Gene disruption by structural mutations drives selection in US rice breeding over the last century.

The genetic basis of general plant vigor is of major interest to food producers, yet the trait is recalcitrant to genetic mapping because of the number of loci involved, their small effects, and linkage. Observations of heterosis in many crops suggests that recessive, malfunctioning versions of genes are a major cause of poor performance, yet we have little information on the mutational spectrum underlying these disruptions. To address this question, we generated a long-read assembly of a tropical japonica rice (Oryza sativa) variety, Carolina Gold, which allowed us to identify structural mutations (>50 bp) and orient them with respect to their ancestral state using the outgroup, Oryza glaberrima. Supporting prior work, we find substantial genome expansion in the sativa branch. While transposable elements (TEs) account for the largest share of size variation, the majority of events are not directly TE-mediated. Tandem duplications are the most common source of insertions and are highly enriched among 50-200bp mutations. To explore the relative impact of various mutational classes on crop fitness, we then track these structural events over the last century of US rice improvement using 101 resequenced varieties. Within this material, a pattern of temporary hybridization between medium and long-grain varieties was followed by recent divergence. During this long-term selection, structural mutations that impact gene exons have been removed at a greater rate than intronic indels and single-nucleotide mutations. These results support the use of ab initio estimates of mutational burden, based on structural data, as an orthogonal predictor in genomic selection.

Improved maize reference genome with single-molecule technologies.

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.

Gramene 2018: unifying comparative genomics and pathway resources for plant research.

Gramene (http://www.gramene.org) is a knowledgebase for comparative functional analysis in major crops and model plant species. The current release, #54, includes over 1.7 million genes from 44 reference genomes, most of which were organized into 62,367 gene families through orthologous and paralogous gene classification, whole-genome alignments, and synteny. Additional gene annotations include ontology-based protein structure and function; genetic, epigenetic, and phenotypic diversity; and pathway associations. Gramene's Plant Reactome provides a knowledgebase of cellular-level plant pathway networks. Specifically, it uses curated rice reference pathways to derive pathway projections for an additional 66 species based on gene orthology, and facilitates display of gene expression, gene-gene interactions, and user-defined omics data in the context of these pathways. As a community portal, Gramene integrates best-of-class software and infrastructure components including the Ensembl genome browser, Reactome pathway browser, and Expression Atlas widgets, and undergoes periodic data and software upgrades. Via powerful, intuitive search interfaces, users can easily query across various portals and interactively analyze search results by clicking on diverse features such as genomic context, highly augmented gene trees, gene expression anatomograms, associated pathways, and external informatics resources. All data in Gramene are accessible through both visual and programmatic interfaces.

Automated update, revision, and quality control of the maize genome annotations using MAKER-P improves the B73 RefGen_v3 gene models and identifies new genes.

The large size and relative complexity of many plant genomes make creation, quality control, and dissemination of high-quality gene structure annotations challenging. In response, we have developed MAKER-P, a fast and easy-to-use genome annotation engine for plants. Here, we report the use of MAKER-P to update and revise the maize (Zea mays) B73 RefGen_v3 annotation build (5b+) in less than 3 h using the iPlant Cyberinfrastructure. MAKER-P identified and annotated 4,466 additional, well-supported protein-coding genes not present in the 5b+ annotation build, added additional untranslated regions to 1,393 5b+ gene models, identified 2,647 5b+ gene models that lack any supporting evidence (despite the use of large and diverse evidence data sets), identified 104,215 pseudogene fragments, and created an additional 2,522 noncoding gene annotations. We also describe a method for de novo training of MAKER-P for the annotation of newly sequenced grass genomes. Collectively, these results lead to the 6a maize genome annotation and demonstrate the utility of MAKER-P for rapid annotation, management, and quality control of grasses and other difficult-to-annotate plant genomes.

A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor.

