Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Field Evaluation of Experimental Maize Hybrids for Resistance to the Fall Armyworm (Lepidoptera: Noctuidae) in a Warm Temperate Climate.

The polyphagous fall armyworm (FAW), Spodoptera frugiperda, has become an invasive pest worldwide in recent years. To develop maize germplasm with multiple pest resistance and understand genetic inheritance, 12 experimental hybrids (six pairs of reciprocal crosses) with diverse genetic backgrounds and four commercial checks were examined for FAW resistance in 2013 and 2014. The experiment utilized a randomized complete block design with four replications as the block factor. FAW injury on maize plants was assessed at 7 and 14 d after the artificial infestation at the V6 stage, and predatory arthropod taxa and abundance on maize seedlings were recorded 7 d after the infestation. Spodoptera frugiperda resistance varied significantly among the 16 hybrids. Two reciprocal crosses ('FAW1430' × 'Oh43' and 'CML333' × 'NC358') showed the least FAW injury. Eleven arthropod predators [i.e., six coleopterans, three hemipterans, earwigs (dermapterans), and spiders (or arachnids)] were also recorded; the two most common predators were the pink spotted ladybeetle, Coleomegilla maculata, and the insidious flower (or minute pirate) bug, Orius spp. Predator abundance was not correlated to FAW injury but varied greatly between 2013 and 2014. Principal component analysis demonstrated that, when compared with FAW resistant (or Bt-transgenic) checks ('DKC69-71', 'DKC67-88', and 'P31P42'), five pairs of the reciprocal crosses had moderate FAW resistance, whereas a pair of reciprocal crosses ('NC350' × 'NC358' and NC358 × NC350) showed the same FAW susceptibility as the non-Bt susceptible check 'DKC69-72'. Both parents contributed similarly to FAW resistance, or no maternal/cytoplasmic effect was detected in the experimental hybrids.

Deciphering age-specific molecular features in cervical cancer and constructing an angio-immune prognostic model.

Cancer incidence is increasingly seen in younger individuals. Molecular distinctions between young and elderly patients at onset are understudied. This study used public databases to explore genomic, transcriptomic, and immune-related features across age groups in cervical cancer. Additionally, it aims to create a prognostic model applicable across diverse age cohorts, enabling precise patient stratification, and personalized therapies. Gene mutations, expression data, and clinicopathological information were obtained from 317 cervical cancer patients. These patients were divided into a young group and an old group based on the median age of onset. The characteristics of differential gene mutation, gene expression, and immune cells analysis were analyzed by R software. Finally, the prognostic model was constructed by univariate Cox, least absolute shrinkage and selection operator, and multivariate Cox regression analyses of angiogenic and immune gene sets. Its validity was further confirmed using an additional 300 cervical squamous cell carcinoma and endocervical adenocarcinoma tissues. Cervical cancer patients at elderly onset age exhibit a significantly higher frequency of NOTCH1 and TP53 driver mutations compared to young patients, along with a notably higher tumor mutational burden. However, there were no significant differences between the 2 groups in terms of genomic instability and age-related mutational signatures. Differential gene expression analysis revealed that the young group significantly upregulated interferon-alpha and gamma responses and exhibited significantly higher activity in multiple metabolic pathways. Immune microenvironment analysis indicated enrichment of dendritic cells and natural killer cells in the young group, while transforming growth factor-ß signature was enriched in the elderly group, indicating a higher degree of immune exclusion. A multigene prognostic model based on angiogenesis and T cell immune gene sets showed excellent prognostic performance independent of clinical factors such as age. High-risk groups identified by the model exhibit significant activation of tumor-promoting processes, such as metastasis and angiogenesis. Our study reveals distinct patterns in cancer-driving mechanisms, biological processes, and immune system status between young and elderly patients at onset with cervical cancer. These findings shed light on the age-specific underlying mechanisms of carcinogenesis. Furthermore, an independent molecular prognostic model is constructed to provide valuable references for patient stratification and the development of potential drug targets.

