively evaded activation of immune response-related genes and completely reprogrammed cellular transcriptional architectures. Knockdown of genes, which gradually upregulated during viral infection but showed distinct expression patterns between ZIKV- and mock infection, discovered novel proviral and antiviral factors. One-third of the 74 drugs found through signature-based drug repositioning and cross-reference with the Drug Gene Interaction Database (DGIdb) were known anti-ZIKV agents. In cellular assays, two promising antiviral candidates (Luminespib/NVP-AUY922, L-161982) were found to reduce viral replication without causing cell toxicity. Overall, our time-series transcriptome-based methods offer a novel and feasible strategy for antiviral drug discovery. Our strategies, which combine conventional and data-driven analysis, can be extended for other pathogens causing pandemics in the future.

LGP2 is a RIG-I-like receptor (RLR) known to bind and recognize the intermediate double-stranded RNA (dsRNA) during virus infection and to induce type-I interferon (IFN)-related antiviral innate immune responses. Here, we find that LGP2 inhibits Zika virus (ZIKV) and tick-borne encephalitis virus (TBEV) replication independent of IFN induction. Co-immunoprecipitation (Co-IP) and confocal immunofluorescence data suggest that LGP2 likely colocalizes with the replication complex (RC) of ZIKV by interacting with viral RNA-dependent RNA polymerase (RdRP) NS5. We further verify that the regulatory domain (RD) of LGP2 directly interacts with RdRP of NS5 by biolayer interferometry assay. Data from in vitro RdRP assays indicate that LGP2 may inhibit polymerase activities of NS5 at pre-elongation but not elongation stages, while an RNA-binding-defective LGP2 mutant can still inhibit RdRP activities and virus replication. Taken together, our work suggests that LGP2 can inhibit flavivirus replication through direct interaction with NS5 protein and downregulates its polymerase pre-elongation activities, demonstrating a distinct role of LGP2 beyond its function in innate immune responses.

Abstract Background JAZ subfamily plays crucial roles in growth and development, stress, and hormone responses in various plant species. Despite its importance, the structural and functional analyses of the JAZ subfamily in Brassica napus are still limited. Results Comparing to the existence of 12 JAZ genes (AtJAZ1-AtJAZ12) in Arabidopsis, there are 28, 31, and 56 JAZ orthologues in the reference genome of B. rapa, B. oleracea, and B. napus, respectively, in accordance with the proven triplication events during the evolution of Brassicaceae. The phylogenetic analysis showed that 127 JAZ proteins from A. thaliana, B. rapa, B. oleracea, and B. napus could fall into five groups. The structure analysis of all 127 JAZs showed that these proteins have the common motifs of TIFY and Jas, indicating their conservation in Brassicaceae species. In addition, the cis-element analysis showed that the main motif types are related to phytohormones, biotic and abiotic stresses. The qRT-PCR of the representative 11 JAZ genes in B. napus demonstrated that different groups of BnJAZ individuals have distinct patterns of expression under normal conditions or treatments with distinctive abiotic stresses and phytohormones. Especially, the expression of BnJAZ52 (BnC08.JAZ1-1) was significantly repressed by abscisic acid (ABA), gibberellin (GA), indoleacetic acid (IAA), polyethylene glycol (PEG), and NaCl treatments, while induced by methyl jasmonate (MeJA), cold and waterlogging. Expression pattern analysis showed that BnC08.JAZ1-1 was mainly expressed in the vascular bundle and young flower including petal, pistil, stamen, and developing ovule, but not in the stem, leaf, and mature silique and seed. Subcellular localization showed that the protein was localized in the nucleus, in line with its orthologues in Arabidopsis. Overexpression of BnC08.JAZ1-1 in Arabidopsis resulted in enhanced seed weight, likely through regulating the expression of the downstream response genes involved in the ubiquitin–proteasome pathway and phospholipid metabolism pathway. Conclusions The systematic identification, phylogenetic, syntenic, and expression analyses of BnJAZs subfamily improve our understanding of their roles in responses to stress and phytohormone in B. napus. In addition, the preliminary functional validation of BnC08.JAZ1-1 in Arabidopsis demonstrated that this subfamily might also play a role in regulating seed weight.

Root system architecture (RSA) is the primary predictor of nutrient intake and significantly influences potassium utilization efficiency (KUE). Uncertainty persists regarding the genetic factors governing root growth in rapeseed. The root transcriptome analysis reveals the genetic basis driving crop root growth. In this study, RNA-seq was used to profile the overall transcriptome in the root tissue of 20 Brassica napus accessions with high and low KUE. 71,437 genes in the roots displayed variable expression profiles between the two contrasting genotype groups. The 212 genes that had varied expression levels between the high and low KUE lines were found using a pairwise comparison approach. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional classification analysis revealed that the DEGs implicated in hormone and signaling pathways, as well as glucose, lipid, and amino acid metabolism, were all differently regulated in the rapeseed root system. Additionally, we discovered 33 transcription factors (TFs) that control root development were differentially expressed. By combining differential expression analysis, weighted gene co-expression network analysis (WGCNA), and recent genome-wide association study (GWAS) results, four candidate genes were identified as essential hub genes. These potential genes were located fewer than 100 kb from the peak SNPs of QTL clusters, and it was hypothesized that they regulated the formation of the root system. Three of the four hub genes’ homologs—BnaC04G0560400ZS, BnaC04G0560400ZS, and BnaA03G0073500ZS—have been shown to control root development in earlier research. The information produced by our transcriptome profiling could be useful in revealing the molecular processes involved in the growth of rapeseed roots in response to KUE.

Silique length influences seed yield in oilseed rape. It shows extensive variation in germplasm resources, and identifying the underlying genes and regulatory mechanisms would advance breeding for the trait. In the present study, a genome-wide association study (GWAS) using 331 core accessions planted in 10 environments revealed 13 loci associated with silique length on chromosomes A01, A04, A07, A09, and C03, explaining 6.2%–19.2% of phenotypic variance. Physiological analysis showed that silique length variation was attributable to differences in silique growth rate and/or duration before four weeks after flowering, with levels of endogenous phytohormones (auxin, ethylene, and GA24, GA12, and GA44) playing an important role. Cytological analysis showed that silique length variation was due mainly to differences in cell number followed by cell size. Transcriptomic analysis of two pools of silique walls with opposite length extremes revealed 3248 differentially expressed genes (DEGs). These DEGs were enriched in several pathways (such as cell wall, cell division, and hormone metabolism) associated with cell proliferation and expansion and silique development. Integrating GWAS, RNA-seq, and functional annotation results revealed 15 candidate genes for the major associated locus qSL.A09-3. Of these, BnaA9.ARF18 and BnaA9.CYP78A9 were validated by haplotype analysis followed by candidate gene association. Sequence variation in the coding region of BnaA9.ARF18 and expression of BnaA9.CYP78A9 in silique walls were strongly associated with silique length. Our results provide an explanation for the natural variation of silique length in oilseed rape germplasm and offer useful information for its improvement.

