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The shoot apical meristem (SAM) orchestrates the balance between stem cell proliferation and organ initiation essential for postembryonic shoot growth. Meristems show a striking diversity in shape and size. How this morphological diversity relates to variation in plant architecture and the molecular circuitries driving it are unclear. By generating a high-resolution gene expression atlas of the vegetative maize shoot apex, we show here that distinct sets of genes govern the regulation and identity of stem cells in maize versus Arabidopsis. Cell identities in the maize SAM reflect the combinatorial activity of transcription factors (TFs) that drive the preferential, differential expression of individual members within gene families functioning in a plethora of cellular processes. Subfunctionalization thus emerges as a fundamental feature underlying cell identity. Moreover, we show that adult plant characters are, to a significant degree, regulated by gene circuitries acting in the SAM, with natural variation modulating agronomically important architectural traits enriched specifically near dynamically expressed SAM genes and the TFs that regulate them. Besides unique mechanisms of maize stem cell regulation, our atlas thus identifies key new targets for crop improvement.

Maize and sorghum are both important crops with similar overall plant architectures, but they have key differences, especially in regard to their inflorescences. To better understand these two organisms at the molecular level, we compared expression profiles of both protein-coding and noncoding transcripts in 11 matched tissues using single-molecule, long-read, deep RNA sequencing. This comparative analysis revealed large numbers of novel isoforms in both species. Evolutionarily young genes were likely to be generated in reproductive tissues and usually had fewer isoforms than old genes. We also observed similarities and differences in alternative splicing patterns and activities, both among tissues and between species. The maize subgenomes exhibited no bias in isoform generation; however, genes in the B genome were more highly expressed in pollen tissue, whereas genes in the A genome were more highly expressed in endosperm. We also identified a number of splicing events conserved between maize and sorghum. In addition, we generated comprehensive and high-resolution maps of poly(A) sites, revealing similarities and differences in mRNA cleavage between the two species. Overall, our results reveal considerable splicing and expression diversity between sorghum and maize, well beyond what was reported in previous studies, likely reflecting the differences in architecture between these two species.

Genetic control of branching is a primary determinant of yield, regulating seed number and harvesting ability, yet little is known about the molecular networks that shape grain-bearing inflorescences of cereal crops. Here, we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. Developmental dynamics of genes targeted in vivo by the transcription factor RAMOSA1, a key regulator of determinacy, revealed potential mechanisms for repressing branches in distinct stem cell populations, including interactions with KNOTTED1, a master regulator of stem cell maintenance. Our results uncover discrete developmental modules that function in determining grass-specific morphology and provide a basis for targeted crop improvement and translation to other cereal crops with comparable inflorescence architectures.

The maize genome, with its large complement of transposons and repeats, is a paradigm for the study of epigenetic mechanisms such as paramutation and imprinting. Here, we present the genome-wide map of cytosine methylation for two maize inbred lines, B73 and Mo17. CG (65%) and CHG (50%) methylation (where H = A, C, or T) is highest in transposons, while CHH (5%) methylation is likely guided by 24-nt, but not 21-nt, small interfering RNAs (siRNAs). Correlations with methylation patterns suggest that CG methylation in exons (8%) may deter insertion of Mutator transposon insertion, while CHG methylation at splice acceptor sites may inhibit RNA splicing. Using the methylation map as a guide, we used low-coverage sequencing to show that parental methylation differences are inherited by recombinant inbred lines. However, frequent methylation switches, guided by siRNA, persist for up to eight generations, suggesting that epigenetic inheritance resembling paramutation is much more common than previously supposed. The methylation map will provide an invaluable resource for epigenetic studies in maize.

Individuals of the same species are generally thought to have very similar genomes. However, there is growing evidence that structural variation in the form of copy number variation (CNV) and presence–absence variation (PAV) can lead to variation in the genome content of individuals within a species. Array comparative genomic hybridization (CGH) was used to compare gene content and copy number variation among 19 diverse maize inbreds and 14 genotypes of the wild ancestor of maize, teosinte. We identified 479 genes exhibiting higher copy number in some genotypes (UpCNV) and 3410 genes that have either fewer copies or are missing in the genome of at least one genotype relative to B73 (DownCNV/PAV). Many of these DownCNV/PAV are examples of genes present in B73, but missing from other genotypes. Over 70% of the CNV/PAV examples are identified in multiple genotypes, and the majority of events are observed in both maize and teosinte, suggesting that these variants predate domestication and that there is not strong selection acting against them. Many of the genes affected by CNV/PAV are either maize specific (thus possible annotation artifacts) or members of large gene families, suggesting that the gene loss can be tolerated through buffering by redundant functions encoded elsewhere in the genome. While this structural variation may not result in major qualitative variation due to genetic buffering, it may significantly contribute to quantitative variation.

Automated evidence-based gene building is a rapid and cost-effective way to provide reliable gene annotations on newly sequenced genomes. One of the limitations of evidence-based gene builders, however, is their requirement for transcriptional evidence—known proteins, full-length cDNAs, or expressed sequence tags (ESTs)—in the species of interest. This limitation is of particular concern for plant genomes, where the rate of genome sequencing is greatly outpacing the rate of EST- and cDNA-sequencing projects. To overcome this limitation, we have developed an evidence-based gene build system (the Gramene pipeline) that can use transcriptional evidence across related species. The Gramene pipeline uses the Ensembl computing infrastructure with a novel data processing scheme. Using the previously annotated plant genomes, the dicot Arabidopsis thaliana and the monocot Oryza sativa, we show that the cross-species ESTs from within monocot or dicot class are a valuable source of evidence for gene predictions. We also find that, using only EST and cross-species evidence, the Gramene pipeline can generate a plant gene set that is comparable in quality to the human genes based on known proteins and full-length cDNAs. We compare the Gramene pipeline to several widely used ab initio gene prediction programs in rice; this comparison shows the pipeline performs favorably at both the gene and exon levels with cross-species gene products only. We discuss the results of testing the pipeline on a 22-Mb region of the newly sequenced maize genome and discuss potential application of the pipeline to other genomes.

Recently there has been a great interest in the identification of microRNAs and their targets as well as understanding the spatial and temporal regulation of microRNA genes. To understand how microRNA genes evolve, we looked at several rapidly evolving families in Arabidopsis thaliana, and found that they arose from a process of genome-wide duplication, tandem duplication, and segmental duplication followed by dispersal and diversification, similar to the processes that drive the evolution of protein gene families. Using multiple expression data sets to examine the transcription patterns of different members of the microRNA families, we find the sequence diversification of duplicated microRNA genes to be accompanied by a change in spatial and temporal expression patterns, suggesting that duplicated copies acquire new functionality as they evolve.