Functional annotation and meta-analysis of maize transcriptomes reveal genes involved in biotic and abiotic stress.

BACKGROUND: Environmental stress factors, such as biotic and abiotic stress, are becoming more common due to climate variability, significantly affecting global maize yield. Transcriptome profiling studies provide insights into the molecular mechanisms underlying stress response in maize, though the functions of many genes are still unknown. To enhance the functional annotation of maize-specific genes, MaizeGDB has outlined a data-driven approach with an emphasis on identifying genes and traits related to biotic and abiotic stress. RESULTS: We mapped high-quality RNA-Seq expression reads from 24 different publicly available datasets (17 abiotic and seven biotic studies) generated from the B73 cultivar to the recent version of the reference genome B73 (B73v5) and deduced stress-related functional annotation of maize gene models. We conducted a robust meta-analysis of the transcriptome profiles from the datasets to identify maize loci responsive to stress, identifying 3,230 differentially expressed genes (DEGs): 2,555 DEGs regulated in response to abiotic stress, 408 DEGs regulated during biotic stress, and 267 common DEGs (co-DEGs) that overlap between abiotic and biotic stress. We discovered hub genes from network analyses, and among the hub genes of the co-DEGs we identified a putative NAC domain transcription factor superfamily protein (Zm00001eb369060) IDP275, which previously responded to herbivory and drought stress. IDP275 was up-regulated in our analysis in response to eight different abiotic and four different biotic stresses. A gene set enrichment and pathway analysis of hub genes of the co-DEGs revealed hormone-mediated signaling processes and phenylpropanoid biosynthesis pathways, respectively. Using phylostratigraphic analysis, we also demonstrated how abiotic and biotic stress genes differentially evolve to adapt to changing environments. CONCLUSIONS: These results will help facilitate the functional annotation of multiple stress response gene models and annotation in maize. Data can be accessed and downloaded at the Maize Genetics and Genomics Database (MaizeGDB).

Enhanced pan-genomic resources at the maize genetics and genomics database.

Pan-genomes, encompassing the entirety of genetic sequences found in a collection of genomes within a clade, are more useful than single reference genomes for studying species diversity. This is especially true for a species like Zea mays, which has a particularly diverse and complex genome. Presenting pan-genome data, analyses, and visualization is challenging, especially for a diverse species, but more so when pan-genomic data is linked to extensive gene model and gene data, including classical gene information, markers, insertions, expression and proteomic data, and protein structures as is the case at MaizeGDB. Here, we describe MaizeGDB's expansion to include the genic subset of the Zea pan-genome in a pan-gene data center featuring the maize genomes hosted at MaizeGDB, and the outgroup teosinte Zea genomes from the Pan-Andropoganeae project. The new data center offers a variety of browsing and visualization tools, including sequence alignment visualization, gene trees and other tools, to explore pan-genes in Zea that were calculated by the pipeline Pandagma. Combined, these data will help maize researchers study the complexity and diversity of Zea, and to use the comparative functions to validate pan-gene relationships for a selected gene model.

PanEffect: a pan-genome visualization tool for variant effects in maize.

SUMMARY: Understanding the effects of genetic variants is crucial for accurately predicting traits and functional outcomes. Recent approaches have utilized artificial intelligence and protein language models to score all possible missense variant effects at the proteome level for a single genome, but a reliable tool is needed to explore these effects at the pan-genome level. To address this gap, we introduce a new tool called PanEffect. We implemented PanEffect at MaizeGDB to enable a comprehensive examination of the potential effects of coding variants across 50 maize genomes. The tool allows users to visualize over 550 million possible amino acid substitutions in the B73 maize reference genome and to observe the effects of the 2.3 million natural variations in the maize pan-genome. Each variant effect score, calculated from the Evolutionary Scale Modeling (ESM) protein language model, shows the log-likelihood ratio difference between B73 and all variants in the pan-genome. These scores are shown using heatmaps spanning benign outcomes to potential functional consequences. In addition, PanEffect displays secondary structures and functional domains along with the variant effects, offering additional functional and structural context. Using PanEffect, researchers now have a platform to explore protein variants and identify genetic targets for crop enhancement. AVAILABILITY AND IMPLEMENTATION: The PanEffect code is freely available on GitHub (https://github.com/Maize-Genetics-and-Genomics-Database/PanEffect). A maize implementation of PanEffect and underlying datasets are available at MaizeGDB (https://www.maizegdb.org/effect/maize/).

Maize protein structure resources at the maize genetics and genomics database.

