Virus-induced gene silencing (VIGS) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology.  However, the investigation of gene silencing and editing in radish remains limited.  In this study, a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus (TRV)- and turnip yellow mosaic virus (TYMV)-mediated gene silencing vectors.  The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish.  The expression level of RsPDS was significantly inhibited using VIGS in ‘NAU-067’ radish leaves.  The rootless seedlings of ‘NAU-067’ were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences.  Nine adventitious roots were blue with GUS staining, and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing.  Furthermore, albino lines were generated with A. tumefaciens-mediated transformation of radish cotyledons.  Five base substitutions and three base deletions occurred at target sequence 2 in Line 1, and three base insertions and three base substitutions occurred at target sequence 1 in Line 2.  This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish, which will facilitate the genetic improvement of vital horticultural traits in radish breeding program

In recent years, Meloidogyne enterolobii has emerged as a major parasitic nematode infesting many plants in tropical or subtropical areas. However, the regions of potential distribution and the main contributing environmental variables for this nematode are unclear. Under the current climate scenario, we predicted the potential geographic distributions of M. enterolobii worldwide and in China using a Maximum Entropy (MaxEnt) model with the occurrence data of this species. Furthermore, the potential distributions of M. enterolobii were projected under three future climate scenarios (BCC-CSM2-MR, CanESM5 and CNRM-CM6-1) for the periods 2050s and 2090s. Changes in the potential distribution were also predicted under different climate conditions. The results showed that highly suitable regions for M. enterolobii were concentrated in Africa, South America, Asia, and North America between latitudes 30° S to 30° N. Bio16 (precipitation of the wettest quarter), bio10 (mean temperature of the warmest quarter), and bio11 (mean temperature of the coldest quarter) were the variables contributing most in predicting potential distributions of M. enterolobii. In addition, the potential suitable areas for M. enterolobii will shift toward higher latitudes under future climate scenarios. This study provides a theoretical basis for controlling and managing this nematode.

With the development of sequencing technology, insertions-deletions (InDels) have been increasingly reported to be involved in the genetic deter mination of agronomical traits.  However, most studies have focused on the identification and application of short-InDels (1–15 bp) for genetic analysis.  The objective of this study was to deeply deploy long-InDels (>15 bp) for the genetic analysis of important agronomic traits in soybean.  A total of 13 573 polymorphic long-InDels were identified between parents Zhongpin 03-5373 (ZP) and Zhonghuang 13 (ZH), which were unevenly distributed on 20 chromosomes of soybean, varying from 321 in Chr11 to 1 246 in Chr18.  Consistent with the distribution pattern of annotated genes, the average density of long-InDels in arm regions was significantly higher than that in pericentromeric regions at the P=0.01 level.  A total of 2 704 (19.9% of total) long-InDels were located in genic regions, including 319 large-effect long-InDels, which resulted in truncated or elongated protein sequences.  A previously identified QTL (qPH16) underlying plant height was further analyzed, and it was found that 26 out of 35 (74.3%) long-InDel markers located in the qPH16 region showed clear polymorphisms between parents ZP and ZH.  Seven markers, including three long-InDels and four previously reported SNP markers, were used to genotype 242 recombinant inbred lines derived from ZP×ZH.  As a result, the qPH16 locus was narrowed from a 960-kb region to a 477.55-kb region, containing 65 annotated genes.  Therefore, these long-InDels are a complementary genetic resource of SNPs and short-InDels for plant height and can facilitate genetic studies and molecular assisted selection breeding in soybean.

Plant height is an important agronomic trait, which is governed by multiple genes with major or minor effects.  Of numerous QTLs for plant height reported in soybean, most are in large genomic regions, which results in a still unknown molecular mechanism for plant height.  Increasing the density of molecular markers in genetic maps will significantly improve the efficiency and accuracy of QTL mapping.  This study constructed a high-density genetic map using 4 011 recombination bin markers developed from whole genome re-sequencing of 241 recombinant inbred lines (RILs) and their bi-parents, Zhonghuang 13 (ZH) and Zhongpin 03-5373 (ZP).  The total genetic distance of this bin map was 3 139.15 cM, with an average interval of 0.78 cM between adjacent bin markers.  Comparative genomic analysis indicated that this genetic map showed a high collinearity with the soybean reference genome.  Based on this bin map, nine QTLs for plant height were detected across six environments, including three novel loci (qPH-b_11, qPH-b_17 and qPH-b_18).  Of them, two environmentally stable QTLs qPH-b_13 and qPH-b_19-1 played a major role in plant height, which explained 10.56–32.7% of the phenotypic variance.  They were fine-mapped to 440.12 and 237.06 kb region, covering 54 and 28 annotated genes, respectively.  Via the function of homologous genes in Arabidopsis and expression analysis, two genes of them were preferentially predicted as candidate genes for further study.

Curvularia leaf spot, caused mainly by Curvularia lunata, is a widespread plant disease in China.  In the recent years, directional host selection by the pathogen, which likely results in the virulence differentiation in pathogens, is widely reported.  Among the hallmarks potentially associated to pathogen variation in virulence, superoxide dismutase gene Sod has been found to be closely related to the enhancement of virulence.  In the present study, the full-length of Sod was obtained via Blastn alignment against GenBank and the whole genome of C. lunata.  In order to understand the role of Sod in the virulence variation in C. lunata, targeted gene disruption was performed to construct Sod mutants.  The cell wall degrading enzyme (CWDE) activities and toxin production of ΔSod were not distinctly different from wild-type strain CX-3 and its complon.  However, at an early stage of infection, ΔSod virulence appeared to be lower than CX-3 and the complon, while at a later stage, its virulence gradually returned to the level of CX-3 and the complon.  Furthermore, the melanin production of ΔSod was significantly reduced compared to CX-3 and the complon, suggesting that Sod gene influences the virulence by regulating melanin production at an early stage of infection but is not essential for pathogenicity.  However, the disruption of Sod did not significantly affect the transcriptional expression of the melanin biosynthesis-associated genes, brn1 and scd.  Therefore, we infer that Sod in C. lunata are involved, to some extent, with the virulence in maize leaf, but still needs further studies to have a clear understanding of its mechanism.

Nutrient deficiency stresses often occur simultaneously in soil.  Thus, it’s necessary to investigate the mechanisms underlying plant responses to multiple stresses through identification of some key stress-responsive genes.  Quantitative real-time PCR (qRT-PCR) is essential for detecting the expression of the interested genes, of which the selection of suitable reference genes is a crucial step before qRT-PCR.  To date, reliable reference genes to normalize qRT-PCR data under different nutrient deficiencies have not been reported in plants.  In this study, expression of ten candidate reference genes was detected in leaves and roots of rapeseed (Brassica napus L.) after implementing different nutrient deficiencies for 14 days.  These candidate genes, included two traditionally used reference genes and eight genes selected from an RNA-Seq dataset.  Two software packages (GeNorm, NormFinder) were employed to evaluate candidate gene stability.  Results showed that VHA-E1 was the highest-ranked gene in leaves of nutrient-deficient rapeseed, while VHA-G1 and UBC21 were most stable in nutrient-deficient roots.  When rapeseed leaves and roots were combined, UBC21, HTB1, VHA-G1 and ACT7 were most stable among all samples.  To evaluate the stabilities of the highest-ranked genes, the relative expression of two target genes, BnTrx1;1 and BnPht1;3 were further determined.  The results showed that the relative expression of BnTrx1;1 depended on reference gene selection, suggesting that it’s necessary to evaluate the stability of reference gene prior to qRT-PCR.  This study provides suitable reference genes for gene expression analysis of rapeseed responses to different nutrient deficiencies, which is essential for elucidation of mechanisms underlying rapeseed responses to multiple nutrient deficiency stresses.

The cultivated soybean (Glycine max (L.) Merr.) was distinguished from its wild progenitor Glycine soja Sieb. & Zucc. in growth period structure, by a shorter vegetative phase (V), a prolonged reproductive phase (R) and hence a larger R/V ratio. However, the genetic basis of the domestication of soybean from wild materials is unclear. Here, a panel of 123 cultivated and 97 wild accessions were genotyped using a set of 24 presence/absence variants (PAVs) while at the same time the materials were phenotyped with respect to flowering and maturity times at two trial sites located at very different latitudes. The major result of this study showed that variation at PAVs is informative for assessing patterns of genetic diversity in Glycine spp. The genotyping was largely consistent with the taxonomic status, although a few accessions were intermediate between the two major clades identified. Allelic diversity was much higher in the wild germplasm than in the cultivated materials. A significant domestication signal was detected at 11 of the PAVs at 0.01 level. In particular, this study has provided information for revealing the genetic basis of photoperiodism which was a prominent feature for the domestication of soybean. A significant marker-trait association with R/V ratio was detected at 14 of the PAVs, but stripping out population structure reduced this to three. These results will provide markers information for further finding of R/V related genes that can help to understand the domestication process and introgress novel genes in wild soybean to broaden the genetic base of modern soybean cultivars.

Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Leaves, especially the flag leaves, as CHA initial recipients play a decisive role in inducing male sterility. To investigate effects of different treatment times of CHA-SQ-1 used, morphological, biochemical and physiological responses of wheat flag leaves were detected in this study. CHA induced programmed cell death (PCD) as shown in terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) and DNA laddering analysis. In the early phase, CHA-SQ-1 triggered organelle changes and PCD in wheat leaves accompanied by excess production of reactive oxygen species (O2 -. and H2O2) and down-regulation of the activities of superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD). Meanwhile, leaf cell DNAs showed ladder-like patterns on agarose gel, indicating that CHA-SQ-1 led to the activation of the responsible endonuclease. The oxidative stress assays showed that lipid peroxidation was strongly activated and photosynthesis was obviously inhibited in SQ-1-induced leaves. However, CHA contents in wheat leaves gradually reduced along with the time CHA-SQ-1 applied. Young flags returned to an oxidative/antioxidative balance and ultimately developed into mature green leaves. These results provide explanation of the relations between PCD and anther abortion and practical application of CHA for hybrid breeding.

The adaptability of soybean to be grown at a wide range of latitudes is attributed to natural variation in the major genes and quantitative trait loci (QTLs) that control flowering time and maturity. Thus, the identification of genes controlling flowering time and maturity and the understanding of their molecular basis are critical for improving soybean productivity. However, due to the great effect of the major maturity gene E1 on flowering time, it is difficult to detect other small-effect QTLs. In this study, aiming to reduce the effect of the QTL, associated with the E1 gene, on the detection of other QTLs, we divided a population of 96 recombinant inbred lines (RILs) into two sub-populations: one with the E1 allele and another with the e1nl allele. Compared with the results of using all 96 recombinant inbred lines, additional QTLs for flowering time were identified in the sub-populations, two (qFT-B1 and qFT-H) in RILs with the E1 allele and one (qFT-J-2) in the RILs with the e1nl allele, respectively. The three QTLs, qFT-B1, qFT-H and qFT-J-2 were true QTLs and played an important role in the regulation of growth period. Our data provides valuable information for the genetic mapping and gene cloning of traits controlling flowering time and maturity and will help a better understanding of the mechanism of photoperiod-regulated flowering and molecular breeding in soybean.

Nitrogen (N) is one of the macronutrients required for plant growth, and reasonable application of N fertilizers can increase crop yields and improve their quality. However, excessive application of N fertilizers will decrease N use efficiency and also lead to increases in N2O emissions from agricultural soils and many other environmental issues. Research on the effects of different N fertilizer management practices on wheat yields and N2O emissions will assist the selection of effective N management measures which enable achieving high wheat yields while reducing N2O emissions. To investigate the effects of different N management practices on wheat yields and soil N2O emissions, we conducted field trials with 5 treatments of no N fertilizer (CK), farmers common N rate (AN), optimal N rate (ON), 20% reduction in optimal rate+dicyandiamide (ON80%+DCD), 20% reduction in optimal rate+nano-carbon (ON80%+NC). The static closed chamber gas chromatography method was used to monitor N2O emissions during the wheat growing season. The results showed that there were obvious seasonal characteristics of N2O emissions under each treatment and N2O emissions were mainly concentrated in the sowing- greening stage, accounting for 54.6–68.2% of the overall emissions. Compared with AN, N2O emissions were decreased by 23.1, 45.4 and 33.7%, respectively, under ON, ON80%+DCD and ON80%+NC, and emission factors were declined by 22.2, 66.7 and 33.3%, respectively. Wheat yield was increased significantly under ON80%+DCD and ON80%+NC by 12.3 and 11.9%, respectively, relative to AN while there was no significant change in yield in the ON treatment. Compared with ON, overall N2O emissions were decreased by 29.1 and 13.9% while wheat yields improved by 18.3 and 17.9% under ON80%+DCD and ON80%+NC, respectively. We therefore recommend that ON80%+DCD and ON80%+NC be referred as effective N management practices increasing yields while mitigating emissions.

