Leaves are the main places for photosynthesis and organic synthesis of cotton.  Leaf shape has important effects on the photosynthetic efficiency and canopy formation, thereby affecting cotton yield.  Previous studies have shown that LMI1 is the main gene regulating leaf shape. In this study, the LMI1 gene (LATE MERISTEM IDENTITY1) was inserted into the 35S promoter expression vector, and cotton plants overexpressing LMI1(OE) were obtained through genetical transformation.  Statistical analysis of the biological traits of T₁ and T₂ populations showed that compared to wild type (WT), OE plants had significant larger leaves, thicker stems and significantly increased dry weight.  Furthermore, plant sections of the main vein and petiole showed that the number of cell in those tissues of OE plants increased significantly.  In addition, RNA-seq analysis revealed differential expression of genes related to gibberellin synthesis and NAC gene family (genes containing the NAC domain) in OE and WT plants, suggesting that LMI1 is involved in secondary wall formation and cell proliferation, and promotes stem thickening.  Moreover, GO (Gene Ontology) analysis enriched the terms of calcium ion binding, and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis enriched the terms of fatty acid degradation, phosphatidylinositol signal transduction system, and cAMP signal pathway.  These results suggested that LMI1 OE plants were responsive to gibberellin hormone signals, and altered messenger signal (cAMP, Ca²⁺) which amplified this function, to promote the stronger above ground vegetative growth.  This study found the LMI1 soared the nutrient growth in cotton, which is the basic for higher yield.

Feed efficiency (FE) is a crucial economic trait that significantly impacts profitability in intensive sheep production, and can be evaluated by the residual feed intake (RFI) and feed conversion ratio (FCR). However, the underlying genetic mechanisms that underlie FE-related traits in sheep are not fully understood. Herein, we measured the FE-related traits of 1280 Hu sheep and conducted the phenotype statistics and correlation analysis, the result showcase that there was a large variation for FE-related traits, and RFI was significant positive correlation with average daily feed intake (ADFI) and FCR. Moreover, a genome-wide association study (GWAS) was conducted using whole-genome resequencing data to investigate the genetic associations of ADFI, FCR and RFI. For ADFI and FCR traits, two and one single nucleotide polymorphisms (SNPs) exceeded the genome-wide significance threshold, whereas ten and five SNPs exceeded the suggestive significance threshold. For RFI traits, only four SNPs exceeded the suggestive significance threshold. Finally, a total of eight genes (LOC101121953, LOC101110202, CTNNA3, IZUMO3, PPM1E, YIPF7, ZSCAN12 and LOC105603808) were identified as potential candidate genes for FE-related traits. Simultaneously, we further analyzed the effects of two candidate SNPs associated with RFI on growth and FE traits in enlarged experimental population, the results demonstrated that these two SNPs was not significantly associated with growth traits (P > 0.05), but significantly related to RFI traits (P < 0.05). These findings will provide valuable reference data and key genetic variants that can be used to effectively select feed-efficient individual in sheep breeding programs.

Starch biosynthesis is a complex process that relies on the coordinated action of multiple enzymes. Resistant starch is not digested in the small intestine, thus preventing the rapid rise of the glycemic index. Starch synthase 2a (SS2a), a key enzyme in amylopectin biosynthesis, has significant effects on starch structure and properties. In this study, we identified an ss2a null mutant (M3-1413) with a single base mutation from an ethyl methane sulfonate (EMS)-mutagenized population of barley. The mutation was located at the 3´ end of the first intron of the RNA splicing receptor (AG) site, resulting in abnormal RNA splicing and two abnormal transcripts of ss2a, which caused the inactivation of the SS2a gene. The starch structure and properties were significantly altered in the mutant, with M3-1413 containing decreased total starch and increased amylose and resistant starch levels. This study sheds light the effect of barley ss2a null mutations on starch properties and helps to guide new applications of barley starch to develop nutritious food products.

Rapeseed (Brassica napus L.) is the second most widely grown premium oilseed crop globally, mainly for its vegetable oil and protein meal.  One of the main goals of breeders is producing high-yield rapeseed cultivars with sustainable production to meet the requirements of the fast-growing population.  Besides the pod number, seeds per silique (SS), and thousand-seed weight (TSW), the ovule number (ON) is a decisive yield determining factor of individual plants and the final seed yield.  In recent years, tremendous efforts have been made to dissect the genetic and molecular basis of these complex traits, but relatively few genes or loci controlling these traits have been reported thus far.  This review highlights the updated information on the hormonal and molecular basis of ON and development in model plants (Arabidopsis thaliana).  It also presents what is known about the hormonal, molecular, and genetic mechanism of ovule development and number, and bridges our understanding between the model plant species (A. thaliana) and cultivated species (B. napus).  This report will open new pathways for primary and applied research in plant biology and benefit rapeseed breeding programs.  This synopsis will stimulate research interest to further understand ovule number determination, its role in yield improvement, and its possible utilization in breeding programs. 

