Maize (Zea mays) is an important model crop for transgenic studies. However, genetic transformation of maize requires embryonic calli derived from immature embryo, and the impact of utilizing tissue culture methods on the maize epigenome is poorly understood. Here, we generated whole-genome MeDIP-seq data examining DNA methylation in dedifferentiated and normal immature maize embryos. We observed that most of the dedifferentiated embryos exhibited a methylation increase compared to normal embryos. Increased methylation at promoters was associated with down-regulated protein-coding gene expression; however, the correlation was not strong. Analysis of the callus and immature embryos indicated that the methylation increase was induced during induction of embryonic callus, suggesting phenotypic consequences may be caused by perturbations in genomic DNA methylation levels. The correlation between the 21-24nt small RNAs and DNA methylation regions were investigated but only a statistically significant correlation for 24nt small RNAs was observed. These data extend the significance of epigenetic changes during maize embryo callus formation, and the methylation changes might explain some of the observed embryonic callus variation in callus formation.

Comparative genomic studies can identify genes under evolutionary constraint or specialized for trait innovation. Growing evidence suggests that evolutionary constraint also acts on non-coding regulatory sequences, exerting significant impacts on fitness-related traits, although it has yet to be thoroughly explored in plants. Using the assay for transposase-accessible chromatin by sequencing (ATAC-seq), we profile over 80,000 maize accessible chromatin regions (ACRs), revealing that ACRs evolve faster than coding genes, with about one-third being maize-specific and regulating genes associated with speciation. We highlight the role of transposable elements (TEs) in driving intraspecific innovation of ACRs and identify hundreds of candidate ACRs potentially involved in transcriptional rewiring during maize domestication. Additionally, we demonstrate the importance of accessible chromatin in maintaining subgenome dominance and controlling complex trait variations. This study establishes a framework for analyzing the evolutionary trajectory of plant regulatory sequences and offers candidate loci for downstream exploration and application in maize breeding.

Copy number variation (CNV) is a major source of genetic variation and often contributes to phenotypic variation in maize. The duplication at the 27-kDa γ-zein locus (qγ27) is essential to convert soft endosperm into hard endosperm in quality protein maize (QPM). This duplication is unstable and generally produces CNV at this locus. We conducted genetic experiments designed to directly measure DNA rearrangement frequencies occurring in males and females of different genetic backgrounds. The average frequency with which the duplication rearranges to single copies is 1.27 × 10⁻³ and varies among different lines. A triplication of γ27 gene was screened and showed a better potential than the duplication for the future QPM breeding. Our results highlight a novel approach to directly determine the frequency of DNA rearrangements, in this case resulting in CNV at the qγ27 locus. Furthermore, this provides a highly effective way to test suitable parents in QPM breeding.

Summary: Maize is a key biomass resource with wide agricultural applications. Anthocyanins, potent antioxidants, offer health benefits like reducing oxidative stress. The biosynthesis of anthocyanins competes with that of lignin for shared metabolic precursors, which can lead to trade‐offs in plant growth and feed quality. Higher lignin content can decrease silage digestibility, posing challenges for livestock feed. The maize brown midrib 6 (bm6) mutant, known for reduced lignin, has an unclear genetic basis. Here, we identify ZmGCH1 as the candidate gene for bm6 through fine mapping. Mutations in ZmGCH1 shift precursors from lignin to anthocyanin biosynthesis. Furthermore, we show that ZmGCH1 interacts with ZmPEBP15 to modulate chalcone synthase activity, thereby stabilizing the allocation of precursors between lignin and anthocyanin pathways. To evaluate the practical implications of our findings, we introduced the bm6 mutation into Zhengdan958 and Xianyu335. In vitro rumen digestion assays confirmed that the introduction of the bm6 mutation significantly improved silage digestibility. This discovery not only holds great potential for enhancing silage digestibility but also provides a broader strategy for optimizing maize production to better meet the increasing demands of both the food and livestock feed.

Hybrid plants are found extensively in the wild, and they often demonstrate superior performance of complex traits over their parents and other selfing plants. This phenomenon, known as heterosis, has been extensively applied in plant breeding for decades. However, the process of decoding hybrid plant genomes has seriously lagged due to the challenges associated with genome assembly and the lack of appropriate methodologies for their subsequent representation and analysis. Here, we present the assembly and analysis of 2 hybrids, an intraspecific hybrid between 2 maize (Zea mays ssp. mays) inbred lines and an interspecific hybrid between maize and its wild relative teosinte (Z. mays ssp. parviglumis), utilizing a combination of PacBio High Fidelity sequencing and chromatin conformation capture sequencing data. The haplotypic assemblies are well phased at chromosomal scale, successfully resolving the complex loci with extensive parental structural variations (SVs). By integrating into a biparental genome graph, the haplotypic assemblies can facilitate downstream short-read–based SV calling and allele-specific gene expression analysis, demonstrating outstanding advantages over a single linear genome. Our work offers a comprehensive workflow that aims to facilitate the decoding of numerous hybrid plant genomes, particularly those with unknown or inaccessible parentage, thereby enhancing our understanding of genome evolution and heterosis.

Heterosis, or hybrid vigor, is a key genetic and agricultural phenomenon driven by non-additive effects, though its mechanisms remain debated. To examine these effects at the transcriptomic level, we integrate population-scale transcriptomic and phenotypic data in maize. We find that ∼30% of expressed seedling genes in many hybrids deviate from mid-parent values, consistent with incomplete dominance. While overall expression is largely controlled by cis-eQTLs, non-additive expression is disproportionately regulated by trans-eQTLs. These trans-eQTLs cluster into hotspots that coordinately control hundreds of genes, often within co-expression networks and enriched for transcription factors. We validate one hotspot involving ZmR1, a bHLH transcription factor, showing its role in regulating widespread incomplete dominance and enhancing heterosis in seedling and agronomic traits. These findings support a model in which trans-acting factors shape expression dominance and contribute to heterosis, providing a framework for crop improvement.