The current limitations in genome sequencing technology require the construction of physical maps for high-quality draft sequences of large plant genomes, such as that of Aegilops tauschii, the wheat D-genome progenitor. To construct a physical map of the Ae. tauschii genome, we fingerprinted 461,706 bacterial artificial chromosome clones, assembled contigs, designed a 10K Ae. tauschii Infinium SNP array, constructed a 7,185-marker genetic map, and anchored on the map contigs totaling 4.03 Gb. Using whole genome shotgun reads, we extended the SNP marker sequences and found 17,093 genes and gene fragments. We showed that collinearity of the Ae. tauschii genes with Brachypodium distachyon, rice, and sorghum decreased with phylogenetic distance and that structural genome evolution rates have been high across all investigated lineages in subfamily Pooideae, including that of Brachypodieae. We obtained additional information about the evolution of the seven Triticeae chromosomes from 12 ancestral chromosomes and uncovered a pattern of centromere inactivation accompanying nested chromosome insertions in grasses. We showed that the density of noncollinear genes along the Ae. tauschii chromosomes positively correlates with recombination rates, suggested a cause, and showed that new genes, exemplified by disease resistance genes, are preferentially located in high-recombination chromosome regions.

MAKER-P: a tool kit for the rapid creation, management, and quality control of plant genome annotations.

We have optimized and extended the widely used annotation engine MAKER in order to better support plant genome annotation efforts. New features include better parallelization for large repeat-rich plant genomes, noncoding RNA annotation capabilities, and support for pseudogene identification. We have benchmarked the resulting software tool kit, MAKER-P, using the Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) genomes. Here, we demonstrate the ability of the MAKER-P tool kit to automatically update, extend, and revise the Arabidopsis annotations in light of newly available data and to annotate pseudogenes and noncoding RNAs absent from The Arabidopsis Informatics Resource 10 build. Our results demonstrate that MAKER-P can be used to manage and improve the annotations of even Arabidopsis, perhaps the best-annotated plant genome. We have also installed and benchmarked MAKER-P on the Texas Advanced Computing Center. We show that this public resource can de novo annotate the entire Arabidopsis and maize genomes in less than 3 h and produce annotations of comparable quality to those of the current The Arabidopsis Information Resource 10 and maize V2 annotation builds.

Disentangling methodological and biological sources of gene tree discordance on Oryza (Poaceae) chromosome 3.

We describe new methods for characterizing gene tree discordance in phylogenomic data sets, which screen for deviations from neutral expectations, summarize variation in statistical support among gene trees, and allow comparison of the patterns of discordance induced by various analysis choices. Using an exceptionally complete set of genome sequences for the short arm of chromosome 3 in Oryza (rice) species, we applied these methods to identify the causes and consequences of differing patterns of discordance in the sets of gene trees inferred using a panel of 20 distinct analysis pipelines. We found that discordance patterns were strongly affected by aspects of data selection, alignment, and alignment masking. Unusual patterns of discordance evident when using certain pipelines were reduced or eliminated by using alternative pipelines, suggesting that they were the product of methodological biases rather than evolutionary processes. In some cases, once such biases were eliminated, evolutionary processes such as introgression could be implicated. Additionally, patterns of gene tree discordance had significant downstream impacts on species tree inference. For example, inference from supermatrices was positively misleading when pipelines that led to biased gene trees were used. Several results may generalize to other data sets: we found that gene tree and species tree inference gave more reasonable results when intron sequence was included during sequence alignment and tree inference, the alignment software PRANK was used, and detectable "block-shift" alignment artifacts were removed. We discuss our findings in the context of well-established relationships in Oryza and continuing controversies regarding the domestication history of O. sativa.

Pervasive gene content variation and copy number variation in maize and its undomesticated progenitor.

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.

Gramene database in 2010: updates and extensions.