Banana is a significant crop, and three banana leaf diseases, including Sigatoka, Cordana and Pestalotiopsis, have the potential to have a serious impact on banana production. Existing studies are insufficient to provide a reliable method for accurately identifying banana leaf diseases. Therefore, this paper proposes a novel method to identify banana leaf diseases. First, a new algorithm called K-scale VisuShrink algorithm (KVA) is proposed to denoise banana leaf images. The proposed algorithm introduces a new decomposition scale k based on the semi-soft and middle course thresholds, the ideal threshold solution is obtained and substituted with the newly established threshold function to obtain a less noisy banana leaf image. Then, this paper proposes a novel network for image identification called Ghost ResNeSt-Attention RReLU-Swish Net (GR-ARNet) based on Resnet50. In this, the Ghost Module is implemented to improve the network's effectiveness in extracting deep feature information on banana leaf diseases and the identification speed; the ResNeSt Module adjusts the weight of each channel, increasing the ability of banana disease feature extraction and effectively reducing the error rate of similar disease identification; the model's computational speed is increased using the hybrid activation function of RReLU and Swish. Our model achieves an average accuracy of 96.98% and a precision of 89.31% applied to 13021 images, demonstrating that the proposed method can effectively identify banana leaf diseases.

Malate dehydrogenase (MDH) is a widely expressed enzyme that plays a key role in plant growth, development, and the stress response.  However, information on MDH genes in the soybean genome is limited. Seventeen members of the soybean MDH family were identified by genome-wide analysis, and the presence of conserved protein motifs was analyzed.  The genes were divided into five clusters according to their phylogenetic relationships.  The intracellular localizations of six GmMDHs were determined by confocal microscopy on Arabidopsis mesophyll protoplasts.  Transcripts of GmMDHs were significantly increased by abiotic stress (drought, salt, and alkalinity) and hormone treatments, as shown by analysis of cis-regulatory elements and quantitative real-time polymerase chain reaction (qRT-PCR).  GmMDHs displayed unique expression patterns in diverse soybean tissues.  It is noteworthy that under salt stress, the expression levels of a chloroplast isoform (GmMDH2) were unusually high, presumably indicating a critical role in soybean responses to salinity.  Expression of GmMDH2 in Escherichia coli showed that the recombinant enzyme had NADP-dependent MDH activity. The redox states of the nicotinamide adenine dinucleotide phosphate (NADPH) pool and antioxidant activities were shown to be modulated by GmMDH2 gene overexpression, which in turn reduced reactive oxygen species (ROS) formation in transgenic soybean, significantly enhancing the salt stress resistance.  Gene-based association analysis showed that variations in GmMDH2 were strongly linked to seedling salt tolerance.  A polymorphism possibly associated with salt tolerance was discovered in the promoter region of GmMDH2.  These findings not only improve our understanding of the stress response mechanism by identifying and characterizing the MDH gene family throughout the soybean genome but it also identified a potential candidate gene for the future enchancement of salt tolerance in the soybean.

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.

Genotyping by target sequencing (GBTS) integrates the advantages of silicon-based technology (high stability and reliability) and genotyping by sequencing (high flexibility and cost-effectiveness). However, GBTS panels are not currently available in pigs. In this study, based on GBTS technology, we first developed a 50K panel, including 52000 SNPs, in pigs, designated GBTS50K. A total of 6032 individuals of Large White, Landrace, and Duroc pigs from ten breeding farms were used to assess the newly developed GBTS50K. Our results showed that GBTS50K obtained a high genotyping ability, the SNP and individual call rates of GBTS50K were 0.997~0.998, and the average consistency rate and genotyping correlation coefficient were 0.997 and 0.993, respectively, in replicate samples. We also evaluated the efficiencies of GBTS50K in the application of population genetic structure analysis, selection signature detection, genome-wide association studies (GWAS), genotyped imputation, genetic selection (GS), etc. The results indicate that GBTS50K is plausible and powerful in genetic analysis and molecular breeding. For example, GBTS50K could gain higher accuracies than the current popular GGP-Porcine bead chip in genomic selection on two important traits of backfat thickness at 100 kg and days to 100 kg in pigs. Particularly, due to the multiple single-nucleotide polymorphisms (mSNPs), GBTS50K generated 100K qualified SNPs without increasing genotyping cost, and our results showed that the haplotype-based method can further improve the accuracies of genomic selection on growth and reproduction traits by 2 to 6%. Our study showed that GBTS50K could be a powerful tool for underlying genetic architecture and molecular breeding in pigs, and it is also helpful for developing SNP panels for other farm animals.

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.

