Improving cotton fiber quality can increase the economic income of cotton farmers, however achieving high fiber quality without decreasing cotton fiber yield remains a major challenge in saline-alkaline cotton fields. A field experiment was conducted in 2020 and 2021 on saline-alkaline soil with cotton under drip irrigation to examine how amount and timing of leaching affected soils salinity, cotton fiber yield and quality. There were five leaching amounts (CK: 0 mm, W1: 75 mm, W2: 150 mm, W3: 225 mm and W4: 300 mm) and three leaching timings (T1: once at the seedling stage, T2: twice at the seedling and budding stages, and T3: thrice at the seedling, budding and pollen-setting stages). Soil salinity, soil nitrate nitrogen (NO₃-N), cotton nitrogen (N) uptake, irrigation water productivity (IWP), cotton fiber yield, fiber length, fiber uniformity, fiber strength, fiber elongation, micronaire and fiber quality index (FQI) were investigated. The results indicated that soil salinity and NO₃-N reduced with increasing leaching amount. The N uptake of cotton bolls was greater than in cotton leaves, stems and roots, and total N accumulation increased with increasing leaching amount. The optimal cotton fiber yield and IWP occurred in treatment W3T2, and were 3,199 and 2,771 kg ha⁻¹, and 0.5482 and 0.4912 kg m⁻³ in 2020 and 2021, respectively. Fiber length, strength, elongation, and uniformity increased with increasing leaching amount, while there was a negative relationship between fiber micronaire and leaching amount. Soil salinity,  NO₃-N and fiber micronaire were negatively correlated with fiber quality (i.e., length, strength, elongation and uniformity) and yield, nitrogen uptake of various organs (i.e., root, stems and leaves) and whole plant nitrogen uptake. Pearson correlation analysis revealed that fiber elongation was most sensitive to soil salinity. The method of Entropy–Order Preference by Similarity to Ideal Solution (EM–TOPSIS) indicated that leaching of 300 mm of water applied equally at the seedling and budding periods was the optimal treatment to maintain soil salinity and nutrient levels and achieve high cotton fiber yield and quality. In conclusion, the optimal level of leaching treatment decreased soil salinity and improved nitrogen uptake and was beneficial to achieve high fiber yield and quality. Our results will be significant for guiding drip irrigation practice of leaching on saline-alkaline soils for sustainable cotton fiber production.

The inflorescence and spikelet structural units of rice significantly impact grain development.  Among the grasses, the sterile lemma represents a unique spikelet organ exclusive to rice.  As most studies on sterile lemma genes primarily focus on Asian cultivated rice (Oryza sativa L.), genes influencing the sterile lemma phenotype in African rice remain unreported.  This study identified a gene, lsl3, located on the short arm of chromosome 7, which controlled the sterile lemma length in African rice.  The key mutation of lsl3 leading to long sterile lemmas in African rice was discovered and differed from that in Asian rice.  The investigation of lsl3 not only provides a reference for the molecular evolution of cultivated rice in Africa but also offers new evidence supporting the parallel domestication of rice and the independent origin of O. glaberrima.

Wheat grain morphology is an important breeding target considering its impact on yield and end-use properties.  However, the genetic basis of grain roundness, a major determinant of grain morphology, remains largely unexplored.  In this study, an F₂ and a recombinant inbred line (RIL) populations from Zhongkemai 138 (ZKM138)×Chinese Spring (CS) cross were employed to analyze the genetic basis of grain shape variation.  Kompetitive Allele Specific PCR (KASP) markers were developed according to single nucleotide polymorphism (SNP) from bulked segregant exome sequencing (BSE-Seq) of F₂ and Wheat 55K SNP array data online, and then were used to construct two genetic maps of F₂ and RIL populations, spanning 148.89 cM (30 KASP markers) and 129.82 cM (25 KASP markers), respectively.  By the traditional QTL mapping method based on these two maps, a stable quantitative trait locus (QTL) for grain roundness (GR), QGr.cib-5A, could be repeatedly highlighted in the interval of 444.8-455.5 Mb on chromosome 5A.  Further conditional QTL mapping analysis revealed that grain width was the major contributor to GR at this locus.  Besides, the utilization of two tightly linked markers 5A4-15 and 55k-31 showed a 96.27% transmissibility of ZKM138-derived alleles in 134 ZKM138 derivatives alongside a 7.38% increase in GR, and a 65.19% distribution of worldwide varieties.  Finally, TraesCS5A02G236400, possibly encoding a hydroxyproline-rich glycoprotein family protein, was deduced to be the candidate gene.  Collectively, these results provided the possibility of facilitating wheat grain shape improvement and enhancing wheat market value.

Cadmium (Cd) contamination in wheat farmland is increasing at an alarming rate, posing threats to food security and public health.  Breeding and utilizing wheat varieties characterized by low Cd accumulation levels constitute an effective strategy in the battle against wheat Cd contamination.  The adoption of molecular marker-assisted approaches can greatly expedite the selection and enhancement of wheat varieties with low Cd accumulation.  Nonetheless, research concerning genes associated with wheat cadmium accumulation remains scarce.  In this study, a high-density 660K SNP array was employed for conducting genome-wide association studies (GWASs) on the grain Cd concentration (GCdC), bioconcentration factor (BCF) and translocation factor (TF) in 175 wheat germplasms.  The findings revealed 401 significant SNPs identified across three diverse environments. Linkage disequilibrium analysis revealed 30 core quantitative trait locus (QTLs)capable of reliably modulating wheat Cd accumulation phenotypes.  Through gene annotation, transcriptomics, and gene molecular features, four candidate genes (TraesCS7B02G000200, TraesCS4A02G035900, TraesCS4A02G040900, TraesCS5D02G564000) were identified as potential constituents of the biological process of wheat Cd accumulation.  Furthermore, in this study 6 wheat germplasms exhibiting low grain Cd accumulation were isolated, and two Kompetitive Allele Specific PCR (KASP) markers conducive to breeding selection were developed.  These findings provide valuable genetic resources for cultivating wheat with low Cd accumulation and establish a foundation for understanding the molecular mechanisms underlying low Cd accumulation in wheat.  The candidate genes and KASP markers elucidated in this research have potential for effective employmentuse in genetic enhancement and marker-assisted selection in the breeding of wheat with low Cd accumulation.

Rapeseed mustard (Brassica juncea L.) is the third most important oilseed crop in the world but the genetic mechanism underlying its massive phenotypic variation remains largely unexplored. In this study, specific length amplified fragment sequencing (SLAF-Seq) was used to resequence a population comprising 197 F₈ recombinant inbred lines (RILs), derived from a cross between vegetable-type Qichi881 and oilseed-type YufengZC in B. juncea. In total, 438,895 high-quality SLAFs were discovered, of which 47,644 were polymorphic, and 3,887 of the polymorphic markers met the requirements for genetic map construction. The final map included 3,887 markers on 18 linkage groups and was 1,830.23 cM in length, with an average distance of 0.47 cM between adjacent markers. Using the newly constructed high-density genetic map, a total of 53 QTLs for erucic acid (EA), oleic acid (OA), and linolenic acid (LNA) were detected and integrated into 8 consensus QTLs with two for each of these traits. For each of these three traits, two candidate genes were cloned and sequence analyzed, indicating colocalization with their respective consensus QTLs. The co-dominant allele-specific markers for Bju.FAD3.A03 and Bju.FAD3.B07 were developed and showed co-localization with their consensus QTL and co-segregation with LNA content, further supporting the results of QTL mapping and bioinformatic analysis. The expression level for the cloned homologous genes was also identified, which was tightly correlated with the EA, OA and LNA contents of different lines. The results would facilitate the improvement of fatty acid traits and molecular breeding of B. juncea. More use of the high-density genetic map created in this study is also discussed. 

Soluble solids content (SSC) plays an important role in determining the flavor of tomato fruits. Tomato fruit SSC has been shown to be transcriptionally regulated via sugar metabolism. Previous studies have been predominantly focused on the role of C2H2-type zinc finger proteins in tomato growth and development. However, the specific regulatory mechanism of C2H2 in the accumulation of soluble solids in tomato fruits are yet to be fully understood. This study involved the selection of eight tomato accessions with varying levels of SSC to study the expression of SlC2H2 family genes in red ripe fruits. The study found that the levels of SlC2H2-71 expressions were significantly reduced in high-SSC accessions compared to low-SSC accessions. The Slc2h2-71 mutant lines were developed using the CRISPR-Cas9 system, leading to elevated levels of soluble solids, fructose, glucose, malic, and citric acids in mature red ripe fruits. However, sucrose content in the edited Slc2h2-71 mutant lines generally decreased. RNA-seq analysis revealed that fruits from the mutant lines had altered expression of genes related to sugar and acid metabolic pathways, which was further confirmed by quantitative real time PCR. Specifically, there was an observed increase in the expression of SlLIN5 encoding the cell wall invertase (CWIN). The yeast one-hybrid (Y1H) assay, 35S::UAS-GUS, dual-luciferase reporter systems and electrophoretic mobility shift assay (EMSA) demonstrated that SlC2H2-71 regulates tomato sugar metabolism by directly binding to the promoter region of SlLIN5, culminating in the repression of its transcriptional activity. The activity of acid invertase in the SlC2H2-71 knock-out lines exhibited a significantly higher level compared to that observed in the control lines. In summary, the regulation of tomato fruit SSC by SlC2H2-71 involves the inhibition of SlLIN5 expression.

Pyrus pyrifolia, commonly known as sand pear, is a key economic fruit tree in temperate regions and possesses highly diverse germplasm resources for pear quality improvement. However, research on the relationship between resistance and fruit quality traits during the breeding of fruit species like pear is limited. Pan-transcriptomes effectively capture genetic information from coding regions and reflect variations in gene expression between individuals. Here, we constructed a pan-transcriptome composed of 506 samples from different tissues of sand pear, and explored the intrinsic relationships among phenotypes and the selection for disease resistance during improvement based on expression presence/absence variation (ePAVs). The pan-transcriptome contains 156,744 transcripts, among which the novel transcripts show significant enrichment in the defense response. Interestingly, disease resistance genes are highly expressed in landraces of pear but have been selected against during the improvement of this perennial tree species. We found that genetically diverse landraces can be divided into two subgroups and inferred that they have undergone different dispersal processes. Through co-expression network analysis, we confirmed that the formation of stone cells in pears, the deposition of fruit anthocyanins, and the ability to resist stress are interrelated. They are jointly regulated by several modules, and the expression of regulatory genes has a significant correlation. Moreover, we identified candidate genes such as HKL1 that may affect sugar content and are missing from the reference genome. Our study provides insights into the associations between complex fruit traits, while providing a database resource for pear disease resistance and fruit quality breeding.

Watermelon (Citrullus lanatus) is an economically important horticultural crop. However, it is susceptible to low-temperature stress, which poses a significant challenge to its production and supply. Despite the great economic importance of watermelon, little is known about its response to low-temperature stress at the transcriptional level. In this study, we performed a time-course transcriptome analysis to systematically investigate the regulatory network of watermelon under low-temperature stress. Six low-temperature-responsive gene clusters representing six expression patterns were identified, revealing diverse regulation of metabolic pathways in watermelon under low-temperature stress. Analysis of temporally specific differentially expressed genes revealed the time-dependent nature of the watermelon response to low temperature. Moreover, ClMYB14 was found to be a negative regulator of low-temperature tolerance as ClMYB14-OE lines were more susceptible to low-temperature stress. Co-expression network analysis demonstrated that ClMYB14 participates in the low-temperature response by regulating the unsaturated fatty acid pathway and heat shock transcription factor. This study provides substantial information for understanding the regulatory network of watermelon in response to low-temperature stress, as well as identifies candidate genes for the genetic improvement of watermelon with higher low-temperature tolerance.