Now in its 10th year, the Gramene database (http://www.gramene.org) has grown from its primary focus on rice, the first fully-sequenced grass genome, to become a resource for major model and crop plants including Arabidopsis, Brachypodium, maize, sorghum, poplar and grape in addition to several species of rice. Gramene began with the addition of an Ensembl genome browser and has expanded in the last decade to become a robust resource for plant genomics hosting a wide array of data sets including quantitative trait loci (QTL), metabolic pathways, genetic diversity, genes, proteins, germplasm, literature, ontologies and a fully-structured markers and sequences database integrated with genome browsers and maps from various published studies (genetic, physical, bin, etc.). In addition, Gramene now hosts a variety of web services including a Distributed Annotation Server (DAS), BLAST and a public MySQL database. Twice a year, Gramene releases a major build of the database and makes interim releases to correct errors or to make important updates to software and/or data.

A genome-wide characterization of microRNA genes in maize.

MicroRNAs (miRNAs) are small, non-coding RNAs that play essential roles in plant growth, development, and stress response. We conducted a genome-wide survey of maize miRNA genes, characterizing their structure, expression, and evolution. Computational approaches based on homology and secondary structure modeling identified 150 high-confidence genes within 26 miRNA families. For 25 families, expression was verified by deep-sequencing of small RNA libraries that were prepared from an assortment of maize tissues. PCR-RACE amplification of 68 miRNA transcript precursors, representing 18 families conserved across several plant species, showed that splice variation and the use of alternative transcriptional start and stop sites is common within this class of genes. Comparison of sequence variation data from diverse maize inbred lines versus teosinte accessions suggest that the mature miRNAs are under strong purifying selection while the flanking sequences evolve equivalently to other genes. Since maize is derived from an ancient tetraploid, the effect of whole-genome duplication on miRNA evolution was examined. We found that, like protein-coding genes, duplicated miRNA genes underwent extensive gene-loss, with approximately 35% of ancestral sites retained as duplicate homoeologous miRNA genes. This number is higher than that observed with protein-coding genes. A search for putative miRNA targets indicated bias towards genes in regulatory and metabolic pathways. As maize is one of the principal models for plant growth and development, this study will serve as a foundation for future research into the functional roles of miRNA genes.

Detailed analysis of a contiguous 22-Mb region of the maize genome.

Most of our understanding of plant genome structure and evolution has come from the careful annotation of small (e.g., 100 kb) sequenced genomic regions or from automated annotation of complete genome sequences. Here, we sequenced and carefully annotated a contiguous 22 Mb region of maize chromosome 4 using an improved pseudomolecule for annotation. The sequence segment was comprehensively ordered, oriented, and confirmed using the maize optical map. Nearly 84% of the sequence is composed of transposable elements (TEs) that are mostly nested within each other, of which most families are low-copy. We identified 544 gene models using multiple levels of evidence, as well as five miRNA genes. Gene fragments, many captured by TEs, are prevalent within this region. Elimination of gene redundancy from a tetraploid maize ancestor that originated a few million years ago is responsible in this region for most disruptions of synteny with sorghum and rice. Consistent with other sub-genomic analyses in maize, small RNA mapping showed that many small RNAs match TEs and that most TEs match small RNAs. These results, performed on approximately 1% of the maize genome, demonstrate the feasibility of refining the B73 RefGen_v1 genome assembly by incorporating optical map, high-resolution genetic map, and comparative genomic data sets. Such improvements, along with those of gene and repeat annotation, will serve to promote future functional genomic and phylogenomic research in maize and other grasses.

Effect of sequence depth and length in long-read assembly of the maize inbred NC358.

Improvements in long-read data and scaffolding technologies have enabled rapid generation of reference-quality assemblies for complex genomes. Still, an assessment of critical sequence depth and read length is important for allocating limited resources. To this end, we have generated eight assemblies for the complex genome of the maize inbred line NC358 using PacBio datasets ranging from 20 to 75 × genomic depth and with N50 subread lengths of 11-21 kb. Assemblies with ≤30 × depth and N50 subread length of 11 kb are highly fragmented, with even low-copy genic regions showing degradation at 20 × depth. Distinct sequence-quality thresholds are observed for complete assembly of genes, transposable elements, and highly repetitive genomic features such as telomeres, heterochromatic knobs, and centromeres. In addition, we show high-quality optical maps can dramatically improve contiguity in even our most fragmented base assembly. This study provides a useful resource allocation reference to the community as long-read technologies continue to mature.

