The stomatal phenotype is a crucial microscopic characteristic of the leaf surface, and modulating the stomata of maize leaves can enhance photosynthetic carbon assimilation and water use efficiency, thereby playing a vital role in maize yield formation. The evolving imaging and image processing technologies offer effective tools for precise analysis of stomatal phenotypes. This study employed Jingnongke 728 and its parental inbred to capture stomatal images from various leaf positions and abaxial surfaces during key reproductive stages using rapid scanning electron microscopy. We uesd a target detection and image segmentation approach based on YOLOv5s and Unet to efficiently obtain 11 phenotypic traits encompassing stomatal count, shape, and distribution. Manual validation revealed high detection accuracies for stomatal density, width, and length, with R2 values of 0.92, 0.97, and 0.95, respectively. Phenotypic analyses indicated a significant positive correlation between stomatal density and the percentage of guard cells and pore area (r=0.36), and a negative correlation with stomatal area and subsidiary cell area (r=-0.34 and -0.46). Additionally, stomatal traits exhibited notable variations with reproductive stages and leaf layers. Specifically, at the monocot scale, stomatal density increased from 74.35 to 87.19 Counts/mm2 from lower to upper leaf layers. Concurrently, the stomatal shape shifted from sub-circular (stomatal roundness = 0.64) to narrow and elongated (stomatal roundness = 0.63). Throughout the growth cycle, stomatal density remained stable during vegetative growth, decreased during reproductive growth with smaller size and narrower shape, and continued to decline while increasing in size and tending towards a rounded shape during senescence. Remarkably, hybrid 728 differed notably from its parents in stomatal phenotype, particularly during senescence. Moreover, the stomatal density of the hybrids showed negative super parental heterosis (heterosis rate = -0.09), whereas stomatal dimensions exhibited positive super parental heterosis, generally resembling the parent MC01. This investigation unveils the dynamic variations in maize stomatal phenotypes, bolstering genetic analyses and targeted improvements in maize, and presenting a novel technological instrument for plant phenotype studies.

The Anthesis-Silking Interval (ASI) is a crucial indicator of the synchrony of reproductive development in maize, reflecting its sensitivity to adverse environmental conditions such as heat stress and drought. This paper presents an automated method for detecting the maize ASI index using a field high-throughput phenotyping platform. Initially, high temporal-resolution visible-light image sequences of maize plants from the tasseling to silking stage are collected using a field rail-based phenotyping platform. Then, the training results of different sizes of YOLOv8 models on this dataset are compared to select the most suitable base model for the task of detecting maize tassels and ear silks. The chosen model is enhanced by incorporating the SENetv2 and the dual-layer routing attention mechanism BiFormer, named SEBi-YOLOv8. The SEBi-YOLOv8 model, with these combined modules, shows improvements of 2.3% and 8.2% in mAP over the original model, reaching 0.989 and 0.886, respectively. Finally, SEBi-YOLOv8 is used for the dynamic detection of maize tassels and ear silks in maize populations. The experimental results demonstrate the method’s high detection accuracy, with a correlation coefficient (R2) of 0.987 and an RMSE of 0.316. Based on these detection results, the ASI indices of different inbred lines are calculated and compared.

Maize Ustilago maydis is a disease that severely affects maize yield and quality. In this paper, we employed transcriptome sequencing and GWAS analysis to identify candidate genes and reveal disease-resistant germplasm resources, thereby laying the foundation for further analysis of the molecular mechanism of maize Ustilago maydis resistance and genetic improvement. The results of transcriptome sequencing revealed that a considerable number of receptor kinase genes, signal-transduction-related protein genes, redox-response-related genes, WRKYs, and P450s genes were significantly upregulated. There was a wide range of mutations of Ustilago maydis in maize inbred lines. Thirty-two high-resistance maize inbred lines were selected, and 16 SNPs were significantly associated with the disease index. By integrating the results of GWAS and RNA-seq, five genes related to disease resistance were identified, encoding the chitinase 1 protein, fatty acid elongase (FAE), IAA9, GATA TF8, and EREB94, respectively. It provides a certain reference for the cloning of maize anti-tumor smut genes and the breeding of new varieties.

The grain moisture content of maize inbred lines at maturity is one of the most important indicators for mechanical harvesting of kernels. In this study, 116 maize inbred lines from a wide range of sources were used as research materials and 30 traits of grain moisture content were analyzed using multivariate statistical analysis. The results showed that all 30 traits had some correlations. Principal component analysis downscaled the 30 traits into 10 principal component factors, reflecting 77.674% of the information in the original traits. Cluster analysis categorized the 116 inbred lines into 5 major groups containing 26, 29, 31, 16 and 14 inbred lines. Based on the D value of the overall evaluation, discriminant analysis reclassified the maize inbred lines by principal component scores and 98 maize inbred lines were correctly discriminated with a probability of 84.48%, which can be regarded as a relatively reliable clustering result. The stepwise regression method was further used to screen seven traits: GMC2, GDR1, HMC3, NH, GDR2, CD and EL and to establish a comprehensive evaluation model for the grain moisture content of maize inbred lines. Among 116 maize inbred lines, 14, represented by H21 and MS71, had the lowest grain moisture content at maturity.

Corn smut, caused by Ustilago maydis, significantly threatens maize production. This study evaluated 199 maize inbred lines at the seedling stage under greenhouse conditions for resistance to U. maydis, identifying 39 highly resistant lines. A genome-wide association study (GWAS) using the mrMLM model detected 19 significant single-nucleotide polymorphism (SNP) loci. Based on a linkage disequilibrium (LD) decay distance of 260 kb, 226 candidate genes were identified. Utilizing the significant loci chr1_244281660 and chr5_220156746, two kompetitive allele-specific PCR (KASP) markers were successfully developed. A PCR-based sequence-specific oligonucleotide probe hybridization technique applied to the 199 experimental lines and 60 validation lines confirmed polymorphism for both markers, with selection efficiencies of 48.12% and 43.33%, respectively. The tested materials were derived from foundational inbred lines of domestic and foreign origin. Analysis of 39 highly resistant lines showed that the advantageous alleles carrying thymine/cytosine (T/C) predominated at frequencies of 94.87% and 53.84%, respectively. The genotype TTCC conferred high resistance, while CCTT was highly susceptible. The resistance exhibited high heritability and significant gene-by-environment interaction. This work systematically dissects the genetic basis of common smut resistance in maize, identifies favorable alleles, and provides a novel KASP marker-based strategy for developing disease-resistant germplasm.