Linkage-Mapping and Genome-wide Association Study Identified Two Peanut Late Leaf Spot Resistance Loci, PLLSR-1 and PLLSR-2, Using a Nested Association Mapping.

Identification of candidate genes and molecular markers for late leaf spot (LLS) disease resistance in peanut (Arachis hypogaea) has been a focus of molecular breeding for the U.S. industry funded peanut genome project. Efforts have been hindered by limited mapping resolution due to low levels of genetic recombination and marker density available in traditional biparental mapping populations. To address this, a multi-parental nested association mapping (NAM) population has been genotyped with the peanut 58 K SNP array and phenotyped for LLS severity in the field for three years. Joint linkage-based QTL mapping identified nine QTLs for LLS resistance with significant phenotypic variance explained (PVE) up to 47.7%. A genome-wide association study (GWAS) identified 13 SNPs consistently associated with LLS resistance. Two genomic regions harboring the consistent QTLs and SNPs were identified from 1,336 Kb to 1,520 Kb (184 Kb) on chromosome B02 and from 1,026.9 Kb to 1,793.2 Kb (767 Kb) on chromosome B03, designated as peanut late leaf spot resistance loci, PLLSR-1 and PLLSR-2, respectively. PLLSR-1 contains 10 NBS-LRR disease resistant genes. An NBS-LRR disease resistance gene Arahy.VKVT6A was also identified on homoeologous chromosome A02. PLLSR-2 contains five significant SNPs associated with five different genes encoding callose synthase, pollen defective in guidance protein, pentatricopeptide repeat (PPR), acyl-activating enzyme, and C2 GRAM domains-containing protein. This study highlights the power of multi-parent populations such as NAM for genetic mapping and marker-trait association studies in peanuts. Validation of these two LLS resistance loci will be needed for marker-assisted breeding.

Nomogram for predicting the risk of nonalcoholic fatty liver disease in older adults in Qingdao, China: A cross-sectional study.

BACKGROUND AND OBJECTIVES: To explore the risk factors for non-alcoholic fatty liver disease (NAFLD) and to establish a non-invasive tool for the screening of NAFLD in an older adult population. METHODS AND STUDY DESIGN: A total of 131,161 participants were included in this cross-sectional study. Participants were randomly divided into training and validation sets (7:3). The least absolute shrinkage and selection operator method was used to screen risk factors. Multivariate logistic regression was employed to develop a nomogram, which was made available online. Receiver operating characteristic curve analysis, calibration plots, and decision curve analysis were used to validate the discrimination, calibration, and clinical practicability of the nomogram. Sex and age subgroup analyses were conducted to further validate the reliability of the model. RESULTS: Nine variables were identified for inclusion in the nomogram (age, sex, waist circumference, body mass index, exercise frequency, systolic blood pressure, fasting plasma glucose, alanine aminotransferase, and low-density lipoprotein cholesterol). The area under the receiver operating characteristic curve values were 0.793 and 0.790 for the training set and the validation set, respectively. The calibration plots and decision curve analyses showed good calibration and clinical utility. Subgroup analyses demonstrated consistent discriminatory ability in different sex and age subgroups. CONCLUSIONS: This study established and validated a new nomogram model for evaluating the risk of NAFLD among older adults. The nomogram had good discriminatory performance and is a non-invasive and convenient tool for the screening of NAFLD in older adults.

Macro-micro exploration on dynamic interaction between aflatoxigenic Aspergillus flavus and maize kernels using Vis/NIR hyperspectral imaging and SEM technology.