A new method to compute K-mer frequencies and its application to annotate large repetitive plant genomes.

BACKGROUND: The challenges of accurate gene prediction and enumeration are further aggravated in large genomes that contain highly repetitive transposable elements (TEs). Yet TEs play a substantial role in genome evolution and are themselves an important subject of study. Repeat annotation, based on counting occurrences of k-mers, has been previously used to distinguish TEs from low-copy genic regions; but currently available software solutions are impractical due to high memory requirements or specialization for specific user-tasks. RESULTS: Here we introduce the Tallymer software, a flexible and memory-efficient collection of programs for k-mer counting and indexing of large sequence sets. Unlike previous methods, Tallymer is based on enhanced suffix arrays. This gives a much larger flexibility concerning the choice of the k-mer size. Tallymer can process large data sizes of several billion bases. We used it in a variety of applications to study the genomes of maize and other plant species. In particular, Tallymer was used to index a set of whole genome shotgun sequences from maize (B73) (total size 109 bp.). We analyzed k-mer frequencies for a wide range of k. At this low genome coverage ( approximately 0.45x) highly repetitive 20-mers constituted 44% of the genome but represented only 1% of all possible k-mers. Similar low-complexity was seen in the repeat fractions of sorghum and rice. When applying our method to other maize data sets, High-C0t derived sequences showed the greatest enrichment for low-copy sequences. Among annotated TEs, the most highly repetitive were of the Ty3/gypsy class of retrotransposons, followed by the Ty1/copia class, and DNA transposons. Among expressed sequence tags (EST), a notable fraction contained high-copy k-mers, suggesting that transposons are still active in maize. Retrotransposons in Mo17 and McC cultivars were readily detected using the B73 20-mer frequency index, indicating their conservation despite extensive rearrangement across cultivars. Among one hundred annotated bacterial artificial chromosomes (BACs), k-mer frequency could be used to detect transposon-encoded genes with 92% sensitivity, compared to 96% using alignment-based repeat masking, while both methods showed 92% specificity. CONCLUSION: The Tallymer software was effective in a variety of applications to aid genome annotation in maize, despite limitations imposed by the relatively low coverage of sequence available. For more information on the software, see http://www.zbh.uni-hamburg.de/Tallymer.

Improved RNA-seq Workflows Using CyVerse Cyberinfrastructure.

RNA-seq is a vital method for understanding gene structure and expression patterns. Typical RNA-seq analysis protocols use sequencing reads of length 50 to 150 nucleotides for alignment to the reference genome and assembly of transcripts. The resultant transcripts are quantified and used for differential expression and visualization. Existing tools and protocols for RNA-seq are vast and diverse; given their differences in performance, it is critical to select an analysis protocol that is scalable, accurate, and easy to use. Tuxedo, a popular alignment-based protocol for RNA-seq analysis, has been updated with HISAT2, StringTie, StringTie-merge, and Ballgown, and the updated protocol outperforms its predecessor. Similarly, new pseudo-alignment-based protocols like Kallisto and Sleuth reduce runtime and improve performance. However, these tools are challenging for researchers lacking command-line experience. Here, we describe two new RNA-seq analysis protocols, in which all tools are deployed on CyVerse Cyberinfrastructure with user-friendly graphical user interfaces, and validate their performance using plant RNA-seq data. © 2018 by John Wiley & Sons, Inc.

Extensive intraspecific gene order and gene structural variations between Mo17 and other maize genomes.

