Exploiting genomic tools for genetic dissection and improving the resistance to Fusarium stalk rot in tropical maize.

KEY MESSAGE: A stable genomic region conferring FSR resistance at ~250 Mb on chromosome 1 was identified by GWAS. Genomic prediction has the potential to improve FSR resistance. Fusarium stalk rot (FSR) is a global destructive disease in maize; the efficiency of phenotypic selection for improving FSR resistance was low. Novel genomic tools of genome-wide association study (GWAS) and genomic prediction (GP) provide an opportunity for genetic dissection and improving FSR resistance. In this study, GWAS and GP analyses were performed on 562 tropical maize inbred lines consisting of two populations. In total, 15 SNPs significantly associated with FSR resistance were identified across two populations and the combinedPOP consisting of all 562 inbred lines, with the P-values ranging from 1.99 × 10-7 to 8.27 × 10-13, and the phenotypic variance explained (PVE) values ranging from 0.94 to 8.30%. The genetic effects of the 15 favorable alleles ranged from -4.29 to -14.21% of the FSR severity. One stable genomic region at ~ 250 Mb on chromosome 1 was detected across all populations, and the PVE values of the SNPs detected in this region ranged from 2.16 to 5.18%. Prediction accuracies of FSR severity estimated with the genome-wide SNPs were moderate and ranged from 0.29 to 0.51. By incorporating genotype-by-environment interaction, prediction accuracies were improved between 0.36 and 0.55 in different breeding scenarios. Considering both the genome coverage and the threshold of the P-value of SNPs to select a subset of molecular markers further improved the prediction accuracies. These findings extend the knowledge of exploiting genomic tools for genetic dissection and improving FSR resistance in tropical maize.

Genome-wide association study of ear tip barrenness in waxy maize.

Ear tip-barrenness (ETB), which results from aborted kernels or infertile florets at the ear tip, is an undesirable factor affecting the yield and quality of waxy maize. To uncover the genetic basis of ETB, a genome-wide association study (GWAS) was conducted using the genotype with 27,354 SNPs and phenotype with three environments. Five SNPs that distributed on chromosomes 1, 3 and 6, were identified to be significantly associated with ETB based on the threshold of false discovery rate (FDR) at 0.05. Among these significant loci, three SNPs were clustered together and colocalized with genomic regions previously reported. The average length of ETB decreased almost linearly from the inbred lines containing no favorable alleles across the three loci (1.75 cm) to those with one (1.18 cm), two (0.94 cm) and three (0.65 cm) favorable alleles. Moreover, three important genes, Zm00001d030028, Zm00001d041510 and Zm00001d038676 were predicted for three significant QTLs, respectively. These results promote the understanding genetic basis for ETB and will be useful for breeding waxy maize varieties with high-quality and high-yield.

Identification and fine mapping of a major QTL (qRtsc8-1) conferring resistance to maize tar spot complex and validation of production markers in breeding lines.

KEY MESSAGE: A major QTL of qRtsc8-1 conferring TSC resistance was identified and fine mapped to a 721 kb region on chromosome 8 at 81 Mb, and production markers were validated in breeding lines. Tar spot complex (TSC) is a major foliar disease of maize in many Central and Latin American countries and leads to severe yield loss. To dissect the genetic architecture of TSC resistance, a genome-wide association study (GWAS) panel and a bi-parental doubled haploid population were used for GWAS and selective genotyping analysis, respectively. A total of 115 SNPs in bin 8.03 were detected by GWAS and three QTL in bins 6.05, 6.07, and 8.03 were detected by selective genotyping. The major QTL qRtsc8-1 located in bin 8.03 was detected by both analyses, and it explained 14.97% of the phenotypic variance. To fine map qRtsc8-1, the recombinant-derived progeny test was implemented. Recombinations in each generation were backcrossed, and the backcross progenies were genotyped with Kompetitive Allele Specific PCR (KASP) markers and phenotyped for TSC resistance individually. The significant tests for comparing the TSC resistance between the two classes of progenies with and without resistant alleles were used for fine mapping. In BC5 generation, qRtsc8-1 was fine mapped in an interval of ~ 721 kb flanked by markers of KASP81160138 and KASP81881276. In this interval, the candidate genes GRMZM2G063511 and GRMZM2G073884 were identified, which encode an integral membrane protein-like and a leucine-rich repeat receptor-like protein kinase, respectively. Both genes are involved in maize disease resistance responses. Two production markers KASP81160138 and KASP81160155 were verified in 471 breeding lines. This study provides valuable information for cloning the resistance gene, and it will also facilitate the routine implementation of marker-assisted selection in the breeding pipeline for improving TSC resistance.

