Plant height (PH), primary lateral branch length (PBL) and branch number (BN) are architectural components impacting peanut pod yield, biomass production and adaptivity to mechanical harvesting. In this study, a recombinant inbred population consisting of 181 individual lines was used to determine genetic controls of PH, PBL and BN across three environments. Phenotypic data collected from the population demonstrated continuous distributions and transgressive segregation patterns. Broad-sense heritability of PH, PBL and BN was found to be 0.87, 0.88 and 0.92, respectively. Unconditional individual environmental analysis revealed 35 additive QTLs with phenotypic variation explained (PVE) ranging from 4.57 to 21.68%. A two-round meta-analysis resulted in 24 consensus and 17 unique QTLs. Five unique QTLs exhibited pleiotropic effects and their genetic bases (pleiotropy or tight linkage) were evaluated. Joint analysis was performed to estimate the QTL by environment interaction (QEI) effects on PH, PBL and BN, which collectively explained phenotypic variations of 10.80, 11.02, and 7.89%, respectively. We identified 3 major and stable QTL regions (uq9-3, uq10-2 and uq16-1) on chromosomes 9, 10 and 16, spanning 1.43-1.53 Mb genomic regions. Candidate genes involved in phytohormones biosynthesis, signaling and cell wall development were proposed to regulate these morphological traits. These results provide valuable information for further genetic studies and development of molecular markers applicable for peanut architecture improvement.
Root-knot nematodes (RKNs) are the most widespread soil-borne obligate endoparasites. They can infect the roots of many crops and cause significant yield losses. In tomato, the only commercially available RKN resistant gene Mi-1.2 fails at soil temperatures above 28°C. We cloned the heat stable RKN-resistant gene Mi-9 from a gene cluster composed of seven nucleotide-binding site and leucine-rich repeat (NBS-LRR) type resistant genes in Solanum arcunum accession LA2157. Screening nematode infections in individual & combinatorial knockouts of five NBS-LRR genes showed that Mi-9 Candidate 4 (MiC-4) alone is sufficient to confer heat stable RKN resistance. Our study identifies a new source of heat stable resistance to RKN in tomato for challenging environmental conditions. We also showcase a roadmap for rapid characterization of resistance genes by combining comparative genomics and genome editing, with the potential to be utilized in other crops.
Mycotoxins are the most widely present pollutants in both dietary provisions and livestock feed, and they pose a series of hazards for humans and animals. Deoxynivalenol (DON) is a prevalent mycotoxin that is primarily produced by Fusarium spp. and commonly found in various cereal products. Feeding swine diets contaminated with trichothecene DON can lead to major adverse effects, including reduced feed intake, diminished weight gains, and compromised immune function. Among all animal species tested, swine were the most sensitive to DON. Here we explored the disruption of gut health by DON, considering aspects such as intestinal histomorphology, epithelial barrier functions, the intestinal immune system, microflora, and short-chain fatty acid production in the intestines. Numerous additives have been documented for their potential in the detoxification of DON. These additives can alleviate the toxic effects of DON on pigs by modulating the Nrf2-Keap1, mitogen-activated protein kinases (MAPKs) and Nuclear factor kappa-B (NF-κB) signaling pathways. Additionally, there are additives capable of mitigating the toxicity of DON through adsorption or biotransformation. This update has novel potential for advancing our comprehension of the mechanisms linked to DON intestinal toxicity and facilitating the formulation of innovative strategies to mitigate the impact of DON.
Southern corn leaf blight (SCLB) caused by Cochlibolus heterostrophus, is a widespread foliar disease that has a substantial impact on maize yield in the Huanghuaihai region of China. Pydiflumetofen (Pyd), a new succinate dehydrogenase inhibitor (SDHI), has been found as a promising fungicide for the efficient control of SCLB, however, resistance of C. heterostrophus to Pyd has not been studied well. Here, five Pyd-resistant mutants were generated through fungicide adaptation. Sequence alignment analysis revealed that these mutants primarily mutated in ChSdhB and ChSdhD, with 3 genotypes: ChSdhBH277Y, ChSdhBI279T and ChSdhDH133Y, exhibiting two distinct categories of resistance: high resistance (HR) and moderate resistance (MR), which resistance factors (RF) is 214.22 and 44.33-53.67, respectively. These mutants were more pathogenic than the wild-type parental strains, but there was a significant reduction in mycelial growth rate and sporulation in the resistant mutants, indicating a significant fitness cost associated with resistance to Pyd. In addition, this study revealed a positive cross-resistance between Pyd and another SDHI fungicide cyclobutrifluram. However, no cross-resistance was found between Pyd and other classes of fungicide, including prochloraz, fludioxonil, iprodioneand pyraclostrobin. Homology modeling and molecular docking further confirmed that point mutation of ChSdhBH277Y, ChSdhBI279T, and ChSdhDH133Y could reduce binding affinity between Pyd and its target subunits from −74.07, −74.07, −152.52 kcal mol-1 to −3.90, −4.95, −9.93 kcal/mol, respectively. These findings not only provided valuable insights for managing SCLB caused by C. heterostrophus, but also enhanced our understanding of molecular mechanism underlying plant pathogen resistance to Pyd.