Stem lodging resistance is a serious problem impairing crop yield and quality. ZS11 is an adaptable and stable yielding rapeseed variety with excellent resistance to lodging. However, the mechanism regulating lodging resistance in ZS11 remains unclear. Here, we observed that high stem mechanical strength is the main factor determining the superior lodging resistance of ZS11 through a comparative biology study. Compared with 4D122, ZS11 has higher rind penetrometer resistance (RPR) and stem breaking strength (SBS) at flowering and silique stages. Anatomical analysis shows that ZS11 exhibits thicker xylem layers and denser interfascicular fibrocytes. Analysis of cell wall components suggests that ZS11 possessed more lignin and cellulose during stem secondary development. By comparative transcriptome analysis, we reveal a relatively higher expression of genes required for S-adenosylmethionine (SAM) synthesis, and several key genes (4-COUMATATE-CoA LIGASE, CINNAMOYL-CoA REDUCTASE, CAFFEATE O-METHYLTRANSFERASE, PEROXIDASE) involved in lignin synthesis pathway in ZS11, which support an enhanced lignin biosynthesis ability in the ZS11 stem. Moreover, the difference in cellulose may relate to the significant enrichment of DEGs associated with microtubule-related process and cytoskeleton organization at the flowering stage. Protein interaction network analysis indicate that the preferential expression of several genes, such as LONESOME HIGHWAY (LHW), DNA BINDING WITH ONE FINGERS (DOFs), WUSCHEL HOMEOBOX RELATED 4 (WOX4), are related to vascular development and contribute to denser and thicker lignified cell layers in ZS11. Taken together, our results provide insights into the physiological and molecular regulatory basis for the formation of stem lodging resistance in ZS11, which will greatly promote the application of this superior trait in rapeseed breeding.

Summary: Upon recognizing danger signals produced by virally infected neurons, macrophages in the central nervous system (CNS) secrete multiple inflammatory cytokines to accelerate neuron apoptosis. The understanding is limited about which key effectors regulate macrophage-neuron crosstalk upon infection. We have used neurotropic-virus-infected murine models to identify that vascular endothelial growth factor receptor 3 (VEGFR-3) is upregulated in the CNS macrophages and that virally infected neurons secrete the ligand VEGF-C. When cultured with VEGF-C-containing supernatants from virally infected neurons, VEGFR-3+ macrophages suppress tumor necrosis factor α (TNF-α) secretion to reduce neuron apoptosis. Vegfr-3ΔLBD/ΔLBD (deletion of ligand-binding domain in myeloid cells) mice or mice treated with the VEGFR-3 kinase inhibitor exacerbate the severity of encephalitis, TNF-α production, and neuron apoptosis post Japanese encephalitis virus (JEV) infection. Activating VEGFR-3 or blocking TNF-α can reduce encephalitis and neuronal damage upon JEV infection. Altogether, we show that the inducible VEGF-C/VEGFR-3 module generates protective crosstalk between neurons and macrophages to alleviate CNS viral infection.

Abstract Background In terms of global demand, rapeseed is the third-largest oilseed crop after soybeans and palm, which produces vegetable oil for human consumption and biofuel for industrial production. Roots are vital organs for plant to absorb water and attain mineral nutrients, thus they are of great importance to plant productivity. However, the genetic mechanisms regulating root development in rapeseed remain unclear. In the present study, seven root-related traits and shoot biomass traits in 280 Brassica napus accessions at five continuous vegetative stages were measured to establish the genetic basis of root growth in rapeseed. Results The persistent and stage-specific genetic mechanisms were revealed by root dynamic analysis. Sixteen persistent and 32 stage-specific quantitative trait loci (QTL) clusters were identified through genome-wide association study (GWAS). Root samples with contrasting (slow and fast) growth rates throughout the investigated stages and those with obvious stage-specific changes in growth rates were subjected to transcriptome analysis. A total of 367 differentially expressed genes (DEGs) with persistent differential expressions throughout root development were identified, and these DEGs were significantly enriched in GO terms, such as energy metabolism and response to biotic or abiotic stress. Totally, 485 stage-specific DEGs with different expressions at specific stage were identified, and these DEGs were enriched in GO terms, such as nitrogen metabolism. Four candidate genes were identified as key persistent genetic factors and eight as stage-specific ones by integrating GWAS, weighted gene co-expression network analysis (WGCNA), and differential expression analysis. These candidate genes were speculated to regulate root system development, and they were less than 100 kb away from peak SNPs of QTL clusters. The homologs of three genes (BnaA03g52990D, BnaA06g37280D, and BnaA09g07580D) out of 12 candidate genes have been reported to regulate root development in previous studies. Conclusions Sixteen QTL clusters and four candidate genes controlling persistently root development, and 32 QTL clusters and eight candidate genes stage-specifically regulating root growth in rapeseed were detected in this study. Our results provide new insights into the temporal genetic mechanisms of root growth by identifying key candidate QTL/genes in rapeseed.

Abstract Fluorescent labeling and dynamic tracking is a powerful tool for exploring virus infection mechanisms. However, for small-sized viruses, virus tracking studies are usually hindered by a lack of appropriate labeling methods that do not dampen virus yield or infectivity. Here, we report a universal strategy for labeling viruses with chemical dyes and Quantum dots (QDs). Enterovirus 71 (EV71) was produced in a cell line that stably expresses a mutant methionyl-tRNA synthetase (MetRS), which can charge azidonorleucine (ANL) to the methionine sites of viral proteins during translation. Then, the ANL-containing virus was easily labeled with DBCO-AF647 and DBCO-QDs. The labeled virus shows sufficient yield and no obvious decrease in infectivity and can be used for imaging the virus entry process. Using the labeled EV71, different functions of scavenger receptor class B, member 2 (SCARB2), and heparan sulfate (HS) in EV71 infection were comparatively studied. The cell entry process of a strong HS-binding EV71 strain was investigated by real-time dynamic visualization of EV71-QDs in living cells. Taken together, our study described a universal biocompatible virus labeling method, visualized the dynamic viral entry process, and reported details of the receptor usage of EV71. Graphic Abstract

Branch architecture is an important factor influencing rapeseed planting density, mechanized harvest, and yield. However, its related genes and regulatory mechanisms remain largely unknown. In this study, branch angle (BA) and branch dispersion degree (BD) were used to evaluate branch architecture. Branch angle exhibited a dynamic change from an increase in the early stage to a gradual decrease until reaching a stable state. Cytological analysis showed that BA variation was mainly due to xylem size differences in the vascular bundle of the branch junction. The phenotypic analysis of 327 natural accessions revealed that BA in six environments ranged from 24.3° to 67.9°, and that BD in three environments varied from 4.20 cm to 21.4 cm, respectively. A total of 115 significant loci were detected through association mapping in three models (MLM, mrMLM, and FarmCPU), which explained 0.53%-19.4% of the phenotypic variations. Of them, 10 loci were repeatedly detected in different environments and models, one of which qBAD.A03-2 was verified as a stable QTL using a secondary segregation population. Totally, 1066 differentially expressed genes (DEGs) were identified between branch adaxial- and abaxial- sides from four extremely large or small BA/BD accessions through RNA sequencing. These DEGs were significantly enriched in the pathways related to auxin biosynthesis and transport as well as cell extension such as indole alkaloid biosynthesis, other glycan degradation, and fatty acid elongation. Four known candidate genes BnaA02g16500D (PIN1), BnaA03g10430D (PIN2), BnaC03g06250D (LAZY1), and BnaC06g20640D (ARF17) were identified by both GWAS and RNA-seq, all of which were involved in regulating the asymmetric distribution of auxins. Our identified association loci and candidate genes provide a theoretical basis for further study of gene cloning and genetic improvement of branch architecture.

