Understanding the frequency and distribution of meiotic crossovers (COs) is critical for both fundamental studies on meiosis and for practical applications in plant breeding, where controlling recombination can accelerate crop improvement. Determining CO rates at specific genomic loci has traditionally relied on labor-intensive methods that require the production and genotyping of large progenies. Here, we present a high-throughput protocol for site-specific quantification of meiotic COs in maize using droplet digital PCR (ddPCR). The method is based on genotyping individual pollen nuclei from hybrid plants to detect recombinant and nonrecombinant alleles at defined chromosomal intervals. By distributing several thousands of pollen nuclei into nanoliter-sized droplets and performing PCR with allele-specific fluorescent probes, this method allows precise quantification of CO frequency with high sensitivity. The protocol provides detailed guidance for nuclei isolation, probe master mix preparation, droplet generation, and data interpretation. This method can be easily adapted for use in other plants.
During meiosis, homologous chromosomes engage in reciprocal exchanges of segments in a process known as crossing over (CO). About 85% of CO events in maize are products of the class I pathway. Class I COs are interference-sensitive, meaning that the formation of one CO reduces the likelihood of another CO forming close by. This protocol describes a chromatin immunoprecipitation-sequencing (ChIP-seq)-based method for mapping meiotic COs in maize, using an antibody against MutL Homolog 3 (MLH3), a key component of the class I CO pathway. CO sites are determined by Illumina sequencing of DNA isolated from MLH3-associated chromatin fragments. Traditionally, COs have been identified through genetic mapping, which relies on the segregation of genetic markers in the progeny of hybrid plants. However, conventional genetic mapping provides limited resolution and requires large numbers of progeny individuals. The MLH3 ChIP-seq approach enables direct detection of COs, providing high-resolution and genome-wide coverage, including genome regions with low DNA sequence polymorphism, which are inaccessible to genetic CO mapping. Furthermore, MLH3 ChIP-seq enables screening of thousands of CO events, greatly accelerating the analysis and reducing its cost. This protocol can also be used to examine any chromatin-bound meiotic proteins and adapted to studying chromatin-associated proteins in somatic cells.
Maize ( Zea mays ) is a cornerstone of agriculture and global genetics research. Every year more than 200 million hectares of maize are grown worldwide for human and animal consumption, and biofuel and energy production. Maize has also been a key research model organism since the 1800s, and over 150 years of research has led to many fundamental discoveries in genetics and developmental biology. These findings include “jumping genes” (transposons) and the first homeobox gene in plants, KNOTTED1 , whose discovery overturned the hypothesis that homeobox genes were unique to animals. Research in maize has not only deepened our fundamental understanding of the world around us but has also driven crop improvements. Maize yield and resilience are intimately linked with plant morphology. Developmental biology aims to understand how morphology arises through the coordinated action of gene expression, hormones, cell behaviors, and biophysics across space and time. This review delves into the foundations of developmental analyses, spanning three components: morphology, time, and space. We also discuss the challenges inherent to maize developmental research, such as the inaccessibility of meristems and developing organs, and introduce techniques that address these challenges.
Zea mays (maize) is a critical global crop, generating more than 1 billion metric tons of grain annually. Plant morphology is tightly associated with final yield and is determined by the process of development. Accurate assessment and recording of mature plant morphology is essential for linking genotype to phenotype and for assessing environmental influences on plant development and yield. In maize, imaging of fully emerged aerial organs provides an accessible and informative developmental end point for comparative phenotypic analyses. Here, we provide a protocol for capturing high-quality images of mature maize plants and detached leaf blades, suitable for publication and downstream quantitative trait extraction. We emphasize standardized imaging conditions, including high-contrast backgrounds, diffuse lighting, fixed camera positioning, and consistent scale calibration, to minimize technical variation and to facilitate automated image processing. Guidance is provided for both laboratory- and field-based imaging setups and uses widely available equipment. Images generated using this protocol can be analyzed using platforms such as FIJI to extract morphometric traits, including length, width, and outline-based shape descriptors. By prioritizing standardization at the point of acquisition, this protocol supports reproducible, scalable phenotyping of mature maize morphology for developmental studies in maize.
The plant kingdom displays a wide array of shapes, and this morphology is tightly linked to function and productivity. For example, leaf size, shape, and arrangement around the stem define canopy architecture, influencing light interception and photosynthetic capacity. Understanding how final morphology is determined is, therefore, not only important for our basic understanding of various plant species, but also an essential tool in the development of new crop varieties. All aerial organs in flowering plants arise on the flanks of meristems throughout development. In Zea mays (maize) , these meristems are deeply enclosed within successive leaf layers, making their identification and isolation technically challenging. Successful dissection requires accurate staging, recognition of anatomical landmarks, and careful tissue handling to avoid damage or desiccation. These challenges can present a significant barrier to researchers attempting maize developmental biology studies. Here, we provide a practical, step-by-step guide for identifying and dissecting vegetative shoot apical, tassel, and ear meristems. The protocol integrates external morphological cues, nodal position, stem texture, and diagnostic cross-sectional features to distinguish cuts made above, through, or below the meristem. Detailed strategies are described to sequentially remove enclosing leaf primordia, while preserving meristem integrity. By formalizing techniques often transmitted informally within laboratories, this protocol lowers the barrier to entry for maize meristem research, supporting downstream applications including imaging, gene expression analysis, and developmental phenotyping.