Fruit spine density is an important commercial trait for cucumber (Cucumis sativus L.). However, most North China-type cucumbers, which are grown over large areas, have a dense-spine phenotype, which directly affects the appearance quality, storage, and transportation of fruits. Here, we isolated a novel few spines mutant (fs2) from the wild-type (WT) inbred line WD1, a North China-type cucumber with high density fruit spines, by ethyl methanesulfonate (EMS) mutagenic treatment. Genetic analysis revealed that the phenotype of fs2 was controlled by a single recessive nuclear gene. We fine-mapped the fs2 locus using F₂ and BC₁ populations (1802 and 420 individuals, respectively) and showed that the candidate gene of FS2 (Csa4G652850) encoded an ARID-HMG transcription factor containing an A/T-rich interaction domain (ARID) and a high mobility group box domain (HMG). One SNP (C to T) and one InDel (a 40-bp deletion) in the coding region of FS2 resulted in amino acid variation and premature translation termination in the fs2 mutant, respectively. FS2 was highly expressed in the apical buds and young ovaries. In addition, the experiments suggest that FS2 participates in the regulation of fruit spine initiation by activating the expression of the Tril gene in cucumber. This work not only provides an important reference for understanding the molecular mechanisms of fruit spine development but also provides an important resource for fruit appearance quality breeding in cucumber.

Apple replant disease (ARD) is mainly caused by biological factors, and it severely restricts the development of the apple industry. The use of biological control measures to alleviate ARD is critically important for the sustainable development of the apple industry. The effects of raw amino acid powder and Trichoderma harzianum fertilizer on plant biomass, leaf and root indexes, soil physical and chemical properties, soil enzyme activity, and the soil fungal community were studied under pot and field conditions using Malus hupehensis Rehd. seedlings and grafted trees (Fuji New 2001/M9T337) as experimental materials. We found that the application of the materials significantly promoted plant growth, increased the leaf photosynthesis and chlorophyll content, root respiration rate, root antioxidant enzyme activity, and soil enzyme activity, significantly decreased the number of Fusarium sp. in soil, and significantly increased the abundance of beneficial bacteria. In conclusion, the mixed application of raw amino acid powder and Trichoderma harzianum fertilizer can is an effective method for the prevention and management of ARD.

CONSTANS (CO) and CONSTANS-LIKE (COL) transcription factors have been known to regulate a series of cellular processes, including the transition from vegetative growth to flower development in plants. However, their role in regulating the content of chlorophyll in fruit is poorly understood. In this study, SlCOL1, the tomato (Solanum lycopersicum) ortholog of Arabidopsis CONSTANS, was shown to play key roles in the control of fruit chlorophyll. The suppression of SlCOL1 expression was found to lead to a reduction in the content of chlorophyll in the immature green fruit. In contrast, the overexpression of SlCOL1 increased the content of chlorophyll in the immature green fruit. An analysis of protein-protein interactions indicated that SlCOL1 forms a complex with GOLDEN2-LIKE (GLK2), which promotes the stability of its protein. The overexpression of SlCOL1 in the glk2 null mutation background of tomato failed to promote the accumulation of chlorophyll in the immature green fruit, which suggests that GLK2 is required for the function of SlCOL1 in the regulation of the content of chlorophyll. These results shed new light into the mechanisms used by COL1 and GLK2 to regulate fruit development and the accumulation of chlorophyll in tomato.

Oxazolidinones, such as linezolid, represent the ‘last resort’ antimicrobial agents used to treat life-threatening human infections caused by multidrug-resistant (MDR) Gram-positive bacteria pathogens, such as Methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant enterococci (VRE) (Brenciani et al. 2022). However, the emergence of transferable oxazolidinone resistance genes including cfr, optrA and poxtA, has led to the widely spread of linezolid-resistant bacteria among healthcare, animal and environmental settings, posing a serious threat to public health. The cfr gene encodes an RNA methyltransferase that targets an adenine residue at position 2503 of 23S rRNA. However, this adenine residue is the binding region for phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A antibiotics. Thus, additional methylation at A2503 mediated by the Cfr protein prevents the binding of the different classes of antimicrobials mentioned above to the ribosome, thereby generating resistance to all of these antimicrobials (Kehrenberg et al. 2005; Long et al. 2006). Since the first description of the cfr gene in a bovine Staphylococcus sciuri isolate from Germany in 2000 (Schwarz et al. 2000), the cfr gene has been found in a wide variety of different Gram-positive and Gram-negative bacteria, including Staphylococcus, Enterococcus, Bacillus, Macrococcus, Jeotgalicoccus, Streptococcus, Escherichia, Proteus, Pasteurella, Morganella, Providencia, Vibrio and Leuconostoc (Brenciani et al. 2022). Proteus vulgaris is an important clinical opportunistic pathogen and widely exists in the soil, sewage and intestinal tracts of human and animals. P. vulgaris is closely associated with clinical diseases and usually causes urinary tract infections, wound and burn infections, and respiratory tract infections (Dutton and Ralston, 1957; Kippaz, 1957; Scott, 1960). Herein, we identified a MDR P. vulgaris strain HJ90 isolated from the lung sample of a pig from Heilongjiang province, China, and characterised a cfr-carrying plasmid pHJ90-cfr. To gain insight into the potential risks posed by the strain HJ90, the genetic background and transferability of the cfr gene located on it was elaborated.

Isolate HJ90 was identified as P. vulgaris by 16S rRNA gene sequencing. Antimicrobial susceptibility testing showed that P. vulgaris HJ90 was resistant to chloramphenicol, florfenicol, gentamicin, sulfamethoxazole, ampicillin and tetracycline according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI 2022). Whole genome sequencing (WGS) was performed using both Illumina NovaSeq and Nanopore MinION platforms. The short- and long-read data were de novo hybrid assembled using Unicycler 0.4.3 (Wick et al. 2017) and then annotated using the online RAST server (https://rast.nmpdr.org/) (Aziz et al. 2008) and BLASTP/BLASTN (https:// blast.ncbi.nlm.nih. gov/Blast.cgi). The complete genome of isolate HJ90 was obtained successfully and deposited in the GenBank database under accession numbers (CP150645-CP150647). The genome of P. vulgaris HJ90 harbored a circular chromosome (4,166,418 bp) and two plasmids, designated pHJ90-1 (93,273 bp with an average G + C content of 47%) and pHJ90-cfr (30,447 bp with an average G + C content of 35%), respectively. Acquired antimicrobial resistance genes (ARGs) were predicted using ResFinder 4.4.3 on the Center for Genomic Epidemiology (CGE) server (http://genomicepidemiology.org/services/) (Bortolaia et al. 2020). The result showed that three ARGs, including aadA1, catB2 and dfrA1 were present in the chromosome. In addition, multiple ARGs were detected on plasmid pHJ90-1, including aph(4)-la, aadA16, aph(6)-ld, aph(3')-la, aph(3")-lb, aac(3)-IV, aac(6')-lb-cr, bla_OXA-1, floR, catB3, arr-3, sul1, sul2, tet(B) and dfrA27, and no ARGs was detected on plasmid pHJ90-cfr except cfr.  

Plasmid pHJ90-cfr consisted of 41 predicted ORFs with an average G + C content of 35% (Fig. 1a). BLAST analysis revealed that no plasmid with significant homology to pHJ90-cfr was found except pJPM35-2 (GenBank accession no. CP053900). Plasmid pHJ90-cfr displayed 99.98% identity with 96% query coverage to plasmid pJPM35-2, which was recovered from Proteus mirabilis strain YPM35 of duck origin in China in 2021 (Zheng et al. 2021). The plasmid replicon of pHJ90-cfr was identified using PlasmidFinder 2.1 (https://cge.food.dtu.dk/services/PlasmidFinder/), but could not be assigned to any known Inc group in the database. Interestingly, sequence analysis showed the rep gene encoding plasmid replication initiation protein was absent on pHJ90-cfr. A plasmid segregation protein-encoding gene parM was present on plasmid pHJ90-cfr. A number of conjugative transfer genes were found on pHJ90-cfr, including virB4, virB5, virB8, virB9, virB10, tfc2, virB1, trbl, virB11 and virB3. To evaluate the transferability of plasmid pHJ90-cfr, conjugative transfer assay was carried out using filter mating as previously described (Yang et al. 2023). The result indicated that pHJ90-cfr was able to transfer into the recipient Escherichia coli C600 (streptomycin resistant), which was consistent with the previous report (Zheng et al. 2021). Additionally, antimicrobial susceptibility testing showed that the positive transconjugant displayed resistance to florfenicol and chloramphenicol. Genetic environment analysis suggested that the cfr gene, together with three hypothetical protein genes hp, were flanked by two copies of IS26 elements located in the same orientation (IS26-hp-hp-hp-cfr-IS26). BLASTN search showed that the segment bracketed by IS26 displayed >99% nucleotide sequence identity to the corresponding region of plasmid pJZ27-cfr from P. mirabilis isolated from chicken meat in China (Fig. 1b). It is suggested that the IS26-flanking segment may have the ability to spread widely across bacterial species, especially among Proteus spp. Based on previous reports (Harmer et al. 2014; Zhu et al. 2021), a circular translocatable unit (TU) can form when two copies of insertion sequence (IS) elements belonging to IS6 family orientated in the same direction and flanking resistance genes. To test whether such a TU was generated, a PCR assay was conducted and the result demonstrated that a circular TU mediated by IS26 was formed. Sequence analysis showed that this TU contained an IS26 copy and the central region between two IS26 copies. Since the sequences of two IS26 copies were identical, it is impossible to determine which one was included in this TU. This finding further supports the view that IS26 plays a vital role in promoting the dissemination of cfr gene in Gram-negative bacteria (Harmer et al. 2014; Liu et al. 2022; Mei et al. 2022; Wang et al. 2012). Furthermore, sequence alignment revealed that compared with plasmid pHJ90-cfr, pJPM35-2 carried an extra ~4-kb fragment, which was flanked by two IS5-like elements copies orientated in the same direction (Fig. 1b). Sequence analysis showed that IS5-like element was 853 bp in length and contained a 756 bp ORF for a transposase of 251 amino acids (aa). Blast searches showed that this transposase shared 57% aa similarity with that of ISAba31, a member of IS5 family. Therefore, it is reasonable to speculate that the IS5-like element was new IS5-family member. This new IS element was submitted to the ISfinder database and received the designation ISPmi4. Immediately up- and downstream of the fragment bounded by ISPmi4, 2-bp direct repeat sequences (5’-TA-3’) were found, which was a typical characteristic of classical composite transposons. This suggested that ISPmi4-flanking segment was inserted into the precursor of plasmid pJPM35-2, resulting in the structure seen on pJPM35-2. A search on PubMed database revealed that all identified cfr-carrying Proteus spp. were found in food-producing animals or retail meat in China until now (Table S1), which implied that Proteus spp. from food-producing animals may be a vital reservoir for cfr gene in China. Additionally, the cfr gene was found mainly located on two kinds of mobile genetic elements, namely plasmids and integrative and conjugative elements (ICEs) in Proteus. The above result suggested that plasmids and ICEs were key vectors for horizontal transmission of cfr gene in Proteus spp.

In summary, a novel conjugative plasmid pHJ90-cfr from P. vulgaris was characterized, which harbored the multiresistance gene cfr. In addition, a new IS5-family member, ISPmi4, was identified. The presence of cfr on plasmids, especially on conjugative plasmids, deserves our attention. Our findings highlight that the prevalence of cfr gene in gram-negative bacteria should be further monitored.

Although African swine fever (ASF) has been prevalent for more than a century, it remains the number one swine disease that seriously endangers the global pig industry, and there is no effective means of prevention and treatment (Wang et al. 2023). Due to its enormous economic and social impact, it is listed as a notifiable animal disease by the World Organization for Animal Health (Costard et al. 2013). ASF has been present in sub-Saharan Africa since its first discovery in Kenya. During the 1950s and 1970s, it spread to Western Europe and Latin America, but most were quickly eradicated. In 2007, it spread to Russia and its neighboring Eastern European countries (Pejsak et al. 2014; Martinez-Lopez et al. 2015), and in 2018, it was first transmitted to China and quickly spread to other Asian countries and regions, posing a serious threat to global pork production (Dixon et al. 2020).