棉花不仅是重要的天然纺织纤维作物，也是一种重要的食用油来源。棉籽油中约含14%的油酸和59%的亚油酸。提高油酸含量有助于增强棉籽油的氧化稳定性和营养价值。磷脂酰胆碱:二酰基甘油胆碱磷酸转移酶(PDCT)是调控磷脂酰胆碱与二酰基甘油转化的关键酶。本研究克隆了四个棉花PDCT同源基因，分别命名为GhPDCT1-4。发现GhPDCT3和GhPDCT4几乎不表达，而GhPDCT1在棉籽油分积累时期显著上调。利用CRISPR/Cas9系统同时敲除序列高度相似的GhPDCT1和GhPDCT2基因后，ghpdct突变体种子中油酸含量从野生型的14.46%增加到16.49%，而亚油酸含量从59.98%减少到52.83%。此外，ghpdct种子中的棕榈酸和硬脂酸含量也有所增加。本研究获得了油酸含量提高的新型棉籽油种质，有望提升棉花作为油料作物的经济和营养价值，推动棉花产业的升级。

Seed aging tolerance during storage is generally an important trait for crop production, yet the role of small auxin-up RNA genes in conferring seed aging tolerance is largely unknown in rice.  In this study, one small auxin-up RNA gene, OsSAUR33, was found to be involved in the regulation of seed aging tolerance in rice.  The expression of OsSAUR33 was significantly induced in aged seeds compared with unaged seeds during the seed germination phase.  Accordingly, the disruption of OsSAUR33 significantly reduced seed vigor compared to the wild type (WT) in response to natural storage or artificial aging treatments.  The rice OsSAUR33 gene promotes the vigor of aged seeds by enhancing their reactive oxygen species (ROS) level during seed germination, and the accumulation of ROS was significantly delayed in the aged seeds of Ossaur33 mutants in comparison with WT during seed germination.  Hydrogen peroxide (H₂O₂) treatments promoted the vigor of aged seeds in various rice varieties.  Our results provide timely theoretical and technical insights for the trait improvement of seed aging tolerance in rice.

Watermelon (Citrullus lanatus) is an economically important horticultural crop.  However, it is susceptible to low-temperature stress, which significantly challenges 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, and identifies candidate genes for the genetic improvement of watermelon with higher low-temperature tolerance.

The germination process of seeds is influenced by the interplay between two opposing factors: pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI), which collectively regulate patterns of pectin methylesterification.  Despite the recognized importance of pectin methylesterification in seed germination, the specific mechanisms that govern this process remain unclear.  In this study, we demonstrated that the overexpression of GhPMEI53 is associated with a decrease in PME activity and an increase in pectin methylesterification.  This leads to the softening of the cell wall in seeds, which positively regulates cotton seed germination.  AtPMEI19, the homologue in Arabidopsis thaliana, plays a similar role in seed germination to GhPMEI53, indicating a conserved function and mechanism of PMEI in seed germination regulation.  Further studies revealed that GhPMEI53 and AtPMEI19 directly contribute to promoting radicle protrusion and seed germination by inducing cell wall softening and reducing mechanical strength.  Additionally, the pathways of ABA and GA in the transgenic materials underwent significant changes, suggesting that GhPMEI53/AtPMEI19-mediated pectin methylesterification serves as a regulatory signal for the related phytohormones involved in seed germination.  In summary, GhPMEI53 and its homologs alter the mechanical properties of cell walls, influencing the mechanical resistance of the endosperm or testa.  Moreover, they impact cellular phytohormone pathways (e.g., ABA, GA) to regulate seed germination.  These findings enhance our understanding of pectin methylesterification in cellular morphological dynamics and signaling transduction, and contribute to a more comprehensive understanding of the PME/PMEI super-gene family in plants.

N6-methyladenosine (m6A) plays a key role in mammalian early embryonic development and cell lineage differentiation. However, the role and mechanisms of 18S ribosomal RNA (rRNA) m6A methyltransferase METTL5 in early embryonic development remain unclear. Here, we found that 18S rRNA m6A methyltransferase METTL5 plays an important role in porcine early embryonic development. METTL5 knockdown and overexpression significantly reduced the developmental efficiency of porcine early embryos and impaired cell lineage allocation. METTL5 knockdown apparently decreased the global translation efficiency in blastocyst, while METTL5 overexpression increased the global translation efficiency. Furthermore, METTL5 knockdown did not affect the abundance of CDX2 mRNA, but resulted in a significant reduction in CDX2 protein levels. Moreover, the low developmental efficiency and abnormal lineage distribution of METTL5 knockdown embryos could be rescued by CDX2 overexpression. Collectively, our results demonstrated that 18S rRNA methyltransferase METTL5 regulates porcine early embryonic development via modulating the translation of CDX2.

The commercialization of genetically modified crops has increased food production, improved crop quality, reduced pesticide use, promoted changes in agricultural production methods, and become an important new productivity to deal with insect pests and weeds while reducing cultivated land area.  This article provides a comprehensive examination of the global distribution of genetically modified crops in 2023.  It discusses the internal factors that are driving this, such as the increasing number of genetically modified crops and the increased variety of commodities.  It also provides information support and application direction guidance for the new productivity of global agricultural science and technology.

Maize (Zea mays L.) is a monoecious grass species with separate male and female inflorescences which form the tassel and ear, respectively.  The mature ear inflorescences usually bear hundreds of grains, thus directly influence maize grain production and yield.  Here, we isolated a recessive maize mutant, tasselseed2016 (ts2016), which exhibits pleiotropic inflorescence defects and a reduction in grain yield.  These defects include loss of determinacy and identity in meristems and floral organs, as well as a lack of the lower floret abortion in maize ear, and the smaller grain size.  Using map-based cloning and allelic test, we identified and confirmed a microRNA gene MIR172e as the target gene controlling these related traits.  Furthermore, our evidence uncovered a new potential miR172e/ETHYLENE RESPONSIVE ELEMENT BINDING197 (EREB197) regulatory module which controls the abortion of lower floret in maize ear. Transcriptome analysis revealed that the mutation of MIR172e represses multiple biological processes, particularly the flower development and hormone-related pathways in maize ear.  Additionally, we found the mutation in the DNA sequence of MIR172e affects in RNA transcription, resulting in elongation blockage at the mutant site.  Our results reveal the function and molecular mechanism of MIR172e in maize inflorescences and grain yield, and this study deepens our knowledge of maize inflorescence development.

