Madelaine Bartlett

Forward genetics is a powerful approach for gene discovery, but identifying causal mutations becomes difficult when mutants are maintained in heterogeneous populations with uncertain pedigrees. This is exemplified by classical tasselseed ( ts ) mutants, which have long served as a genetic model for studying sex determination and carpel suppression. Decades of repeated outcrossing to diverse inbred lines have created substantial genetic heterogeneity, limiting the effectiveness of conventional bulked-segregant analysis sequencing (BSA-Seq). To address this, we developed a BSA-Seq framework that integrates flexible experimental designs, multiple reference genomes, and complementary statistical methods tailored for genetically heterogeneous populations. Applying this framework revealed that reference genome selection is critical for mapping success and that Euclidean distance raised to the fourth power (ED ⁴ ) outperformed homozygosity mapping (HM). Furthermore, the framework enables simultaneous mapping of multiple mutations within a single population, eliminating the need for additional mapping populations. Applying this framework to 26 ts mutant stocks from the Maize Genetics Cooperation Stock Center, we successfully mapped 24 mutants to genomic intervals containing known ts genes, while the remaining mutants mapped to distinct genomic intervals, defining novel candidate regions underlying carpel suppression. Together, these results demonstrate that historical mutant collections represent an underutilized resource for gene discovery and establish a generalizable mapping strategy for unlocking their genetic potential across diverse species. Author Summary: Forward genetics has been instrumental in uncovering genes that control important biological processes, but mapping the specific causal mutation responsible for a phenotype can be difficult when mutants are maintained in mixed genetic backgrounds with uncertain pedigrees. This challenge is common in historical mutant collections preserved in public stock centers. Here, we demonstrate that bulked-segregant analysis coupled to whole genome sequencing (BSA-Seq) can effectively map these mutants when combined with flexible experimental designs, multiple reference genomes, and alternative statistical methods. We tested this approach in classical maize tasselseed ( ts ) mutants, which alter carpel development and sex determination. Using this approach, we mapped 24 out of 26 mutants to genomic intervals containing known sex determination genes and identified candidate novel intervals for the remaining mutants. Additionally, we showed that multiple independent mutations can be mapped simultaneously from a single population, reducing the time and cost required for genetic mapping. These results demonstrate that historical mutant collections are an untapped resource for gene discovery and provide a clear path for unlocking their genetic potential across diverse species.

In plants, final organ shape and position influence function and whole plant productivity. In Zea mays (maize), leaf, ear, and tassel morphology are all key agronomic features influencing yield. These aerial structures originate from stem cell populations within meristems. Understanding how meristems are regulated and how stem cells differentiate over developmental time to form mature leaves, ears, and tassels is important for identifying novel routes for maize improvement. High-resolution imaging of meristem morphology is a key component of developmental studies. Scanning electron microscopy (SEM) is widely used to visualize meristem topology; however, conventional SEM preparation requires fixation, dehydration, drying, and conductive coating. These steps are time-consuming, technically demanding, and can introduce damage. Here, we present a rapid protocol for imaging freshly dissected meristems from maize using a benchtop scanning electron microscope. Fresh tissues are mounted directly onto conductive adhesive and imaged under high vacuum at low accelerating voltage, enabling high-quality micrographs to be captured within minutes of dissection. Although dehydration and tissue collapse can occur under vacuum, rapid sample handling and image acquisition minimize these effects and produce images comparable to those obtained from conventionally prepared specimens. This streamlined workflow substantially reduces preparation time, lowers material requirements, and facilitates medium-throughput morphological analysis. The method provides a practical and reproducible approach for rapid developmental phenotyping and is readily adaptable to other plant species and other tissues.

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.

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.

Developmental gene function is often conserved over deep time, but cis-regulatory sequence conservation is difficult to identify. Rapid sequence turnover, paleopolyploidy, structural variation, and limited phylogenomic sampling have impeded conserved noncoding sequence (CNS) discovery. Using Conservatory, an algorithm that leverages microsynteny and iterative alignments to map CNS-gene associations over evolution, we uncovered ~2.3 million CNSs, including more than 3000 predating angiosperms, from 284 plant species spanning 300 million years of diversification. Ancient CNSs were enriched near developmental regulators, and mutating CNSs near HOMEOBOX genes produced strong phenotypes. Tracing CNS evolution uncovered key principles: CNS spacing varies, but order is conserved; genomic rearrangements form new CNS-gene associations; and ancient CNSs are preferentially retained among paralogs but are often lost as cohorts or evolve into lineage-specific CNSs.