Protein structures play an important role in bioinformatics, such as in predicting gene function or validating gene model annotation. However, determining protein structure was, until now, costly and time-consuming, which resulted in a structural biology bottleneck. With the release of such programs AlphaFold and ESMFold, this bottleneck has been reduced by several orders of magnitude, permitting protein structural comparisons of entire genomes within reasonable timeframes. MaizeGDB has leveraged this technological breakthrough by offering several new tools to accelerate protein structural comparisons between maize and other plants as well as human and yeast outgroups. MaizeGDB also offers bulk downloads of these comparative protein structure data, along with predicted functional annotation information. In this way, MaizeGDB is poised to assist maize researchers in assessing functional homology, gene model annotation quality, and other information unavailable to maize scientists even a few years ago.

A pan-genomic approach to genome databases using maize as a model system.

Research in the past decade has demonstrated that a single reference genome is not representative of a species' diversity. MaizeGDB introduces a pan-genomic approach to hosting genomic data, leveraging the large number of diverse maize genomes and their associated datasets to quickly and efficiently connect genomes, gene models, expression, epigenome, sequence variation, structural variation, transposable elements, and diversity data across genomes so that researchers can easily track the structural and functional differences of a locus and its orthologs across maize. We believe our framework is unique and provides a template for any genomic database poised to host large-scale pan-genomic data.

MaizeMine: A Data Mining Warehouse for the Maize Genetics and Genomics Database.

MaizeMine is the data mining resource of the Maize Genetics and Genome Database (MaizeGDB; http://maizemine.maizegdb.org). It enables researchers to create and export customized annotation datasets that can be merged with their own research data for use in downstream analyses. MaizeMine uses the InterMine data warehousing system to integrate genomic sequences and gene annotations from the Zea mays B73 RefGen_v3 and B73 RefGen_v4 genome assemblies, Gene Ontology annotations, single nucleotide polymorphisms, protein annotations, homologs, pathways, and precomputed gene expression levels based on RNA-seq data from the Z. mays B73 Gene Expression Atlas. MaizeMine also provides database cross references between genes of alternative gene sets from Gramene and NCBI RefSeq. MaizeMine includes several search tools, including a keyword search, built-in template queries with intuitive search menus, and a QueryBuilder tool for creating custom queries. The Genomic Regions search tool executes queries based on lists of genome coordinates, and supports both the B73 RefGen_v3 and B73 RefGen_v4 assemblies. The List tool allows you to upload identifiers to create custom lists, perform set operations such as unions and intersections, and execute template queries with lists. When used with gene identifiers, the List tool automatically provides gene set enrichment for Gene Ontology (GO) and pathways, with a choice of statistical parameters and background gene sets. With the ability to save query outputs as lists that can be input to new queries, MaizeMine provides limitless possibilities for data integration and meta-analysis.

BonnMu: A Sequence-Indexed Resource of Transposon-Induced Maize Mutations for Functional Genomics Studies.

Sequence-indexed insertional libraries in maize (Zea mays) are fundamental resources for functional genetics studies. Here, we constructed a Mutator (Mu) insertional library in the B73 inbred background designated BonnMu A total of 1,152 Mu-tagged F2-families were sequenced using the Mu-seq approach. We detected 225,936 genomic Mu insertion sites and 41,086 high quality germinal Mu insertions covering 16,392 of the annotated maize genes (37% of the B73v4 genome). On average, each F2-family of the BonnMu libraries captured 37 germinal Mu insertions in genes of the Filtered Gene Set (FGS). All BonnMu insertions and phenotypic seedling photographs of Mu-tagged F2-families can be accessed via MaizeGDB.org Downstream examination of 137,410 somatic and germinal insertion sites revealed that 50% of the tagged genes have a single hotspot, targeted by Mu By comparing our BonnMu (B73) data to the UniformMu (W22) library, we identified conserved insertion hotspots between different genetic backgrounds. Finally, the vast majority of BonnMu and UniformMu transposons was inserted near the transcription start site of genes. Remarkably, 75% of all BonnMu insertions were in closer proximity to the transcription start site (distance: 542 bp) than to the start codon (distance: 704 bp), which corresponds to open chromatin, especially in the 5' region of genes. Our European sequence-indexed library of Mu insertions provides an important resource for functional genetics studies of maize.

MaizeDIG: Maize Database of Images and Genomes.