EPSPS is a key gene in the shikimic acid synthesis pathway and has been widely used in breeding crops with herbicide resistance.  However, its role in regulating cell elongation is poorly understood.  Through the overexpression of EPSPS genes, we generated lines resistant to glyphosate that exhibited an unexpected dwarf phenotype.  A representative line, DHR1, exhibits a stable dwarf phenotype throughout its entire growth period.  Except for plant height, the other agronomic traits of DHR1 were similar to its transgenic explants ZM24.  Paraffin section experiments showed that DHR1 internodes were shortened due to reduced elongation and division of internode cells.  Exogenous hormones confirmed that DHR1 is not a classical BR- or GA-related dwarfing mutant.  Hybridization analysis and fine mapping confirmed that the EPSPS gene is the causal gene for dwarfism, and the phenotype can be inherited in different genotypes.  Transcriptome and metabolome analyses showed that genes associated with the phenylpropanoid synthesis pathway were enriched in DHR1 when compared with ZM24.  Flavonoid metabolites were enriched in DHR1, whereas lignin metabolites were decreased.  The enhancement of flavonoids likely resulted in differential expression of auxin signal pathway genes and altered the auxin response, subsequently affecting cell elongation.  This study provides a new strategy for generating dwarfs and will accelerate advancements in light simplification of cultivation and mechanized harvesting for cotton.

Cotton fiber quality is a persistent concern that determines planting benefits and the quality of finished textile products.  However, the limitations of measurement instruments have hindered the accurate evaluation of some important fiber characteristics (such as fiber maturity, fineness, neps), which in turn has impeded the genetic improvement and industrial utilization of cotton fiber.  Here, twelve single fiber quality traits were measured using Advanced Fiber Information System (AFIS) equipment among 383 accessions of upland cotton (Gossypium hirsutum L.).  Also, eight conventional fiber quality traits were assessed by the High Volume Instrument (HVI) system.  Genome-Wide Association Study (GWAS), linkage disequilibrium (LD) block genotyping and functional identification were conducted sequentially to uncover the associated elite loci and candidate genes of fiber quality traits.  As a result, the pleiotropic locus FL_D11 regulating fiber length related traits was again identified in this study.  More importantly, three novel pleiotropic loci (FM_A03, FF_A05, FN_A07) regulating fiber maturity, fineness and neps respectively were detected on the basis of AFIS traits.  Numerous highly-promising candidate genes were screened out by integrating RNA-seq and qRT-PCR analyses, including the reported GhKRP6 for fiber length and newly identified GhMAP8 for maturity and GhDFR for fineness.  The origin and evolution analysis of pleiotropic loci indicated that the selection pressure on FL_D11, FM_A03 and FF_A05 increased as the breeding period approached and the origins of FM_A03 and FF_A05 were traced back to cotton landraces.  These findings reveal the genetic basis underlying fiber quality and provide insight into genetic improvements and textile utilization of fiber in G. hirsutum.

Planting density is a major limiting factor for maize yield, and breeding for density tolerance breeding has become an urgent issue.  The leaf structure of the maize ear leaf is the main factor that restricts planting density and yield composition.  In this study, a natural population of 201 maize inbred lines was used for genome-wide association analysis, which identified nine SNPs on chromosomes 2, 5, 8, 9, and 10 that were significantly associated with ear leaf type structure.  Further verification through qRT-PCR confirmed the association of five candidate genes with these SNPs, with the Zm00001d008651 gene showing significant differential expression in compact and flat maize inbred lines.  Enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) suggested that this gene is involved in the glycolysis process.  The analysis of the basic properties of this gene revealed that it encodes a stable, basic protein consisting of 593 amino acids with some hydrophobic ability.  The promoter region contains stress and hormone (ABA) related elements.  The mutant of this gene increased the uppermost ear leaf angle (eLA) and the first leaf below the uppermost ear (bLA) by 4.96° and 0.97° compared with normal inbred lines.  Overall, this research sheds light on the regulatory mechanism of ear and leaf structure that influence density tolerance and provides solid foundational work for the development of new varieties.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight in rice, which reduces crop yield and leads to significant economic losses.  Bacterial sigma (σ) factors are highly specialized proteins that allow RNA polymerase to recognize and bind to specific promoters.  σ⁷⁰ factors also regulate the expression of genes involved in stress response and virulence.  However, the role of RpoD in Xoo is still unclear.  In this study, we found that σ⁷⁰ factor RpoD is quite conservative among phytopathogenic bacteria, especially in Xanthomonas sp.  In Xoo, PXO_RpoD plays an important role in oxidative stress tolerance and cell motility, as well as being essential for full virulence.  Cleavage under targets and tagmentation (CUT&Tag) analyses indicated that RpoD mediates the type three secretion system (T3SS) by regulating the regulation of hrpG and hrpX.  By performing bacterial one-hybrid and electrophoretic mobility assay (EMSA), we observed that RpoD directly bound to the promoters of hrpG and hrpX.  Collectively, these results demonstrate the transcriptional mechanism and pathogenic functions of RpoD in regulating cell motility and oxidative stress response, providing novel insights into potential targets for disease control.