Functions and regulatory framework of ZmNST3 in maize under lodging and drought stress.

The growth and development of maize are negatively affected by various abiotic stresses including drought, high salinity, extreme temperature, and strong wind. Therefore, it is important to understand the molecular mechanisms underlying abiotic stress resistance in maize. In the present work, we identified that a novel NAC transcriptional factor, ZmNST3, enhances maize lodging resistance and drought stress tolerance. ChIP-Seq and expression of target genes analysis showed that ZmNST3 could directly regulate the expression of genes related to cell wall biosynthesis which could subsequently enhance lodging resistance. Furthermore, we also demonstrated that ZmNST3 affected the expression of genes related to the synthesis of antioxidant enzyme secondary metabolites that could enhance drought resistance. More importantly, we are the first to report that ZmNST3 directly binds to the promoters of CESA5 and Dynamin-Related Proteins2A (DRP2A) and activates the expression of genes related to secondary cell wall cellulose biosynthesis. Additionally, we revealed that ZmNST3 directly binds to the promoters of GST/GlnRS and activates genes which could enhance the production of antioxidant enzymes in vivo. Overall, our work contributes to a comprehensive understanding of the regulatory network of ZmNST3 in regulating maize lodging and drought stress resistance.

Kaempferol Regulates miR-15b/Bcl-2/TLR4 to Alleviate OGD-Induced Injury in H9c2 Cells.

Ischemic heart disease (IHD) is one of the world's leading causes of human death. Kaempferol (Kae) was proved to have anti-inflammatory, antioxidant, and anticancer effects. Such properties suggested that it might play protective roles in IHD. In this study, we have attempted to disclose the potential regulating mechanisms of Kae in primary cardiomyocytes and H9c2 cells.Cells were first stimulated by oxygen-glucose deprivation (OGD) and then exposed to Kae. CCK-8 assay and flow cytometry were used to examine cell characteristics. Quantitative reverse-transcription polymerase chain reaction was utilized to test the expression levels of miR-15b and TLR4. Afterward, cell transfection, dual-luciferase activity assay, and western blot were used to explore the potential mechanisms.OGD treatment suppressed cell viability, whereas it enhanced cell apoptosis. Besides, OGD treatment enhanced the expression of apoptosis-associated proteins. Kae exposure, however, attenuated the effects that OGD-induced. Further experiments showed that Kae exposure promoted down-regulation of miR-15b, Bcl-2 and TLR4 were a target of miR-15b. Moreover, Kae enhanced the expression of key factors involved in PI3K/AKT and Wnt/ß-catenin pathways, whereas miR-15b mimic reversed the Kae-triggered effects.This investigation revealed that Kae diminished OGD-triggered cell damage through down-regulating miR-15b expression via activating PI3K/AKT and Wnt3a/ß-catenin pathways.

Uncommon Peptide Bond Cleavage of Glucagon from a Specific Vendor under near Neutral to Basic Conditions.