African swine fever virus (ASFV), the causative agent of ASF, is an icosahedral nucleocytoplasmic large DNA virus belonging to the genus Asfivirus in the family Asfarviridae (Alonso et al. 2018). To date, more than 150 proteins encoded by ASFV have been identified; however, the functions of most of them remain unknown. The structure of ASFV virion is extremely complex, consisting of five layers, namely outer envelope, capsid, inner envelope, core shell, and nucleoid (Liu et al. 2019). Among them, the core shell is mainly composed of two polyproteins, pp62 and pp220. Both play an important role in the correct assembly of ASFV core shell and viral replication (Andrés et al. 1997; Suárez et al. 2010). The pp62, encoded by the CP530R gene, is located in the viral factory and can be proteolytically cleaved into mature proteins p35 and p15 by the ASFV cysteine protease pS273R in the late stage of infection (Simón-Mateo et al. 1997; Andrés et al. 2002; Jia et al. 2017). The pp62 derivatives p35 and p15 are important structural proteins of ASFV, which participate in the formation of icosahedral capsid structure. Moreover, pp62 was demonstrated to be a late protein that is incredibly immunogenic and is highly conserved among different ASFV strains (Simón-Mateo et al. 1997). Considering the important properties of pp62 and its critical role in the ASFV replication cycle, development of monoclonal antibodies (mAbs) against pp62 can provide valuable tools for the diagnosis and basic research of ASFV.

In this study, multiple bioinformatics analyses of pp62 showed that its 1−180 amino acid (aa) region has good antigenicity and solubility, and contains abundant antigenic epitopes (Appendix A). Therefore, this region was prokarytically expressed as a His-tagged fusion protein (designated pp62_(1−180aa)-His) by constructing a recombinant plasmid pET-28a(+)-pp62_(1−180aa)-His, transforming it into E. coli Rosette (DE3) cells and then inducing with isopropyl β-D-1-thiogalactopyranoside. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a protein band of ~21 kDa was detected in the supernatant of transformed cell lysates (Appendix B), and its size was consistent with the theoretical calculation. The soluble pp62_(1−180aa)-His was affinity-purified from the supernatants of bacterial lysates using Ni-NTA agarose under native conditions. SDS-PAGE analysis indicated that the purity of purified pp62_(1−180aa)-His was about 95%, and western blot (WB) analysis further revealed that the protein could be recognized by mouse anti-His-tag mAb and swine ASFV antiserum (Fig. 1-A). The purified pp62_(1−180aa)-His was used to immunize 6-week-old BALB/c mice subcutaneously using 60 μg protein emulsified in Freund's complete adjuvant, followed by two booster immunizations with 30 μg protein emulsified in Freund's incomplete adjuvant at two-week interval. Splenocytes from the immunized mice were fused with SP2/0 cells. By means of indirect ELISA screening combined with three rounds of subcloning, two monoclonal hybridoma cell clones (4B5-19-7 and 4C5-11-6) stably secreting anti-pp62 mAbs were obtained, and then intraperitoneally injected into BALB/c mice to produce ascites. The titers of 4B5-19-7 and 4C5-11-6 in ascites were 1:204800 and 1:409600, respectively, as determined by indirect ELISA (Appendix C). The isotypes of mAbs 4B5-19-7 and 4C5-11-6 were determined to be IgG2b/κ and IgG1/κ, respectively (Appendix D). Furthermore, RT-PCR was performed to amplify the heavy- and light-chain variable regions of each mAb using the RNA extracted from hybridoma cells as the template and with the specific primers (Appendix E, F). The amplicons were sequenced and the results were submitted to the GenBank database under accession numbers PQ037594−PQ037597 (Table 1). The corresponding aa sequences were annotated using the Kabat Antibody Numbering Scheme online tool to show the complementarity-determining regions (CDR), including CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The results showed that aa sequences of the heavy-chain CDR regions of pp62 mAbs are different from each other, while their light-chain CDR region aa sequences are completely identical (Table 1). Next, the reaction of mAbs with pp62 protein was analyzed by an indirect immunofluorescence assay (IFA), which showed that both mAbs react well with the natural pp62 protein in ASFV-infected WSL cells (Fig. 1-B). However, the two mAbs showed no cross reactivity with other important porcine pathogens, such as porcine reproductive and respiratory syndrome virus and Getah virus (Appendix G), indicating that they have good specificity. WB analysis further demonstrated that pp62 mAbs could recognize a ~62 kDa protein band in ASFV-infected WSL cells (Fig. 1-C), which matches the theoretical molecular weight of the natural pp62. Considering that the protein denaturation condition was used when analyzing the reactivity of mAbs with pp62 by WB, it was concluded that the epitopes recognized by two mAbs are linear. Notably, the expression of pp62 was detectable from 12 h post-infection onward, which is consistent with the previous reports that pp62 is a late protein (Andrés et al. 2002; Simón-Mateo et al. 1997).

To our knowledge, there are very few reports on preparation of mAbs against the ASFV pp62 protein. Bai and colleagues prepared eighteen mAbs against pp62, of which fifteen recognize p15 and the remaining three recognize p35 (Bai et al. 2020). In addition, Chu et al. generated four mAbs against pp62, of which two recognize pp62 and the remaining recognize p15 and p35, respectively (Chu et al. 2022). Interestingly, when using WB to analyze the reaction of pp62 mAbs with the total proteins of ASFV-infected WSL cells, we found that in addition to producing a clear protein band at ~62 kDa, the 4B5-19-7 also produced a clear band at ~15 kDa, while 4C5-11-6 at ~35 kDa (Fig. 1-D). This suggests that the epitopes recognized by pp62 mAbs should be located at different positions in pp62. Previous studies have shown that pp62 needs to be proteolytically cleaved into two mature structural proteins, p15 and p35, when assembled into the core shell in the late stage of ASFV infection (Simón-Mateo et al. 1997; Andrés et al. 2002; Jia et al. 2017). The cleavage of pp62 has been demonstrated to be mediated by the ASFV pS273R, with cleavage sites located at G158-G159 and G463-G464 (Appendix H). The resultant p15 and p35 are composed of aa at positions 1−158 and 159−462 of pp62, respectively (Simón-Mateo et al. 1997; Suárez et al. 2010). To verify whether 4B5-19-7 and 4C5-11-6 recognize p15 and p35, respectively, the eukaryotic recombinant plasmids pCMV-HA-p15 and pCMV-HA-p35 constructed with the primers shown in Appendix F were individually transfected into WSL cells, which were then analyzed by IFA. The results showed that 4B5-19-7 only reacted with p15, while 4C5-11-6 only reacted with p35 (Appendix I). This indicates that pp62 mAbs recognize different antigenic epitopes.

To identify the epitopes recognized by pp62 mAbs, a series of recombinant prokaryotic plasmids respectively expressing GST-tagged pp62_(1−180aa) and 19 additional truncated pp62_(1−180aa) were constructed (Appendix F, J). Fig. 1-E shows that only the truncated proteins containing 95–105 aa (SYTGVKLEVEK) were able to react with both 4B5-19-7 and GST polyclonal antibody, while those lacking this domain could not. This indicate that the epitope recognized by 4B5-19-7 is located at 95–105 aa of pp62. Similarly, the epitope recognized by 4C5-11-6 was shown to be located at 171–180 aa (YTPRTRIAIE). As illustrated in Appendix H, the epitopes recognized by mAbs 4B5-19-7 and 4C5-11-6 are located exactly within the aa range of p15 and p35.

To analyze whether the epitopes recognized by pp62 mAbs are conserved, a total of 36 pp62 aa sequences of representative ASFV strains of different genotypes currently available in the GenBank database were downloaded and analyzed. These ASFV strains used for analysis originate from 21 countries in Africa, Europe, and Asia, involving 12 genotypes. The results showed that the pp62 is highly conserved among ASFV strains of different genotypes, and the epitopes recognized by pp62 mAbs are more conserved, without any mutations in the aa regions where the epitopes are located (Fig. 1-F).

In summary, we successfully prepared two mAbs that can specifically recognize the ASFV pp62. Among them, 4B5-19-7 and 4C5-11-6 can further recognize the p15 and p35 of pp62 cleavage products, respectively. The epitopes recognized by 4B5-19-7 and 4C5-11-6 are highly conserved and located at amino acids 95−105 (SYTGVKLEVEK) and 171−180 (YTPRTRIAIE) of pp62, respectively. The mAbs provide valuable materials for pp62 functional study and development of improved serological diagnostic agents and assays.

The Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in innate immunity by inducing antiviral proteins in response to interferon signals. Marek’s disease virus (MDV), a member of the alpha-herpes virus family, exerts potent tumorigenic and immunosuppressive effects. Recent studies have primarily focused on the tumorigenic mechanisms of MDV, and the mechanism of immune evasion has not been fully understood. In this study, we showed that MDV reduced the production of interferon-stimulated gene (ISGs) by inhibiting the phosphorylation and nuclear translocation of STAT1. Using a dual-luciferase reporter system, we screened for viral proteins that significantly suppress interferon-stimulated response element (ISRE) promoter activity. Meq overexpression markedly reduced ISRE promoter activity and ISG expression, whereas infection with Meq-deficient MDV induced higher ISG production in vitro and in vivo than infection with wild-type MDV. Meq also inhibited the phosphorylation and nuclear translocation of STAT1. Further experiments showed that Meq interacted with JAK1 and tyrosine kinase 2 (TYK2) and thereby inhibited JAK1–STAT1 interactions. Meq degraded TYK2 via a caspase-mediated pathway. The Meq-deficient MDV mutant replicated less efficiently than the wild-type MDV, both in vitro and in vivo. Collectively, these findings demonstrate that Meq played an immunosuppressive role in MDV by attenuating the JAK–STAT signaling pathway, which facilitated escape from innate immune-surveillance mechanisms.

Nipah virus (NiV) is a member of the genus Henipavirus of the family Paramyxoviridae, and is an emerging zoonotic virus (Singh et al. 2019). NiV was first observed in 1998 and this outbreak triggered an uncontrollable epidemic lasting until 1999, during which more than a million pigs were destroyed (Ang et al. 2018). Since then, several South and Southeast Asian countries have experienced relatively small sporadic outbreaks almost every year, with mortality rates of up to 40-75 % in humans infected with NiV (Raj Kumar et al. 2019). Therefore, NiV not only does terrible damage to the farming industry but is also a serious threat to human public health and safety. Fruit bats are recognized as natural reservoir hosts for NiV, and other species of bats, as well as pigs, horses, dogs, and humans can also be infected (Soman Pillai et al. 2020). Bats act as asymptomatic carriers of the virus, but they excrete the virus through their saliva, urine, semen, and feces (Soman Pillai et al. 2020). NiV infection in humans and animals primarily occurs through the consumption of date palm juice and fruits that have been contaminated by fruit bats (Bruno et al. 2022). Human-to-human transmission is also possible (Garbuglia et al. 2023). NiV infection in humans and pigs can result in fatal encephalitis and severe respiratory disease (Ma et al. 2019, Ming-Yen et al. 2020). Because of its high pathogenicity and mortality, as well as the lack of effective treatments or vaccines for humans or animals (Gómez Román et al. 2022), NiV is included in the list of epidemiological threats requiring urgent R&D action in the World Health Organization (WHO) R&D Blueprint as one of the pathogens. WHO has therefore prepared a technical brief aimed at guiding countries in their preparedness planning for a NiV event, particularly in countries that have not yet reported a NiV event. The technical brief suggests that early diagnosis of NiV can enhance the survival chances of an infected individual and can also prevent transmission to others.