CRISPR-Cas9 emerged as a powerful tool for gene editing, which has been widely used in plant functional genomics research and crop genetic breeding (Chen et al. 2019). The target specificity of CRISPR-Cas9 relies on the 20-base-pair single guide RNA (sgRNA), making it relatively quick and straightforward to create plant-specific mutant libraries through large-scale synthesis of sgRNAs targeting multiple genes or even the whole genome. Several CRISPR-Cas9 mutant libraries have been developed for crops such as rice (Lu et al. 2017; Meng et al. 2017), soya bean (Bai et al. 2020), Brassica napus (He et al. 2023), and cotton (Sun et al. 2023). However, no CRISPR-Cas9 mutant library has yet been generated in woody crop plants. Grape (Vitis vinifera L.) is one of the oldest and most economically valuable fruit crops worldwide. The MYB family is one of the most abundant and versatile transcription factor families in plant (Wu et al. 2022). Here, we described a strategy for generating a collection of MYB mutant lines in grape using a sgRNA library.

We obtained 138 grape MYB transcription factor sequences from the Plant Transcription Factor Database (PlantTFDB, http://planttfdb.gao-lab.org/family.php?sp=Vvi&fam=MYB) (Appendix A). Since genes with similar sequences often share similar functions, a phylogenetic tree was constructed and divided all MYBs into 30 sets based on the sequence similarity (Fig. 1-A and Appendix B-a). A total of 127 sgRNAs were designed, each targeting conserved regions shared by two or more MYB transcription factors with fewer than three base-pair differences. This approach aimed to simultaneously mutate multiple MYB members within a cluster using a single sgRNA, addressing the challenge of genetic redundancy (Appendix B-b). These shared target sites were designated as Target1 (T1). In addition, specific sgRNAs targeting individual MYB transcription factors were designed using the  CRISPR-P 2.0 online tool (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR) (Liu et al. 2017). The design criteria included selecting target sites within exons near the start of open reading frames (ORFs), a GC content of at least 40%, and an off-target efficiency below 0.4. Finally, 138 sgRNAs targeting specific sites for each MYB transcription factor were designed, referred to as Target2 (T2). In total, a comprehensive sgRNA library comprising 265 sgRNA was developed to target 138 MYBs, with an average  of 1.92 sgRNA per MYB (Fig. 1-A and Appendix C).

The sgRNA fragments from the same set were mixed as one sgRNA pool, ligated into the pKSE401 vector using Gibson ligation, and subsequently transformed into the Escherichia coli  TOP10 competence cells (Fig. 1-B and Appendix D-a). To evaluate the ligation efficiency of the sgRNA pool with the vector, 90 E. coli clones from sets #1-3 were randomly selected and sequenced. The results demonstrated that the ligation efficiency exceeded 90% and the sgRNA coverage ratio over 80%, confirming the feasibility of this method (Appendix D-b). Using this approach, approximately 1300 (~5×) positive E. coli clones were obtained across the 30 sets (Fig. 1-D). Plasmids extracted from each set were mixed in equal proportions and transformed into Agrobacterium tumefaciens GV3101 competence cells. Finally, all Agrobacterium colonies were collected and verified with next-generation sequencing (NGS). The results revealed that 95.31% of the sequences in the library were accurate, and 178 of 265 sgRNAs were represented by at least one read, targeting 125 (90.58%) MYB transcription factors. Most (83.93%) sgRNA read counts fell within the range of 2⁸-2¹⁵. These results indicated that the sgRNAs library in Agrobacterium exhibited high accuracy and gene coverage, which is usable for grape transformation (Fig.1-E).

Vitis vinifera L. cv. Cabernet Sauvignon is one of the most renowned red wine grape varieties, widely cultivated worldwide. In this study, pro-embryonic masses of ‘Cabernet Sauvignon’ were used as recipient material for Agrobacterium-mediated transformation (Fig. 1-C and Appendix E). A total of 1354 kanamycin-resistant seedlings were obtained, and which 341 were confirmed as transgenic lines (PCR positive). And, all the lines were determined to harbor a single correct sgRNA, representing 13 unique sgRNAs targeting 18 MYB transcription factors. Target site DNA was amplified and sequenced, revealing only 67 gene-edited lines with mutations in 8 MYB transcription factors (Fig. 1F and Appendix F and G). Among these, 56 lines were chimeric mutants, nine were biallelic mutants, one was a homozygous mutant and one was a heterozygous mutant (Appendix H). Five Target1 type transgenic lines were obtained, three of them did not mutate in all of the targeted genes, in which the sgRNA was targeting two or more completely conserved sites. Additionally, gene-edited lines for GSVIVT01032467001-T1 and GSVIVT01014770001-T1 were producted. However, the sgRNA harbored in these transgenic lines only caused the mutations in GSVIVT01032467001 and GSVIVT01014770001, without off-target effects on genes with similar sequences (Appendix I and J). All MYB-edited lines were subsequently transplanted into a greenhouse for observation (Appendix K). Phenotypic analysisrevealed that the GSVIVT01026481001 edited lines exhibited significantly enhanced tolerance to drought stress (Fig. 1-G).

