Accurate Phenotypic Identification and Genetic Analysis of the Ear Leaf Veins in Maize (<i>Zea mays</i> L.)

The ear leaf veins are an important transport structure in the maize "source" organ; therefore, the microscopic phenotypic characteristics and genetic analysis of the leaf veins are particularly essential for promoting the breeding of ideal maize varieties with high yield and quality. In this study, the microscopic image of the complete blade cross section was realized using X-ray micro-computed tomography (micro-CT) technology with a resolution of 13.5 µm. Moreover, the veins’ phenotypic traits in the cross section of the complete maize leaf, including the number of leaf veins, midvein area, leaf width, and density of leaf veins, were automatically and accurately detected by a deep-learning-integrated phenotyping pipeline. Then, we systematically collected vein phenotypes of 300 inbred lines at the silking stage of the ear leaves. It was found that the leaf veins’ microscopic characteristics varied among the different subgroups. The number of leaf veins, the density of leaf veins, and the midvein area in the stiff-stalk (SS) subgroup were significantly higher than those of the other three subgroups, but the leaf width was the smallest. The leaf width in the tropical/subtropical (TST) subgroup was the largest, but there was no significant difference in the number of leaf veins between the TST subgroup and other subgroups. Combined with a genome-wide association study (GWAS), 61 significant single-nucleotide polymorphism markers (SNPs) and 29 candidate genes were identified. Among them, the candidate gene <i>Zm00001d018081</i> regulating the number of leaf veins and <i>Zm00001d027998</i> regulating the midvein area will provide new theoretical support for in-depth analysis of the genetic mechanism of maize leaf veins.