Oilseed rape is one of the world’s largest oil and industrial crops, providing humans with various products, such as vegetable oil and biofuel. Ovules are the direct precursors of seeds, and ovule number per ovary (ONPO) largely determines seed number per fruit that affects both yield and fitness of seed crops. The ONPO shows wide variation in oilseed rape, whereas the underlying genes and mechanisms are poorly known. The present study performed the genetic, physiological and transcriptomic analyses of ovule number per ovary using an association panel and the extreme lines. The ONPO of 327 accessions planted in four environments showed a large variation from 19.2 to 43.8, indicating a great potential for the further genetic improvement of ovule number. The genome-wide association study (GWAS) identified a total of 43 significant SNP markers. Further, these SNPs were integrated into 18 association loci, which were distributed on chromosomes A01, A03, A06, A07, A09, C01, C03, C06, C07, and C09, explaining 4.3–11.5% of the phenotypic variance. The ONPO decreased as their appearance order on the inflorescence and was associated with the level of several types of endogenous phytohormones but not related to leaf area and photosynthetic rate. Comparative transcriptomic analysis identified a total of 4,449 DEGs enriched in 30 classes, including DNA, RNA, protein, signaling, transport, development, cell wall, lipid metabolism, and secondary metabolism. Nearly half of DEGs were involved in the known pathways in regulating ovule number, of which 12 were homologous to know ovule number regulating genes, indicating a strong link between the identified DEGs and ovule number. A total of 73 DEGs were located within the genomic regions of association loci, of which six were identified as candidates based on functional annotation. These results provide useful information for the further genetic improvement of ovule and seed number in oilseed rape.

Plants rely on root systems for nutrient uptake from soils. Marker-assisted selection helps breeders to select desirable root traits for effective nutrient uptake. Here, 12 root and biomass traits were investigated at the seedling stage under low nitrogen (LN), low phosphorus (LP), and low potassium (LK) conditions, respectively, in a recombinant inbred line (RIL) population, which was generated from Brassica napus L. Zhongshuang11 and 4D122 with significant differences in root traits and nutrient efficiency. Significant differences for all the investigated traits were observed among RILs, with high heritabilities (0.43–0.74) and high correlations between the different treatments. Quantitative trait loci (QTL) mapping identified 57, 27, and 36 loci, explaining 4.1–10.9, 4.6–10.8, and 4.9–17.4% phenotypic variances under LN, LP, and LK, respectively. Through QTL-meta analysis, these loci were integrated into 18 significant QTL clusters. Four major QTL clusters involved 25 QTLs that could be repeatedly detected and explained more than 10% phenotypic variances, including two NPK-common and two specific QTL clusters (K and NK-specific), indicating their critical role in cooperative nutrients uptake of N, P, and K. Moreover, 264 genes within the four major QTL clusters having high expressions in roots and SNP/InDel variations between two parents were identified as potential candidate genes. Thirty-eight of them have been reported to be associated with root growth and development and/or nutrient stress tolerance. These key loci and candidate genes lay the foundation for deeper dissection of the NPK starvation response mechanisms in B. napus.

Zika virus (ZIKV) infection in the human central nervous system (CNS) causes Guillain–Barre syndrome, cerebellum deformity, and other diseases. Astrocytes are immune response cells in the CNS and an important component of the blood–brain barrier. Consequently, any damage to astrocytes facilitates the spread of ZIKV in the CNS. Connective tissue growth factor/Nephroblastoma overexpressed gene family 1 (CCN1), an important inflammatory factor secreted by astrocytes, is reported to regulate innate immunity and viral infection. However, the mechanism by which astrocyte viral infection affects CCN1 expression remains undefined. In this study, we demonstrate that ZIKV infection up-regulates CCN1 expression in astrocytes, thus promoting intracellular viral replication. Other studies revealed that the cAMP response element (CRE) in the CCN1 promoter is activated by the ZIKV NS3 protein. The cAMP-responsive element-binding protein (CREB), a transacting factor of the CRE, is also activated by NS3 or ZIKV. Furthermore,a specific inhibitor of CREB, i.e. SGC-CBP30, reduced ZIKV-induced CCN1 up-regulation and ZIKV replication. Moreover, co-immunoprecipitation, overexpression, and knockdown studies confirmed that the interaction between NS3 and the regulatory domain of CaMKIIα could activate the CREB pathway, thus resulting in the up-regulation of CCN1 expression and enhancement of virus replication. In conclusion, the findings of our investigations on the NS3-CaMKIIα-CREB-CCN1 pathway provide a foundation for understanding the infection mechanism of ZIKV in the CNS.

Abstract Background Brassica napus provides approximately 13–16% of global vegetable oil for human consumption and biodiesel production. Plant height (PH) is a key trait that affects plant architecture, seed yield and harvest index. However, the genetic mechanism of PH in B. napus is poorly understood. Results A dwarf mutant df59 was isolated from a large-scale screening of an ethyl methanesulphonate-mutagenized rapeseed variety Ningyou 18. A genetic analysis showed that the dwarfism phenotype was controlled by one semi-dominant gene, which was mapped on C9 chromosome by quantitative trait loci sequencing analysis and designated as BnaDwf.C9. To fine-map BnaDwf.C9, two F2 populations were constructed from crosses between conventional rapeseed cultivars (Zhongshuang 11 and Holly) and df59. BnaDwf.C9 was fine-mapped to the region between single-nucleotide polymorphism (SNP) markers M14 and M4, corresponding to a 120.87-kb interval of the B. napus ‘Darmor-bzh’ genome. Within this interval, seven, eight and nine annotated or predicted genes were identified in “Darmor-bzh”, “Ningyou 7” and “Zhongshuang 11” reference genomes, respectively. In addition, a comparative transcriptome analysis was performed using stem tips from Ningyou 18 and df59 at the stem elongation stage. In total, 3995 differentially expressed genes (DEGs) were identified. Among them, 118 DEGs were clustered in plant hormone-related signal transduction pathways, including 81 DEGs were enriched in auxin signal transduction. Combining the results of fine-mapping and transcriptome analyses, BnaC09g20450D was considered a candidate gene for BnaDwf.C9, which contains a SNP that co-segregated in 4746 individuals. Finally, a PCR-based marker was developed based on the SNP in BnaC09g20450D. Conclusions The combination of quantitative trait loci sequencing, fine-mapping and genome-wide transcriptomic analysis revealed one candidate gene located within the confidence interval of 120.87-kb region. This study provides a new genetic resource for semi-dwarf breeding and new insights into understanding the genetic architecture of PH in B. napus.

ABSTRACT Tick-borne encephalitis virus (TBEV) is the causative agent of a potentially fatal neurological infection affecting humans. The host factors required for viral entry have yet to be described. Here, we found that T-cell immunoglobulin and mucin domain 1 (TIM-1) acted as the cellular entry factor for TBEV. Using a virus overlay protein binding assay, TIM-1 was identified as a virion-interacting protein. Cells that were relatively resistant to TBEV infection became highly susceptible to infection when TIM-1 was ectopically expressed. TIM-1 knockout and viral RNA bypass assays showed that TIM-1 functioned in the entry phase of TBEV infection. TIM-1 mediated TBEV uptake and was cointernalized with virus particles into the cell. Antibodies for TIM-1, soluble TIM-1, or TIM-1 knockdown significantly inhibited TBEV infection in permissive cells. Furthermore, in TIM-1 knockout mice, TIM-1 deficiency markedly lowered viral burden and reduced mortality and morbidity, highlighting the functional relevance of TIM-1 in vivo. With TIM-1, we have identified a key host factor for TBEV entry and a potential target for antiviral intervention. IMPORTANCE TBEV is a tick-transmitted flavivirus that causes serious diseases in the human central nervous system in Eurasia. The host determinants required for viral entry remain poorly understood. Here, we found that TIM-1 is a cellular entry factor for TBEV. Antibodies directed at TIM-1 or soluble TIM-1 treatment decreased virus infection in cell cultures. TIM-1 was cointernalized with virus particles into cells. TIM-1 deficiency significantly lowered viral burden and attenuated pathogenesis in the murine TBEV infection model. The demonstration of TIM-1 as a cellular entry factor for TBEV will improve understanding of virus infection and provide a target for antiviral development.