Currently, the detection methods for NiV are mainly based on traditional serological methods and molecular diagnostic methods. Of the serological detection methods, ELISA, phage plaque assay, and immunofluorescence staining do not require special instruments or equipment, but the NiV-infected samples need to be processed in a Biosafety-Level-4 (BSL-4) laboratory, which is risky for operators and is not available in all regions (Diwakar D et al. 2015, Fischer et al. 2018, Gary et al. 2001). Real-time reverse transcriptase polymerase chain reaction (RT-PCR) tests are the preferred method for viral detection due to their high sensitivity and ability to detect the virus in patients at the earliest stages of infection. However, Real-time RT-PCR requires sophisticated instruments and equipment, as well as specialized operator personnel, and cannot fully satisfy the need for early diagnosis of NiV infection in situ or in poor areas lacking these conditions (Wu et al. 2022). Therefore, the development of a rapid, specific, and sensitive assay that does not require complex instrumentation is critical for the early detection of NiV.

Nucleic acid isothermal amplification technology, characterized by its rapidity, sensitivity, and specificity, is capable of meeting the demands for speed and simplicity detection, and has significant practical application value (Zhao et al. 2015). Of these methods, recombinase-aided amplification (RAA) technology, with its simplicity and ease of use, is ideal for early diagnosis in resource-limited settings (James et al. 2018). Recombinant enzyme-based isothermal amplification tests have been used to develop three rapid assays for NiV that can detect 1,000 copies μL^-1 for synthetic NiV RNA in less than 30 min (Pollak et al. 2023). However, these assays require the transfer of the RPA amplification products, exposing the nucleic acid-rich samples to the environment, and leading to potential aerosol contamination. In the present study, a fully enclosed device was employed, combining reverse transcription recombinase-aided amplification technology with lateral flow immunochromatography (RT-RAA-VF), to establish a rapid and sensitive assay for the detection of NiV. This assay avoids false positives caused by aerosols, does not require sophisticated instruments and equipment, does not require specialized personnel to operate, and holds the potential to fulfill the requirements for early diagnosis during the initial stages of a NiV infection outbreak even in poor and remote areas.

To detect all known NiV strains, 55 P gene sequences of different NiV strains, including NiV-B (transmitted in Bangladesh and India), NiV-M (transmitted in Malaysia), and NiV-T (transmitted in Thailand) were retrieved from GenBank (https: //www.ncbi.nlm.nih.gov/) and compared using MAFFT version 7. The highly conserved region of the P gene was selected as the target and was used to design the specific RAA primers and probe by the Prime Primer 5.0 software. The probe contained 55 nucleotides with the tetrahydrofuran (THF) residue replacing the adenine at position 37. The 5' end of the probe was labeled with the fluorescent marker FAM, while the 3' end was labeled with a blocking group comprising a C3 spacer. The 5' end of the reverse primer was labeled with biotin (Table 1). The primers and probe were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The RNA transcript of NiV P (Genebank: NC_002728) and the positive plasmid pcDNA3.1-NiV-P were also synthesized by Sangon Biotech. In the presence of NiV RNA, the reverse transcriptase is activated to reverse transcribe the NiV RNA to cDNA, and the RAA amplification reaction is followed activated by two priming oligonucleotides to produce targets for probe annealing hybridization. The THF site of the probe can then be cleaved by the nfo enzyme to create a new 3'-hydroxyl group, which is used as a start site for polymerase extension, thus converting the probe into a primer that, together with a reverse primer with biotin, produces double-stranded amplification products labeled with two antigens (FAM and biotin). Subsequently, the reaction tube, which contains the amplified products, is positioned within a sealed and disposable nucleic acid visualization paper device. The amplified DNA products are capable of adhering to streptavidin-coated gold nanoparticles, thereby forming a conjugate that is subsequently immobilized by the anti-FAM antibody present on the test line (T line), which manifests as a red stripe. Meanwhile, any streptavidin-labeled gold nanoparticles that are not associated with the amplified DNA are trapped by the anti-streptavidin antibody at the control line (C line) (Fig. 1-A).

To find the optimal RT-RAA reaction time, 10-fold gradient dilutions of the plasmid pcDNA3.1-NiV-P (5×10¹⁰ copies μL^-1) were used as targets for RT-RAA-VF assay. The reaction was performed at three distinct temperatures—42 °C, 39 °C, and 37 °C—for a consistent period of 20 min. The outcomes indicated that at 42 °C, the emergence of discernible bands on both the C and T lines, indicating of a positive result, was observed at plasmid concentrations of 5 copies μL^-1 or greater. At 39 °C and 37 °C, the positive results were only observed at plasmid concentrations of 500 copies μL^-1 or more. These results suggested that 42 °C was the optimal reaction temperature for the NiV RT-RAA assay (Fig. 1-B). To determine the most efficient amplification time, the assay was executed for time intervals of 15, 20, 25, and 30 min. Analysis of the data provided in Appendix A confirmed that the optimal amplification duration for the assay was 20 min.

Next, we assessed the sensitivity of the assay using 10-fold gradient dilutions of NiV P RNA transcripts, reacting at 42 °C for 20 min. The results showed that this assay could detect as few as 5 copies μL^-1 of RNA transcripts (Fig. 1-C). We further compared the sensitivity of this assay with that of the real-time RT-PCR method currently recommended by the World Organisation for Animal Health (WOAH), using 10-fold serial dilutions of RNA extracted from NiV-infected Vero E6 cells as templates.  The results showed that the two methods shared an identical detection limit, and both were capable of detecting NiV RNA from infected cells diluted 10,000 times (Appendix B). To evaluate the specificity of this assay, the nucleic acids of pathogens that can cause neurological symptoms similar to NiV were also tested, including Hendra virus (HeV), herpes simplex virus type 1 (HSV-1), rabies virus (RABV) and Streptococcus suis. The results showed that our established assay only recognized the nucleic acids of NiV, with no cross-reactivity observed with other pathogens (Fig. 1-D).

It has been reported that NiV can be transmitted by droplets, or by contact with throat or nasal secretions, from the respiratory tract of patients or sick pigs. To validate the clinical applicability of this assay, we mixed NiV RNA with RNA extracted from human saliva samples to simulate NiV-infected clinical samples. The simulated NiV clinical samples were evaluated using the RT-RAA-VF assay as well as real-time RT-PCR. Of all 47 samples tested, 25 samples were prepared by 3-, 4-, and 5-fold gradient dilutions of NiV RNA using healthy human RNA as the diluent, and 22 samples were healthy human RNA samples without NiV RNA. The RT-RAA-VF assay was able to distinguish between the 25 positive and 22 negative samples, and the real-time RT-PCR assay also detected 25 positive and 22 negative samples, with Ct values ranging from 25 to 40 for positive samples (Fig. 1-E to G). We also simulated the clinical NiV infected swine samples. Among all 28 samples, 15 samples were obtained by 3-, 4- and 5-fold gradient dilutions of NiV RNA with healthy swine RNA as the diluent, and 13 samples were healthy swine RNA samples without NiV RNA. The results demonstrated that the RT-RAA-VF assay was in accordance with the real-time RT-PCR and was capable of effectively differentiating between 15 positive samples and 13 negative samples (Fig 1-H to J). The assay is therefore expected to be an alternative assay for the clinical detection of NiV, being faster, more specific, and more sensitive than real-time RT-PCR and without the need for complex instrumentation.

As human societies evolve, the combination of urbanization and climate change has resulted in the destruction of bat habitats, leading to increased contact between bats and humans. This has further elevated the risk of NiV infection for both humans and animals (Jonathan A et al. 2004, Kessler et al. 2018). NiV outbreaks have been reported in four countries - Bangladesh, India, Malaysia, and Singapore. However, fruit bats (the natural reservoir host of NiV) have been found in several Southeast Asian countries, suggesting the potential for NiV outbreaks in previously unaffected areas (Mangesh et al. 2022). Given the incubation period associated with NiV, the potential for cross-regional spread due to the movement of infected individuals cannot be dismissed. Consequently, it is imperative to focus on the rapid detection of NiV infections to enhance the survival prospects for those affected and to mitigate the risk of further transmission of the virus to others. In the present study, we developed an RT-RAA-VF assay for the detection of NiV using a sealed disposable nucleic acid visualization test paper device which effectively eliminates false positives caused by aerosol contamination. The assay demonstrated a sensitivity of 5 copies μL^-1 for detecting NiV RNA at 42 °C for 20 minutes. The assay exhibits high specificity, with no cross-reactivity observed against other paramyxoviruses or neurological pathogens. Moreover, when evaluated on simulated clinical samples, it demonstrated 100% concordance with the results obtained from the real-time RT-PCR method. With its rapidity, specificity, and sensitivity features, this assay holds promise as an effective tool for early diagnosis of NiV infection.

The "Greater Food" approach has replaced the older "taking grain production as a top priority" approach. The importance of feed and forage as the material basis for guaranteeing high-quality development of the livestock industry has gradually become prominent. However, owing to the tradition of "both human staple food and animal feed relying on grain production" in China and the decoupling of feed crop planting and livestock farming, the risk of feed grain security has increased, especially as it relates to the supply of high-quality protein feed ingredients from abroad, which is facing a bottleneck. To ensure food security, effective domestic agricultural production should be adopted. Nevertheless, guaranteeing the supply of high-quality protein feed through domestic soybean production is difficult because of limited arable land; furthermore, pressure on the staple food supply is still extreme. In this article, the historical and realistic implications for the security risks of feed grain in China are analyzed. Proposals are made to separate staple food grains for humans from the feed grain supply for animals and to develop high-quality forage to reduce feed grain use. High-quality forage can be supplied via intercropping with grain crops in arable land and reseeding perennial legumes or grasses into natural grasslands. However, “managing forage for grain” needs to be supported via technical paths and policies as the forage industry develops to effectively increase the capacity to ensure feed grain security.

The role of β-hydroxybutyric acid (BHBA) includes providing energy, regulating signaling pathways, and ameliorating the gut microbiota in the host, while its nutrient mechanism to improve rumen epithelium development in young ruminants is still unclear. In this study, a total of 12 female Haimen goats with 30 days of age were chosen and divided into two groups. One group was fed with basic diet (CON), and the other group was fed a basal diet supplemented with 6 g d^-1 dietary β-hydroxybutyrate sodium (BHBA-Na). The experimental period was 30 days, and all goats were slaughtered at 60 days of age. The joint analysis of multi-omics, including rumen microbiota, rumen epithelial transcriptome and rumen epithelial metabolomics in young goat model, was performed to systematically investigate the effect of dietary BHBA-Na on rumen development in young goats. As the results, we found that dietary BHBA-Na improved the growth performance of young goat including body weight, average daily gain (ADG) and dry matter intake (DMI) (P<0.05). Dietary BHBA-Na also increased the weight of rumen, and promoted the growth of rumen epithelium development (P < 0.05). The abundance of several beneficial bacteria was increased (Fibrobacter, Succinivibrio, Clostridiales, etc.,). The rumen epithelium transcriptome and metabolomics indicated that BHBA-Na supplementation showed a remarkable effect on the nutrient metabolism of the rumen epithelium. Specifically, the pathways of “fatty acid metabolism”, “cholesterol homeostasis”, “reactive oxygen species (ROS) pathway” and “peroxisome” were activated in response to BHBA-Na addition (P < 0.05). Moreover, the genes (HMGCS2, ECSH1, ACAA2, ECH1, ACADS etc.) and metabolites (succinic acid, alpha-ketoisovaleric acid, etc.) involved in these pathways were also regulated positively (P < 0.05). The rumen epithelium obtained the energy for its development from the process of volatile fatty acids (VFAs) decomposition. Finally, we observed the close correlations among the phenotypes, ruminal microbiota, host genes and epithelial metabolites. Overall, our results revealed that the BHBA-Na promoted the growth and rumen development of young goats possibly by enhancing DMI and regulating the rumen microbiota and the metabolisms of VFA and amino acid in the rumen epithelium.

