Maize (Zea mays L.) is a crucial global crop that serves as a primary source of food and feed. However, its kernels are susceptible to infection by Aspergillus flavus, a fungus known for producing aflatoxins-which are highly carcinogenic compounds harmful to human and animal health. Identifying quantitative trait loci (QTLs) for aflatoxin resistance and developing aflatoxin-resistant maize varieties are essential for mitigating aflatoxin contamination. In this study, a genome-wide association study (GWAS) using an enlarged genotypic panel of 311 maize inbred lines was used to identify genetic loci associated with A. flavus resistance. Phenotypic data on A. flavus resistance were collected through controlled inoculation experiments conducted under controlled conditions. The results revealed that the resistance to A. flavus follows a normal distribution. In addition, temperate inbreds exhibited stronger resistance to A. flavus than tropical/subtropical materials. This study identified 13 novel QTLs encompassing 47 highly expressed genes, with each QTL explaining 8.22–27.71% of the phenotypic variation, indicating that the higher marker density improved statistical power. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that these genes are related to fatty acid synthesis, glycoside decomposition, and root growth and development. One specific gene located on ZmAFR16, ZmFUC1, displayed clustered peaks and accounted for an average of 10.21% of the phenotypic variation. This gene was found to play a role in cell membrane formation and possess alpha-L-fucosidase activity, so it promotes glycoside metabolism and contributes to polysaccharide degradation. Haplotype analysis showed significant differences in resistance to A. flavus among the different haplotypes of elo1 and ZmFUC1. Inbreds carrying the favorable haplotype combination of these two genes exhibited strong resistance to A. flavus. A select sweep analysis indicated that ZmFUC1 was selected during the domestication of teosinte (Zea mays ssp. mexicana) to modern maize, as well as during the adaptation from tropical/subtropical maize to temperate maize. Importantly, this study developed molecular markers in the promoter region of ZmFUC1 to efficiently identify maize germplasm with beneficial haplotypes for resistance to A. flavus. These findings not only enhance our understanding of the genetic factors influencing maize kernel resistance to A. flavus but also offer valuable insights for improving existing germplasm and developing new maize varieties with enhanced resistance to this pathogen.

Plant height (PH) is associated with lodging resistance and planting density, which is regulated by a complicated gene network. In this study, we identified a spontaneous dwarfing mutation in maize, m30, with decreased internode number and length but increased internode diameter. A candidate gene, ZmCYP90D1, which encodes a member of the cytochrome P450 family, was isolated by map-based cloning. ZmCYP90D1 was constitutively expressed and showed highest expression in basal internodes, and its protein was targeted to the nucleus. A G-to-A substitution was identified to be the causal mutation, which resulted in a truncated protein in m30. Loss of function of ZmCYP90D1 changed expression of hormone-responsive genes, in particular brassinosteroid (BR)-responsive genes which is mainly involved in cell cycle regulation and cell wall extension and modification in plants. The concentration of typhasterol (TY), a downstream intermediate of ZmCYP90D1 in the BR pathway, was reduced. A haplotype conferring dwarfing without reducing yield was identified. ZmCYP90D1 was inferred to influence plant height and stalk diameter via hormone-mediated cell division and cell growth via the BR pathway.

Mitochondrial protein translation that is essential for aerobic energy production includes four essential steps of the mitochondrial ribosome cycle, namely, initiation, elongation, termination of the polypeptide, and ribosome recycling. Translation termination initiates when a stop codon enters the A site of the mitochondrial ribosome where it is recognized by a dedicated peptide release factor (RF). However, RFs and mechanisms involved in translation in plant mitochondria, especially in monocotyledons, remain largely unknown. Here, we identified a crumpled kernel (crk5 allele) mutant, with significantly decreased kernel size, 100-kernel weight, and an embryo-lethal phenotype. The Crk5 allele was isolated using map-based cloning and found to encode a mitochondrial localization RF2a. As it is an ortholog of Arabidopsis mitochondrial RF2a, we named the gene ZmmtRF2a. ZmmtRF2a is missing the 5th–7th exons in the crk5 resulting in deletion of domains containing motifs GGQ and SPF that are essential for release activity of RF, mitochondrial ribosome binding, and stop codon recognition. Western blot and qRT-PCR analyses indicate that the crk5 mutation results in abnormal mitochondrion structure and function. Intriguingly, we observed a feedback loop in the crk5 with up-regulated transcript levels detected for several mitochondrial ribosome and mitochondrial-related components, in particular mitochondrial complexes CI, CIV, and a ribosome assembly related PPR. Together, our data support a crucial role for ZmmtRF2a in regulation of mitochondrial structure and function in maize.

Normal microsporogenesis is determined by both nuclear and mitochondrial genes. In maize C-type cytoplasmic male sterility, it is unclear how the development of meiocytes and microspores is affected by the mitochondrial sterility gene and the nuclear restorer gene. In this study, we sequenced the transcriptomes of single meiocytes (tetrad stage) and early mononucleate microspores from sterile and restorer lines. The numbers of expressed genes varied in individual cells and fewer than half of the expressed genes were common to the same cell types. Four comparisons revealed 3379 differentially expressed genes (DEGs), with 277 putatively associated with mitochondria, 226 encoding transcription factors, and 467 possibly targeted by RF4. KEGG analysis indicated that the DEGs in the two lines at the tetrad stage were involved predominantly in carbon metabolism and in amino acid biosynthesis and metabolism, whereas the DEGs during the transition from the tetrad stage to the early mononucleate stage were associated mostly with regulation of protein metabolism, fatty acid metabolism, and anatomical structure morphogenesis. Thus, meiocyte and microspore development was affected by the surrounding cells and the restorer gene, and the restorer gene helped restore the redox homeostasis of microspores and the normal cellular reconstruction during the transition.

Southern corn rust (SCR) is a destructive maize disease caused by Puccinia polysora Underw. To investigate the mechanism of SCR resistance in maize, a highly resistant inbred line, L119A, and a highly susceptible line, Lx9801, were subjected to gene mapping and transcriptome analysis. Bulked-segregant analysis coupled with whole-genome sequencing revealed several quantitative trait loci (QTL) on chromosomes 1, 6, 8, and 10. A set of 25 genes, including two coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) genes, were identified as candidate genes for a major-effect QTL on chromosome 10. To investigate the mechanism of SCR resistance in L119A, RNA-seq of P. polysora-inoculated and non-inoculated plants of L119A and Lx9801 was performed. Unexpectedly, the number of differentially expressed genes in inoculated versus non-inoculated L119A plants was about 10 times that of Lx9801, with only 29 common genes identified in both lines, suggesting extensive gene expression changes in the highly resistant but not in the susceptible line. Based on the transcriptome analysis, one of the CC-NBS-LRR candidate genes was confirmed to be upregulated in L119A relative to Lx9801 independently of P. polysora inoculation. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that transcription factors, as well as genes involved in defense responses and metabolic processes, were dominantly enriched, with the phenylpropanoid biosynthesis pathway most specifically activated. Consistently, accumulation of phenylpropanoid-derived lignin, especially S lignin, was drastically increased in L119A after P. polysora inoculation, but remained unchanged in Lx9801, suggesting a critical role of lignin in SCR resistance. A regulatory network of defense activation and metabolic change in SCR-resistant maize upon P. polysora infection is described.

Seed aging decreases the quality and vigor of crop seeds, thereby causing substantial agricultural and economic losses in crops. To identify genetic differences in seed aging between homozygotes and heterozygotes in maize, the seeds of a set of recombinant inbred lines (RILs) and an immortalized F₂ (IF₂) population were subjected to artificial aging treatments for 0, 2, 3, and 4 days under 45 ℃ and 85% relative humidity and seed vigor was then evaluated in a field experiment. Seed vigor of all entries tested decreased sharply with longer aging treatment and seed vigor decreased more slowly in heterozygotes than in homozygotes. Forty-nine QTL were detected for four measured seed vigor traits in the RIL (28 QTL) and IF₂ (21 QTL) populations. Only one QTL, qGP5, was detected in both populations, indicating that the genes involved in anti-aging mechanisms differed between inbred lines and hybrids. Several QTL were identified to be responsible for multiple seed vigor traits simultaneously in the RIL and IF₂ populations under artificial aging conditions. These QTL may include major genes for seed vigor or seed aging. QTL qVI4b and qGE3a detected in the RIL population coincided with genes ZmLOX1 and ZmPLD1 in the same respective chromosomal regions. These QTL would be useful for screening for anti-aging genes in maize breeding.