Enterovirus 71 (EV71) is one of the most important etiological agents for hand–foot–mouth disease. Compared with coxsackievirus A16 infection, EV71 infection is often associated with severe central nervous system complications, such as encephalitis, encephalomyelitis, and acute flaccid paralysis in infants and young children. In this study, we constructed a recombinant baculovirus with T7 ribonucleic acid polymerase under the control of a cytomegalovirus promoter and simultaneously engineered the T7 promoter upstream of a full-length EV71 complementary deoxyribonucleic acid. After transduction into mammalian cells, typical cytopathic effects (CPEs) and VP1 signals were detected in cells transfected with recombinant baculovirus. Additionally, viral particles located in the cytoplasm of human rhabdomyosarcoma cells (Rd) and Vero cells were observed by electron microscope, indicating that EV71 was recovered using a Bac-to-Bac expression system in vitro. After four passages, the rescued virus had a growth curve and plaque morphology similar to those of the parental virus. Furthermore, the Vp1 gene and the protein from the mouse brain were detected by reverse transcription polymerase chain reaction and immunohistochemistry after intracerebral injection of purified recombinant baculovirus. Typical CPEs were observed after inoculation of the supernatant from mouse brain to Rd cells, revealing a reconstruction of EV71 in vivo. Thus, we established a new approach to rescue EV71 based on a baculovirus expression system in vitro and in vivo, which may provide a safe and convenient platform for fundamental research and a strategy to rescue viruses that currently lack suitable cell culture and animal models.

Roots are essential organs for a plant’s ability to absorb water and obtain mineral nutrients, hence they are critical to its development. Plants use root architectural alterations to improve their chances of absorbing nutrients when their supply is low. Nine root traits of a Brassica napus association panel were explored in hydroponic-system studies under low potassium (K) stress to unravel the genetic basis of root growth in rapeseed. The quantitative trait loci (QTL) and candidate genes for root development were discovered using a multilocus genome-wide association study (ML-GWAS). For the nine traits, a total of 453 significant associated single-nucleotide polymorphism (SNP) loci were discovered, which were then integrated into 206 QTL clusters. There were 45 pleiotropic clusters, and qRTA04-4 and qRTC04-7 were linked to TRL, TSA, and TRV at the same time, contributing 5.25–11.48% of the phenotypic variance explained (PVE) to the root traits. Additionally, 1360 annotated genes were discovered by examining genomic regions within 100 kb upstream and downstream of lead SNPs within the 45 loci. Thirty-five genes were identified as possibly regulating root-system development. As per protein–protein interaction analyses, homologs of three genes (BnaC08g29120D, BnaA07g10150D, and BnaC04g45700D) have been shown to influence root growth in earlier investigations. The QTL clusters and candidate genes identified in this work will help us better understand the genetics of root growth traits and could be employed in marker-assisted breeding for rapeseed adaptable to various conditions with low K levels.

Rapeseed is the third largest oil crop in the world and the largest oil crop in China. The multi-functional development and utilization of rapeseed is an effective measure for the high-quality development of rapeseed industry in China. In this study, several basic nutrients of eight rapeseed sprouts and five bean sprouts (3–5 varieties each) were determined, including sugar, crude protein, crude fiber, vitamin E, minerals, fatty acids, amino acids, and glucosinolates. Data analysis revealed that compared with bean sprouts, rapeseed sprouts were nutritionally balanced and were richer in active nutrients such as glucose, magnesium, selenium, vitamin E, and glucosinolate. Moreover, rapeseed sprouts exhibited reasonable amino acid composition and abundant unsaturated fatty acids (accounting for 90.32% of the total fatty acids). All these results indicated the potential of rapeseed sprout as a functional vegetable. Subsequently, three dominant nutrients including vitamin E, glucosinolate, and selenium were investigated in seeds and sprouts of 44 B. napus L. varieties. The results showed that germination raised the ratio of α-tocopherol/γ-tocopherol from 0.53 in seeds to 9.65 in sprouts, greatly increasing the content of α-tocopherol with the strongest antioxidant activity among the eight isomers of vitamin E. Furthermore, germination promoted the conversion and accumulation of glucosinolate components, especially, glucoraphanin with strong anti-cancer activity with its proportion increased from 1.06% in seeds to 1.62% in sprouts. In addition, the contents of selenium, vitamin E, and glucosinolate in rapeseed sprouts were highly correlated with those in seeds. Furthermore, these three dominant nutrients varied greatly within B. napus varieties, indicating the great potential of rapeseed sprouts to be further bio-enhanced. Our findings provide reference for the multi-purpose development and utilization of rapeseed, lay a theoretical foundation for the development of rapeseed sprout into a functional vegetable, and provide a novel breeding direction.

Zika virus (ZIKV) infections can cause microcephaly and neurological disorders. However, the early infection events of ZIKV in neural cells remain to be characterized. Here, by using a combination of pharmacological and molecular approaches and the human glioblastoma cell T98G as a model, we first observed that ZIKV infection was inhibited by chloroquine and NH4Cl, indicating a requirement of low intracellular pH. We further showed that dynamin is required as the ZIKV entry was affected by the specific inhibitor dynasore, small interfering RNA (siRNA) knockdown of dynamin, or by expressing the dominant-negative K44A mutant. Moreover, the ZIKV entry was significantly inhibited by chlorpromazine, pitstop2, or siRNA knockdown of clathrin heavy chain, indicating an involvement of clathrin-mediated endocytosis. In addition, genistein treatment, siRNA knockdown of caveolin-1, or overexpression of a dominant-negative caveolin mutant impacted the ZIKV entry, with ZIKV particles being observed to colocalize with caveolin-1, implying that caveola endocytosis can also be involved. Furthermore, we found that the endocytosis of ZIKV is dependent on membrane cholesterol, microtubules, and actin cytoskeleton. Importantly, ZIKV infection was inhibited by silencing of Rab5 and Rab7, while confocal microscopy showed that ZIKV particles localized in Rab5- and Rab7-postive endosomes. These results indicated that, after internalization, ZIKV likely moves to Rab5-positive early endosome and Rab7-positive late endosomes before delivering its RNA into the cytoplasm. Taken together, our study, for the first time, described the early infection events of ZIKV in human glioblastoma cell T98G.

Seed number is a key character/trait tightly related to the plant fitness/evolution and crop domestication/improvement. The seed number per silique (SNPS) shows a huge variation from several to more than 30, however the underlying regulatory mechanisms are poorly known, which has hindered its improvement. To answer this question, several representative lines with extreme SNPS were previously subjected to systematic genetic and cytological analyses. The results showed that the natural variation of seed number per silique is mainly controlled by maternal and embryonic genotype, which are co-determined by ovule number per ovary, fertile ovule ratio, ovule fertilization rate, and fertilized ovule development rate. More importantly, we also mapped two repeatable quantitative trait loci (QTLs) for SNPS using the F2:3 population derived from Zhongshuang11 and No. 73290, of which the major QTL qSN.A6 has been fine-mapped. In the current study, the near-isogenic lines (NILs) of qSN.A7 were successfully developed by the successive backcross of F1 with Zhongshuang11. First, the effect of qSN.A7 was validated by evaluating the SNPS of two types of homozygous NILs from BC3F2 population, which showed a significant difference of 2.23 on average. Then, qSN.A7 was successfully fine-mapped from the original 4.237 to 1.389 Mb, using a BC4F2 segregating population of 2,551 individuals. To further clarify the regulatory mechanism of qSN.A7, the two types of homologous NILs were subjected to genetic and cytological analyses. The results showed that the difference in SNPS between the two homologous NILs was determined by the embryonic genotypic effect. Highly accordant with this, no significant difference was observed in ovule number per ovary, ovule fertility, fertilization rate, and pollen fertility between the two homologous NILs. Therefore, the regulatory mechanism of qSN.A7 is completely different from the cloned qSS.C9 and qSN.A6. These results will advance the understanding of SNPS and facilitate gene cloning and molecular breeding in Brassica napus.