Cover cropping is a diversifying agricultural practice that can improve soil structure and function by altering the underground litter diversity and soil microbial communities. Here, we tested how a wheat cover crop alters the decomposition of cucumber root litter. A three-year greenhouse litterbag decomposition experiment showed that a wheat cover crop accelerates the decomposition of cucumber root litter. A microcosm litterbag experiment further showed that wheat litter and the soil microbial community could improve cucumber root litter decomposition. Moreover, the wheat cover crop altered the abundances and diversities of soil bacterial and fungal communities, and enriched several putative keystone OTUs, such as Bacillus spp. OTU1837 and Mortierella spp. OTU1236, that were positively related to the mass loss of cucumber root litter. The representative bacterial and fungal strains B186 and M3 were isolated and cultured. In vitro decomposition tests demonstrated that both B186 and M3 had cucumber root litter decomposition activity and a stronger effect was found when they were co-incubated. Overall, a wheat cover crop accelerated cucumber root litter decomposition by altering the soil microbial communities, particularly by stimulating certain putative keystone taxa, which provides a theoretical basis for using cover crops to promote sustainable agricultural development.
With the development of international trade and frequent personnel exchanges, biological invasion is showing a rapidly growing trend worldwide. Insects are ectothermic animals, so their geographical distribution is due largely to their high and low temperature tolerances. To study the temperature response mechanisms of Bemisia tabaci MED cryptic species, miRNA-seq technology was used to unravel the miRNA library of B. tabaci MED in three field populations (TP, HB, and HK) from cities with different environmental temperatures. We identified 12 differentially expressed miRNAs in response to temperature stress, and Bta-miR-998 and Bta-miR-129 were shown to be associated with temperature tolerance. In addition, we predicted and verified the target genes associated with the temperature tolerance imparted by Bta-miR-998 and Bta-miR-129. The results showed that the down-regulated target gene of Bta-miR-129, BtMGAT3, significantly reduced the heat tolerance and another down-regulated target gene, BtRGS7, affected the cold tolerance of B. tabaci MED. These results indicate that gene expression regulated by miRNAs is an important temperature response mechanism in B. tabaci MED. This study reveals the important regulatory role of miRNA in insect temperature adaptation and provides a new avenue for studying the regulation of insect gene expression by miRNA.
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 6th exon and 6th 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.
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, blaOXA-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.
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.