PURPOSE: The main purposes of this manuscript are to report a surprising and interesting degradation reaction of glucagon from a specific vendor in which glucagon underwent cleavage among several peptide bonds quickly under near neutral to basic conditions, and to propose the root cause of mechanism for the degradation reaction. METHODS: The degradation reaction was monitored by HPLC and the fragment structures were confirmed by LC-MS. Possible impurities responsible for the degradation were either confirmed or excluded by a variety of techniques such as addition of chelator EDTA and transitional metal ions or separation by ultrafiltration. RESULTS: This type of degradation was rarely reported in literature, especially considering its extreme cleavage efficiency. Contamination by a thermostable high molecular impurity (such as a peptidase with molecular weight between 10 and 30 KDa) during the manufacturing process was the main reason for this interesting phenomenon. CONCLUSIONS: The degradation phenomenon described here could be used as an excellent example showing that products ordered from vendors meeting the rudimentary quality standards might contain impurities which could cause significant degradation. We suggest that a simple solution, i.e. additional tests of stability under real or accelerated conditions by manufacturers and inclusion of the "accelerated stability criteria" in the Certificate of Analysis (CoAs), especially for sensitive biological reagents prone to faster degradation, would be very helpful for avoiding losses for both vendors and users.

Combined linkage and association mapping reveal QTL for host plant resistance to common rust (Puccinia sorghi) in tropical maize.

BACKGROUND: Common rust, caused by Puccinia sorghi, is an important foliar disease of maize that has been associated with up to 50% grain yield loss. Development of resistant maize germplasm is the ideal strategy to combat P. sorghi. RESULTS: Association mapping performed using a mixed linear model (MLM), integrating population structure and family relatedness identified 25 QTL (P < 3.12 × 10- 5) that were associated with resistance to common rust and distributed on chromosomes 1, 3, 5, 6, 8, and 10. We identified three QTLs associated with all three disease parameters (final disease rating, mean disease rating, and area under disease progress curve) located on chromosomes 1, 3, and 8. A total of 5 QTLs for resistance to common rust were identified in the RIL population. Nine candidate genes located on chromosomes 1, 5, 6, 8, and 10 for resistance to common rust associated loci were identified through detailed annotation. CONCLUSIONS: Using a diverse set of inbred lines genotyped with high density markers and evaluated for common rust resistance in multiple environments, it was possible to identify QTL significantly associated with resistance to common rust and several candidate genes. The results point to the need for fine mapping common rust resistance by targeting regions identified in common between this study and others using diverse germplasm.

TAK-242 suppresses lipopolysaccharide-induced inflammation in human coronary artery endothelial cells.

TAK-242 (resatorvid), a novel small-molecule cyclohexene derivative, inhibits TLR4 signaling selectively. TAK-242 blocked the Toll-like receptor (TLR) 4-triggered inflammatory signaling by binding directly to a specific amino acid Cys747 in the intracellular domain of TLR4. The present study was designed to examine the effects of TAK-242 on vascular inflammatory responses in human coronary artery endothelial cells (HCAECs) challenged by lipopolysaccharide (LPS, a TLR4 ligand). The results show that TAK-242 attenuated the LPS-induced expression of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein 1 both at the transcription and translation levels in HCAECs. LPS-induced endothelial cell adhesion molecules, intercellular adhesion molecular-1 and vascular cell adhesion molecule-1 expressions were also reduced by treatment with TAK-242. In addition, coincubation with TAK-242 did not effect the expression of TLR4 in LPS-activated HCAECs. Furthermore, TAK-242 efficiently suppressed LPS-induced phosphorylation of nuclear factor κB (NF-κB) and IL-1 associated kinase-1 (IRAK-1) in HCAECs. These findings show that TAK-242 can suppress endothelial cell inflammation, suggesting that TAK-242 might be suitable for development as a therapeutic agent for inflammatory cardiovascular disease.

Effect of sypQ gene on poly-N-acetylglucosamine biosynthesis in Vibrio parahaemolyticus and its role in infection process.

The syp locus includes four genes encoding putative regulators, six genes encoding glycosyltransferases, two encoding export proteins, and six other genes encoding unidentified functional proteins associated with biofilm formation and symbiotic colonization. However, the individual functions of the respective genes remain unclear. Amino acid alignment indicates that sypQ is presumably involved in biosynthesizing poly-N-acetylglucosamine (PNAG), which is proposed to be a critical virulence factor in pathogen infection and is regarded as a target for protective immunity against a variety of Gram-negative/positive pathogens. However, no evidence showing that Vibrio parahaemolyticus also produces PNAG has been reported. Herein, the V. parahaemolyticus is confirmed to possess potential for producing PNAG for the first time. Our results indicated that gene sypQ is associated with PNAG biosynthesis and PNAG is involved in pathogen colonization. We propose that the function of pgaC in Escherichia coli could be taken over by sypQ from V. parahaemolyticus. We also tested whether PNAG can be used as a target against V. parahaemolyticus when it infects Pseudosciaena crocea. Our results showed that PNAG isolated from V. parahaemolyticus is an effective agent for decreasing V. parahaemolyticus invasion, implying that PNAG could be used to develop an effective vaccine against V. parahaemolyticus infection.

Genetic architecture of kernel-related traits in sweet and waxy maize revealed by genome-wide association analysis.

Introduction: Maize (Zea mays L.) is one of the most important crops worldwide, the kernel size-related traits are the major components of maize grain yield. Methods: To dissect the genetic architecture of four kernel-related traits of 100-kernel weight, kernel length, kernel width, and kernel diameter, a genome-wide association study (GWAS) was conducted in the waxy and sweet maize panel comprising of 447 maize inbred lines re-sequenced at the 5× coverage depth. GWAS analysis was carried out with the mixed linear model using 1,684,029 high-quality SNP markers. Results: In total, 49 SNPs significantly associated with the four kernel-related traits were identified, including 46 SNPs on chromosome 3, two SNPs on chromosome 4, and one SNP on chromosome 7. Haplotype regression analysis identified 338 haplotypes that significantly affected these four kernel-related traits. Genomic selection (GS) results revealed that a set of 10,000 SNPs and a training population size of 30% are sufficient for the application of GS in waxy and sweet maize breeding for kernel weight and kernel size. Forty candidate genes associated with the four kernel-related traits were identified, including both Zm00001d000707 and Zm00001d044139 expressed in the kernel development tissues and stages with unknown functions. Discussion: These significant SNPs and important haplotypes provide valuable information for developing functional markers for the implementation of marker-assisted selection in breeding. The molecular mechanism of Zm00001d000707 and Zm00001d044139 regulating these kernel-related traits needs to be investigated further.

Genome-Wide Association Study and Genomic Prediction on Plant Architecture Traits in Sweet Corn and Waxy Corn.

Sweet corn and waxy corn has a better taste and higher accumulated nutritional value than regular maize, and is widely planted and popularly consumed throughout the world. Plant height (PH), ear height (EH), and tassel branch number (TBN) are key plant architecture traits, which play an important role in improving grain yield in maize. In this study, a genome-wide association study (GWAS) and genomic prediction analysis were conducted on plant architecture traits of PH, EH, and TBN in a fresh edible maize population consisting of 190 sweet corn inbred lines and 287 waxy corn inbred lines. Phenotypic data from two locations showed high heritability for all three traits, with significant differences observed between sweet corn and waxy corn for both PH and EH. The differences between the three subgroups of sweet corn were not obvious for all three traits. Population structure and PCA analysis results divided the whole population into three subgroups, i.e., sweet corn, waxy corn, and the subgroup mixed with sweet and waxy corn. Analysis of GWAS was conducted with 278,592 SNPs obtained from resequencing data; 184, 45, and 68 significantly associated SNPs were detected for PH, EH, and TBN, respectively. The phenotypic variance explained (PVE) values of these significant SNPs ranged from 3.50% to 7.0%. The results of this study lay the foundation for further understanding the genetic basis of plant architecture traits in sweet corn and waxy corn. Genomic selection (GS) is a new approach for improving quantitative traits in large plant breeding populations that uses whole-genome molecular markers. The marker number and marker quality are essential for the application of GS in maize breeding. GWAS can choose the most related markers with the traits, so it can be used to improve the predictive accuracy of GS.