Summary: Meristem activity is controlled by the CLAVATA (CLV) signaling pathway, which involves a suite of leucine‐rich receptor (LRR) receptors, receptor‐like proteins, and CLV‐EMBRYO SURROUNDING REGION (CLE) peptide ligands. FASCIATED EAR 3 (FEA3) is a leucine‐rich receptor (LRR) receptor‐like protein important for meristem maintenance in maize and acts independently of canonical CLV receptors. To identify FEA3's interaction network, we used TurboID‐based proximity labeling in Zea mays meristems and identified a putative co‐receptor, BARELY ANY MERISTEM 1D (BAM1D). BAM1D and FEA3 proximity labeling proteomes shared over 40 proteins, including many signaling proteins, suggesting they feed into a common signaling pathway. fea3 was epistatic to bam1d in the control of inflorescence meristem (IM) size, supporting the idea that FEA3 and BAM1D interact physically. However, fea3 and bam1d act antagonistically because fea3 mutants had larger IMs, whereas bam1d mutants produced smaller IMs. This study demonstrates how in vivo TurboID‐based proximity labeling clarifies complex genetic interactions between CLV receptors and expands our knowledge of downstream signaling components of CLV signaling pathways, which are largely uncharacterized. Our findings support the notion that multiple, partially overlapping CLV receptor complexes coordinately control meristem maintenance.

In maize, several yield-related traits are associated with meristem activity, regulated by CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptide signals perceived by CLAVATA(CLV) receptors in the CLAVATA-WUSCHEL (CLV-WUS) pathway. However, additional signaling pathways in maize meristem development remain poorly understood. Here, we identify three receptor-like kinases, ZmERECTA1 (ZmER1), ZmER2 and ZmER1-like (ZmERL), and their ligands, EPIDERMAL PATTERNING FACTOR-like (ZmEPFL), as critical regulators of meristem activity, plant architecture, and ear development. We demonstrate that ZmER receptors act redundantly, with ZmER1 playing a primary role. Zmer1 mutants have compact architecture, enlarged inflorescence meristems (IMs), and increased kernel row numbers (KRNs), while higher-order Zmer mutants display exacerbated phenotypes. We further reveal that ZmER1 specifically binds to five EPFL peptides, which act redundantly in ear development regulation. Furthermore, we find that ZmWUS1 is upregulated in Zmer mutants and mutation in Zmwus1 partially suppress the enlarged IM of Zmer1 mutants. We also generate weak Zmer1 alleles with enhanced yield traits, including reduced leaf angles and increased KRN. These findings offer valuable insights into ER-EPFL signaling in maize meristem development and provide promising genetic targets for breeding high-yield maize varieties through optimized plant and ear architecture.

Developmental gene function is often conserved over deep time, but cis-regulatory sequence conservation is difficult to identify. Rapid sequence turnover, paleopolyploidy, structural variation, and limited phylogenomic sampling have impeded conserved non-coding sequence (CNS) discovery. Using Conservatory, an algorithm that leverages microsynteny and iterative alignments to map CNS-gene associations over evolution, we uncovered ∼2.3 million CNSs, including over 3,000 predating angiosperms, from 284 plant species spanning 300 million years of diversification. Ancient CNSs were enriched near developmental regulators, and mutating CNSs near HOMEOBOX genes produced strong phenotypes. Tracing CNS evolution uncovered key principles: CNS spacing varies, but order is conserved; genomic rearrangements form new CNS-gene associations; and ancient CNSs are preferentially retained among paralogs, but are often lost as cohorts or evolve into lineage-specific CNSs. One Sentence Summary: Conservatory maps ancient cis-regulatory elements and uncovers regulatory evolution dynamics.

Changes in form are driven by the differential, context-dependent regulation of pleiotropic genes. How genetic pleiotropy itself emerges, however, remains unclear. The maize genes GRASSY TILLERS1 ( GT1 ) and RAMOSA3 ( RA3 ) are required for axillary meristem suppression, a deeply conserved trait across angiosperms, and for floral organ suppression, a trait which evolved within the grass family. To determine how these pleiotropic functions are regulated, we first established a high-throughput method for quantitative phenotyping of grass flowers. Using this method, we show that distinct environmental mechanisms regulate axillary meristem versus floral organ suppression. In line with these differences, we find upstream regulation of GT1 and RA3 has diverged, consistent with their redeployment in flowers. Our results show that, rather than wholesale adoption of genetic networks, developmental genes can retain ancient functions and be recruited into other programs in the evolution of form, thereby increasing genetic pleiotropy.