Aspergillus flavus and its toxic metabolites-aflatoxins infect and contaminate maize kernels, posing a threat to grain safety and human health. Due to the complexity of microbial growth and metabolic processes, dynamic mechanisms among fungal growth, nutrient depletion of maize kernels and aflatoxin production is still unclear. In this study, visible/near infrared (Vis/NIR) hyperspectral imaging (HSI) combined with the scanning electron microscope (SEM) was used to elucidate the critical organismal interaction at kernel (macro-) and microscopic levels. As kernel damage is the main entrance for fungal invasion, maize kernels with gradually aggravated damages from intact to pierced to halved kernels with A. flavus were cultured for 0-120 h. The spectral fingerprints of the A. flavus-maize kernel complex over time were analyzed with principal components analysis (PCA) of hyperspectral images, where the pseudo-color score maps and the loading plots of the first three PCs were used to investigate the dynamic process of fungal infection and to capture the subtle changes in the complex with different hardness of the maize matrix. The dynamic growth process of A. flavus and the interactions of fungus-maize complexes were explained on a microscopic level using SEM. Specifically, fungus morphology, e.g., hyphae, conidia, and conidiophore (stipe) was accurately captured on the microscopic level, and the interaction process between A. flavus and nutrient loss from the maize kernel tissues (i.e., embryo, and endosperm) was described. Furthermore, the growth stage discrimination models based on PLSDA with the results of CCRC = 100 %, CCRV = 97 %, CCRIV = 93 %, and the prediction models of AFB1 based on PLSR with satisfactory performance (R2C = 0.96, R2V = 0.95, R2IV = 0.93 and RPD = 3.58) were both achieved. In conclusion, the results from both macro-level (Vis/NIR-HSI) and micro-level (SEM) assessments revealed the dynamic organismal interactions in A. flavus-maize kernel complex, and the detailed data could be used for modeling, and quantitative prediction of aflatoxin, which would establish a theoretical foundation for the early detection of fungal or toxin contaminated grains to ensure food security.

[Different calcium ion concentrations affect epithelial mesenchymal transformation of human peritoneal mesothelial cells via endoplasmic reticulum stress].