Genome-wide association study presents insights into the genetic architecture of drought tolerance in maize seedlings under field water-deficit conditions.

Introduction: Drought stress is one of the most serious abiotic stresses leading to crop yield reduction. Due to the wide range of planting areas, the production of maize is particularly affected by global drought stress. The cultivation of drought-resistant maize varieties can achieve relatively high, stable yield in arid and semi-arid zones and in the erratic rainfall or occasional drought areas. Therefore, to a great degree, the adverse impact of drought on maize yield can be mitigated by developing drought-resistant or -tolerant varieties. However, the efficacy of traditional breeding solely relying on phenotypic selection is not adequate for the need of maize drought-resistant varieties. Revealing the genetic basis enables to guide the genetic improvement of maize drought tolerance. Methods: We utilized a maize association panel of 379 inbred lines with tropical, subtropical and temperate backgrounds to analyze the genetic structure of maize drought tolerance at seedling stage. We obtained the high quality 7837 SNPs from DArT's and 91,003 SNPs from GBS, and a resultant combination of 97,862 SNPs of GBS with DArT's. The maize population presented the lower her-itabilities of the seedling emergence rate (ER), seedling plant height (SPH) and grain yield (GY) under field drought conditions. Results: GWAS analysis by MLM and BLINK models with the phenotypic data and 97862 SNPs revealed 15 variants that were significantly independent related to drought-resistant traits at the seedling stage above the threshold of P < 1.02 × 10-5. We found 15 candidate genes for drought resistance at the seedling stage that may involve in (1) metabolism (Zm00001d012176, Zm00001d012101, Zm00001d009488); (2) programmed cell death (Zm00001d053952); (3) transcriptional regulation (Zm00001d037771, Zm00001d053859, Zm00001d031861, Zm00001d038930, Zm00001d049400, Zm00001d045128 and Zm00001d043036); (4) autophagy (Zm00001d028417); and (5) cell growth and development (Zm00001d017495). The most of them in B73 maize line were shown to change the expression pattern in response to drought stress. These results provide useful information for understanding the genetic basis of drought stress tolerance of maize at seedling stage.

Integrative transcriptome and metabolome analysis reveals the mechanisms of light-induced pigmentation in purple waxy maize.

Introduction: Waxy maize, mainly consumed at the immature stage, is a staple and vegetable food in Asia. The pigmentation in the kernel of purple waxy maize enhances its nutritional and market values. Light, a critical environmental factor, affects anthocyanin biosynthesis and results in pigmentation in different parts of plants, including in the kernel. SWL502 is a light-sensitive waxy maize inbred line with purple kernel color, but the regulatory mechanism of pigmentation in the kernel resulting in purple color is still unknown. Methods: In this study, cyanidin, peonidin, and pelargonidin were identified as the main anthocyanin components in SWL502, evaluated by the ultra-performance liquid chromatography (UPLC) method. Investigation of pigment accumulation in the kernel of SWL502 was performed at 12, 17, and 22 days after pollination (DAP) under both dark and light treatment conditions via transcriptome and metabolome analyses. Results: Dark treatment affected genes and metabolites associated with metabolic pathways of amino acid, carbohydrate, lipid, and galactose, biosynthesis of phenylpropanoid and terpenoid backbone, and ABC transporters. The expression of anthocyanin biosynthesis genes, such as 4CL2, CHS, F3H, and UGT, was reduced under dark treatment. Dynamic changes were identified in genes and metabolites by time-series analysis. The genes and metabolites involved in photosynthesis and purine metabolism were altered in light treatment, and the expression of genes and metabolites associated with carotenoid biosynthesis, sphingolipid metabolism, MAPK signaling pathway, and plant hormone signal transduction pathway were induced by dark treatment. Light treatment increased the expression level of major transcription factors such as LRL1, myc7, bHLH125, PIF1, BH093, PIL5, MYBS1, and BH074 in purple waxy maize kernels, while dark treatment greatly promoted the expression level of transcription factors RVE6, MYB4, MY1R1, and MYB145. Discussion: This study is the first report to investigate the effects of light on waxy maize kernel pigmentation and the underlying mechanism at both transcriptome and metabolome levels, and the results from this study are valuable for future research to better understand the effects of light on the regulation of plant growth.