Asian citrus psyllid (ACP) is a significant pest of citrus crops that can transmit citrus Huanglongbing (HLB) by feeding on the phloem sap of citrus plants, which poses a significant threat to citrus production. Volatile signal chemicals with plant communication functions can effectively enhance the resistance of recipient plants to herbivorous insects with minimal impacts on plant growth. While (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), (E,E)-4,8,12-trimethyl-1,3,7,11-tridecene (TMTT), (E)-β-caryophyllene, and dimethyl disulfide (DMDS), are known as signaling molecules in guava-sweet orange communication, whether these four chemical signals can enhance the resistance of Citrus sinensis to feeding by ACP adults with no apparent costs in terms of plant growth remains unclear. Therefore, this study measured the effect of non-damaging induction by DMNT, TMTT, (E)-β-caryophyllene, and DMDS on the ability of C. sinensis to resist feeding by ACP, as well as their impacts on the defensive phytochemicals, defensive enzymes, functional nutrients, Photosystem II's utilization and allocation of light energy, photosynthetic pigments, growth conditions, and leaf stomatal aperture in C. sinensis. The results indicate that non-damaging induction by these four chemicals can enhance the activity of the defensive enzyme polyphenol oxidase (PPO) and increase the contents of total phenols, tannins, and terpenoid defensive phytochemicals within C. sinensis, thereby enhancing the resistance of C. sinensis to ACP feeding. Specifically, DMNT and DMDS exhibit more significant effects in inducing resistance compared to TMTT and (E)-β-caryophyllene. The characteristics of chlorophyll fluorescence parameters and changes in photosynthetic pigments in C. sinensis during different post-exposure induction periods revealed these chemicals can maintain the stability of the photosynthetic system in C. sinensis and regulate its capacity to capture, transmit, and distribute light energy, which significantly enhances the non-photochemical quenching ability (Y(NPQ)) of C. sinensis. In addition, detailed measurements of the water content, specific leaf mass (LMA), functional nutrients (soluble protein, soluble sugar, and amino acids), and stomatal parameters in C. sinensis leaves further indicated that the non-destructive induction by these chemicals can optimize the levels of functional nutrients in C. sinensis, primarily manifesting as the upregulation of soluble sugars, proline, or soluble proteins, and reduction of stomatal area and aperture, which maintains a stable leaf water content and LMA, thereby enhancing resistance to ACP while sustaining the healthy growth of C. sinensis. These results fully substantiate that the non-damaging induction by the signal chemicals DMNT, TMTT, (E)-β-caryophyllene, and DMDS can enhance the resistance of C. sinensis to ACP feeding while maintaining the balance between pest resistance and growth. This balance prevents any catastrophic effects on the growth of C. sinensis, so these agents can potentially be integrated with other pest management strategies for the collective protection of crops. This study provides theoretical support and assistance for the development of signal chemical inducers for the prevention and management of ACP in agricultural systems.
Chestnuts are important economic forest tree species with enormous application value in the wood, medicine, and chemical industries. Currently, the limited genome-wide SSR molecular marker information on chestnut resources significantly limits research on genetic diversity and identification of chestnut resources. To address this issue, we used GMATA to screen simple sequence repeat (SSR) markers throughout the Chinese chestnut genome. A total of 312,302 molecular markers were obtained with a density of 434.38/Mb. Subsequently, all SSR markers were examined for polymorphism using the HipSTR program and 138,208 polymorphic loci were finally obtained. To verify the identification ability of the developed SSR, we randomly selected 36 markers on 12 chromosomes to construct fingerprint maps of 96 ancient chestnut resources from the Yanshan Mountains. The results showed that only 6 pairs of primers were required to create a unique DNA fingerprint of the tested ancient trees, showing that the developed markers have high identification potential. We then evaluated the inter-specific universality and polymorphism of these markers using three species, including 91 chestnut plants. The molecular markers amplified 94% of the interspecies with a PIC value of 0.859. Cluster analysis revealed that testing resources using these developed markers can be well differentiated and these markers have been widely used to identify interspecific boundaries. These results proved that the developed molecular markers have the potential for genotypic diversity, which can provide references for genetic diversity research, variety identification, kinship analysis, selection of good products, and construction of core germplasm resources of chestnut and even chestnut plants. They lay a solid foundation for the molecular design of hybrids to improve breeding and develop germplasm resources.
Melon (Cucumis melo) is an important economic horticulture crop cultivated worldwide. NAC (NAM, ATAC, and CUC) transcription factors play crucial roles in the transcriptional regulation of various developmental stages in plant growth and fruit development, but little about their gene function is known in melon. Here, we identified 78 CmNAC family genes containing integrated and conserved NAM (no apical meristem) domain in the melon genome by performing genome-wide identification and bioinformatics analysis. Transcriptome data analysis and qRT-PCR results showed that most CmNACs are specifically enriched in the vegetative organ or the reproductive organ in melon. Through genetic transformation, we found that overexpression of CmNAC34 in melons led to the early ripening fruits, suggesting its positive role in promoting fruit maturation. Through performing yeast two-hybrid and bimolecular fluorescence complementation assays, we verified the direct protein interaction between CmNAC34 and CmNAC-NOR. The expression pattern of CmNAC34 and CmNAC-NOR were similar in melon tissues, and subcellular localization also revealed their nuclear protein characteristic. We transformed CmNAC-NOR in melon and found that its overexpression resulted in the early ripening fruits. Then, the yeast one-hybrid and dual luciferase reporter gene assay explored that CmNAC34 protein can bind to the promoters of two Glyoxalase (GLY) genes, which were involved in the abscisic acid signal pathway and associated with the fruit regulation. These findings revealed the molecular characteristics, expression profile, and functional pattern of the NAC family transcription factors genes and provided an insight into the molecular mechanism of CmNAC34 in regulating climacteric fruit ripening.