Background: An organism can be described by its observable features (phenotypes) and the genes and genomic information (genotypes) that cause these phenotypes. For many decades, researchers have tried to find relationships between genotypes and phenotypes, and great strides have been made. However, improved methods and tools for discovering and visualizing these phenotypic relationships are still needed. The maize genetics and genomics database (MaizeGDB, www.maizegdb.org) provides an array of useful resources for diverse data types including thousands of images related to mutant phenotypes in Zea mays ssp. mays (maize). To integrate mutant phenotype images with genomics information, we implemented and enhanced the web-based software package BioDIG (Biological Database of Images and Genomes). Findings: We developed a genotype-phenotype database for maize called MaizeDIG. MaizeDIG has several enhancements over the original BioDIG package. MaizeDIG, which supports multiple reference genome assemblies, is seamlessly integrated with genome browsers to accommodate custom tracks showing tagged mutant phenotypes images in their genomic context and allows for custom tagging of images to highlight the phenotype. This is accomplished through an updated interface allowing users to create image-to-gene links and is accessible via the image search tool. Conclusions: We have created a user-friendly and extensible web-based resource called MaizeDIG. MaizeDIG is preloaded with 2,396 images that are available on genome browsers for 10 different maize reference genomes. Approximately 90 images of classically defined maize genes have been manually annotated. MaizeDIG is available at http://maizedig.maizegdb.org/. The code is free and open source and can be found at https://github.com/Maize-Genetics-and-Genomics-Database/maizedig.

MaizeGDB 2018: the maize multi-genome genetics and genomics database.

Since its 2015 update, MaizeGDB, the Maize Genetics and Genomics database, has expanded to support the sequenced genomes of many maize inbred lines in addition to the B73 reference genome assembly. Curation and development efforts have targeted high quality datasets and tools to support maize trait analysis, germplasm analysis, genetic studies, and breeding. MaizeGDB hosts a wide range of data including recent support of new data types including genome metadata, RNA-seq, proteomics, synteny, and large-scale diversity. To improve access and visualization of data types several new tools have been implemented to: access large-scale maize diversity data (SNPversity), download and compare gene expression data (qTeller), visualize pedigree data (Pedigree Viewer), link genes with phenotype images (MaizeDIG), and enable flexible user-specified queries to the MaizeGDB database (MaizeMine). MaizeGDB also continues to be the community hub for maize research, coordinating activities and providing technical support to the maize research community. Here we report the changes MaizeGDB has made within the last three years to keep pace with recent software and research advances, as well as the pan-genomic landscape that cheaper and better sequencing technologies have made possible. MaizeGDB is accessible online at https://www.maizegdb.org.

Maize Genomes to Fields: 2014 and 2015 field season genotype, phenotype, environment, and inbred ear image datasets.

OBJECTIVES: Crop improvement relies on analysis of phenotypic, genotypic, and environmental data. Given large, well-integrated, multi-year datasets, diverse queries can be made: Which lines perform best in hot, dry environments? Which alleles of specific genes are required for optimal performance in each environment? Such datasets also can be leveraged to predict cultivar performance, even in uncharacterized environments. The maize Genomes to Fields (G2F) Initiative is a multi-institutional organization of scientists working to generate and analyze such datasets from existing, publicly available inbred lines and hybrids. G2F's genotype by environment project has released 2014 and 2015 datasets to the public, with 2016 and 2017 collected and soon to be made available. DATA DESCRIPTION: Datasets include DNA sequences; traditional phenotype descriptions, as well as detailed ear, cob, and kernel phenotypes quantified by image analysis; weather station measurements; and soil characterizations by site. Data are released as comma separated value spreadsheets accompanied by extensive README text descriptions. For genotypic and phenotypic data, both raw data and a version with outliers removed are reported. For weather data, two versions are reported: a full dataset calibrated against nearby National Weather Service sites and a second calibrated set with outliers and apparent artifacts removed.

Surveying the Maize community for their diversity and pedigree visualization needs to prioritize tool development and curation.

The Maize Genetics and Genomics Database (MaizeGDB) team prepared a survey to identify breeders' needs for visualizing pedigrees, diversity data and haplotypes in order to prioritize tool development and curation efforts at MaizeGDB. The survey was distributed to the maize research community on behalf of the Maize Genetics Executive Committee in Summer 2015. The survey garnered 48 responses from maize researchers, of which more than half were self-identified as breeders. The survey showed that the maize researchers considered their top priorities for visualization as: (i) displaying single nucleotide polymorphisms in a given region for a given list of lines, (ii) showing haplotypes for a given list of lines and (iii) presenting pedigree relationships visually. The survey also asked which populations would be most useful to display. The following two populations were on top of the list: (i) 3000 publicly available maize inbred lines used in Romay et al. (Comprehensive genotyping of the USA national maize inbred seed bank. Genome Biol, 2013;14:R55) and (ii) maize lines with expired Plant Variety Protection Act (ex-PVP) certificates. Driven by this strong stakeholder input, MaizeGDB staff are currently working in four areas to improve its interface and web-based tools: (i) presenting immediate progenies of currently available stocks at the MaizeGDB Stock pages, (ii) displaying the most recent ex-PVP lines described in the Germplasm Resources Information Network (GRIN) on the MaizeGDB Stock pages, (iii) developing network views of pedigree relationships and (iv) visualizing genotypes from SNP-based diversity datasets. These survey results can help other biological databases to direct their efforts according to user preferences as they serve similar types of data sets for their communities. Database URL: https://www.maizegdb.org.