CRISPR-Cas9 has greatly accelerated gene function research and breeding in plants. In this study, we developed a strategy for generating a collection of MYB mutant lines in grape using a CRISPR-Cas9 library. However, the relatively lower transformation efficiency in grape limited the number of mutants obtained. Factors affecting grape transformation efficiency primarily included the regeneration rate of the recipient material and the efficiency of Agrobacterium infection. Numerous studies have demonstrated that plant regeneration efficiency enhanced using developmental regulators such as BABY BOOM (BBM), WUSCHEL (WUS), GROWTH-REGULATING FACTOR (GRF), and REGENERATION FACTOR (REF) (Debernardi et al. 2020; Yang et al. 2022). Additionally, plant transformation and gene editing efficiency improved through optimized genetic transformation methods and gene editing vector designs (Debernardi et al. 2024; Yan et al. 2024). We confirmed that a large number of edited plants could be obtained simultaneously using a sgRNA mixed-pool library, provided that grape transformation efficiency is improved. This strategy holds significant potential for constructing genome-wide mutant libraries in woody crop plants in the future.

Phthalate esters (PAEs) are an emerging pollutant due to widespread distribution in environmental mediums that have attracted widespread attention over recent years.  However, there is little information about tea plantation soil PAEs.  A total of 270 soil samples collected from 45 tea plantations in the major high-quality tea-producing regions of Jiangsu, Zhejiang, and Anhui provinces in China were analyzed for seven PAEs.  The detection frequency of PAEs in tea plantation soil was 100%.  DBP, DEHP, and DiBP were the main congeners in tea plantation soil.  The PAEs concentrations in the upper soil were significantly higher than those in the lower soil.  The concentration of tea plantation soil PAEs in Jiangsu Province was significantly lower than those in Zhejiang and Anhui provinces.  Intercropping with chestnuts can effectively reduce the contamination level of PAEs in tea plantation soil.  Correlation analysis, redundancy analysis, partial correlation analysis, and structural equation modeling methods further confirmed the strong direct influence of factors such as chestnut–tea intercropping, temperature, and agricultural chemicals on the variation of PAEs in tea plantation soil.  The health and ecological risk assessments indicated that non-carcinogenic risk was within a safe range and that there was a high carcinogenic risk via the dietary pathway, with DBP posing the highest ecological risk. 

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.

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.

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.

The sugar beet cyst nematode (Heterodera schachtii) is one of the most destructive pathogens in sugar beet production, which causes serious economic losses every year.  Few molecular details of effectors of H. schachtii parasitism are known.  We analyzed the genome and transcriptome data of H. schachtii and identified multiple potential predicted proteins.  After filtering out predicted proteins with high homology to other plant-parasitic nematodes, we performed functional validation of the remaining effector proteins.  37 putative effectors of H. schachtii were screened based on the Nicotiana benthamiana system for identifying the effectors that inhibit plant immune response, eventually determines 13 candidate effectors could inhibit cell death caused by Bax.  Among the 13 effectors, nine have the ability to inhibit GPA2/RBP1-induced cell death.  All 13 effector-triggered immunity (ETI) suppressor genes were analyzed by qRT-PCR and confirmed to result in a significant downregulation of one or more defense genes during infection compared to empty vector.  For in situ hybridization, 13 effectors were specifically expressed and located in esophageal gland cells.  These data and functional analysis set the stage for further studies on the interaction of H. schachtii with host and H. schachtii parasitic control.

Cattle carcass traits are economically important in the beef industry. In the present study, we identified 184 significant genes and 822 alternative genes for 7 carcass traits using genome-wide association studies (GWASs) in 1566 Huaxi beef cattle. We then identified 5,860 unique cis-genes and 734 trans-genes in 227 longissimus dorsi muscle (LDM) samples to better understand the genetic regulation of gene expression. Our integration study of the GWAS and cis-eQTL analysis detected 13 variants regulating 12 identical genes, in which one variant was also detected in fine-mapping analysis. Moreover, using a transcriptome-wide association study (TWAS), we identified 4 genes (TTC30B, HMGA1, PRKD3 and FXN) that were significantly related to carcass chest depth (CCD), carcass length (CL), carcass weight (CW) and dressing percentage (DP). This study identified variants and genes that may be useful for understanding the molecular mechanism of carcass traits in beef cattle.

Soil microorganisms play critical roles in ecosystem function. However, the relative impact of the potassium (K) fertilizer gradient on the microbial community in wheat‒maize double-cropping systems remains unclear. In this long-term field experiment (2008-2019), we researched bacterial and fungal diversity, composition, and community assemblage in the soil along a K fertilizer gradient (in the wheat season: K0, no K fertilizer; K1, 45 kg ha⁻¹ K₂O; K2, 90 kg ha⁻¹ K₂O; K3, 135 kg ha⁻¹ K₂O; and in the maize season: K0, no K fertilizer; K1, 150 kg ha⁻¹ K₂O; K2, 300 kg ha⁻¹ K₂O; K3, 450 kg ha⁻¹ K₂O) using bacterial 16S rRNA and fungal ITS data. We observed that environmental variables (such as mean annual soil temperature (MAT) and precipitation, available K, ammonium, nitrate, and organic matter) impacted the soil bacterial and fungal communities, and their impacts varied with fertilizer treatments and crop species. Furthermore, the relative abundance of bacteria involved in soil nutrient transformation (phylum Actinobacteria and class Alphaproteobacteria) in the wheat season was significantly increased compared to the maize season, and the optimal K fertilizer dosage (K2 treatment) boosted the relative bacterial abundance of soil nutrient transformation (genus Lactobacillus) and soil denitrification (phylum Proteobacteria) bacteria in the wheat season. The abundance of the soil bacterial community promoting root growth and nutrient absorption (genus Herbaspirillum) in the maize season was improved compared to the wheat season, and the K2 treatment enhanced the bacterial abundance of soil nutrient transformation (genus MND1) and soil nitrogen cycling (genus Nitrospira) genera in the maize season. The results indicated that the bacterial and fungal communities in the double-cropping system exhibited variable sensitivities and assembly mechanisms along a K fertilizer gradient, and microhabitats explained the largest amount of the variation in crop yields, and improved wheat‒maize yields by 11.2-22.6 and 9.2-23.8% with K addition, respectively. These modes are shaped contemporaneously by the different meteorological factors and soil nutrient changes in the K fertilizer gradients.

The impact of drought stress on crop yield and quality is substantial.  Drought priming during the early growth stage of plants has been shown to improve tolerance to drought stress during the reproductive stage, although its effects on grain quality remains elusive.  This study aimed to investigate the influence of drought priming on starch and protein levels in grains under drought stress during grain filling.  Our results reveal that drought stress results in a reduction in starch content and its constituents, while simultaneously increasing glutenin macropolymers and protein fractons.  Notably,, drought primed plants under drought stress (PD) exhibit mitigated declines in starch content and its components, leading to improvements in starch swelling power and pasting properties.  Additionally, PD results in a slight increase in protein fractions, limiting the overall rise in total protein content compared to drought stress alone.  Collectively, our study underscores the efficacyof drought priming as a strategy to counteract the negative effects of drought stress on grain quality, particularly by minimizing starch losses and restraining protein content elevation.

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.

穗部小花结实率是影响谷类作物产量的关键因素,相关的调控机制至今仍不清楚。本研究鉴定到一个谷子花器官败育无籽突变体（sinog1），解剖学分析表明sinog1突变体的穗部小花在抽穗期发生褐变并逐渐败育。通过构建BC₃F₂群体，将sinog1的候选基因定位在5号染色体32.44Mb至34.29Mb的1.89Mb区间；RNA-seq分析表明野生型和突变体的差异表达基因主要富集在ABC转运体通路，结合BSA-seq和RNA-seq分析发现候选区间内的3个ABC转运体通路相关候选基因在差异表达基因中富集，推测ABC转运体通路可能是影响sinog1表型的关键通路。本研究为深入了解谷子以及近缘禾谷类作物的小花发育机制奠定了理论基础。

Cotton breeding for the development of early maturing varieties is an effective way to improve the multiple cropping indexes and alleviate the conflict in cultivated fields between grains and cotton in China.  In the present study, we aimed to find upland cotton quantitative trait loci (QTLs) and candidate genes related to early maturity traits, including whole growth period (WGP), flowering timing (FT), node of the first fruiting branch (NFFB), height of the node of the first fruiting branch (HNFFB) and plant height (PH).  An early-maturing variety, CCRI50, and a late-maturing variety, Guoxinmian 11 were crossed to obtain biparental populations. These populations were used to map QTLs for the early-maturity traits for two years (2020 and 2021).  With BSA-seq analysis based on the data of population 2020, the candidate regions related to early maturity were found to be located on chromosome D03.  Then, we developed 22 polymorphic Indel makers to further narrow down the candidate regions, resulting in detection of five and four QTLs in the 2020 and 2021 populations, respectively.  According to the results of QTL mapping, two candidate regions (InDel_G286-InDel_G144 and InDel_G24-InDel_G43) were detected.  In these regions, three genes (GH_D03G0451, GH_D03G0649, and GH_D03G1180) have non-synonymous mutations in the exon and one gene (GH_D03G0450) has SNP variations in the upstream sequence between CCRI50 and Guoxinmian11.  These four genes also showed a dominant expression in the floral organs.  The expression of GH_D03G0451, GH_D03G0649 and GH_D03G1180 in CCRI50 was significantly higher than in Guoxinmian11 during the bud differentiation stages, while GH_D03G0450 showed an opposite trend.  Further functional verification of GH_D03G0451 showed that the GH_D03G0451-silenced plants showed a delay in the flowering time.  These results may suggest that these are the candidate genes for cotton early maturity and may further be used for cotton breeding aiming for early-maturity.