Abstract Background Many viruses depend on the extensive membranous network of the endoplasmic reticulum (ER) for their translation, replication, and packaging. Certain membrane modifications of the ER can be a trigger for ER stress, as well as the accumulation of viral protein in the ER by viral infection. Then, unfolded protein response (UPR) is activated to alleviate the stress. Zika virus (ZIKV) is a mosquito-borne flavivirus and its infection causes microcephaly in newborns and serious neurological complications in adults. Here, we investigated ER stress and the regulating model of UPR in ZIKV-infected neural cells in vitro and in vivo. Methods Mice deficient in type I and II IFN receptors were infected with ZIKV via intraperitoneal injection and the nervous tissues of the mice were assayed at 5 days post-infection. The expression of phospho-IRE1, XBP1, and ATF6 which were the key markers of ER stress were analyzed by immunohistochemistry assay in vivo. Additionally, the nuclear localization of XBP1s and ATF6n were analyzed by immunohistofluorescence. Furthermore, two representative neural cells, neuroblastoma cell line (SK-N-SH) and astrocytoma cell line (CCF-STTG1), were selected to verify the ER stress in vitro. The expression of BIP, phospho-elF2α, phospho-IRE1, and ATF6 were analyzed through western blot and the nuclear localization of XBP1s was performed by confocal immunofluorescence microscopy. RT-qPCR was also used to quantify the mRNA level of the UPR downstream genes in vitro and in vivo. Results ZIKV infection significantly upregulated the expression of ER stress markers in vitro and in vivo. Phospho-IRE1 and XBP1 expression significantly increased in the cerebellum and mesocephalon, while ATF6 expression significantly increased in the mesocephalon. ATF6n and XBP1s were translocated into the cell nucleus. The levels of BIP, ATF6, phospho-elf2α, and spliced xbp1 also significantly increased in vitro. Furthermore, the downstream genes of UPR were detected to investigate the regulating model of the UPR during ZIKV infection in vitro and in vivo. The transcriptional levels of atf4, gadd34, chop, and edem-1 in vivo and that of gadd34 and chop in vitro significantly increased. Conclusion Findings in this study demonstrated that ZIKV infection activates ER stress in neural cells. The results offer clues to further study the mechanism of neuropathogenesis caused by ZIKV infection.

Roots are complicated quantitative characteristics that play an essential role in absorbing water and nutrients. To uncover the genetic variations for root-related traits in rapeseed, twelve mature root traits of a Brassica napus association panel were investigated in the field within three environments. All traits showed significant phenotypic variation among genotypes, with heritabilities ranging from 55.18% to 79.68%. Genome-wide association studies (GWAS) using 20,131 SNPs discovered 172 marker-trait associations, including 103 significant SNPs (−log10 (p) > 4.30) that explained 5.24–20.31% of the phenotypic variance. With the linkage disequilibrium r2 > 0.2, these significant associations were binned into 40 quantitative trait loci (QTL) clusters. Among them, 14 important QTL clusters were discovered in two environments and/or with phenotypic contributions greater than 10%. By analyzing the genomic regions within 100 kb upstream and downstream of the peak SNPs within the 14 loci, 334 annotated genes were found. Among these, 32 genes were potentially associated with root development according to their expression analysis. Furthermore, the protein interaction network using the 334 annotated genes gave nine genes involved in a substantial number of interactions, including a key gene associated with root development, BnaC09g36350D. This research provides the groundwork for deciphering B. napus’ genetic variations and improving its root system architecture.

Abstract Human bocavirus 1 (HBoV1) is an autonomous parvovirus in the Bocaparvovirus genus. The multifunctional nuclear protein NP1 is involved in viral replication. In the present study, we found that the mutations in the C-terminus of NS1 affected NP1 function in viral replication. Knocking out NP1 expression in the recombinant infectious clone, on which the C-terminus of NS1 was mutated based on the clinical samples from nasopharyngeal aspirates, resulted in different degrees of decreased replication. The result suggested that NP1 facilitated the replication of viral genome but was not necessary, which is different from the minute virus of canines, where NP1 is essential for viral replication. Further studies showed that clinical mutations in the NP1 region did not affect viral genome replication, and UP1 promoted viral DNA replication. Our results suggested that the C-terminus of NS1 is important for viral replication and may be a target for regulating the replication of the viral genome.

Rapeseed (Brassica napus L.) is the largest oilseed crop in China and accounts for about 20% of world production. For the last 10 years, the production, planting area, and yield of rapeseed have been stable, with improvement of seed quality and especially seed oil content. China is among the leading countries in rapeseed genomic research internationally, having jointly with other countries accomplished the whole genome sequencing of rapeseed and its two parental species, Brassica oleracea and Brassica rapa. Progress on functional genomics including the identification of QTL governing important agronomic traits such as yield, seed oil content, fertility regulation, disease and insect resistance, abiotic stress, nutrition use efficiency, and pod shattering resistance has been achieved. As a consequence, molecular markers have been developed and used in breeding programs. During 2005–2014, 215 rapeseed varieties were registered nationally, including 210 winter- and 5 spring-type varieties. Mechanization across the whole process of rapeseed production was investigated and operating instructions for all relevant techniques were published. Modern techniques for rapeseed field management such as high-density planting, controlled-release fertilizer, and biocontrol of disease and pests combined with precision tools such as drones have been developed and are being adopted in China. With the application of advanced breeding and production technologies, in the near future, the oil yield and quality of rapeseed varieties will be greatly increased, and more varieties with desirable traits, especially early maturation, high yield, high resistance to biotic and abiotic stress, and suitability for mechanized harvesting will be developed. Application of modern technologies on the mechanized management of rapeseed will greatly increase grower profit.

In May 2016, a highly pathogenic avian influenza A(H5N8) virus strain caused deaths among 3 species of wild migratory birds in Qinghai Lake, China. Genetic analysis showed that the novel reassortant virus belongs to group B H5N8 viruses and that the reassortment events likely occurred in early 2016.

Enterovirus 71 (EV71) is the major causative pathogen of human hand, foot, and mouth disease (hHFMD) and has evolved to use various cellular receptors for infection. However, the relationship between receptor preference and EV71 virulence has not been fully revealed. By using reverse genetics, we identified that a single E98K mutation in VP1 is responsible for rapid viral replication in vitro. The E98K mutation enhanced binding of EV71-GZCII to cells in a heparan sulfate (HS)-dependent manner, and it attenuated the virulence of EV71-GZCII in BALB/c mice, indicating that the HS-binding property is negatively associated with viral virulence. HS is widely expressed in vascular endothelial cells in different mouse tissues, and weak colocalization of HS with scavenger receptor B2 (SCARB2) was detected. The cGZCII-98K virus bound more efficiently to mouse tissue homogenates, and the cGZCII-98K virus titers in mouse tissues and blood were much lower than the cGZCII virus titers. Together, these findings suggest that the enhanced adsorption of the cGZCII-98K virus, which likely occurs through HS, is unable to support the efficient replication of EV71 in vivo. Our study confirmed the role of HS-binding sites in EV71 infection and highlighted the importance of the HS receptor in EV71 pathogenesis.