Rapeseed (Brassica napus L.) is one of the most important oilseed crops worldwide.  Development of rapeseed varieties with high-quality oil is a long-term breeding goal.  Reducing the contents of palmitic acid, the main saturated fatty acid in rapeseed oil, could greatly improve oil quality.  Here, we performed genome-wide association study (GWAS) and transcriptome-wide association study (TWAS) of seed palmitic acid content (SPAC) using 393 diverse B. napus accessions.  Four genes (BnaA08.DAP, BnaA08.PAA1, BnaA08. DUF106, and BnaC03.DAP) were identified by both GWAS and TWAS.  The transcripts per million (TPM) values of these candidate genes at 20 and 40 days after flowering (DAF) were significantly correlated with SPAC in this association panel.  Based on genetic variation in the candidate genes, we identified four low-SPAC haplotypes by combining candidate gene association analysis and haplotype analysis.  Brassica napus accessions carrying low-SPAC haplotypes had lower SPAC than those carrying high-SPAC haplotypes without affecting seed oil content, seed protein content, or seed yield.  Based on the functional single-nucleotide polymorphism (SNP) chrA08_9529850 (C/A) in the promoter of BnaA08.DUF106, we developed a molecular marker (Bn_A8_SPAC_Marker) that could be used to facilitate breeding for low SPAC in B. napus.  Our findings provide valuable information for studying the genetic control of SPAC in B. napus.  Moreover, the candidate genes, favorable haplotypes, and molecular marker identified in this study will be useful for breeding low-SPAC B. napus varieties.

Crude fat is an important nutritional component of maize kernels.  However, the genetic mechanisms underlying crude fat content in maize kernels remain elusive.  Previous studies used single-model genome-wide association studies (GWAS) with limited population sizes, which can result in false positives of loci and hinder the identification of functional genes.  Therefore, this study used a population consisting of 495 maize inbred lines, combined with 1.25 million single nucleotide polymorphisms (SNPs), and implemented GWAS using six models to identify quantitative trait nucleotides (QTNs) controlling crude fat content and to mine key genes.  The results revealed a wide variation in crude fat content (0.62–16.03%) and broad-sense heritability (96.23%).  In total, 744 significant QTNs were detected, with 147 co-located across different models, environments, and methods.  Based on the 147 co-located QTNs, candidate genes were searched at 50 kb up- and downstream intervals of each QTN.  We finally screened eight candidate genes (GRMZM2G169089, GRMZM2G117935, GRMZM2G002075, GRMZM2G368838, GRMZM2G058496, GRMZM2G090669, GRMZM2G001241, and GRMZM2G333454) related to crude fat content that exhibited high expression levels during kernel development in maize inbred line B73.  Notably, GRMZM2G169089, GRMZM2G117935, GRMZM2G002075, and GRMZM2G368838 are involved in the linoleic acid metabolic pathway, oil metabolism, kernel growth, and development in maize.  Furthermore, co-expression network analysis revealed that the eight candidate genes exhibited strong correlations with 30 known genes.  Proteins encoded by candidate genes interact with various other proteins and play an important role in oil content and oleic acid metabolism in maize kernels.  The best haplotypes of candidate genes might increase crude fat content without decreasing maize yield.  These results broaden the understanding of the genetic mechanism of crude fat content and facilitate marker-assisted selection for high-crude fat breeding programs for maize.

Rapeseed (Brassica napus L.) is a major oil crop worldwide that is vigorously promoted for cultivation in China.  Boron (B) is an essential micronutrient for plant growth and development.  However, agricultural soils in rapeseed planting areas often show either B deficiency or severe B deficiency.  Increasing the resistance to B deficiency is a pivotal goal in the breeding of rapeseed, yet the genetic basis for variations in B efficiency-related traits remains unclear. In this study, a natural population with 391 rapeseed accessions was used to investigate B efficiency-related traits through a nutrient solution system, including relative root length (RRL), shoot dry weight (SDW), root dry weight (RDW), and B efficiency coefficient (BEC), which exhibited extensive phenotypic variations under B deficiency.  Through a genome-wide association study (GWAS) of B efficiency-related traits using high-density SNP markers obtained from whole-genome resequencing, we identified 106 significantly associated SNPs by employing both the general linear model and the mixed linear model.  Among these SNP loci, two prominent SNP clusters were detected on chrA03:14087835–14764672 and chrC03:20110319–22135492 at low B level across three repeated experiments of multiple traits.  Integrated with the transcriptome analysis, four genes, BnaA03g29020D, BnaA03g29440D, BnaC03g33010D, and BnaC03g34490D, exhibiting higher differentially expressed fold-change along with favorable haplotypes within the promoter or coding region were identified as candidate genes that could potentially be involved in B efficient utilization, and their favorable haplotypes improved seedling growth and productivity under B deficiency.  In view of the lack of B mineral resources in China, rapid and accurate identification of more B-efficient alleles and the study of the genetic mechanism underlying crops in response to B deficiency have important theoretical and practical significance for cultivating B-efficient varieties and maintaining green, sustainable agriculture quickly and accurately.

Hydraulic theory predicts a positive coupling between leaf hydraulic conductance (K_leaf) and stomatal conductance (g_s); however, this theory has not been fully supported by observations, and the underlying mechanisms remain unclear.  Currently, subdividing K_leaf into leaf hydraulic conductance inside xylem (K_x) and outside xylem (K_ox) offers a new perspective for elucidating the regulatory mechanism of K_leaf on g_s.  Optimal planting density can enhance water use efficiency (WUE) by optimizing g_s; however, the changes in leaf hydraulic properties during this process and its regulation of g_s and WUE remain unclear.  We examined the relationships between K_x and K_ox with g_s, photosynthetic rate (A_N), and WUE, and investigated the structural basis determining K_ox in cotton under eight planting densities of 12, 18, 24, 36, 48, 60, 72, and 84 plant m^-⊃2;.  The results showed that as the increase of planting density, K_leaf and A_N remained consistent while K_ox and g_s decreased significantly.  K_ox was significantly influenced by leaf thickness and the volume fraction of inter-cellular air space.  K_leaf and K_x showed no correlation with A_N or g_s, but K_ox exhibited a significant positive correlation with g_s.  Furthermore, K_ox is significantly negatively correlated with WUE.  These findings suggest that K_ox modulates g_s to reduce water loss while maintaining A_N, thereby enhancing WUE in cotton under various planting densities.

DNA methylation, a key epigenetic modification, plays a crucial role in regulating lipid metabolism. Consistent correlations have been observed between aberrant DNA methylation patterns and lipid metabolic disorders. Emerging evidence indicates that methyl donor micronutrients could influence DNA methylation patterns, consequently exerting an influence on lipid metabolism. Specifically, the deficiency or excesses of methyl donor micronutrients (folate, choline, betaine, B vitamins and methionine) have been associated with altered DNA methylation patterns linked to lipid metabolism. These alteration in DNA methylation levels, occurring globally and within promoter regions, could affect gene expression related to lipid metabolism. However, the mechanisms through which methyl donor micronutrients regulate lipid metabolism via the DNA methylation modification and the role of methyl donor micronutrients supplementation on DNA methylation profiles remain unclear. In this review, we summarized the regulatory role of DNA methylation in lipid metabolism, and highlighted recent findings investigating the impact of methyl donor micronutrients on lipid metabolism, as well as DNA methylation-mediated adipogenesis and adipose deposition. Taken together, this review deepened our understanding of how the complex interplay between methyl donor micronutrients, DNA methylation, and lipid metabolism, and provides valuable information for accurately regulating lipid metabolism of livestock and poultry, thereby improving meat quality, and promoting the development of animal husbandry.

Cultivar mixtures increases crop diversification and grain yield stability.  It is a major challenge to achieve high grain yield and nitrogen use efficiency with environmentally friendly practices.  However, it is currently unclear whether the cultivar mixtures of maize can improve nitrogen use efficiency.  A two-year field experiment was conducted using two maize cultivars with different roots angles and leaf angles planted in monoculture or in mixtures under four nitrogen levels N0 (0 kg N ha^-1), N140 (140 kg N ha^-1), N280 (280 kg N ha^-1) and N340 (340kg N ha^-1).  Cultivar mixtures significantly increased light interception of middle canopy, dry matter accumulation and total roots length under N0, N140, and N280 conditions.  Light interception of middle canopy positively related to dry matter accumulation and thus increased grain yield.  And light interception of whole canopy positively related to total lateral root length, while the increased total lateral root length of outer nodal roots significantly improved nitrogen accumulation and nitrogen use efficiency.  Thus, cultivar mixtures promoted an optimal canopy structure and good root growth, then improved grain yield and nitrogen use efficiency.  These findings could deepen our understanding of the facilitating effect of canopy structure and root traits of cultivar mixtures on the collaborative promotion of grain yield and nitrogen use efficiency. 

Nitrogen (N) is a limiting factor that determines the yield and quality of Chrysanthemum. Genetic variation in N use efficiency (NUE) has been reported among Chrysanthemum genotypes. We performed a transcriptome analysis of two Chrysanthemum genotypes, ‘Nannonglihuang’ (‘LH’, N-efficient genotype) and ‘Nannongxuefeng’ (‘XF’, N-inefficient genotype), under low N (0.4 mmol·L^-1 N) and normal N (8 mmol·L^-1 N) treatments for 15 d and an N recovery treatment for 12 h (low N treatment for 15 d and then normal N treatment for 12 h) to understand the genetic factors impacting NUE in Chrysanthemum. The two genotypes exhibited contrasting responses to the different N treatments. The N-efficient genotype ‘LH’ had significant superiority in agronomic traits, N accumulation and glutamine synthase activity under both normal N and low N treatments. Low N treatment promoted root growth in ‘LH’, but inhibited root growth in ‘XF’. Transcriptome analysis revealed that the low N treatment increased the expression of some N metabolism genes, genes related to auxin and abscisic acid signal transduction in the roots of both genotypes, as well as genes related to gibberellin signal transduction in roots of ‘LH’. The N recovery treatment just increased the expression of genes related to cytokinin signal transduction in roots of ‘LH’. The expression levels of the NRT2.1, AMT1.1, and Gln1 genes related to gibberellin and cytokinin signal transduction were higher in roots of ‘LH’ than in ‘XF’ under different N treatments, suggesting that the genes related to N metabolism and hormone (auxin, abscisic acid, gibberellin, and cytokinin) signal transduction in roots of ‘LH’ are more sensitive to different N treatments than those of ‘XF’. Co-expression network analysis (WGCNA) also identified hub genes like bZIP43, bHLH93, NPF6.3, IBR10, MYB62, PP2C, PP2C06 and NLP7, which may be the key regulators of N-mediated responses in Chrysanthemum and play crucial roles in enhancing NUE and resistance to low N stress in the N-efficient Chrysanthemum genotype. These results revealed the key factors involved in regulating NUE in Chrysanthemum at the genetic level, which provides new insights into the complex mechanism of efficient nitrogen utilization in Chrysanthemum, and can be useful for the improvement and breeding of high NUE Chrysanthemum genotypes.