Ketosis, a common metabolic disease during early lactation, is associated with high circulating levels of β-hydroxybutyrate (BHB). A portion of BHB that reaches the mammary gland is utilized as precursor for synthesis of fatty acids. Recent findings from nonruminant studies revealed that long chain fatty acyl-CoA ligase 4 (ACSL4) could play a role in the regulation of cellular fatty acid metabolism, but the mechanisms by which ACSL4 mediates cellular lipid metabolism in response to BHB remains unclear. To achieve the aims, we conducted in vivo or in vitro analyses using bovine mammary gland biopsies and the immortalized mammary epithelial cell line (MAC-T). The in vivo study (n = 6 cows group-1) involved healthy cows (plasma BHB < 0.60 mmol L-1) or ketotic cows (plasma BHB > 2.0 mmol L-1) from which mammary gland tissue was biopsied. In vitro, MAC-T cells were challenged with 0, 0.3, 0.6, 1.2, or 2.4 mmol L-1 BHB for 24 h to determine an optimal dose. Subsequently, MAC-T were incubated with 1.2 mmol L-1 BHB for 0, 3, 6, 12, 24, or 48 h. Furthermore, MAC-T cells were treated with small interfering ACSL4 (siACSL4) for 24 h or ACSL4 overexpression plasmid (pcACSL4) for 36 h followed by a challenge with 1.2 mmol L-1 BHB for 24 h. Results showed that increased mRNA and protein abundance of lipogenic genes were linked to both mammary gland and in vitro challenge with BHB. BHB increased fatty acid content by activating ACSL4 expression, whereas inhibition of ACSL4 reduced BHB-induced reactive oxygen species (ROS) overproduction, enhancement of mitochondrial membrane potential, increase in fatty acid content, and lipid droplet accumulation. Furthermore, we also elevated ACSL4 expression with an overexpression plasmid to clarify its molecular role in response to BHB challenge. ACSL4 overexpression enhances BHB-induced lipid droplet accumulation by increased fatty acid content. Overall, the information showed that ACSL4 is crucial for the process of producing fatty acids from exogenous BHB. Reduced ACSL4 decreased fatty acid content and lipid droplet accumulation, improved mitochondrial function, directed more fatty acids towards oxidation. Thus, ACSL4 plays an important role in determining the fate of intracellular fatty acids and BHB in BMECs.
Genotype imputation is essential for increasing marker density and maximizing the utility of existing SNP array data in animal breeding. Although a wide range of software is available for genotype imputation, a comprehensive benchmark in pigs is still lacking. In this study, we benchmarked 24 combinations of genotype imputation software for SNP arrays in pigs, comprising six independent pre-phasing software (fastPHASE, MaCH, BIMBAM, Eagle, SHAPEIT, Beagle) and four distinct imputation software (pbwt, Minimac, IMPUTE, Beagle), using 1,602 whole-genome sequencing (WGS) pigs from a multibreed pig genomics reference panel (PGRP) in PigGTEx. Our results indicated that the combination of Beagle for pre-phasing and Minimac for imputation achieves the highest imputation accuracy with a concordance of 0.983, especially for low-frequency SNPs (MAF<0.05). Finally, we proposed three recommended strategies: i) the combination of Beagle and Minimac is optimal for achieving the highest accuracy; ii) the combination of Beagle and Beagle is recognized for its convenience and relatively high accuracy despite it being memory-intensive; iii) the combination of Eagle and pbwt is feasible for its minimal computational cost with relatively high accuracy. This study provides valuable insights for implementing genotype imputation for pig SNP arrays toward sequence data and offers a basis for applications in livestock and poultry breeding.
