Zhiyuan Fu

SUMMARY: Stomatal patterning is a fundamental aspect of plant photosynthetic performance and water‐use efficiency, yet its genetic basis in C ₄ grasses such as maize remains poorly understood. Here, we integrated linkage analysis across three recombinant inbred line populations and a genome‐wide association study in a diverse maize panel to dissect the genetic architecture of six stomatal traits. This approach identified 33 quantitative trait loci and 51 association loci for 6 stomatal traits and revealed a significant negative genetic correlation between stomatal density (SD) and size, with multiple genomic intervals showing co‐localization of effects on both trait categories. By integrating reported cell‐type‐specific transcriptomic data, we prioritized high‐confidence candidate genes, including conserved stomatal regulators as well as previously uncharacterized candidates involved in processes such as cell wall biosynthesis. Functional analyses of EMS‐induced mutants for candidates ZmbHLH140 and ZmCESA6 demonstrated that loss of ZmbHLH140 reduces SD, whereas mutation of ZmCESA6 has the opposite effect. In addition, the signature of selection at the ZmbHLH140 locus is consistent with a role for stomatal regulation during maize domestication. Together, our results establish a comprehensive genetic framework for stomatal development in maize, highlighting both conserved and grass‐specific mechanisms that shape stomatal patterning and offering a foundation for future functional studies of stomatal development in cereals.

Integrated QTL GWAS identified three key genes, whose heat ind uction during maize germination was verified via RNA seq and qRT PCR analysis. Heat stress during germination critically impairs maize seedling establishment and yield stability, yet its genetic basis remains poorly understood. Here, 21 quantitative trait loci (QTLs) for heat tolerance were identified in a recombinant inbred line (RIL) population under heat stress (39 °C vs. 28 °C), including a major hypocotyl diameter QTL (qHDT2-1, LOD = 23.68). Concurrently, genome-wide association study (GWAS) in an association panel detected 213 significant SNPs associated with heat tolerance. Population-stable SNPs, pleiotropic SNPs and high-impact QTLs were integrated to identify candidate genes. Transcriptome analysis and GO enrichment prioritized 612 candidate genes, among which six heat-induced genes were selected. Subsequent validation confirmed Zm00001d005300, Zm00001d023524, and Zm00001d026563 as core regulators forming a coordinated heat-response network. We also found that heat stress had a relatively minor impact on germination rates, but significantly affects biomass accumulation and tissue morphology. This study identifies key genetic targets for breeding heat tolerant maize.

Sweet–waxy maize is a highly valuable specialty maize type with an increasing market demand, but conventional breeding methods for producing sweet–waxy maize are restricted by severe bottlenecks, such as long breeding cycles and linkage drag. This study was conducted to rapidly create sweet–waxy maize germplasm using CRISPR/Cas9 genome-editing technology. We used a CRISPR/Cas9 system to target maize Sh2 (regulating the super-sweet kernel trait) and Wx (controlling the waxy kernel trait), which are two key genes in the starch biosynthesis pathway. Two small-guide RNAs (sgRNAs) designed for each gene were incorporated into CRISPR/Cas9 vectors, which were then introduced into maize via Agrobacterium-mediated transformation. We obtained Cas9-free T₃ homozygous sh2 and wx mutant lines with significant increases in kernel soluble sugar and amylopectin contents, respectively, but no adverse changes to major agronomic traits. Using these Cas9-free lines, we developed a new type of sweet–waxy maize germplasm, in which waxy and sweet kernels on the same ear segregated at a 3:1 ratio. Our results indicate that CRISPR/Cas9-mediated editing of Sh2 and Wx can efficiently generate sweet–waxy maize germplasm with no detectable linkage drag. The study methods would be useful for optimizing the molecular breeding of novel and innovative maize germplasm.