Seed number is one of the key traits related to plant evolution/domestication and crop improvement/breeding. In rapeseed germplasm, the seed number per pod (SNPP) shows a very wide variation from several to nearly 30; however, the underlying causations/mechanisms for this variation are poorly known. In the current study, the genetic and cytological bases for the natural variation of SNPP in rapeseed was firstly and systematically investigated using the representative four high-SNPP and five low-SNPP lines. The results of self- or cross-pollination experiment between the high- and low-SNPP lines showed that the natural variation of SNPP was mainly controlled by maternal effect (mean = 0.79), followed by paternal effect (mean = 0.21). Analysis of the data using diploid seed embryo–cytoplasmic–maternal model further showed that the maternal genotype, embryo, and cytoplasm effects, respectively, explained 47.6, 35.2, and 7.5% of the genetic variance. In addition, the analysis of combining ability showed that for the SNPP of hybrid F1 was mainly determined by the general combining ability of parents (63.0%), followed by special combining ability of parental combination (37.0%). More importantly, the cytological observation showed that the SNPP difference between the high- and low-SNPP lines was attributable to the accumulative differences in its components. Of which, the number of ovules, the proportion of fertile ovules, the proportion of fertile ovules to be fertilized, and the proportion of fertilized ovules to develop into seeds accounted for 30.7, 18.2, 7.1, and 43.9%, respectively. The accordant results of both genetic and cytological analyses provide solid evidences and systematic insights to further understand the mechanisms underlying the natural variation of SNPP, which will facilitate the development of high-yield cultivars in rapeseed.

As the major determinant for nutrient uptake, root system architecture (RSA) has a massive impact on nitrogen use efficiency (NUE). However, little is known the molecular control of RSA as related to NUE in rapeseed. Here, a rapeseed recombinant inbred line population (BnaZNRIL) was used to investigate root morphology (RM, an important component for RSA) and NUE-related traits under high-nitrogen (HN) and low-nitrogen (LN) conditions by hydroponics. Data analysis suggested that RM-related traits, particularly root size had significantly phenotypic correlations with plant dry biomass and N uptake irrespective of N levels, but no or little correlation with N utilization efficiency (NUtE), providing the potential to identify QTLs with pleiotropy or specificity for RM- and NUE-related traits. A total of 129 QTLs (including 23 stable QTLs, which were repeatedly detected at least two environments or different N levels) were identified and 83 of them were integrated into 22 pleiotropic QTL clusters. Five RM-NUE, ten RM-specific and three NUE-specific QTL clusters with same directions of additive-effect implied two NUE-improving approaches (RM-based and N utilization-based directly) and provided valuable genomic regions for NUE improvement in rapeseed. Importantly, all of four major QTLs and most of stable QTLs (20 out of 23) detected here were related to RM traits under HN and/or LN levels, suggested that regulating RM to improve NUE would be more feasible than regulating N efficiency directly. These results provided the promising genomic regions for marker-assisted selection on RM-based NUE improvement in rapeseed.

Plant architecture is crucial for rapeseed yield and is determined by plant height (PH), branch initiation height (BIH), branch number (BN) and leaf and inflorescence morphology. In this study, we measured three major factors (PH, BIH, and BN) in a panel of 333 rapeseed accessions across 4 years. A genome-wide association study (GWAS) was performed via Q + K model and the panel was genotyped using the 60 k Brassica Infinium SNP array. We identified seven loci for PH, four for BIH, and five for BN. Subsequently, by determining linkage disequilibrium (LD) decay associated with 38 significant SNPs, we gained 31, 15, and 17 candidate genes for these traits, respectively. We also showed that PH is significantly correlated with BIH, while no other correlation was revealed. Notably, a GA signaling gene (BnRGA) and a flowering gene (BnFT) located on chromosome A02 were identified as the most likely candidate genes associated with PH regulation. Furthermore, a meristem initiation gene (BnLOF2) and a NAC domain transcriptional factor (BnCUC3) that may be associated with BN were identified on the chromosome A07. This study reveals novel insight into the genetic control of plant architecture and may facilitate marker-based breeding for rapeseed.

Silique shattering resistance is one of the most important agricultural traits in oil crop breeding. Seed shedding from siliques prior to and during harvest causes devastating losses in oilseed yield. Lignin biosynthesis in the silique walls is thought to affect silique-shattering resistance in oil crops. Here, we identified and characterized B. napus LATE FLOWERING (BnLATE), which encodes a Cys2/His2-type zinc-finger protein. Heterologous expression of BnLATE under the double enhanced CaMV 35S promoter (D35S) in wild-type Arabidopsis plants resulted in a marked decrease in lignification in the replum, valve layer (carpel) and dehiscence zone. pBnLATE::GUS activity was strong in the yellowing silique walls of transgenic lines. Furthermore, the expression pattern of BnLATE and the lignin content gradient in the silique walls at 48 days after pollination (DAP) of 73290, a B. napus silique shattering-resistant line, are similar to those in transgenic Arabidopsis lines expressing BnLATE. Transcriptome sequencing of the silique walls revealed that genes encoding peroxidases, which polymerize monolignols and lignin in the phenylpropanoid pathway, were down-regulated at least two-fold change in the D35S::BnLATE transgenic lines. pBnLATE::BnLATE transgenic lines were further used to identify the function of BnLATE, and the results showed that lignification in the carpel and dehiscence zone of yellowing silique also remarkably decreased compared with the wild-type control, the silique shattering-resistance and expression pattern of peroxidase genes are very similar to results with D35S::BnLATE. These results suggest that BnLATE is a negative regulator of lignin biosynthesis in the yellowing silique walls, and promotes silique-shattering resistance in B. napus through restraining the polymerization of monolignols and lignin.

Enterovirus A71 (EVA71) is a human enterovirus belonging to the Picornaviridae family and mostly causes hand-foot-and-mouth disease in infants. Viperin is an important interferon-stimulated gene with a broad antiviral activity against various viruses. However, the effect of viperin on human enteroviruses and the interaction mechanism between EVA71 and viperin remains elusive. Here, we confirmed the EVA71-induced expression of viperin in a mouse model and cell lines and showed that viperin upregulation by EVA71 infection occurred on both the mRNA and protein level. Viperin knockdown and overexpression in EVA71-infected cells indicated that this protein can markedly inhibit EVA71 infection. Interestingly, immunofluorescent confocal microscopy and co-immunoprecipitation assays indicated that viperin interacts and colocalizes with the EVA71 protein 2C in the endoplasmic reticulum. Furthermore, amino acids 50–60 in the N-terminal domain of viperin were the key residues responsible for viperin interaction with 2C. More importantly, the N-terminal domain of viperin was found responsible for inhibiting EVA71 replication. Our findings can potentially aid future research on the prevention and treatment of nervous system damage caused by EVA71 and may provide a potential target for antiviral therapy.