The combined application of organic manure and chemical fertilizers is an effective way to enhance soil organic carbon (SOC) sequestration through its influences on organic carbon (OC) input and the stability of SOC fractions. However, there is limited information on the carbon sequestration efficiency (CSE) of chemically separated SOC fractions and its response to OC input under long-term fertilization regimes, especially at different sites. This study used three long-term fertilization experiments in Gongzhuling, Zhengzhou and Qiyang spanning 20 years to compare the stocks and CSE in four different OC fractions (very labile OC, labile OC, less labile OC, and non-labile OC) and their relationships with annual OC input. Three treatments of no fertilization (CK), chemical nitrogen, phosphorous, and potassium fertilizers (NPK), and chemical NPK combined with manure (NPKM) were employed. The results showed that compared with CK, NPKM resulted in enhanced SOC stocks and sequestration rates as well as CSE levels of all fractions irrespective of experimental site. Specifically for the very labile and non-labile OC fractions, NPKM significantly increased the SOC stocks by 43 and 83%, 77 and 86%, and 73 and 82% in Gongzhuling, Qiyang, and Zhengzhou relative to CK, respectively. However, the greatest changes in SOC stock relative to the initial value were associated with non-labile OC fractions in Gongzhuling, Zhengzhou, and Qiyang, which reached 6.65, 7.16, and 7.35 Mg ha-1 under NPKM. Similarly, the highest CSE was noted for non-labile OC fractions under NPKM followed sequentially by the very labile OC, labile OC, and less-labile OC fractions, however a CSE of 8.56% in the non-labile OC fraction for Gongzhuling was higher than the values of 6.10 and 4.61% in Zhengzhou and Qiyang, respectively. In addition, the CSE for the passive pool (very labile+labile OC fractions) was higher than the active pool (less-labile+non-labile OC fractions), with the highest value in Gongzhuling. The redundancy analysis revealed that the CSEs of fractions and pools were negatively influenced by annual OC input, mean annual precipitation and temperature, but positively influenced by the initial SOC and total nitrogen contents. This suggests that differential stability of sequestered OC is further governed by indigenous site characteristics and variable amounts of annual OC input.
Deep learning-based intelligent recognition algorithms are increasingly recognized for their potential to address the labor-intensive challenge of manual pest detection. However, their deployment on mobile devices has been constrained by high computational demands. Here, we developed GBiDC-PEST, a mobile application that incorporates an improved, lightweight detection algorithm based on the you only look once (YOLO) series single-stage architecture, for real-time detection of four tiny pests (wheat mites, sugarcane aphids, wheat aphids, and rice planthoppers). GBiDC-PEST incorporates several innovative modules, including GhostNet for lightweight feature extraction and architecture optimization by reconstructing the backbone, the Bi-directional Feature Pyramid Network (BiFPN) for enhanced multiscale feature fusion, Depthwise convolution (DWConv) layers to reduce computational load, and the Convolutional Block Attention Module (CBAM) to enable precise feature focus. The newly developed GBiDC-PEST was trained and validated using a multitarget agricultural tiny pest dataset (Tpest-3960) that covered various field environments. GBiDC-PEST (2.8 MB) significantly reduced the model size to only 20% of the original model size, offering a smaller size than the YOLO series (v5 ~ v10), higher detection accuracy than YOLOv10n and v10s, and faster detection speed than v8s, v9c, v10m and v10b. In Android deployment experiments, GBiDC-PEST demonstrated enhanced performance in detecting pests against complex backgrounds, and the accuracy for wheat mites and rice planthoppers was improved by 4.5-7.5% compared with the original model. The GBiDC-PEST optimization algorithm and its mobile deployment proposed in this study offer a robust technical framework for the rapid, onsite identification and localization of tiny pests. This advancement provides valuable insights for effective pest monitoring, counting, and control in various agricultural settings.
Due to their sessile nature, plants require strong adaptability to complex environments, with stress tolerance often associated with trade-offs in growth and development (Major et al. 2020). This antagonistic relationship between defense and growth has been interpreted as a competition for limited resources that are allocated to defense at the expense of growth, or vice versa. Recent studies have demonstrated that hormone-based signaling networks trigger transcriptional changes in key genes, leading to trade-offs between growth rates and stress defense (Huot et al. 2014). Several genes involved in biotic and abiotic stress response have been identified. These genes contain nonsynonymous variants that show convergent changes in allele frequency across different breeding eras in both China and the United States (Wang et al. 2020), which may reflect the selection of biotic and abiotic stress response genes during modern maize breeding.
Transcription factors (TFs) play vital roles in regulation of gene expression in plant cells, with specific key TFs exhibiting multifunctionality by coordinating various regulatory pathways to promote plant growth (Hufford et al. 2021). Jasmonates (JAs) are identified among phytohormones for their significant roles in regulating various plant processes, particularly in defense mechanisms against pests. MYC2 is a central transcription factor that orchestrates the JA signaling pathway and defense responses in plants by regulating the expression of numerous genes (Du et al. 2022). Although MYC2 has been extensively characterized in Arabidopsis, studies in crops have revealed the complexity of MYC2’s function, with reports addressing different aspects, such as growth in wheat (Li et al. 2023) or stress defense in maize (Ma et al. 2023). However, lack of systematic understanding of the complex regulatory network of MYC2 in crops, particularly in maize constrain the further utilization of MYC2 and its downstream genes in maize genetic modification for breeding elite varieties. Here, we reported that ZmMYC2 had undergone selection during domestication and modern breeding; it acts as a key regulator of the trade-off between development and defense gene expression in maize, elucidating its regulatory network, which holds significant importance in balancing yield and resistance.
Given that some resistance genes have been selected during modern breeding, we analyzed the history of ZmMYC2 over the processes of maize evolution and artificial selection. According to maize Haplotype Map v3 (HapMap3) database consisting of 1164 modern maize accessions, 25 landraces, and 21 teosintes (Zea mays. parviglumis) (Bukowski et al. 2018), nucleotide diversity strongly decreased sharply at the promoter region (2000 bp upstream of transcription start site) of ZmMYC2 during breeding, while the coding region and 3’-downstream region of ZmMYC2 showed less dramatic changes in nucleotide diversity changes (Fig. 1-A). Thus, we hypothesize that the genetic diversity within the promoter region of ZmMYC2 has decreased during the breeding process, with favorable variations being selected. Moreover, the frequency of three polymorphisms underwent convergent changes during modern breeding in both the United States and China (Fig. 1-B–D). These three polymorphisms constituted three principal haplotypes: pZmMYC2Hap1, pZmMYC2Hap2, and pZmMYC2Hap3, of which the frequency of pZmMYC2Hap1 showed an increasing trend during modern maize breeding (Fig. 1-E). The rare haplotype pZmMYC2Hap3 (n=4) emerged only during the breeding era of China in 2000. LUC signal activity for pZmMYC2Hap3 was significantly lower than that of the other two haplotypes in the promoter region (pZmMYC2Hap1, pZmMYC2Hap2) (Fig. 1-F–H), suggesting a differential regulatory potential among the haplotypes. These data indicate that ZmMYC2 was under-selected during maize evolution and breeding processes of maize. Next, we investigated the expression level of genome-wide association studies of ZmMYC2 based on 368 maize inbred lines using RNA-seq and genome resequencing data (Fu et al. 2013; Li et al. 2013). The results showed a strong peak signal containing the genomic region of ZmMYC2 on chromosome 1 (Fig. 1-I).
To mine the genes downstream of ZmMYC2, we performed protoplast transient expression-based RNA-sequencing (PER-seq) analysis to facilitate the discovery of new downstream genes utilizing a consistent protoplast system (Zhu et al. 2023). In total， 281.6 million clean reads were generated, among which an average of approximately 87% of reads were mapped uniquely to the reference genome (Appendices A and B). The results demonstrated a significant increase in the expression level of ZmMYC2 in each of three replicates of the pRTL2-ZmMYC2-GFP (MYC2-GFP) construct, exceeding a 500-fold increase compared to the pRTL2-GFP-empty (GFP-empty) construct (Fig. 1-J). Furthermore, upon analyzing differentially expressed genes (DEGs) with a false discovery rate (FDR) <0.05 as the threshold, it was found that 4480 unique DEGs of MYC2-GFP, among which 2,677 were up-regulated compared to GFP-empty (Appendix C). These up-regulated genes are enriched in circadian rhythm, cell cycles, plant growth, and in response to stress, indicating that these genes are regulated directly or indirectly by ZmMYC2 (Appendix D-A–B).
Several potential candidate genes were selected in an unbiased manner based on their log2(fold-change) ≥2.5 (Fig. 1-J). Gene expression profiling analysis of ZmMYC2 and its potential targets revealed essential coincidence (Appendix E). The interaction between MYC2 and targets observed in the PER-seq system, were further confirmed through expression quantitative trait loci (eQTL) analysis, dual-luciferase reporter assay (DLR), and electrophoretic mobility shift assay (EMSA). Among the target genes, the members of cytochrome P450 (CYP) gene family are widely distributed in plants involving in various biological processes, such as detoxification of xenobiotics, secondary metabolites production, and terpenoid synthesis (Chakraborty et al. 2023; Sun et al. 2024). Our results identified an unreported gene of cytochrome P450 family ZmCYP709H1 as a target of ZmMYC2. Additionally, eQTL analysis of ZmCYP709H1 revealed a strong trans-eQTL signal in the region of chromosome 1, which contains the genomic region of ZmMYC2 (Fig. 1-K). Subsequent validation through DLR and EMSA confirmed that ZmMYC2 interacts with the promoter region of ZmCYP709H1 and stimulates its expression (Fig. 1-L; Appendices F-A and G-A). Moreover, the transcriptional activation effect of ZmMYC2 on the promoter of ZmCYP709H1 was suppressed by ZmJAZ8 (Fig. 1-L). A recent report showed reduced expression of ZmCYP709H1 in three maize dwarf mutants compared to the wild-type, reflecting its potential role in regulating growth, particularly plant height. This result supports our proposed function of the ZmMYC2-ZmCYP709H1 model (Gao et al. 2024). Additionally, two other CYP genes, ZmBX5 and ZmBX6, were identified as potential downstream genes of ZmMYC2 that participate in benzoxazinoid synthesis, which is consistent with the findings of a previous study (Ma et al. 2023). We further confirmed that ZmMYC2 can physically bind to the promoter region of these two genes and activate their expression (Appendix H-A–F). Besides, the result showed that ZmMYC2 can activate ZmBRD1 expression, which is a member of the CYP gene family and responsible for the final step of brassinosteroid synthesis (Tian et al. 2019) (Fig. 4-A and B; Appendix I-A–D).
The AUXIN RESPONSE FACTOR (ARF) family consists of plant-specific TFs that are key regulators of gene expression in response to the plant hormone auxin (AUX), and participated in various developmental processes such as vascular tissue differentiation, root and shoot development, and environmental stimuli responses (Hagen and Guilfoyle 2002; Salmon et al. 2008). However, little evidence has been found to support the regulation of ARF gene expression by the core factor ZmMYC2 in the JA signal transduction pathway in maize. Our data showed that the expression of ZmARF3 was regulated by a trans-eQTL signal involving the gene region of ZmMYC2 (Appendix F-B). In addition, ZmMYC2 can bind to the promoter region of the ZmARF3 gene and activate its transcription (Fig. 1-M; Appendix G-B). Besides, MYC2 can activate expressions of senescence-associated genes in rice and wheat, which could be repressed by physical interactions with TaARF15-A1 (Li et al. 2023). These data demonstrate the key role of MYC2 in regulating the stress resistance and growth of maize through the synergistic regulation of JA and AUX hormone signaling pathways.
Tonoplast intrinsic proteins (TIPs), a subgroup of the aquaporin family, are integral membrane proteins that are crucial for transporting water and small solutes across cellular membrane to maintain water balance (Chaumont et al. 2001). We found that ZmTIP3c was activated by ZmMYC2 (Fig. 1-N; Appendices F-C and G-C), which supports the potential role of ZmMYC2 in jointly regulating drought stress and JA signal transduction. The CER2 gene, which is a member of the ECERIFERUM family, is critical for the synthesis of epicuticular wax (Bourdenx et al. 2011; Zhao et al. 2024). A recent study demonstrated that wounding-induced wax accumulation was primarily regulated by the JA signaling pathway in Arabidopsis, suggesting the potential of JA signaling in wax synthesis (Huang et al. 2024). We identified ZmCER2 as a ZmMYC2 target (Fig. 1-O; Appendices F-D and G-D). Additionally, we confirmed the upregulation of ZmCER2 in response to drought stress in five elite inbred lines representing distinct heterotic groups of maize (Fig. 1-P), as observed by previous studies (Zhang et al. 2018, 2020; Jiang et al. 2023). The result indicates that the drought-induced trait of ZmCER2 can be observed across different genetic backgrounds, thus supporting the potential role of ZmMYC2 in modulating JA signaling and response to drought stress in maize mediated by ZmCER2.