Smart breeding driven by big data, artificial intelligence, and integrated genomic-enviromic prediction.

The first paradigm of plant breeding involves direct selection-based phenotypic observation, followed by predictive breeding using statistical models for quantitative traits constructed based on genetic experimental design and, more recently, by incorporation of molecular marker genotypes. However, plant performance or phenotype (P) is determined by the combined effects of genotype (G), envirotype (E), and genotype by environment interaction (GEI). Phenotypes can be predicted more precisely by training a model using data collected from multiple sources, including spatiotemporal omics (genomics, phenomics, and enviromics across time and space). Integration of 3D information profiles (G-P-E), each with multidimensionality, provides predictive breeding with both tremendous opportunities and great challenges. Here, we first review innovative technologies for predictive breeding. We then evaluate multidimensional information profiles that can be integrated with a predictive breeding strategy, particularly envirotypic data, which have largely been neglected in data collection and are nearly untouched in model construction. We propose a smart breeding scheme, integrated genomic-enviromic prediction (iGEP), as an extension of genomic prediction, using integrated multiomics information, big data technology, and artificial intelligence (mainly focused on machine and deep learning). We discuss how to implement iGEP, including spatiotemporal models, environmental indices, factorial and spatiotemporal structure of plant breeding data, and cross-species prediction. A strategy is then proposed for prediction-based crop redesign at both the macro (individual, population, and species) and micro (gene, metabolism, and network) scales. Finally, we provide perspectives on translating smart breeding into genetic gain through integrative breeding platforms and open-source breeding initiatives. We call for coordinated efforts in smart breeding through iGEP, institutional partnerships, and innovative technological support.

Regulation of angiotensin-(1-7) and angiotensin II type 1 receptor by telmisartan and losartan in adriamycin-induced rat heart failure.

AIM: To investigate the possible effects of telmisartan and losartan on cardiac function in adriamycin (ADR)-induced heart failure in rats, and to explore the changes in plasma level of angiotensin-(1-7)[Ang-(1-7)] and myocardial expression of angiotensin II type 1/2 receptors (AT(1)R / AT(2)R) and Mas receptor caused by the two drugs. METHODS: Male Sprague-Dawley rats were randomly divided into 4 groups: the control group, ADR-treated heart failure group (ADR-HF), telmisartan plus ADR-treated group (Tel+ADR) and losartan plus ADR-treated group (Los+ADR). ADR was administrated (2.5 mg/kg, ip, 6 times in 2 weeks). The rats in the Tel+ADR and Los+ADR groups were treated orally with telmisartan (10 mg/kg daily po) and losartan (30 mg/kg daily), respectively, for 6 weeks. The plasma level of Ang-(1-7) was determined using ELISA. The mRNA and protein expression of myocardial Mas receptor, AT(1)R and AT(2)R were measured using RT-PCR and Western blotting, respectively. RESULTS: ADR significantly reduced the plasma level of Ang-(1-7) and the expression of myocardial Mas receptor and myocardial AT(2)R, while significantly increased the expression of myocardial AT(1)R. Treatment with telmisartan and losartan effectively increased the plasma level of Ang-(1-7) and suppressed myocardial AT(1)R expression, but did not influence the expression of Mas receptor and AT(2)R. CONCLUSION: The protective effects of telmisartan and losartan in ADR-induced heart failure may be partially due to regulation of circulating Ang-(1-7) and myocardial AT(1)R expression.

Proteomic analysis of ubiquitinated proteins in maize immature kernels.