Primary root (PR) development is a crucial developmental process that is essential for plant survival. The elucidation of the PR transcriptome provides insight into the genetic mechanism controlling PR development in crops. In this study, we performed a comparative transcriptome analysis to investigate the genome-wide gene expression profiles of the seedling PRs of four Brassica napus genotypes that were divided into two groups, short group (D43 and D61) and long group (D69 and D72), according to their extremely different primary root lengths (PRLs). The results generated 55,341,366–64,631,336 clean reads aligned to 62,562 genes (61.9% of the current annotated genes) in the B. napus genome. We provide evidence that at least 44,986 genes are actively expressed in the B. napus PR. The majority of the genes that were expressed during seedling PR development were associated with metabolism, cellular processes, response to stimulus, biological regulation and signaling. Using a pairwise comparison approach, 509 differentially expressed genes (DEGs) (absolute value of log2 fold-change ≥1 and p ≤0.05) between the long and short groups were revealed, including phytohormone-related genes, protein kinases and phosphatases, oxygenase, cytochrome P450 proteins, etc. Combining GO functional category, KEGG and MapMan pathway analyses indicated that the DEGs involved in cell wall metabolism, carbohydrate metabolism, lipid metabolism, secondary metabolism, protein modification and degradation, hormone pathways and signaling pathways were the main causes of the observed PRL differences. We also identified 16 differentially expressed transcription factors (TFs) involved in PR development. Taken together, these transcriptomic datasets may serve as a foundation for the identification of candidate genes and may provide valuable information for understanding the molecular and cellular events related to PR development.

Seed weight is a very important and complex trait in rapeseed (Brassica napus L.). The seed weight of rapeseed shows great variation in its natural germplasm resources; however, the morphological, cytological and genetic causes of this variation have remained unclear. In the present study, nine highly pure inbred rapeseed lines with large seed weight variation and different genetic backgrounds were selected for morphological, cytological and genetic studies on seed weight. The results showed the following: (1) Seed weight showed an extremely significant correlation and coordinated variation with seed size (including seed diameter, seed surface area and seed volume), but it showed no significant correlation with bulk density, which suggests that seed weight is determined by size rather than bulk density. (2) Seed weight showed a higher correlation with the cell numbers of seed coats and cotyledons than the cell sizes of seed coats and cotyledons, which suggests that cell number is more tightly correlated with final seed weight. (3) Seed weight was mainly controlled by the maternal genotype, with little or no xenia and cytoplasmic effects. This is the first report on the morphological and cytological causes of seed weight natural variation in rapeseed. We concluded that the natural variation of seed weight is mainly controlled by maternal genotype. This finding lays a foundation for genetic and breeding studies of seed weight in rapeseed and opens a new field of research on the regulation of seed traits in plants.

In conventional approaches to plastid and mitochondrial genome sequencing, the sequencing steps are performed separately; thus, plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) should be prepared independently. However, it is difficult to extract pure ptDNA and mtDNA from plant tissue. Following the development of high-throughput sequencing technology, many researchers have attempted to obtain plastid genomes or mitochondrial genomes using high-throughput sequencing data from total DNA. Unfortunately, the huge datasets generated consume massive computing and storage resources and cost a great deal, and even more importantly, excessive pollution reads affect the accuracy of the assembly. Therefore, it is necessary to develop an effective method that can generate base sequences from plant tissue and that is suitable for all plant species. Here, we describe a highly effective, low-cost method for obtaining plastid and mitochondrial genomes simultaneously.First, we obtained high-quality DNA employing Partial Concentration Extraction. Second, we evaluated the purity of the DNA sample and determined the sequencing dataset size employing Vector Control Quantitative Analysis. Third, paired-end reads were obtained using a high-throughput sequencing platform. Fourth, we obtained scaffolds employing Two-step Assembly. Finally, we filled in gaps using specific methods and obtained complete plastid and mitochondrial genomes. To ensure the accuracy of plastid and mitochondrial genomes, we validated the assembly using PCR and Sanger sequencing. Using this method,we obtained complete plastid and mitochondrial genomes with lengths of 153,533 nt and 223,412 nt separately.A simple method for extracting, evaluating, sequencing and assembling plastid and mitochondrial genomes was developed. This method has many advantages: it is timesaving, inexpensive and reproducible and produces high-quality sequence. Furthermore, this method can produce plastid and mitochondrial genomes simultaneously and be used for other plant species. Due to its simplicity and extensive applicability, this method will support research on plant cytoplasmic genomes.

The effect of the number of pods on the main inflorescence (NPMI) on seed yield in Brassica napus plants grown at high density is a topic of great economic and scientific interest. Here, we sought to identify patterns of gene expression that determine the NPMI during inflorescence differentiation. We monitored gene expression profiles in the main inflorescence of two B. napus F6 RIL pools, each composed of nine lines with a low or high NPMI, and their parental lines, Zhongshuang 11 (ZS11) and 73290, using a Brassica 90K elements oligonucleotide array. We identified 4,805 genes that were differentially expressed (≥1.5 fold-change) between the low- and high-NPMI samples. Of these, 82.8% had been annotated and 17.2% shared no significant homology with any known genes. About 31 enriched GO clusters were identified amongst the differentially expressed genes (DEGs), including those involved in hormone responses, development regulation, carbohydrate metabolism, signal transduction, and transcription regulation. Furthermore, 92.8% of the DEGs mapped to chromosomes that originated from B. rapa and B. oleracea, and 1.6% of the DEGs co-localized with two QTL intervals (PMI10 and PMI11) known to be associated with the NPMI. Overexpression of BnTPI, which co-localized with PMI10, in Arabidopsis suggested that this gene increases the NPMI. This study provides insight into the molecular factors underlying inflorescence architecture, NPMI determination and, consequently, seed yield in B. napus.

Of 322 stool specimens collected from children with diarrhea from October 2005 through September 2006 in Wuhan, China, group A rotavirus was identified in 101 (31.4%). The most prevalent group A rotavirus genotype was G3P[8] (62.6%), followed by G1P[8](17.6%), G1+G3P[8](8.8%), G3P[4](6.6%), G1P[4](2.2%), and G9P[8](2.2%). The G9 strains were first detected in Wuhan.

Despite their ubiquity and functional importance, microsatellites have been largely ignored in comparative genomics, mostly due to the lack of genomic information. In the current study, microsatellite distribution was characterized and compared in the whole genomes and both the coding and non-coding DNA sequences of the sequenced Brassica, Arabidopsis and other angiosperm species to investigate their evolutionary dynamics in plants. The variation in the microsatellite frequencies of these angiosperm species was much smaller than those for their microsatellite numbers and genome sizes, suggesting that microsatellite frequency may be relatively stable in plants. The microsatellite frequencies of these angiosperm species were significantly negatively correlated with both their genome sizes and transposable elements contents. The pattern of microsatellite distribution may differ according to the different genomic regions (such as coding and non-coding sequences). The observed differences in many important microsatellite characteristics (especially the distribution with respect to motif length, type and repeat number) of these angiosperm species were generally accordant with their phylogenetic distance, which suggested that the evolutionary dynamics of microsatellite distribution may be generally consistent with plant divergence/evolution. Importantly, by comparing these microsatellite characteristics (especially the distribution with respect to motif type) the angiosperm species (aside from a few species) all clustered into two obviously different groups that were largely represented by monocots and dicots, suggesting a complex and generally dichotomous evolutionary pattern of microsatellite distribution in angiosperms. Polyploidy may lead to a slight increase in microsatellite frequency in the coding sequences and a significant decrease in microsatellite frequency in the whole genome/non-coding sequences, but have little effect on the microsatellite distribution with respect to motif length, type and repeat number. Interestingly, several microsatellite characteristics seemed to be constant in plant evolution, which can be well explained by the general biological rules.