OBJECTIVE: To study the effects of different calcium ion concentrations on epithelial mesenchymal transformation (EMT) of human peritoneal mesothelial cell (HPMC) via endoplasmic reticulum stress (ERS). METHODS: HPMC cell line HMrSV5 was cultured in vitro and treated in groups. The cells in the control group, high calcium group 1, and high calcium group 2 were treated with medium containing calcium ion concentrations of 1.25, 1.75, and 2.25 mmol/L, respectively. The solvent control group was treated with medium containing 1.25 mmol/L physiological calcium ion concentration and 0.1% dimethyl sulfoxide (DMSO), the high calcium+solvent group was treated with medium containing 2.25 mmol/L calcium ion concentration and 0.1% DMSO, the high calcium+4-phenylbutyric acid (4-PBA) group was treated with medium containing 2.25 mmol/L calcium ion concentration and 1 mmol/L ERS inhibitor 4-PBA, and each group was treated for 48 hours. Morphological changes of cells in each group were observed under light microscope. The expressions of epithelial cell phenotype marker zonula occluden-1 (ZO-1) and mesenchymal cell phenotype marker α-smooth muscle actin (α-SMA) in the cells were observed by immunofluorescence staining. The expressions of EMT marker genes E-cadherin, ZO-1, α-SMA and Vimentin were detected by fluorescence quantitative polymerase chain reaction (PCR). The expressions of ERS marker proteins phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), transcription activating factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by Western blotting. RESULTS: Compared with the control group, the morphology of HMrSV5 cells became slender and fibrotic, the fluorescence intensity of ZO-1 increased, and the fluorescence intensity of α-SMA decreased in high calcium 1 and high calcium 2 groups, indicating that the cells transformed from epithelial cells to mesenchyme cells. The mRNA expressions of E-cadherin and ZO-1 were significantly decreased, while the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly increased, moreover, the expressions of the above marker genes or proteins in the high calcium 2 group was more obvious than those in the high calcium 1 group [E-cadherin mRNA (2-ΔΔCt): 0.53±0.05 vs. 0.75±0.09, ZO-1 mRNA (2-ΔΔCt): 0.42±0.06 vs. 0.69±0.06, α-SMA mRNA (2-ΔΔCt): 1.81±0.16 vs. 1.32±0.14, Vimentin mRNA (2-ΔΔCt): 2.05±0.22 vs. 1.48±0.16, p-PERK protein (p-PERK/ß-actin): 0.81±0.09 vs. 0.59±0.06, p-eIF2α protein (p-eIF2α/ß-actin): 0.87±0.10 vs. 0.50±0.06, ATF4 protein (ATF4/ß-actin): 0.93±0.10 vs. 0.72±0.06, CHOP protein (CHOP/ß-actin): 0.79±0.09 vs. 0.46±0.04, all P < 0.05]. Compared with the solvent control group, the morphological changes of cells, the expressions of EMT marker genes and ERS marker proteins after high calcium ion concentration of 2.25 mmol/L were consistent with those in the high calcium 2 group than control group. Compared with the high calcium+solvent group, the cell morphology recovered the characteristics of polygonal and pebble-like epithelial cells in the high calcium+4-PBA group, the fluorescence intensity of ZO-1 increased, the fluorescence intensity of α-SMA decreased, and the mRNA expressions of E-cadherin and ZO-1 in the cells were significantly increased [E-cadherin mRNA (2-ΔΔCt): 0.86±0.09 vs. 0.57±0.04, ZO-1 mRNA (2-ΔΔCt): 0.81±0.06 vs. 0.48±0.05, both P < 0.05], the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly decreased [α-SMA mRNA (2-ΔΔCt): 1.21±0.13 vs. 1.77±0.15, Vimentin mRNA (2-ΔΔCt): 1.30±0.14 vs. 1.94±0.20, p-PERK protein (p-PERK/ß-actin): 0.38±0.04 vs. 0.92±0.11, p-eIF2α protein (p-eIF2α/ß-actin): 0.34±0.05 vs. 1.05±0.13, ATF4 protein (ATF4/ß-actin): 0.57±0.06 vs. 0.97±0.11, CHOP protein (CHOP/ß-actin): 0.51±0.04 vs. 0.90±0.12, all P < 0.05]. CONCLUSIONS: High calcium ion concentrations of 1.75 mmol/L and 2.25 mmol/L promote EMT of HPMC via activating ERS.

Retrospective study on the effect of Niaoduqing particles on outcome of non-diabetic patients with stage IV chronic kidney disease.

This study was to investigate the improvement value of Niaoduqing particles in the outcome of non-diabetic patients with stage IV chronic kidney disease (CKD). The non-diabetic patients with stage IV CKD who were to receive Niaoduqing particles were set as the study group (252 cases), and the patients with the same disease who only received Western medicine in the public database were set as the control group (220 cases). The follow-up visits were 3 months/time for 1 year. Deaths due to various causes, doubling of creatinine levels, and end-stage renal disease were used as hard end points to stop follow-up. The clinical indexes of the 2 groups were observed and compared. The results showed that the rate of compound outcome was significantly lower in the study group (28.17%) than in the control group (36.82%), the glomerular filtration rate was significantly higher than that in the control group, and the levels of uric acid and urea were significantly lower than that in the control group (P < .05). Niaoduqing particles can reduce creatinine and urea nitrogen, stabilize renal function, delay dialysis time, and improve the incidence of compound outcome in patients with non-diabetic stage IV CKD, which is worthy of clinical promotion.

Evaluation of Available Energy and Standardized Ileal Digestibility of Amino Acids in Fermented Flaxseed Meal for Growing Pigs.