Protein ubiquitination is a dynamic post-translational modification involved in various biological processes in eukaryotes. To understand the function of ubiquitinated proteins in maize kernels, we used the specific K-GG antibody coupled with high-resolution LC-MS/MS to identify the ubiquitinated proteins in maize immature kernels. A total of 1999 lysine ubiquitination sites in 881 proteins were identified in maize kernels. Eight conserved ubiquitination motifs included KubD, GKub, EKub, KubXXXE, AKub, NXKub, KubXXXXXN, and KKub were found in ubiquitinated peptides. The ubiquitinated lysine neighborhoods are more frequently presented in ordered structures. Go and KEGG analysis showed the proteins involved in carbohydrate metabolism and protein processing were identified to be the targets of lysine ubiquitination. Other proteins, which related to RNA transport, spliceosome, endocytosis, ubiquitin-mediated proteolysis, proteasome, and MAPK signaling, were also found to be ubiquitinated. Protein-protein interaction network and KEGG analysis indicated that protein ubiquitination plays a major role in regulating many cellular processes and modulating diverse interactions in maize kernel development. The identification of the 881 ubiquitinated proteins in maize kernels provides a foundation for understanding the physiological roles of these ubiquitinated proteins. Our finding also provides a new insight view into the function of ubiquitinated proteins involved in maize kernel development. SIGNIFICANCE: We reported here the comprehensive proteomic analysis of the ubiquitin-modified proteome in maize kernel. We found that there are some new characteristics of them in ubiquitome of maize immature kernels. The results suggested that protein ubiquitination, as a post-translation modification, plays an essential role in regulating many cellular processes in maize kernel development. This study expands our knowledge on the regulatory roles and mechanisms of protein ubiquitination in maize. and other plants.

Weighted gene co-expression network analysis unveils gene networks regulating folate biosynthesis in maize endosperm.

Folates are essential elements for human growth and development, and their deficiency can lead to serious disorders. Waxy maize is a rich source of folates; however, the regulatory mechanism underlying folate biosynthesis in the endosperm remains unclear. Here, we examined changes in the folate content of maize endosperm collected at 15, 18, 21, 24, and 27 days after pollination (DAP) using liquid chromatograph-mass spectrometry and identified genes related to folate biosynthesis using transcriptome sequencing data. The results showed that 5-methyl-tetrahydrofolate and 5,10-methylene tetrahydrofolate were the main storage forms of folates in the endosperm, and their contents were relatively high at 21-24 days. We also identified 569, 3183, 4365, and 5513 differentially expressed genes (DEGs) in different days around milk stage. Functional annotation revealed 518 transcription factors (TFs) belonging to 33 families exhibiting specific expression in at least one sampling time. The key hub genes involved in folate biosynthesis were identified by weighted gene co-expression network analysis. In total, 24,976 genes were used to construct a co-expression network with 29 co-expression modules, among which the brown and purple modules were highly related to folate biosynthesis. Further, 187 transcription factors in the brown and purple modules were considered potential transcription factors related to endosperm folate biosynthesis. These results may improve the understanding of the molecular mechanism underlying folate biosynthesis in waxy maize and lead to the development of nutritionally fortified varieties. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02974-7.

Protein Sub-Visible Particle and Free Radical formation of a Freeze-Dried Monoclonal Antibody Formulation During Dropping.

Dropping during shipping and handling of liquid biopharmaceutical formulations has long been known to cause protein degradation and aggregation. On the other hand, accidental dropping of freeze-dried protein formulations is generally considered not a major issue for biopharmaceutical quality. Reports of stability and especially the underling degradation mechanism(s) during shipping and handling of freeze-dried protein formulations were rarely seen in literature. In this manuscript, we report an interesting phenomenon in which repeated dropping of freeze-dried monoclonal antibody X (mAb-X) formulation powder resulted in significant protein sub-visible particles (SbVPs) in the reconstituted liquid as determined by the sensitive particle analyzing technique micro-flow imaging (MFI). Free radicals were observed after repeated dropping by electron paramagnetic resonance (EPR). Formation of SbVPs could be partially inhibited by the free radical scavengers methionine and 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidin-yloxy free radical (CTPO). The amount of free radicals and SbVPs was correlated to the sample temperature during dropping. Therefore we propose that the high temperature formed during dropping was probably the root cause for protein aggregation and free radical formation, which could further cause protein aggregation. Our observations suggest that similar to liquid protein formulations, dropping of freeze-dried protein formulations should also be avoided or mitigated.