Maize is an important crop with a high level of genome diversity and heterosis. The genome sequence of a typical female line, B73, was previously released. Here, we report a de novo genome assembly of a corresponding male representative line, Mo17. More than 96.4% of the 2,183 Mb assembled genome can be accounted for by 362 scaffolds in ten pseudochromosomes with 38,620 annotated protein-coding genes. Comparative analysis revealed large gene-order and gene structural variations: approximately 10% of the annotated genes were mutually nonsyntenic, and more than 20% of the predicted genes had either large-effect mutations or large structural variations, which might cause considerable protein divergence between the two inbred lines. Our study provides a high-quality reference-genome sequence of an important maize germplasm, and the intraspecific gene order and gene structural variations identified should have implications for heterosis and genome evolution.

The maize W22 genome provides a foundation for functional genomics and transposon biology.

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.

Publisher Correction: Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza.

This article was not made open access when initially published online, which was corrected before print publication. In addition, ORCID links were missing for 12 authors and have been added to the HTML and PDF versions of the article.

Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza.

The genus Oryza is a model system for the study of molecular evolution over time scales ranging from a few thousand to 15 million years. Using 13 reference genomes spanning the Oryza species tree, we show that despite few large-scale chromosomal rearrangements rapid species diversification is mirrored by lineage-specific emergence and turnover of many novel elements, including transposons, and potential new coding and noncoding genes. Our study resolves controversial areas of the Oryza phylogeny, showing a complex history of introgression among different chromosomes in the young 'AA' subclade containing the two domesticated species. This study highlights the prevalence of functionally coupled disease resistance genes and identifies many new haplotypes of potential use for future crop protection. Finally, this study marks a milestone in modern rice research with the release of a complete long-read assembly of IR 8 'Miracle Rice', which relieved famine and drove the Green Revolution in Asia 50 years ago.

Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing.

Zea mays is an important genetic model for elucidating transcriptional networks. Uncertainties about the complete structure of mRNA transcripts limit the progress of research in this system. Here, using single-molecule sequencing technology, we produce 111,151 transcripts from 6 tissues capturing ∼70% of the genes annotated in maize RefGen_v3 genome. A large proportion of transcripts (57%) represent novel, sometimes tissue-specific, isoforms of known genes and 3% correspond to novel gene loci. In other cases, the identified transcripts have improved existing gene models. Averaging across all six tissues, 90% of the splice junctions are supported by short reads from matched tissues. In addition, we identified a large number of novel long non-coding RNAs and fusion transcripts and found that DNA methylation plays an important role in generating various isoforms. Our results show that characterization of the maize B73 transcriptome is far from complete, and that maize gene expression is more complex than previously thought.

Whole genome de novo assemblies of three divergent strains of rice, Oryza sativa, document novel gene space of aus and indica.

BACKGROUND: The use of high throughput genome-sequencing technologies has uncovered a large extent of structural variation in eukaryotic genomes that makes important contributions to genomic diversity and phenotypic variation. When the genomes of different strains of a given organism are compared, whole genome resequencing data are typically aligned to an established reference sequence. However, when the reference differs in significant structural ways from the individuals under study, the analysis is often incomplete or inaccurate. RESULTS: Here, we use rice as a model to demonstrate how improvements in sequencing and assembly technology allow rapid and inexpensive de novo assembly of next generation sequence data into high-quality assemblies that can be directly compared using whole genome alignment to provide an unbiased assessment. Using this approach, we are able to accurately assess the "pan-genome" of three divergent rice varieties and document several megabases of each genome absent in the other two. CONCLUSIONS: Many of the genome-specific loci are annotated to contain genes, reflecting the potential for new biological properties that would be missed by standard reference-mapping approaches. We further provide a detailed analysis of several loci associated with agriculturally important traits, including the S5 hybrid sterility locus, the Sub1 submergence tolerance locus, the LRK gene cluster associated with improved yield, and the Pup1 cluster associated with phosphorus deficiency, illustrating the utility of our approach for biological discovery. All of the data and software are openly available to support further breeding and functional studies of rice and other species.