Flaxseed meal (FSM) is a byproduct of flaxseed oil extraction which has rich nutritional value and can be used as a high-quality new protein ingredient. However, the anti-nutrient factor (ANF) in FSM restricts its potential application in feed. The strategy of microbial fermentation is a highly effective approach to reducing ANF in FSM and enhancing its feeding value. However, evaluation of the nutritional value of fermented flaxseed meal (FFSM) in growing pigs has not yet been conducted. Thus, the purpose of this study was to investigate the nutritional value of FFSM in growing pigs and comparison of the effect of fermentation treatment on improving the nutritional value of FSM. Two experiments were conducted to determine the available energy value, apparent digestibility of nutrients, and standard ileal digestibility of amino acids of FSM and FFSM in growing pigs. The results showed as follows: (1) Fermentation treatment increased the levels of crude protein (CP), Ca and P in FSM by 2.86%, 9.54% and 4.56%, while decreasing the concentration of neutral detergent fiber (NDF) and acid detergent fiber (ADF) by 34.09% and 12.71%, respectively (p < 0.05); The degradation rate of CGs in FSM was 54.09% (p < 0.05); (2) The digestible energy (DE) and metabolic energy (ME) of FSM and FFSM were 14.54 MJ/kg, 16.68 MJ/kg and 12.85 MJ/kg, 15.24 MJ/kg, respectively; (3) Compared with FSM, dietary FFSM supplementation significantly increased the apparent digestibility of CP, NDF, ADF, Ca, and P of growing pigs (p < 0.05) and significantly increased the standard ileal digestibility of methionine (p < 0.05). These results indicate that fermentation treatment could effectively enhance the nutritional value of FSM and provide basic theoretical data for the application of FFSM in pig production.

Analysis of gut microbiota in chinese donkey in different regions using metagenomic sequencing.

BACKGROUND: Gut microbiota plays a significant role in host survival, health, and diseases; however, compared to other livestock, research on the gut microbiome of donkeys is limited. RESULTS: In this study, a total of 30 donkey samples of rectal contents from six regions, including Shigatse, Changdu, Yunnan, Xinjiang, Qinghai, and Dezhou, were collected for metagenomic sequencing. The results of the species annotation revealed that the dominant phyla were Firmicutes and Bacteroidetes, and the dominant genera were Bacteroides, unclassified_o_Clostridiales (short for Clostridiales) and unclassified_f_Lachnospiraceae (short for Lachnospiraceae). The dominant phyla, genera and key discriminators were Bacteroidetes, Clostridiales and Bacteroidetes in Tibet donkeys (Shigatse); Firmicutes, Clostridiales and Clostridiales in Tibet donkeys (Changdu); Firmicutes, Fibrobacter and Tenericutes in Qinghai donkeys; Firmicutes, Clostridiales and Negativicutes in Yunnan donkeys; Firmicutes, Fibrobacter and Fibrobacteres in Xinjiang donkeys; Firmicutes, Clostridiales and Firmicutes in Dezhou donkeys. In the functional annotation, it was mainly enriched in the glycolysis and gluconeogenesis of carbohydrate metabolism, and the abundance was the highest in Dezhou donkeys. These results combined with altitude correlation analysis demonstrated that donkeys in the Dezhou region exhibited strong glucose-conversion ability, those in the Shigatse region exhibited strong glucose metabolism and utilization ability, those in the Changdu region exhibited a strong microbial metabolic function, and those in the Xinjiang region exhibited the strongest ability to decompose cellulose and hemicellulose. CONCLUSION: According to published literature, this is the first study to construct a dataset with multi-regional donkey breeds. Our study revealed the differences in the composition and function of gut microbes in donkeys from different geographic regions and environmental settings and is valuable for donkey gut microbiome research.

Prospects for developing allergen-depleted food crops.

In addition to the challenge of meeting global demand for food production, there are increasing concerns about food safety and the need to protect consumer health from the negative effects of foodborne allergies. Certain bio-molecules (usually proteins) present in food can act as allergens that trigger unusual immunological reactions, with potentially life-threatening consequences. The relentless working lifestyles of the modern era often incorporate poor eating habits that include readymade prepackaged and processed foods, which contain additives such as peanuts, tree nuts, wheat, and soy-based products, rather than traditional home cooking. Of the predominant allergenic foods (soybean, wheat, fish, peanut, shellfish, tree nuts, eggs, and milk), peanuts (Arachis hypogaea) are the best characterized source of allergens, followed by tree nuts (Juglans regia, Prunus amygdalus, Corylus avellana, Carya illinoinensis, Anacardium occidentale, Pistacia vera, Bertholletia excels), wheat (Triticum aestivum), soybeans (Glycine max), and kidney beans (Phaseolus vulgaris). The prevalence of food allergies has risen significantly in recent years including chance of accidental exposure to such foods. In contrast, the standards of detection, diagnosis, and cure have not kept pace and unfortunately are often suboptimal. In this review, we mainly focus on the prevalence of allergies associated with peanut, tree nuts, wheat, soybean, and kidney bean, highlighting their physiological properties and functions as well as considering research directions for tailoring allergen gene expression. In particular, we discuss how recent advances in molecular breeding, genetic engineering, and genome editing can be used to develop potential low allergen food crops that protect consumer health.

Application of CRISPR/Cas9-mediated gene editing for abiotic stress management in crop plants.

Abiotic stresses, including drought, salinity, cold, heat, and heavy metals, extensively reducing global agricultural production. Traditional breeding approaches and transgenic technology have been widely used to mitigate the risks of these environmental stresses. The discovery of engineered nucleases as genetic scissors to carry out precise manipulation in crop stress-responsive genes and associated molecular network has paved the way for sustainable management of abiotic stress conditions. In this context, the clustered regularly interspaced short palindromic repeat-Cas (CRISPR/Cas)-based gene-editing tool has revolutionized due to its simplicity, accessibility, adaptability, flexibility, and wide applicability. This system has great potential to build up crop varieties with enhanced tolerance against abiotic stresses. In this review, we summarize the latest findings on understanding the mechanism of abiotic stress response in plants and the application of CRISPR/Cas-mediated gene-editing system towards enhanced tolerance to a multitude of stresses including drought, salinity, cold, heat, and heavy metals. We provide mechanistic insights on the CRISPR/Cas9-based genome editing technology. We also discuss applications of evolving genome editing techniques such as prime editing and base editing, mutant library production, transgene free and multiplexing to rapidly deliver modern crop cultivars adapted to abiotic stress conditions.

Genetic mapping identifies genomic regions and candidate genes for seed weight and shelling percentage in groundnut.

Seed size is not only a yield-related trait but also an important measure to determine the commercial value of groundnut in the international market. For instance, small size is preferred in oil production, whereas large-sized seeds are preferred in confectioneries. In order to identify the genomic regions associated with 100-seed weight (HSW) and shelling percentage (SHP), the recombinant inbred line (RIL) population (Chico × ICGV 02251) of 352 individuals was phenotyped for three seasons and genotyped with an Axiom_Arachis array containing 58K SNPs. A genetic map with 4199 SNP loci was constructed, spanning a map distance of 2708.36 cM. QTL analysis identified six QTLs for SHP, with three consistent QTLs on chromosomes A05, A08, and B10. Similarly, for HSW, seven QTLs located on chromosomes A01, A02, A04, A10, B05, B06, and B09 were identified. BIG SEED locus and spermidine synthase candidate genes associated with seed weight were identified in the QTL region on chromosome B09. Laccase, fibre protein, lipid transfer protein, senescence-associated protein, and disease-resistant NBS-LRR proteins were identified in the QTL regions associated with shelling percentage. The associated markers for major-effect QTLs for both traits successfully distinguished between the small- and large-seeded RILs. QTLs identified for HSW and SHP can be used for developing potential selectable markers to improve the cultivars with desired seed size and shelling percentage to meet the demands of confectionery industries.

Transcriptional networks orchestrating red and pink testa color in peanut.

BACKGROUND: Testa color is an important trait of peanut (Arachis hypogaea L.) which is closely related with the nutritional and commercial value. Pink and red are main color of peanut testa. However, the genetic mechanism of testa color regulation in peanut is not fully understood. To elucidate a clear picture of peanut testa regulatory model, samples of pink cultivar (Y9102), red cultivar (ZH12), and two RNA pools (bulk red and bulk pink) constructed from F4 lines of Y9102 x ZH12 were compared through a bulk RNA-seq approach. RESULTS: A total of 2992 differential expressed genes (DEGs) were identified among which 317 and 1334 were up-regulated and 225 and 1116 were down-regulated in the bulk red-vs-bulk pink RNA pools and Y9102-vs-ZH12, respectively. KEGG analysis indicates that these genes were divided into significantly enriched metabolic pathways including phenylpropanoid, flavonoid/anthocyanin, isoflavonoid and lignin biosynthetic pathways. Notably, the expression of the anthocyanin upstream regulatory genes PAL, CHS, and CHI was upregulated in pink and red testa peanuts, indicating that their regulation may occur before to the advent of testa pigmentation. However, the differential expression of down-stream regulatory genes including F3H, DFR, and ANS revealed that deepening of testa color not only depends on their gene expression bias, but also linked with FLS inhibition. In addition, the down-regulation of HCT, IFS, HID, 7-IOMT, and I2'H genes provided an alternative mechanism for promoting anthocyanin accumulation via perturbation of lignin and isoflavone pathways. Furthermore, the co-expression module of MYB, bHLH, and WRKY transcription factors also suggested a fascinating transcriptional activation complex, where MYB-bHLH could utilize WRKY as a co-option during the testa color regulation by augmenting anthocyanin biosynthesis in peanut. CONCLUSIONS: These findings reveal candidate functional genes and potential strategies for the manipulation of anthocyanin biosynthesis to improve peanut varieties with desirable testa color.

Chromosome-length genome assemblies of six legume species provide insights into genome organization, evolution, and agronomic traits for crop improvement.

INTRODUCTION: Legume crops are an important source of protein and oil for human health and in fixing atmospheric N2 for soil enrichment. With an objective to accelerate much-needed genetic analyses and breeding applications, draft genome assemblies were generated in several legume crops; many of them are not high quality because they are mainly based on short reads. However, the superior quality of genome assembly is crucial for a detailed understanding of genomic architecture, genome evolution, and crop improvement. OBJECTIVES: Present study was undertaken with an objective of developing improved chromosome-length genome assemblies in six different legumes followed by their systematic investigation to unravel different aspects of genome organization and legume evolution. METHODS: We employed in situ Hi-C data to improve the existing draft genomes and performed different evolutionary and comparative analyses using improved genome assemblies. RESULTS: We have developed chromosome-length genome assemblies in chickpea, pigeonpea, soybean, subterranean clover, and two wild progenitor species of cultivated groundnut (A. duranensis and A. ipaensis). A comprehensive comparative analysis of these genome assemblies offered improved insights into various evolutionary events that shaped the present-day legume species. We highlighted the expansion of gene families contributing to unique traits such as nodulation in legumes, gravitropism in groundnut, and oil biosynthesis in oilseed legume crops such as groundnut and soybean. As examples, we have demonstrated the utility of improved genome assemblies for enhancing the resolution of "QTL-hotspot" identification for drought tolerance in chickpea and marker-trait associations for agronomic traits in pigeonpea through genome-wide association study. Genomic resources developed in this study are publicly available through an online repository, 'Legumepedia'. CONCLUSION: This study reports chromosome-length genome assemblies of six legume species and demonstrates the utility of these assemblies in crop improvement. The genomic resources developed here will have significant role in accelerating genetic improvement applications of legume crops.

Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9.