The transition from vegetative to reproductive growth is critical in flowering plants. INDETERMINATE1 (ID1), a monocot-specific zinc finger transcription factor (TF), is thought to promote flowering by indirectly influencing ZCN8, the encoded protein of which transmits the florigen signal to downstream floral identity genes. However, the precise mechanism by which ID1 regulates floral transition in day-neutral maize remains poorly understood. Here, we characterized a floral transition-defective (ftd1) mutant that produces excess leaves and exhibits impaired reproductive development. Map-based cloning and complementation confirmed that ftd1 is an allele of id1. We demonstrated that ID1 is a nucleus-localized transcriptional activator that preferentially binds to the canonical GTC-core motif. Genome-wide binding and transcriptome analyses of ftd1 immature leaves identified potential target genes, including the flowering regulator gene MADS67 and the SPL gene SBP20. We further showed that ID1 physically interacts with MYB31 and TCP transcription factors, which synergistically enhance ID1-mediated transactivation of the MADS67 promoter. Genetic analyses indicated that ID1 acts epistatically to MADS67, and overexpression of MADS67 only partially rescued the id1 phenotype, indicating that MADS67 is necessary but insufficient for flowering. In parallel, the ID1–TCP20 complex activated SBP20, which in turn directly upregulated the florigen gene ZCN8. Collectively, our findings propose a dual-pathway model for ID1-mediated flowering regulation, in which ID1 orchestrates the floral transition through both a MADS67-dependent module (ID1 + MYB31/TCP14/16/20–MADS67) and a parallel SBP20–ZCN8 module (ID1 + TCP20–SBP20–ZCN8), thereby ensuring robust regulation of maize flowering time.

Key message This study mapped and screened three candidate genes related to kernel dehydration in maize. Abstract The slow development rate of maize kernels during later stages leads to high kernel moisture content at harvest, posing a challenge for mechanized maize harvesting in China. This study utilized a recombinant inbred line population derived from Zheng 58 (slow dehydration) and PH6WC (fast dehydration) as parents. After four years of trait investigation and analysis, 25 quantitative trait loci (QTLs) associated with kernel dehydration rate and moisture content were identified, with six QTLs showing a significant contribution value exceeding 10% in the phenotype. Furthermore, a comparison was made between the QTLs identified in this study and those from previous research on maize kernel moisture content and dehydration rate, followed by screening through the omics analysis of the parental lines. Three candidate genes related to kernel dehydration rate were identified, primarily involving carbohydrate metabolism, energy metabolism processes (Zm00001d014030 and Zm00001d006476), and stimulus resistance (Zm00001d040113). These findings provide valuable insights to assist and guide future breeding efforts for mechanical harvesting of maize.

SUMMARY: Maize is one of the world's most important staple crops, yet its production is increasingly threatened by the rising frequency of high‐temperature stress (HTS). To investigate the genetic basis of anther thermotolerance under field conditions, we performed linkage and association analysis to identify HTS response quantitative trait loci (QTL) using three recombinant inbred line (RIL) populations and an association panel containing 375 diverse maize inbred lines. These analyses resulted in the identification of 16 co‐located large QTL intervals. Among the 37 candidate genes identified in these QTL intervals, five have rice or Arabidopsis homologs known to influence pollen and filament development. Notably, one of the candidate genes, ZmDUP707, has been subject to selection pressure during breeding. Its expression is suppressed by HTS, leading to pollen abortion and barren seeds. We also identified several additional candidate genes potentially underly QTL previously reported by other researchers. Taken together, our results provide a pool of valuable candidate genes that could be employed by future breeding programs aiming at enhancing maize HTS tolerance.

Mercury (Hg) pollution not only poses a threat to the environment but also adversely affects the growth and development of plants, with potential repercussions for animals and humans through bioaccumulation in the food chain. Maize, a crucial source of food, industrial materials, and livestock feed, requires special attention in understanding the genetic factors influencing mercury accumulation. Developing maize varieties with low mercury accumulation is vital for both maize production and human health. In this study, a comprehensive genome-wide association study (GWAS) was conducted using an enlarged SNP panel comprising 1.25 million single nucleotide polymorphisms (SNPs) in 230 maize inbred lines across three environments. The analysis identified 111 significant SNPs within 78 quantitative trait loci (QTL), involving 169 candidate genes under the Q model. Compared to the previous study, the increased marker density and optimized statistical model led to the discovery of 74 additional QTL, demonstrating improved statistical power. Gene ontology (GO) enrichment analysis revealed that most genes participate in arsenate reduction and stress responses. Notably, GRMZM2G440968, which has been reported in previous studies, is associated with the significant SNP chr6.S_155668107 in axis tissue. It encodes a cysteine proteinase inhibitor, implying its potential role in mitigating mercury toxicity by inhibiting cysteine. Haplotype analyses provided further insights, indicating that lines carrying hap3 exhibited the lowest mercury content compared to other haplotypes. In summary, our study significantly enhances the statistical power of GWAS, identifying additional genes related to mercury accumulation and metabolism. These findings offer valuable insights into unraveling the genetic basis of mercury content in maize and contribute to the development of maize varieties with low mercury accumulation.