Peanut kernels rich in oil, particularly those with oleic acid as their primary fatty acid, are sought after by consumers, the food industry, and farmers due to their superior nutritional content, extended shelf life, and health benefits.  The oil content and fatty acid composition are governed by multiple genetic factors.  Identifying the quantitative trait loci (QTL) related to these attributes would facilitate marker-assisted selection or genomic selection, thus enhancing the quality-focused peanut breeding program.  For this purpose, we developed a population of 521 recombinant inbred lines (RIL) and tested their kernel quality traits across five different environments. We identified two major and stable QTLs for oil content (qOCAh12.1 and qOCAh16.1).  The markers linked to these QTLs were designed by competitive allele-specific PCR (KASP) and were subsequently validated.  Moreover, we found that the superior haplotype of oil content in the qOCAh16.1 region was conserved within the PI germplasm cluster, as evidenced by a diverse peanut accession panel.  In addition, we determined that qAh09 and qAh19.1, which harbor the key gene encoding fatty acid desaturase 2 (FAD2), influence all seven fatty acids, including palmitic, stearic, oleic, linoleic, arachidic, gadoleic, and behenic acids.  As for protein content and the long-chain saturated fatty acid behenic acid, qAh07 emerged as the major and stable QTLs, accounting for over 10% of the phenotypic variation explained (PVE).  These findings would enhance marker-assisted selection in peanut breeding, aiming to improve oil content, and deepen our understanding of the genetic mechanisms that shape fatty acid composition. 

The Rpd3 histone deacetylase complex is a multiple-subunit complex that mediates the regulation of chromatin accessibility and gene expression. Sin3, the largest subunit of Rpd3 complex, is conserved in a broad range of eukaryotes. Despite being a molecular scaffold for complex assembly, the functional sites and mechanism of action of Sin3 remain unexplored. In this study, we functionally characterized a glutamate residue (E810) in FgSin3, the ortholog of yeast Sin3 in Fusarium graminearum (known as wheat scab fungus). Our findings indicate that E810 was important for the functions of FgSin3 in regulating vegetative growth, sexual reproduction, wheat infection, and DON biosynthesis. Furthermore, the E810K missense mutation restored the reduced H4 acetylation caused by the deletion of FNG1, the ortholog of the human inhibitor of growth (ING1) gene in F. graminearum. Correspondingly, the defects of the fng1 mutant were also partially rescued by the E810K mutation in FgSin3. Sequence alignment and evolutionary analysis revealed that E810 residue is well-conserved in fungi, animals, and plants. Based on Alphafold2 structure modeling, E810 localized on the FgRpd3-FgSin3 interface for the formation of a hydrogen bond with FgRpd3. Mutation of E810 disrupts the hydrogen bond and likely affects the FgRpd3-FgSin3 interaction. Taken together, E810 of FgSin3 is functionally associated with Fng1 in the regulation of H4 acetylation and related biological processes, probably by affecting the assembly of the Rpd3 complex.

The application of organic fertilizers has become an increasingly popular substitution in maize production to reduce gaseous nitrogen (N) loss and soil degradation caused by inorganic fertilizers.  Organic fertilizer plays a key role in improving soil quality and stabilizing maize yields, but studies that refine different substitution rates remain poorly documented.  A field study was carried out in 2021 and 2022 based on a long-term trial initiated in 2016.  The experiment included five organic fertilizer N substitution rates with equal input of 200 kg N ha^–1: 0% organic fertilizer (T1, 100% inorganic fertilizer), 50.0% organic+50.0% inorganic fertilizer (T2), 37.5% organic+62.5% inorganic fertilizer (T3), 25.0% organic+75.0% inorganic fertilizer (T4), 12.5% organic+87.5% inorganic fertilizer (T5), and no fertilizer control (T6).  The average result of two years showed that T3 and T1 had the highest grain yield and biomass, respectively, and there was no significant difference between T1 and T3.  Compared with T1, 12.5, 25.0, 37.5, and 50.0% substitution rates (T5, T4, T3, and T2) significantly reduced total nitrogen loss (NH₃、N₂O) by 8.3, 16.1, 18.7, and 27.0%, respectively.  Nitrogen use efficiency (NUE) was higher in T5, T3, and T1, and there was no significant difference among them.  The organic fertilizer substitution directly reduced NH₃ volatilization and N₂O emission from farmland by lowering ammonium nitrogen and alkali-dissolved N content and by increasing soil moisture.  These substitution treatments reduced N₂O emissions indirectly by regulating the abundance of AOB and nirK-harboring genes by promoting soil moisture.  The 37.5% of organic fertilizer substitution reduces NH₃ volatilization and N₂O emission from farmland by decreasing ammonium nitrogen and alkali-dissolved N content and increasing moisture which negatively regulate the abundance of AOB and nirK-harboring genes to reduce N₂O emissions indirectly in rainfed maize fields on the Loess Plateau of China.