Peel color is an important appearance quality of melons that greatly affects consumer preferences. In this study, a near-isogenic line NIL-G (dark green peel) was generated from B8 (grey-green peel) and B15 (white peel). The F₂ population constructed by crossing NIL-G and B15 was used to study the inheritance pattern of peel color, and bulked-segregant analysis sequencing (BSA-seq) was employed to identify the interval in which the target gene was located. Genetic analysis results showed that the dark green peel trait at maturity is controlled by a dominant gene. BSA-seq and molecular markers were used to localize the candidate gene in a 263.7 kb interval of chromosome 4, which contained the CmAPRR2 gene with known functions. Moreover, allelic sequence analysis revealed four SNP variations of the CmAPRR2 gene in B15, of which SNP.G614331A was located at the junction of the 6^th exon and 6^th intron. The G-to-A mutation caused alternative splicing of the transcript of CmAPRR2 in B15, generating two transcripts (CmAPRR2-A and CmAPRR2-B) with premature termination codons. Furthermore, the Kompetitive Allele Specific PCR (KASP) marker, APRR2-G/A, was developed based on this SNP and shown to co-segregate with the peel color phenotype in the F₂ population. Compared to white-peel B15, the expression level of CmAPRR2 in dark green peel NIL-G was higher at each growth stage. Therefore, CmAPRR2 may be the key gene controlling the fruit color of melons. Overall, this study identified a novel allelic variant of CmAPRR2 that leads to white peel formation in mature melons. The study also provides a theoretical basis for further research on the gene regulatory mechanism of melon peel colors and may promote the future use of molecular marker-assisted selection to modify melon peel colors.

Red fruit peel is one of the most valuable economic traits in pear and is mainly determined by anthocyanins. Many pear cultivars with a red peel originated from bud sports; however, little is known about the genetic mechanisms underlying this trait. We have previously identified a mutant PpBBX24 containing a 14-nucleotide deletion in the coding region (Ppbbx24-del) as the only known variant associated with the red coloration of the mutant 'Red Zaosu' pear (Pyrus pyrifolia White Pear Group). Herein, we analyzed the role of the mutant gene in red coloration and its mechanism of action. The results showed that light promoted red peel coloration in 'Red Zaosu' pear, and Ppbbx24-del had positive effects on light-induced anthocyanin biosynthesis, while normal PpBBX24 had the opposite effects. Transient and stable transformation experiments confirmed that Ppbbx24-del could promote anthocyanin accumulation in pear fruit peels, calli, and tobacco flowers. Due to the loss of NLS and VP domains, Ppbbx24-del co-localized in the nucleus and cytoplasm, whereas PpBBX24 localized only in the nucleus. Real-time PCR and transcriptome analyses indicated that PpMYB10 and PpHY5 is highly expressed in 'Red Zaosu' pear. In yeast one-hybrid and dual luciferase assays, Ppbbx24-del and PpHY5 independently promoted the expression of PpCHS, PpCHI, and PpMYB10 by binding to their promoters; however, PpBBX24 did not affect the expression of these genes. Additionally, we found that Ppbbx24-del and PpHY5 had additive effects on the expression of PpCHS, PpCHI, and PpMYB10, as they promote the expression of anthocyanin synthesis genes separately. The co-expression of PpBBX24 and PpHY5 inhibited the activation of downstream genes by PpHY5, and this was attributed to the interaction between the two loci. In conclusion, our results clarify the molecular mechanism by which mutant Ppbbx24-del and PpBBX24 exert opposite effects in the regulation of anthocyanin accumulation in pear. These findings lay an important theoretical foundation for the use of Ppbbx24-del to create red pear cultivars.

The evaluation of plant stress tolerance and the screening of key regulatory genes under the combined stresses of high temperature and drought are important for the study of plant stress tolerance mechanisms. In this study, the drought tolerance of five grape varieties was evaluated under high-temperature conditions to screen key genes for further exploration of resistance mechanisms. By comparing and analysing the morphological characteristics and physiological indicators associated with the response of grapevines to drought stress and integrating them with the membership function to assess the strength of their drought tolerance, the order of drought tolerance was found to be as follows: 420A>110R>CS>fercal>188-08. To further analyse the mechanism of differences in drought tolerance, transcriptomic sequencing was performed on the drought-tolerant cultivar 420A, the drought-sensitive cultivar 188-08 and the control cultivar CS. The functional analysis of differential metabolic pathways indicated that the differentially expressed genes were enriched mainly in biological that 420A had higher antioxidant activity. Moreover, the transcription factors which differentially expressed were also analysed in the five grape varieties, and several genes, such as VvAGL15, VvLBD41, and VvMYB86, appeared to be closely related to drought tolerance, suggesting their potential involvement in the regulation of grapevine drought tolerance and their value in drought tolerance research.

Oxytetracycline (OTC) is used extensively in animal husbandry and enters the soil in different forms, causing severe environmental pollution. Previous studies have shown that the genus Pseudomonas can potentially degrade antibiotics in the soil environment. Environmental conditions, such as the initial concentration of antibiotics, incubation temperature and others, have significant impacts on the activity of antibiotic-degrading bacteria. However, few reports have clarified the environmental impacts on the effectiveness of Pseudomonas spp. In the present study, we investigated the effects of different initial concentrations of OTC and incubation temperatures, as well as soil sterilization, on OTC degradation by Pseudomonas strain T4. We also focused on the microbial degradation pathways of OTC, and variations in both antibiotic resistance genes (ARGs) and microbial communities with T4 functioning under optimal conditions. The results showed that the most effective degradation occurred under an initial OTC concentration of 2.5 mg kg^-1 at 30°C in unsterilized soil spiked with T4. These conditions yielded an OTC degradation rate of 69.53% within 63 days. The putative degradation pathways of OTC in the presence of T4 included dehydration, demethylation, deamination, hydroxylation, oxidation and ring opening. Bacteroidetes, Proteobacteria and Acidobacteria played key roles in the biodegradation of OTC with T4 in the soil. The results also showed that tet(G) was the most frequently detected among the 13 common tetracycline ARGs that were investigated. The bacterial community shift observed in this study may provide new insights into the microbial degradation of OTC in soil.

Grassland degradation presents overwhelming challenges to biodiversity, ecosystem services, and the socio-economic sustainability of dependent communities. However, a comprehensive synthesis of global knowledge on the frontiers and key areas of grassland degradation research has not been achieved due to the limitations of traditional scientometrics methods. The present synthesis of information employed BERTopic, an advanced natural language processing tool, to analyze the extensive ecological literature on grassland degradation. We compiled a dataset of 4,504 publications from the Web of Science core collection database and used it to evaluate the geographic distribution and temporal evolution of different grassland types and available knowledge on the subject. Our analysis identified key topics in the global grassland degradation research domain, including the effects of grassland degradation on ecosystem functions, grassland ecological restoration and biodiversity conservation, erosion processes and hydrological models in grasslands, and others. The BERTopic analysis significantly outperforms traditional methods in identifying complex and evolving topics in large datasets of literature. Compared to traditional scientometrics analysis, BERTopic provides a more comprehensive perspective on the research areas, revealing not only popular topics but also emerging research areas that traditional methods may overlook, although scientometrics offers more specificity and detail. Therefore, we argue for the simultaneous use of both approaches to achieve more systematic and comprehensive assessments of specific research areas. This study represents an emerging application of BERTopic algorithms in ecological research, particularly in the critical research focused on global grassland degradation. It also highlights the need for integrating advanced computational methods in ecological research in this era of data explosion. Tools like the BERTopic algorithm are essential for enhancing our understanding of complex environmental problems, and it marks an important stride towards more sophisticated, data-driven analysis in ecology.

 Drought is one of the major environmental constraints that significantly affects seedling emergence, yield, and quality of Tartary buckwheat, thereby hindering the development of its industry.  However, the molecular mechanisms underlying drought tolerance genes in Tartary buckwheat remain largely unexplored.  Alcohol dehydrogenase (ADH), one of the essential plant proteins, plays a crucial role in growth, development, and stress responses, but its specific role in drought resistance is still unclear.  In this study, we identified an ADH gene FtADH1, using a membership function value of drought tolerance (MFVD) combined with a genome-wide association study (GWAS) and transcriptomic profiles that confers drought tolerance in Tartary buckwheat. Our findings demonstrated that the overexpression of FtADH1 in Arabidopsis and Tartary buckwheat hairy roots enhances drought tolerance by promoting root elongation and mitigating elevated levels of reactive oxygen species (ROS).  Our findings demonstrated that FtADH1 can enhanced tolerance to drought stresses in both Tartary buckwheat and Arabidopsis.  This study identifies the FtADH1 as a new player in affecting ROS level and the stress response of Tartary buckwheat by regulating protective enzyme activities at a high level to scavenge ROS and modulating root growth under drought stress.  Further, we identified proteins interacting with FtADH1 through a prokaryotic expression pull-down assay combined with mass spectrometry, revealing that FtADH1 specifically interacts with the S-adenosyl-L-methionine (SAM) synthetase protein, FtSAMS1.  Overexpression of FtSAMS1 was found to enhance ADH enzymatic activity, leading to increased SAM content in overexpressing Tartary buckwheat hairy roots under water-deficit conditions.  Additionally, FtSAMS1 overexpression induced a drought-resistant phenotype in Arabidopsis and Tartary buckwheat hairy roots under drought stress, revealing the biological function of FtADH1. Evolutionary analysis indicates that ADH1 in Fagopyrum species has undergone significant evolutionary events, including duplication and purifying selection, which may contribute to functional diversification and adaptive advantages such as drought resistance in cultivated buckwheat.  In summary, this study proposes that FtADH1 is a key contributor to drought tolerance, and its interaction with FtSAMS1 holds potential for the development of drought-resistant varieties in Tartary buckwheat and its relative species.

The phenylpropane metabolic pathway is one of the most significant metabolic pathways in plants, synthesizing more than 8,000 metabolites, including flavonoids, lignans, lignin, coumarins, and other metabolites through multiple branching pathways (Gao et al. 2024, Zhang and Liu 2015).  This pathway is crucial in plant growth, development, and plant-environment interactions (Dong and Lin 2021).  The essential functions of flavonoids in UV protection (Gaberscik et al. 2002), disease resistance (He et al. 2023a), and salt tolerance (Ismail et al. 2015) highlight the significance of developing a rapid detection method for phenylpropane pathway compounds and their associated flavonoids, facilitating gene function validation, genetic breeding, and metabolic engineering for enhanced flavonoid production.

Currently, several liquid-phase methods, particularly LC-Q-TOF-MS/MS, are available to detect different bioactive compounds, especially flavonoids in the phenylpropane metabolic pathway.  For example, it has been reported in kiwifruit (Wojdyło and Nowicka 2019), citrus (Xing et al. 2017), chili (Mi et al. 2020), grapes (Mayr et al. 2018), soybeans, and ginkgo biloba (Meng et al. 2022).  While several liquid-phase methods exist for detecting phenylpropane pathway substances and flavonoids, prior studies have certain limitations, such as fewer detected compounds, lack of systematicity and comprehensiveness, low detection efficiency, and high cost.  Therefore, developing a systematic and comprehensive method to detect flavonoid compounds from initiating the phenylpropane pathway to rutin becomes necessary.

Addressing this need, we have established a rapid method for detecting flavonoids, facilitating a deeper understanding of the dynamics and gene functions within the phenylpropane metabolic pathway.  Briefly, the freeze-dried material was pulverized into a dry powder, sieved through an 80 mesh sieve, and precisely weighed 0.1 g.  Subsequently, 10 mL of 80 % (v/v) methanol/water solution was added, followed by vortexing and shaking for 1 min.  The extraction process was performed using an ultrasonic bath at 80 kHz for 40 min at a constant temperature of 50°C.  Finally, the extracts underwent filtration through a 0.22 µm organofiltration membrane, followed by identification and quantification of compounds using LC-QqQ-MS/MS (Agilent 1290-6495).  An analytical system comprising an Ultra-High Performance Liquid Chromatography (UHPLC) Agilent 1290 Infinity coupled with an Agilent 1290 Infinity G4226A autosampler, in conjunction with an accurate-mass Quadrupole-Time of Flight (Q/TOF) Mass Spectrometer Agilent 6495 (nominal resolution 40000) equipped with an Agilent Dual Jet Stream Ionization source (Agilent Technologies, Santa Clara, CA, USA) was used.  Chromatographic separation was performed utilizing a Zorbax reverse-phase column (RRHD SB-C18 3×150 mm, 1.8 µm, Agilent Technologies, Santa Clara, CA, USA) with a solvent composition consisting of 0.1% (v/v) aqueous formic acid (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B).  The elution gradient program entailed an initial 2% B isocratic hold for 2 mins, followed by a linear increase from 2 to 10% B over 2 mins, a subsequent gradient from 10 to 80% B over 7 mins, a further increase to 98% B over 2 mins, a return to 2% B over 2 mins, and concluding with a 2% B isocratic hold for 2 mins.  The flow rate was set at 0.4 mL/min, with a sample injection volume of 2 µL and a column temperature of 40°C.  Data acquisition was facilitated using Agilent MassHunter version B.04.00 (B4033.2) software.  Data analysis was performed utilizing Agilent MassHunter Qualitative Analysis software version B.07.00.  Compound identification relied on accurate mass and isotope pattern, with compound identification scores expressed as “overall identification score”, calculated as the weighted average of the isotopic pattern signal.

The flavonoid biosynthesis pathway has been well studied in model plant Arabidopsis and various crop species, including rice, maize, bean, and tomato (Tohge et al. 2017).  Flavonoids are mainly synthesized through the phenylpropane biosynthesis pathway (Li et al. 2021).  Therefore, this study constructs a detection method for flavonoids related to the phenylpropanoid metabolism pathway (Appendix A).  In addition to the major flavonoids such as rutin, our assay encompasses a range of related compounds such as kaempferol, astragalin, nicotiflorin, afzelin, etc. (Appendices B and C).  Furthermore, our method extends beyond flavonoids to include the detection of additional substance classes, including phenolic acids such as trans-cinnamic acid, 4-hydroxycinnamic acid, and chlorogenic acid, as well as quinones like emodin. Moreover, coumarins such as esculetin and scopoletin, proanthocyanidins like procyanidin A1/B1/C1, and anthocyanins such as keracyanin chloride and cyanidin chloride are also included (Fig. 1-A).  Although some compounds have been previously reported (El-Najjar et al. 2011, Matos 2021), their integration into the flavonoid assay enhances the assay’s comprehensiveness.  Thus, our method facilitates a more thorough assessment of flavonoid-related compounds, elucidating a more complete pathway from phenylpropane to rutin.

The validation of the assay in this study encompassed a comprehensive series of experiments, including linearity, limit of detection (LOD), limit of quantification (LOQ), precision, stability, and repeatability (Appendix D).  Notably, the established calibration curves for all analytes demonstrated good linear regression with high coefficients of determination (R²≥0.9935).  The LOD and LOQ were higher for five substances, including chlorogenic acid, proanthocyanidins, procyanidin C1, ampelopsin, and trans-cinnamic acid, while for the remaining analytes, they were below 8.54 and 28.47 ng mL^-1, respectively.  Precision, stability, and repeatability tests demonstrated relative standard deviations below 4.85, 4.98, and 4.96%, respectively, collectively indicating the reliability of the method.  In summary, our LC-MS/MS method demonstrates high sensitivity, precision, and accuracy, enabling simultaneous and rapid determination of the targeted 37 compounds.

We tested the method’s feasibility by taking a flavonoid-rich plant (buckwheat) as the research object. Tartary buckwheat is known for its diverse flavonoid content and various health-promoting attributes, including antioxidant properties, balanced amino acid composition, richness in resistant starch, and other functions (Joshi et al. 2020, Kreft et al. 2020, Huda et al. 2021).  Utilizing this method to detect flavonoids in buckwheat is, therefore, crucial and can serve as validation of its applicability.  Consequently, various tissues (roots, stems, leaves, flowers, and seeds) from three widely distributed buckwheat varieties (Tartary buckwheat, common buckwheat, and golden buckwheat) were utilized as samples for analysis (Fig. 1-B-D).  Moreover, constituents within the golden buckwheat rhizome were also detected.  The findings revealed that, overall, the flowers of all three buckwheat species exhibited the highest flavonoid content, encompassing compounds such as quercitrin, naringenin, afzelechin, rutin, nicotiflorin, afzelin, and others (Fig. 1-E-G).  This observation aligns with existing literature indicating that buckwheat flowers are notably rich in flavonoids (Zhang et al. 2017, He et al. 2023b), thereby validating the effectiveness of the established methodology in accurately reflecting the sample's composition.  Besides, catechin, epicatechin, procyanidin C1, procyanidin B1, and chlorogenic acid exhibit higher concentrations in leaves and flowers than in other tissues, consistent with previous findings (Hou et al. 2021).  These findings demonstrate the potential of the method to analyze flavonoid-rich plants, such as buckwheat, while also affirming its ability to provide an objective reflection of sample composition.

The successful application of this method in buckwheat makes us consider whether this method can be extended to other model plants.  Arabidopsis thaliana and tobacco were used to broaden its application.  The findings showed a significant abundance of chlorogenic acid in tobacco leaves, constituting approximately 2% of the sample composition (Fig. 1-H-J), consistent with prior studies attributing chlorogenic acid as the most abundant polyphenol in tobacco (Wang et al. 2023, Zou et al. 2021), thereby affirming the reliability of our method to a certain extent.  A large number of flavonoids were detected in both model plants, including rutin, nicotiflorin, afzelin, procyanidin, procyanidin C1, procyanidin B1, and ampelopsin.  Additionally, several compounds were identified at lower levels in tobacco and Arabidopsis, such as kaempferol, isoquercitrin, quercitrin, apigenin, catechin, epicatechin, afzelechin, linarin, keracyanin chloride, 4-hydroxycinnamic acid, vitexin-glucoside, and kaempferol-3-glucuronide, with concentrations at approximately 4 µg g^-1 or less.  Besides, tobacco exhibited notably higher levels of quercitrin, epicatechin, and keracyanin compared to Arabidopsis.  On the other hand, some substances were not detected in either tobacco or Arabidopsis, which potentially indicates that these substances are extremely rare in these model plants or that this method may be more suitable for application in flavonoid-rich plants.  However, the majority of compounds (20) included in the method were detected in both Arabidopsis and tobacco, indicating that the method is effective for flavonoid detection across diverse plant species.

In summary, this study presents a comprehensive method for the rapid detection and evaluation of flavonoids along the phenylpropane pathway in plants, encompassing 37 substances ranging from phenylpropanes to rutin and its derivatives, including phenolic acids (4), flavonoids (21), anthocyanins (4), coumarins (3), proanthocyanidins (4), and quinones (1).  The method's effectiveness was successfully validated in flavonoid-rich buckwheat plants, yielding positive results. In addition, the application of the method was extended to Arabidopsis and tobacco leaves.  Our method has several main advantages: focused on the phenylpropane pathway, abundant detection substances, short time consuming, lower cost, and accurate quantification.  This method provides valuable support for assessing flavonoid contents in the phenylpropanoid metabolic pathway of plants, facilitating gene function validation and advancing crop genetic improvement.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a globally quarantine disease that infect nearly all Citrus cultivars. Remarkably, Citron C-05 has been identified with complete and active resistance to Xcc. However, the mechanism underlying Citron C-05’s resistance to Xcc remains elusive. In this study, we identified a gene cluster on the chromosome 8 of citrus genome comprising five pathogenesis-related 4-like genes. PR4A was up-regulated in Citron C-05 leaves under Xcc infection, exhibiting the highest expression among these PR4-like genes. In addition, PR4A expression was higher in leaves of disease-resistant genotypes compared to susceptible genotypes under Xcc invasion. Bimolecular fluorescence complementation (BiFC) and Split-Luc assays indicated that CmWRKY75, a positive regulator of PR4A, interacted with pthA4 and up-regulated expression of PR4A in Citron C-05 leaves. Segmentation of the CmPR4A promoter revealed that P_CmPR4A-P_-516 may be involved in regulating PR4A expression. Transient overexpression of CmPR4A improved resistance to Xcc in sweet orange, and three transgenic lines of OE-CmPR4A exhibited resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in Arabidopsis. Furthermore, CmSMU2 was identified through yeast two-hybrid library using CmPR4A as bait, BiFC and Split-Luc assays further verified their interaction. Transient overexpression of CmSMU2 in the sweet orange increased resistance to Xcc. Co-expression of CmSMU2 and CmPR4A enhanced accumulation of reactive oxygen species compared to CmSMU2 or CmPR4A, indicating that they may synergistically enhance resistance to Xcc in citrus. These findings lay the groundwork for a theoretical analysis of the mechanism underlying the resistance of Citron C-05 against citrus canker.

Lodging is a primary factor limiting rice grain yield. How to achieve the synergistic improvement of high yield and nitrogen use efficiency without lodging has always been the focus worldwide.  In this study, Yongyou 2640 (indica-japonica hybrid rice) and Jinxiangyu 1 (inbred japonica rice) were used as materials for field experiments across two years. Six different nitrogen managements were set up, including no nitrogen (T1), conventional urea (T2), controlled-release nitrogen (T3), reduction of controlled-release nitrogen (T4), controlled-release nitrogen combined with one-time basal conventional urea (T5), controlled-release nitrogen combined with split conventional urea (T6).  The results showed that compared with T2, the combined application strategy of controlled-release nitrogen (T5 and T6) could improve nitrogen use efficiency and grain yield by 4.89–5.69% and 3.41–4.65%, respectively.  The carbohydrate contents of the second basal internode, the internode breaking strength, the thickness of the epidermal silicon layer, the number of large and small vascular bundles, and the thickness of parenchymatous tissue and mechanical tissue were increased, whereas the internode length, bending moment and lodging index were reduced under the combined application strategy of controlled-release nitrogen.  These results indicated that the combined application strategy of controlled-release nitrogen could achieve the goal of high yield and nitrogen use efficiency with synchronously increased stem strength due to the improvement in the morphological, mechanical, physicochemical and anatomical properties of second basal stem.

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.

Tree peony is an emerging woody oilseed crop that has the potential to help meet the growing demand for edible oil and biofuel, and breeding high-yield varieties is the key to increasing production. The DA1 gene regulates seed weight and number and effectively improves crop yield; however, its function in tree peonies has not yet been reported. In this study, three PrDA1 genes were first identified in the whole genome of 'Jing Hong' flare tree peony (Paeonia rockii). PrDA1-1, which contains three types of conserved structural domains UIM (ubiquitin-interacting motif), LIM (Lin11, Isl- 1, and Mec-3), and DA-like, is homologous to AtDA1. PrDA1-1 was strongly expressed during both the early and late stages of seed development, and its constitutive expression in Arabidopsis reduced seed number but increased seed weight, which ultimately led to a decrease in yield. PrDA1-1 interacted with PrTCP1/PrTCP9 in yeast and plants, and the constitutive expression of PrTCP1/PrTCP9 in Arabidopsis increased seed weight and number, which resulted in high yield. We showed that PrDA1-1 and PrTCP1/PrTCP9 regulate seed weight and number to affect yield; these new genetic resources will help improve the seed yield of tree peonies.

Soil aggregates profoundly impact soil sustainability and crop productivity, and they are influenced by complex interactions between minerals and organics. This study aimed to elucidate the alterations in mineralogy and soil organic carbon (SOC) following long-term green manure incorporation and the effect on soil aggregates. Based on 5- and 36-year field experiments, surface soil samples (0-20 cm) were collected from Alfisol and Ferrisol soils subjected to rice-rice-winter fallow (CK) and rice-rice-Chinese milk vetch (MV) treatments to investigate aggregate stability, mineralogy, SOC composition, and soil microstructural characteristics. The results showed that high clay-content Ferrisol exhibited greater aggregate stability than low clay-content Alfisol. The phyllosilicates in Alfisol primarily comprised illite and vermiculite, whereas those in Ferrisol with high-content free-form Fe oxides (Fed) were dominated by kaolinite. Additionally, the clay fraction in Ferrisol contained more aromatic-C than the clay fraction in Alfisol. The 36-year MV incorporation significantly increased the Ferrisol macroaggregate stability (9.57-13.37%), and it also facilitated the transformation of vermiculite into kaolinite and significantly increased the clay, Fed, and aromatic-C contents in Ferrisol. Backscattered electron (BSE)-scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) revealed a compact aggregate structure in Ferrisol with co-localization of Fe oxides and kaolinite. Moreover, the partial least path model (PLS-PM) revealed that clay content directly improved macroaggregate stability, and that kaolinite and Fed positively and directly affected clay or indirectly modulated clay formation by increasing the aromatic-C levels. Overall, long-term MV incorporation promotes clay aggregation by affecting mineral transformation to produce more kaolinite and Fe oxides and retain aromatic-C, and it ultimately improves aggregate stability. 