The novel cry1Ai gene that cloned from Bacillus thuringiensis strain SC6H8 encoded a protein exhibiting strong toxicity against Plutella xylostella and Chilo suppressalis in our previous study. Using the available information for the active fragments of other Cry toxins, eight truncated fragments were constructed to identify the minimal active fragment of Cry1Ai. All truncated fragments were expressed in Escherichia coli strain BL21 (DE3), and the insecticidal activity against 2nd- instar P. xylostella larvae was assessed using full-length Cry1Ai as a positive control. The results indicate that the minimal active fragment of the Cry1Ai toxin against P. xylostella is located between amino acid residues 36I and 605I, which is smaller than the regions previously reported for Cry1A. The first two amino acids (34T and 35P) on helix α-1 and whole helix α-2 of domain I and sheet β-32 of domain III are necessary for Cry1Ai toxin to keep its toxicity against P. xylostella.

In order to establish double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for detection of duck or goose flavivirus, polyclonal antibody against the flavivirus strain JS804 in geese and monoclonal antibody against the E protein of flavivirus strain JS804 in geese were used as the capture antibody and detection antibody, respectively. The optimal dilution of the capture antibody and detecting antibody capable of detecting the flavivirus strain JS804 in geese were 1:3 200 and 1:160 in the check-board titration, respectively. The reaction time of sample was 1 h, and the optimal working dilution of HRP-labeled goat-anti-mouse IgG was 1:10 000. The positive standard value was 0.247 (OD450 nm). The geese flavivirus could be detected at a minimal concentration of 1.875 μg mL-1. The ELISA had no cross-reaction with Newcastle disease virus (NDV), Avian influenza virus (AIV), Infectious bronchitis virus (IBV), Infectious bursal disease virus (IBDV), Duck hepatitis virus (DHV), and Gosling plague virus (GPV). Twenty clinical samples were detected by the DAS-ELISA and RT-PCR respectively, with the agreement rate of 75%. The results revealed that the DAS-ELISA possessed favorable specificity and higher sensitivity, indicating a suitable method for rapid detection of the duck or goose flavivirus.

Supercritical carbon dioxide (SC-CO2) extraction was employed to extract oil from Nigella glandulifera Freyn seed in this study. Response surface methodology (RSM) was applied to evaluate the effects of the process parameters (pressure, temperature, and CO2 flow rate) on oil yield of N. glandulifera seed. A Box-Behnken design was used to optimize the extraction parameters. The analysis of variance indicated that the linear coefficients of pressure and CO2 flow rate, the quadratic term coefficients of pressure and temperature and the interactions between pressure and temperature, as well as temperature and CO2 flow rate, had significant effects on the oil yield (P<0.05). The optimal conditions to obtain the maximum oil yield from N. glandulifera seed were pressure 30.84 MPa, temperature 40.57°C, and CO2 flow rate 22.00 L h-1. Under these optimal conditions, the yield of oil was predicted to be 38.19%. The validation experiment results agreed with the predicted values. The fatty acid composition of N. glandulifera seed oil extracted using SC-CO2 was compared with that of oil obtained by Soxhlet method. The results showed that the fatty acid compositions of oil extracted by the two methods were similar. Identification of oil compounds with gas chromatography-mass spectrometry (GC-MS) showed that the contents of unsaturated fatty acids linoleic acid (48.30%), oleic acid (22.28%) and saturated fatty acids palmitic acid (16.65%), stearic acid (4.17%) were the most abundant fatty acids in seed oil from N. glandulifera.

The investigation was carried out to study the response of winter rapeseed to potassium (K) feritlization and the critical soil available K level for current winter rapeseed production in the Yangtze River Valley (YRV) of China. A total of 132 field experiments were conducted in fields of farmers in the major winter rapeseed growing areas in YRV in 2000/2001 and 2004/2005 to 2006/2007 during growing season. Results of these field experiments showed that the average rapeseed yield increment resulting from 100 kg K ha^-1 application was 358 kg ha^-1, an increase over the control CK (no K) of 18.0% in 2005/2006 and 2006/2007. The average internal use efficiency (IE) of K was higher in the CK treatment (21.9 kg grain, kg-1 K uptake) than in the +K (100 kg K ha^-1) treatment (17.7 kg grain, kg^-1 K uptake). Winter rapeseed required 68.1 kg of K to produce 1 000 kg seed. The recovery efficiency of K fertilizer in rapeseed production averaged 39.3%. The K balance was negative, with an average net removal of 117.6 kg K ha^-1 in the CK treatment annually, and 56.8 kg K ha^-1 in the +K treatment. The results indicated that there was a significant negative relationship between yield increments by K application and soil available K content. Based on the relative yield of CK/+K at 90% level, the critical level of soil available K(NH₄OA_c-extractable K) was 135 mg kg^-1.

This study was carried out to investigate the transfection effect of exogenous gene into plant protoplast cell mediated by polyethylenimine (PEI) nanovector, based on PEI gene delivery system in the field of medical science. PEI/DNA complexes were prepared by using PEI polymer to bind the plant expression plasmid, pCMl205-GFPn. The ability of PEI combining and protecting DNA was investigated by agarose gel electrophoresis retardation assay. The surface characteristics of PEI/DNA complexes were observed with transmission electron microscope. The transfection efficiency of Arabidopsis thaliana protoplasts mediated by PEI/DNA complexes at different N/P ratios was analyzed based on observation of transient expression of green fluorescent protein with confocal laser scanning microscope. PEI could bind and condense DNA, and form stable 100-200 nm PEI/DNA complexes when the proportion of PEl and DNA is in the range of 5:1-1:4. Transfection efficiency of PEI/DNA complexes increased with N/P ratios in range of N/P<5 and reached the highest at N/P=5, and began to decrease beyond N/P>5 as higher toxicity to cells. The transfection efficiency of PEI/DNA complexes at N/P=5 was higher than PEG. This study confirmed that PEI nanovector could effectively mediate foreign gene entering into A. thaliana protoplast cell to obtain transient expression, which may be developed as a hopeful and novel transgenic method combined with plant protoplast regeneration.

Kernel length (KL) is one of the components determining grain weight (GW) in wheat.  In this study, we firstly detected a putative locus on chromosome arm 2BL from a mutant BLS2 with long kernels using a Bulked Segregant Analysis (BSA) combined with a 60 K SNP array.  This putative locus was then confirmed as a major and stable QTL based on linkage mapping.  The locus, Qkl.sau-BC-2B.1, was mapped in an interval of 0.4 cM, and phenotypic variance explained by it varied from 17.01 to 30.53% across different environments.  Effects of this locus was further verified in a second population.  The positive allele of the locus could significantly increase hundred-kernel weight and prolong anthesis date, but it did not affect plant height, tiller number, spike length, and spikelet number per spike.  Expression and sequencing analyses identified TraesCS2B02G478100, possessing a G to C transition variation leading to an amino acid change, as the likely candidate gene underlying the locus.  Further, a new model for analyzing the genetic basis of yield-related traits was proposed. Taken together, our results provide a foundation for subsequent gene mining and breeding utilization of this promising QTL for KL.

African swine fever (ASF) is an acute, hemorrhagic disease caused by the African swine fever virus (ASFV), with a mortality up to 100%. The disease poses a seriously threat to the global swine industry, yet no commercial vaccines or antiviral drugs are available other than in Vietnam. ASFV attenuation through serial passages is a key approach for vaccine development. In this study, a cell-adapted virus, named HLJ18/BK33, was successfully generated by serially passaging the ASFV Pig/HLJ/18 in wild boar kidney cells (BK2258). This adapted virus exhibited clear cytopathic effects (CPE) and replicated stably and efficiently in BK2258 cells and porcine alveolar macrophages. Whole-genome sequence analysis revealed that, compared with the Pig/HLJ/18 virus, HLJ18/BK33 had a large deletion of 6162 bp from sites 181,027 to 187,188, and four single nucleotide deletions that led to frameshift mutations, resulting in the truncated expression of three open reading frames (ORFs) (ASFV_G_ACD_00120, ASFV_G_ACD_00350, and A179L), and the fusion expression of two ORFs (MGF_110-14L and MGF_110-11L). Additionally, four genes exhibited missense mutations, leading to single amino acid changes. Five pigs intramuscularly inoculated with 10⁶ TCID₅₀ of HLJ18/BK33 remained healthy with normal body temperatures and no clinical signs, indicating a high attenuation of virulence for HLJ18/BK33 in pigs. Upon challenge with the parental Pig/HLJ/18 virus, four of the five inoculated pigs developed persistent high fever and ASF-related clinical signs and died within 13 days of the challenge; the remaining pig developed transient fever but survived until the end of the observation period. These results indicate that the HLJ18/BK33 virus is highly attenuated but cannot induce protection against the parental virulent virus. Even though the HLJ18/BK33 virus is not a good vaccine candidate, its stable replication and distinct CPE in BK2258 cells as well as its low biosafety risk make it a valuable resource for studies on virus-host interactions, antiviral drug screening, diagnostic methods, and biological characteristics.