CRISPR-mediated genome editing has become a powerful tool for the genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3' end of the MKK2 MAP kinase gene to generate a BleoR-MKK2 fusion protein. Based on fully edited nucleotides evaluated by TaqMan real-time PCR, the HDR-mediated knock-in efficiency was up to 48% when using XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared with no HDR enhancement or NHEJ silencing. Furthermore, this combination of HDR enhancer overexpression and NHEJ repression also increased genome targeting efficiency and gave 7-fold fewer CRISPR-induced insertions and deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammals for improving CRISPR-mediated gene knock-in efficiency.

Spatio-temporal patterns of Aspergillus flavus infection and aflatoxin B1 biosynthesis on maize kernels probed by SWIR hyperspectral imaging and synchrotron FTIR microspectroscopy.

The dynamics mechanisms regulating the growth and AFB1 production of Aspergillus flavus during its interactions with maize kernels remain unclear. In this study, shortwave infrared hyperspectral imaging (SWIR-HSI) and synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy were combined to investigate chemical and spatial-temporal changes in incremental damaged maize kernels induced by A. flavus infection at macroscopic and microscopic levels. SWIR-HSI was employed to extract spectral information of A. flavus growth and quantitatively detect AFB1 levels. Satisfactory full-spectrum models and simplified multispectral models were obtained respectively by partial least squares regression (PLSR) for three types of samples. Furthermore, SR-FTIR microspectroscopy coupled with two-dimensional correlation spectroscopy (2DCOS) was utilized to reveal the possible sequence of dynamic changes of nutrient loss and trace AFB1 in maize kernels. It exhibited new insights on how to quantify the spatio-temporal patterns of fungal infection and AFB1 accumulation on maize and provided theoretical basis for online sorting.

Two decades of association mapping: Insights on disease resistance in major crops.

Climate change across the globe has an impact on the occurrence, prevalence, and severity of plant diseases. About 30% of yield losses in major crops are due to plant diseases; emerging diseases are likely to worsen the sustainable production in the coming years. Plant diseases have led to increased hunger and mass migration of human populations in the past, thus a serious threat to global food security. Equipping the modern varieties/hybrids with enhanced genetic resistance is the most economic, sustainable and environmentally friendly solution. Plant geneticists have done tremendous work in identifying stable resistance in primary genepools and many times other than primary genepools to breed resistant varieties in different major crops. Over the last two decades, the availability of crop and pathogen genomes due to advances in next generation sequencing technologies improved our understanding of trait genetics using different approaches. Genome-wide association studies have been effectively used to identify candidate genes and map loci associated with different diseases in crop plants. In this review, we highlight successful examples for the discovery of resistance genes to many important diseases. In addition, major developments in association studies, statistical models and bioinformatic tools that improve the power, resolution and the efficiency of identifying marker-trait associations. Overall this review provides comprehensive insights into the two decades of advances in GWAS studies and discusses the challenges and opportunities this research area provides for breeding resistant varieties.

Chromatin spatial organization of wild type and mutant peanuts reveals high-resolution genomic architecture and interaction alterations.

BACKGROUND: Three-dimensional (3D) chromatin organization provides a critical foundation to investigate gene expression regulation and cellular homeostasis. RESULTS: Here, we present the first 3D genome architecture maps in wild type and mutant allotetraploid peanut lines, which illustrate A/B compartments, topologically associated domains (TADs), and widespread chromatin interactions. Most peanut chromosomal arms (52.3%) have active regions (A compartments) with relatively high gene density and high transcriptional levels. About 2.0% of chromosomal regions switch from inactive to active (B-to-A) in the mutant line, harboring 58 differentially expressed genes enriched in flavonoid biosynthesis and circadian rhythm functions. The mutant peanut line shows a higher number of genome-wide cis-interactions than its wild-type. The present study reveals a new TAD in the mutant line that generates different chromatin loops and harbors a specific upstream AP2EREBP-binding motif which might upregulate the expression of the GA2ox gene and decrease active gibberellin (GA) content, presumably making the mutant plant dwarf. CONCLUSIONS: Our findings will shed new light on the relationship between 3D chromatin architecture and transcriptional regulation in plants.