In summary, our findings support the selection of ZmMYC2 during domestication and breeding, highlighting its critical role in regulating genes involving plant growth and development. Collectively, our eQTL, DLR, and EMSA data successfully validated several targets (ZmCER2, ZmARF3, ZmBRD1 ZmTIP3c, ZmCYP709H1, ZmBX5, and ZmBX6) of ZmMYC2, that encode diverse proteins and participate in various metabolic pathways (Fig. 1-Q). Of these, ZmCER2 was confirmed to be induced by drought stress and activated by ZmMYC2, suggesting that ZmMYC2 may play a role in the drought response by regulating synthesizing epicuticular wax. These findings underscore the diverse functions of ZmMYC2 in maintaining the balance between plant development and defense-response, primarily via the JA signaling pathway. Our data represent a foundation for the further function and mechanism elucidation of of ZmMYC2 and its “Yin-Yang” roles in regulating plant defense and growth (Fig. 1-Q). Given the crucial role of ZmMYC2 in balancing development and resistance, further work is needed to confirm to unlock the full potentials of ZmMYC2 in mediating yield and resistance through JA signaling pathway by exploring the function of those downstream targets, which is a significant step toward crop precision breeding.
Cadmium (Cd) stress is a serious threat to apple growth and development. Ethylene response factors (ERFs) are a major family of transcription factors (TFs) that play a key role in the resistance to Cd stress. In this study, we found that the ERF TF MdERF114 was induced in response to Cd stress. The overexpression of MdERF114 in apple (Malus domestica) roots reduced the accumulation of Cd in the plants and enhanced their tolerance to Cd stress. Yeast one-hybrid (Y1H) assays, dual-luciferase assays, and electrophoretic mobility shift assays indicated that MdERF114 directly binds to the promoter of MdATG16 and activates its expression to increase autophagic activity, which leads to higher resistance to Cd stress. In addition, MdMYB306 interacts with MdERF114 and enhances the resistance to Cd stress by promoting the binding of MdERF114 to the promoter of MdATG16. Our findings reveal an important mechanism by which MdMYB306-MdERF114-MdATG16 influences the resistance of apple to Cd stress.
This investigation evaluated the impact of as-is biochar (BC) and phosphorous (P)-loaded biochar (PBC) (3%) on the growth and biochemical characteristics of rice under exposure to vanadium (V) (60 mg L–1). The results indicate that rice plants exposed to a V-only treatment experienced declines in several growth parameters. Conversely, the inclusion of BC and PBC caused noteworthy increases in physiological traits. PBC performed well in stress environments. Specifically, the shoot and root fresh weights increased by 82.86 and 53.33%, respectively, when compared to the V-only treatment. In addition, the SPAD chlorophyll of the shoot increased by 13.05% relative to the V-amended plants. Moreover, including BC and PBC improved the antioxidant enzyme traits of plant shoot and root, such as significant increases in superoxide dismutase (SOD by 56.11 and 117.35%), catalase (CAT by 34.19 and 35.77%), and peroxidase (POD by 25.90 and 18.74%) when compared to V-only amended plants, respectively. These findings strongly suggest that the application of BC and PBC can trigger biochemical pathways that facilitate biomass accumulation in meristematic cells. However, further investigations are required to elucidate the underlying mechanisms responsible for this growth promotion.
A critical challenge for global food security and sustainable agriculture is enhancing crop yields while reducing chemical N inputs. Improving N use efficiency in crops is essential for increasing agricultural productivity. The aim of this study was to evaluate the impacts of intercropping maize with leguminous green manure on grain yield and N utilization under reduced N-fertilization conditions. A field experiment with a split-plot design was conducted in northwestern China from 2018 to 2021. The main plots consisted of two cropping systems: maize–common vetch intercropping (IM) and sole maize (SM). The subplots had three N levels: zero N application (N0, 0 kg ha–1), a 25% reduction from the traditional chemical N supply (N1, 270 kg ha–1), and the traditional chemical N supply (N2, 360 kg ha–1). The results showed that the negative effects of N reduction on maize grain yield and N uptake were compensated by intercropping leguminous green manure, and the improvements increased with cultivation years. The integrated system involving maize–leguminous green manure intercropping and a reduced N supply enhanced N translocation from maize vegetative organs to grains and increased the nitrate reductase and glutamine synthetase activities in maize leaves. The supercompensatory effect in maize leaves increased year by year, reaching values of 16.1, 21.3, and 25.5% in 2019, 2020, and 2021, respectively. These findings suggest that intercropping maize with leguminous green manure under reduced chemical N input can enhance N assimilation and uptake in maize. By using this strategy, chemical fertilizer is effectively replaced by leguminous green manure, thereby improving N use efficiency and maintaining stable yields in the maize-based intercropping system.