Rapeseed (Brassica napus L.) is one of most important oilseed crops in the world. There are now various rapeseed cultivars in nature that differ in their seed oil content because they vary in oil-content alleles and there are high-oil alleles among the high-oil rapeseed cultivars. For these experiments, we generated doubled haploid (DH) lines derived from the cross between the specially high-oil cultivar zy036 whose seed oil content is approximately 50% and the specially low-oil cultivar 51070 whose seed oil content is approximately 36%. First, to address the deficiency in polymorphic markers, we designed 5944 pairs of newly developed genome-sourced primers and 443 pairs of newly developed primers related to oil-content genes to complement the 2244 pairs of publicly available primers. Second, we constructed a new DH genetic linkage map using 527 molecular markers, consisting of 181 publicly available markers, 298 newly developed genome-sourced markers and 48 newly developed markers related to oil-content genes. The map contained 19 linkage groups, covering a total length of 2,265.54 cM with an average distance between markers of 4.30 cM. Third, we identified quantitative trait loci (QTL) for seed oil content using field data collected at three sites over 3 years, and found a total of 12 QTL. Of the 12 QTL associated with seed oil content identified, 9 were high-oil QTL which derived from the specially high-oil cultivar zy036. Two high-oil QTL on chromosomes A2 and C9 co-localized in two out of three trials. By QTL mapping for seed oil content, we found four candidate genes for seed oil content related to four gene markers: GSNP39, GSSR161, GIFLP106 and GIFLP046. This information will be useful for cloning functional genes correlated with seed oil content in the future.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are an important class of genetic marker for target gene mapping. As of yet, there is no rapid and effective method to identify SNPs linked with agronomic traits in rapeseed and other crop species. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate a novel method for identifying SNP markers in rapeseed by deep sequencing a representative library and performing bulk segregant analysis. With this method, SNPs associated with rapeseed pod shatter-resistance were discovered. Firstly, a reduced representation of the rapeseed genome was used. Genomic fragments ranging from 450-550 bp were prepared from the susceptible bulk (ten F2 plants with the silique shattering resistance index, SSRI 0.90), and also Solexa sequencing-produced 90 bp reads. Approximately 50 million of these sequence reads were assembled into contigs to a depth of 20-fold coverage. Secondly, 60,396 'simple SNPs' were identified, and the statistical significance was evaluated using Fisher's exact test. There were 70 associated SNPs whose -log(10)p value over 16 were selected to be further analyzed. The distribution of these SNPs appeared a tight cluster, which consisted of 14 associated SNPs within a 396 kb region on chromosome A09. Our evidence indicates that this region contains a major quantitative trait locus (QTL). Finally, two associated SNPs from this region were mapped on a major QTL region. CONCLUSIONS/SIGNIFICANCE: 70 associated SNPs were discovered and a major QTL for rapeseed pod shatter-resistance was found on chromosome A09 using our novel method. The associated SNP markers were used for mapping of the QTL, and may be useful for improving pod shatter-resistance in rapeseed through marker-assisted selection and map-based cloning. This approach will accelerate the discovery of major QTLs and the cloning of functional genes for important agronomic traits in rapeseed and other crop species.

Pathogens have evolved sophisticated mechanisms to evade detection and destruction by the host immune system. Large DNA viruses encode homologues of chemokines and their receptors, as well as chemokine-binding proteins (CKBPs) to modulate the chemokine network in host response. The SECRET domain (smallpox virus-encoded chemokine receptor) represents a new family of viral CKBPs that binds a subset of chemokines from different classes to inhibit their activities, either independently or fused with viral tumor necrosis factor receptors (vTNFRs). Here we present the crystal structures of the SECRET domain of vTNFR CrmD encoded by ectromelia virus and its complex with chemokine CX3CL1. The SECRET domain adopts a β-sandwich fold and utilizes its β-sheet I surface to interact with CX3CL1, representing a new chemokine-binding manner of viral CKBPs. Structure-based mutagenesis and biochemical analysis identified important basic residues in the 40s loop of CX3CL1 for the interaction. Mutation of corresponding acidic residues in the SECRET domain also affected the binding for other chemokines, indicating that the SECRET domain binds different chemokines in a similar manner. We further showed that heparin inhibited the binding of CX3CL1 by the SECRET domain and the SECRET domain inhibited RAW264.7 cell migration induced by CX3CL1. These results together shed light on the structural basis for the SECRET domain to inhibit chemokine activities by interfering with both chemokine-GAG and chemokine-receptor interactions.

The pathogenesis of SARS coronavirus (CoV) remains poorly understood. In the current study, two recombinant baculovirus were generated to express the spike (S) protein of SARS-like coronavirus (SL-CoV) isolated from bats (vAcBS) and the envelope (E) and membrane (M) proteins of SARS-CoV, respectively. Co-infection of insect cells with these two recombinant baculoviruses led to self-assembly of virus-like particles (BVLPs) as demonstrated by electron microscopy. Incorporation of S protein of vAcBS (BS) into VLPs was confirmed by western blot and immunogold labeling. Such BVLPs up-regulated the level of CD40, CD80, CD86, CD83, and enhanced the secretion of IL-6, IL-10 and TNF-alpha in immature dendritic cells (DCs). Immune responses were compared in immature DCs inoculated with BVLPs or with VLPs formed by S, E and M proteins of human SARS-CoV. BVLPs showed a stronger ability to stimulate DCs in terms of cytokine induction as evidenced by 2 to 6 fold higher production of IL-6 and TNF-alpha. Further study indicated that IFN-gamma+ and IL-4+ populations in CD4+ T cells increased upon co-cultivation with DCs pre-exposed with BVLPs or SARS-CoV VLPs. The observed difference in DC-stimulating activity between BVLPs and SARS CoV VLPs was very likely due to the S protein. In agreement, SL-CoV S DNA vaccine evoked a more vigorous antibody response and a stronger T cell response than SARS-CoV S DNA in mice. Our data have demonstrated for the first time that SL-CoV VLPs formed by membrane proteins of different origins, one from SL-CoV isolated from bats (BS) and the other two from human SARS-CoV (E and M), activated immature DCs and enhanced the expression of co-stimulatory molecules and the secretion of cytokines. Finding in this study may provide important information for vaccine development as well as for understanding the pathogenesis of SARS-like CoV.

China's open pit mining industry faces the dual challenge of increasing production and preventing disasters. In order to ensure the safe exploitation of mineral resources, the stability of slopes must be assessed. In light of the fact that over 1,950 landslide accidents have occurred over the past decade, accounting for 15% of all safety incidents, the evaluation of slope stability has become a critical research focus in the fields of geo-resources and geo-engineering. Traditional slope stability evaluation methods rely on empirical tools and the expertise of professionals to assess slope stability. In contrast, machine learning (ML) methods offer a more comprehensive approach, analyzing the intricate features present in diverse sampling data. As a novel extension of ML, this paper presents a single-valued neutrosophic number-based ensemble learning (SVNN-EL) model. This model employs binary coding groups (1, 0, 0), (0, 1, 0) and (0, 0, 1) to express the learning outcomes of the slope stability, quasi-stability and instability statuses. Subsequently, a similarity measure is employed to determine the classification results of slopes. Finally, the proposed SVNN-EL model is applied to a case study in Yunnan province, China, the proposed model's four performance metrics, namely accuracy, precision, recall, and F1-score (the harmonic mean of precision and recall), are 0.915, 0.894, 0.948, and 0.921, respectively. A comparison with the k-nearest neighbor, support vector machine and random forest methods reveals that the performance metrics of the proposed SVNN-EL model are superior to those of existing methods. [ABSTRACT FROM AUTHOR]