Flowering is one of the most important phenological periods, as it determines the timing of fruit maturation and seed dispersal. To date, both nitric oxide (NO) and DNA demethylation have been reported to regulate flowering in plants. However, there is no compelling experimental evidence of the relationship between NO and DNA demethylation during plant flowering. In this study, an NO donor and a DNA methylation inhibitor were used to investigate the involvement of DNA demethylation in NO-mediated tomato (Solanum lycopersicum cv. Micro-Tom) flowering. The results showed that the promoting effect of NO on tomato flowering was dose-dependent, with the most positive role observed at 10 μmol L^-1 of the NO donor S-nitrosoglutathione (GSNO). Treatment with 50 μmol L^-1 of the DNA methylation inhibitor 5-azacitidine (5-AzaC) also significantly promoted tomato flowering. Moreover, GSNO and 5-AzaC increased the peroxidase (POD) and catalase (CAT) activity and cytokinin (CTK) and proline contents, while they decreased the gibberellic acid (GA3) and indole-3-acetic acid (IAA) contents. Co-treatment with GSNO and 5-AzaC accelerated the positive effects of GSNO and 5-AzaC in promoting tomato flowering. Meanwhile, compared with GSNO or 5-AzaC treatment alone, co-treatment with GSNO+5-AzaC significantly increased the global DNA demethylation levels in different tissues in tomato. The results also indicate that DNA demethylation may be involved in NO-induced flowering. The expression of flowering genes was significantly altered by the GSNO+5-AzaC treatment. Among these genes, five flowering induction genes-ARGONAUTE 4 (AGO4A), SlSP3D/SINGLE FLOWER TRUSS (SFT), MutS HOMOLOG 1 (MSH1), ZINC FINGER PROTEIN 2 (ZFP2), and FLOWERING LOCUS D (FLD) were selected as candidate genes for further study. Then, McrBC-PCR analysis showed that the DNA demethylation of the SFT gene in the apex and the FLD gene in the stem might be involved in NO-induced flowering. Therefore, our study shows that NO might promote tomato flowering by mediating the DNA demethylation of flowering induction genes. This study provides direct evidence of a synergistic effect of NO and DNA demethylation in promoting tomato flowering. 

Maize (Zea mays L.) is an important food and silage crop with high production potential and economic benefits (Erenstein et al. 2022).  Understanding the maize yield potential can provide reliable theoretical and practical support for achieving breakthroughs in grain yield (Meng et al. 2013).  It was one of the effective ways to explore the grain yield potential based on the model on a national scale.

In China, maize was widely cultivated. The four major maize regions span from 21° to 53°N in latitude and 73° to 135°E in longitude and cover the most complex climatic conditions suitable for maize growing in the world (Xu et al. 2017).  The regional climate heterogeneity tends to induce ecological adaptability responses of maize, which can also lead to variations in the interregional adaptability of crop models (Abbas et al. 2023).  In addition, in the context of global climate change, crop production has been severely affected by extreme weather events, especially maize and wheat (Schmitt et al. 2022).  Clarifying the regional adaptability of the crop growth models is an important basis for analyzing the response of maize to climate change.

Nowadays, crop growth models have been increasingly developed and applied, which play a crucial role in regional simulation, future climate scenario simulation, optimization of cultivation measures, and assessment of meteorological disaster impacts.  Currently, the main crop models applied in China include AquaCrop, APSIM, and WOFOST models, most of them were mainly focused on wheat and rice and the application status varied with regions (Jin et al. 2016; Kheir et al. 2021).  The Hybrid-Maize model is a maize-specific process model developed on the studies of high-yielding maize in the United States, which has been tested and widely used in the United States and South Asia (Yang et al. 2004; Timsina et al. 2010).  It can simulate the maize grain yield potential in specific years and regions by inputting the required relevant parameters including meteorological data, the tested cultivars, and field management information (Yang et al. 2004).  Most importantly, it provides a basis for clearing the maize grain yield improvement space and technical approach to reduce the yield gap.  Currently, under the background of dense panting conditions to increase maize grain yield, the application of this model in China has gradually attracted attention.  However, previous studies on the Hybrid-Maize model were mainly limited to specific regions or sites and applied under low planting conditions (Liu et al. 2012; Meng et al. 2013; Hou et al. 2014).  There were few reports assessing its adaptability at large spatial scales and under dense planting conditions. The climatic conditions were diverse in different maize growing regions across China.  Therefore, it is crucial to evaluate the adaptability of models under dense planting conditions in different regions.  In this study, we evaluated the performance of the Hybrid-Maize model in the major maize growing regions of China based on field data at 22 experimental sites under high planting density (7.5×10⁴ plants ha^-1) during the period of 2011-2015.  The maize growing information and climatic conditions of the experimental sites were listed in the Appendix A.  The findings can provide a reference for the application of the Hybrid-Maize model under dense planting conditions in different regions of China.

The results of this study showed that the normalized root mean square error (NRMSE) were all below 30.0% and the index of agreement (D) were approached to 1, which indicated that the simulated yield was within an acceptable range (Fig. 1-A).  Particularly, the model showed the best adaptability in the NW (Northwestern maize growing region, NRMSE=9.8%).  A similar performance in the prediction of grain yield (NRMSE=7.1%) was observed in the United States under high density (Abimbola et al. 2022).  It was mainly that the Hybrid-Maize model was developed based on high-yielding fields in the United States, where the planting densities commonly exceed 7.5×10⁴ plants ha^-1 (Yang et al. 2004).  It was also shown that the average maize grain yield in the NW was significantly higher than that in the SW (Southwestern maize growing region), HHH (Huang-Huai-Hai maize growing region), and NM (Northern Spring maize growing region).  The average measured grain yield in the NW (18.2 Mg ha^-1) was higher than that in the SW (9.4 Mg ha^-1), HHH (11.0 Mg ha^-1), and NM (13.2 Mg ha^-1).

Aboveground biomass and harvest index (HI) are the bases for maize grain yield formation.  It was indicated that the model had better simulation effects in aboveground biomass with a lower NRMSE value in the NW (17.2%) than that of SW (24.8%), HHH (22.9%), and NM (26.2%) (Fig, 1-B).  The simulation accuracy of this model for aboveground biomass in the NM (NRMSE=26.2%) was similar to a previous study conducted in the Northeast region under sufficient irrigation conditions (NRMSE=24.4%) (Liu et al. 2012).  However, the performance of the model was slightly different in these growing stages with a similar trend in simulated accuracy (i.e., the jointing stage>silking stage>maturity stage) for these four regions.  As for the harvest index, it was shown that the Hybrid-Maize model perform well in the NM and HHH under dense planting conditions (Table 1).  The simulated HI for all regions with the trend of HHH>NM>NW>SW that differed from the spatial distribution (NW>SW>NM>HHH) in the previous study (Liu et al. 2020), which may be related to the overestimation of the HI in the HHH and NM and underestimation in the NW and SW by this model.  Therefore, further optimization in the HI is required for this model when applied across different regions.

The dynamic changes in leaf area index (LAI) are shown in Fig. 1-C.  There were significant differences in the performance of the Hybrid-Maize model between regions under dense planting conditions.  The performance of the model in simulating LAI was better in the HHH (NRMSE=28.8%) and NM (NRMSE=22.0%) than that in the NW (NRMSE=33.4%) and SW (NRMSE=44.2%).  Additionally, it was observed that the Hybrid-Maize model showed a good fitting degree in the jointing and mature stage period in most of these regions.  Overall, this simulated values were higher than the measured with an average overestimation of 37.0% for the whole growth season.  Specifically, the simulated values in the SW were on average 43.6% higher than the measured.  In the NM, the simulated values were on average 12.5% lower than the measured after silking.

In summary, the Hybrid-Maize model showed good adaptability in the simulation of grain yield and dynamic changes of aboveground biomass in the four major maize growing regions of China under moderate planting density conditions, especially in the NW and SW.  However, there was a significant underestimation of HI in the NW and SW.  Conversely, there was an overestimation of LAI in these regions.  Further evaluation of the model can be calibrated by adjusting the LAI and HI to refine the prediction of grain yield potential.  Overall, the Hybrid-Maize model can provide relatively acceptable simulation references in the HHH and NM for all parameters under dense planting conditions. 

Two Cotton Research Institute (CRI) near-isogenic lines, CRI-12 glanded and CRI-12 glandless, were used to pinpoint potential genes and metabolic pathways linked to gossypol biosynthesis through transcriptome sequencing.  We discovered more than 235 million clean reads and 1,184 differentially expressed genes (DEGs).  Consecutively, we conducted a weighted gene co-expression network analysis and found strong correlation of white and yellow modules containing GhTPS (GH_D09G0090) and GhCYP (GH_D05G2016) hub genes with the gossypol content.  The importance of the GhTPS and GhCYP genes was demonstrated using RT‒qPCR, virus-induced gene silencing (VIGS) and target metabolite analysis.  Silencing of these genes resulted in fewer glands on both leaves and stems two weeks after the infection, as compared to the wild type.  In addition, a total of 152 metabolites were identified through targeted metabolite profiling.  Differential metabolite screening revealed 12 and 18 significantly different metabolites in TRV:GhTPS and TRV:GhCYP plants vs. control group, respectively, showing reduction in accumulation of metabolites compared to the control.  The content of hemigossypol, the final product of gossypol biosynthesis, was also reduced, as revealed by target metabolite analysis, suggesting the role of these genes in the gossypol biosynthetic pathway.  Furthermore, a highly significant difference in gossypol content between the glanded and glandless lines was recorded.  The findings of this study reveal a strong link between the gossypol content and GhTPS and GhCYP hub genes, suggesting their role in the gossypol biosynthetic pathway to reduce the accumulation of hemigossypol, which may offer new comprehension into the regulatory checkpoints of the gossypol biosynthesis pathway in cotton.

Greenhouse gas (GHG) production during ensiling not only causes the nutrient losses of silage but also promotes climate warming. However, there is little information on the production of GHG and strategies for mitigating GHG emissions during ensiling. This work aimed to study the gas production characteristics and techniques for reducing gas emissions during ensiling. Oats and triticale, with Lactiplantibacillus plantarum (LP) or corn meal (CM) addition, were ensiled. The cumulative gas volume rapidly increased and reached to the peak within the first 9 days of ensiling for both forage crops. The highest cumulative gas volume of triticale silage was twice as much as that of oats silage. Triticale silage produced lower carbon dioxide (CO₂) concentration, higher methane (CH₄) and nitrous oxide (N₂O) concentrations than oats silage within the 28 days of ensiling. Adding LP or CM significantly improved the fermentation quality and decreased the gas volume and GHG concentrations of two silages on d56 (except CH₄ of triticale). At the early stage of ensiling, more Enterobacter, Lactococcus and Leuconostoc related to gas production were observed, and adding LP increased the abundance of Lactobacillus and decreased the abundance of bacteria like Kosakonia, Pantoea, Enterobacter and Lactococcus positively correlated with gas volume, CO₂ and N₂O concentrations. These results suggest that gas formation during ensiling mainly occurs in the first 9 days. Adding LP or CM can significantly improve the fermentation quality and decrease the gas volume. This would benefit to reducing GHG emissions in silage production.