MaizeGDB update: new tools, data and interface for the maize model organism database.

MaizeGDB is a highly curated, community-oriented database and informatics service to researchers focused on the crop plant and model organism Zea mays ssp. mays. Although some form of the maize community database has existed over the last 25 years, there have only been two major releases. In 1991, the original maize genetics database MaizeDB was created. In 2003, the combined contents of MaizeDB and the sequence data from ZmDB were made accessible as a single resource named MaizeGDB. Over the next decade, MaizeGDB became more sequence driven while still maintaining traditional maize genetics datasets. This enabled the project to meet the continued growing and evolving needs of the maize research community, yet the interface and underlying infrastructure remained unchanged. In 2015, the MaizeGDB team completed a multi-year effort to update the MaizeGDB resource by reorganizing existing data, upgrading hardware and infrastructure, creating new tools, incorporating new data types (including diversity data, expression data, gene models, and metabolic pathways), and developing and deploying a modern interface. In addition to coordinating a data resource, the MaizeGDB team coordinates activities and provides technical support to the maize research community. MaizeGDB is accessible online at http://www.maizegdb.org.

MaizeGDB: curation and outreach go hand-in-hand.

First released in 1991 with the name MaizeDB, the Maize Genetics and Genomics Database, now MaizeGDB, celebrates its 20th anniversary this year. MaizeGDB has transitioned from a focus on comprehensive curation of the literature, genetic maps and stocks to a paradigm that accommodates the recent release of a reference maize genome sequence, multiple diverse maize genomes and sequence-based gene expression data sets. The MaizeGDB Team is relatively small, and relies heavily on the research community to provide data, nomenclature standards and most importantly, to recommend future directions, priorities and strategies. Key aspects of MaizeGDB's intimate interaction with the community are the co-location of curators with maize research groups in multiple locations across the USA as well as coordination with MaizeGDB's close partner, the Maize Genetics Cooperation--Stock Center. In this report, we describe how the MaizeGDB Team currently interacts with the maize research community and our plan for future interactions that will support updates to the functional and structural annotation of the B73 reference genome.

The MaizeGDB Genome Browser tutorial: one example of database outreach to biologists via video.

Video tutorials are an effective way for researchers to quickly learn how to use online tools offered by biological databases. At MaizeGDB, we have developed a number of video tutorials that demonstrate how to use various tools and explicitly outline the caveats researchers should know to interpret the information available to them. One such popular video currently available is 'Using the MaizeGDB Genome Browser', which describes how the maize genome was sequenced and assembled as well as how the sequence can be visualized and interacted with via the MaizeGDB Genome Browser. Database

Identification of epigenetic regulators of a transcriptionally silenced transgene in maize.

Transcriptional gene silencing is a gene regulatory mechanism essential to all organisms. Many transcriptional regulatory mechanisms are associated with epigenetic modifications such as changes in chromatin structure, acetylation and methylation of core histone proteins, and DNA methylation within regulatory regions of endogenous genes and transgenes. Although several maize mutants have been identified from prior forward genetic screens for epigenetic transcriptional silencing, these screens have been far from saturated. Herein, the transcriptionally silent b1 genomic transgene (BTG-silent), a stable, epigenetically silenced transgene in Zea mays (maize), is demonstrated to be an effective phenotype for a forward genetic screen. When the transgene is reactivated, a dark purple plant phenotype is evident because the B1 transcription factor activates anthocyanin biosynthesis, making loss of silencing mutants easy to identify. Using BTG-silent, ten new putative mutants were identified and named transgene reactivated1 through 11 (tgr1-6 and tgr8-11). Three of these mutants have been examined in more detail, and molecular and genetic assays demonstrated that these mutants have both distinct and overlapping phenotypes with previously identified maize mutants that relieve epigenetic transcriptional silencing. Linkage analysis suggests that tgr2 and tgr3 do not correspond to a mutation at previously identified maize loci resulting from other forward genetic screens, while tgr1 shows linkage to a characterized gene. These results suggest that the mutants are a valuable resource for future studies because some of the mutants are likely to reveal genes that encode products required for epigenetic gene regulation in maize but are not currently represented by sequenced mutations.