Genetic Dissection of Quantitative Resistance to Common Rust (Puccinia sorghi) in Tropical Maize (Zea mays L.) by Combined Genome-Wide Association Study, Linkage Mapping, and Genomic Prediction.

Common rust is one of the major foliar diseases in maize, leading to significant grain yield losses and poor grain quality. To dissect the genetic architecture of common rust resistance, a genome-wide association study (GWAS) panel and a bi-parental doubled haploid (DH) population, DH1, were used to perform GWAS and linkage mapping analyses. The GWAS results revealed six single-nucleotide polymorphisms (SNPs) significantly associated with quantitative resistance of common rust at a very stringent threshold of P-value 3.70 × 10-6 at bins 1.05, 1.10, 3.04, 3.05, 4.08, and 10.04. Linkage mapping identified five quantitative trait loci (QTL) at bins 1.03, 2.06, 4.08, 7.03, and 9.00. The phenotypic variation explained (PVE) value of each QTL ranged from 5.40 to 12.45%, accounting for the total PVE value of 40.67%. Joint GWAS and linkage mapping analyses identified a stable genomic region located at bin 4.08. Five significant SNPs were only identified by GWAS, and four QTL were only detected by linkage mapping. The significantly associated SNP of S10_95231291 detected in the GWAS analysis was first reported. The linkage mapping analysis detected two new QTL on chromosomes 7 and 10. The major QTL on chromosome 7 in the region between 144,567,253 and 149,717,562 bp had the largest PVE value of 12.45%. Four candidate genes of GRMZM2G328500, GRMZM2G162250, GRMZM2G114893, and GRMZM2G138949 were identified, which played important roles in the response of stress resilience and the regulation of plant growth and development. Genomic prediction (GP) accuracies observed in the GWAS panel and DH1 population were 0.61 and 0.51, respectively. This study provided new insight into the genetic architecture of quantitative resistance of common rust. In tropical maize, common rust could be improved by pyramiding the new sources of quantitative resistance through marker-assisted selection (MAS) or genomic selection (GS), rather than the implementation of MAS for the single dominant race-specific resistance gene.

Development of high-resolution multiple-SNP arrays for genetic analyses and molecular breeding through genotyping by target sequencing and liquid chip.

Genotyping platforms, as critical supports for genomics, genetics, and molecular breeding, have been well implemented at national institutions/universities in developed countries and multinational seed companies that possess high-throughput, automatic, large-scale, and shared facilities. In this study, we integrated an improved genotyping by target sequencing (GBTS) system with capture-in-solution (liquid chip) technology to develop a multiple single-nucleotide polymorphism (mSNP) approach in which mSNPs can be captured from a single amplicon. From one 40K maize mSNP panel, we developed three types of markers (40K mSNPs, 251K SNPs, and 690K haplotypes), and generated multiple panels with various marker densities (1K-40K mSNPs) by sequencing at different depths. Comparative genetic diversity analysis was performed with genic versus intergenic markers and di-allelic SNPs versus non-typical SNPs. Compared with the one-amplicon-one-SNP system, mSNPs and within-mSNP haplotypes are more powerful for genetic diversity detection, linkage disequilibrium decay analysis, and genome-wide association studies. The technologies, protocols, and application scenarios developed for maize in this study will serve as a model for the development of mSNP arrays and highly efficient GBTS systems in animals, plants, and microorganisms.