POPcorn: An Online Resource Providing Access to Distributed and Diverse Maize Project Data.

The purpose of the online resource presented here, POPcorn (Project Portal for corn), is to enhance accessibility of maize genetic and genomic resources for plant biologists. Currently, many online locations are difficult to find, some are best searched independently, and individual project websites often degrade over time-sometimes disappearing entirely. The POPcorn site makes available (1) a centralized, web-accessible resource to search and browse descriptions of ongoing maize genomics projects, (2) a single, stand-alone tool that uses web Services and minimal data warehousing to search for sequence matches in online resources of diverse offsite projects, and (3) a set of tools that enables researchers to migrate their data to the long-term model organism database for maize genetic and genomic information: MaizeGDB. Examples demonstrating POPcorn's utility are provided herein.

Gene expression analyses in maize inbreds and hybrids with varying levels of heterosis.

BACKGROUND: Heterosis is the superior performance of F1 hybrid progeny relative to the parental phenotypes. Maize exhibits heterosis for a wide range of traits, however the magnitude of heterosis is highly variable depending on the choice of parents and the trait(s) measured. We have used expression profiling to determine whether the level, or types, of non-additive gene expression vary in maize hybrids with different levels of genetic diversity or heterosis. RESULTS: We observed that the distributions of better parent heterosis among a series of 25 maize hybrids generally do not exhibit significant correlations between different traits. Expression profiling analyses for six of these hybrids, chosen to represent diversity in genotypes and heterosis responses, revealed a correlation between genetic diversity and transcriptional variation. The majority of differentially expressed genes in each of the six different hybrids exhibited additive expression patterns, and approximately 25% exhibited statistically significant non-additive expression profiles. Among the non-additive profiles, approximately 80% exhibited hybrid expression levels between the parental levels, approximately 20% exhibited hybrid expression levels at the parental levels and ~1% exhibited hybrid levels outside the parental range. CONCLUSION: We have found that maize inbred genetic diversity is correlated with transcriptional variation. However, sampling of seedling tissues indicated that the frequencies of additive and non-additive expression patterns are very similar across a range of hybrid lines. These findings suggest that heterosis is probably not a consequence of higher levels of additive or non-additive expression, but may be related to transcriptional variation between parents. The lack of correlation between better parent heterosis levels for different traits suggests that transcriptional diversity at specific sets of genes may influence heterosis for different traits.

A BAC pooling strategy combined with PCR-based screenings in a large, highly repetitive genome enables integration of the maize genetic and physical maps.

BACKGROUND: Molecular markers serve three important functions in physical map assembly. First, they provide anchor points to genetic maps facilitating functional genomic studies. Second, they reduce the overlap required for BAC contig assembly from 80 to 50 percent. Finally, they validate assemblies based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy in combination with a high-throughput PCR-based screening method to anchor the maize genetic and physical maps. RESULTS: A total of 110,592 maize BAC clones (approximately 6x haploid genome equivalents) were pooled into six different matrices, each containing 48 pools of BAC DNA. The quality of the BAC DNA pools and their utility for identifying BACs containing target genomic sequences was tested using 254 PCR-based STS markers. Five types of PCR-based STS markers were screened to assess potential uses for the BAC pools. An average of 4.68 BAC clones were identified per marker analyzed. These results were integrated with BAC fingerprint data generated by the Arizona Genomics Institute (AGI) and the Arizona Genomics Computational Laboratory (AGCoL) to assemble the BAC contigs using the FingerPrinted Contigs (FPC) software and contribute to the construction and anchoring of the physical map. A total of 234 markers (92.5%) anchored BAC contigs to their genetic map positions. The results can be viewed on the integrated map of maize 12. CONCLUSION: This BAC pooling strategy is a rapid, cost effective method for genome assembly and anchoring. The requirement for six replicate positive amplifications makes this a robust method for use in large genomes with high amounts of repetitive DNA such as maize. This strategy can be used to physically map duplicate loci, provide order information for loci in a small genetic interval or with no genetic recombination, and loci with conflicting hybridization-based information.

Characterization of three maize bacterial artificial chromosome libraries toward anchoring of the physical map to the genetic map using high-density bacterial artificial chromosome filter hybridization.

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the 185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage lambda. The results indicate that the libraries are of high quality with low contamination by organellar and lambda-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6x coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 x Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 +/- 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction.