Low-Density Reference Fingerprinting SNP Dataset of CIMMYT Maize Lines for Quality Control and Genetic Diversity Analyses.

CIMMYT maize lines (CMLs), which represent the tropical maize germplasm, are freely available worldwide. All currently released 615 CMLs and fourteen temperate maize inbred lines were genotyped with 180 kompetitive allele-specific PCR single nucleotide polymorphisms to develop a reference fingerprinting SNP dataset that can be used to perform quality control (QC) and genetic diversity analyses. The QC analysis identified 25 CMLs with purity, identity, or mislabeling issues. Further field observation, purification, and re-genotyping of these CMLs are required. The reference fingerprinting SNP dataset was developed for all of the currently released CMLs with 152 high-quality SNPs. The results of principal component analysis and average genetic distances between subgroups showed a clear genetic divergence between temperate and tropical maize, whereas the three tropical subgroups partially overlapped with one another. More than 99% of the pairs of CMLs had genetic distances greater than 0.30, showing their high genetic diversity, and most CMLs are distantly related. The heterotic patterns, estimated with the molecular markers, are consistent with those estimated using pedigree information in two major maize breeding programs at CIMMYT. These research findings are helpful for ensuring the regeneration and distribution of the true CMLs, via QC analysis, and for facilitating the effective utilization of the CMLs, globally.

Scalable Sparse Testing Genomic Selection Strategy for Early Yield Testing Stage.

To enable a scalable sparse testing genomic selection (GS) strategy at preliminary yield trials in the CIMMYT maize breeding program, optimal approaches to incorporate genotype by environment interaction (GEI) in genomic prediction models are explored. Two cross-validation schemes were evaluated: CV1, predicting the genetic merit of new bi-parental populations that have been evaluated in some environments and not others, and CV2, predicting the genetic merit of half of a bi-parental population that has been phenotyped in some environments and not others using the coefficient of determination (CDmean) to determine optimized subsets of a full-sib family to be evaluated in each environment. We report similar prediction accuracies in CV1 and CV2, however, CV2 has an intuitive appeal in that all bi-parental populations have representation across environments, allowing efficient use of information across environments. It is also ideal for building robust historical data because all individuals of a full-sib family have phenotypic data, albeit in different environments. Results show that grouping of environments according to similar growing/management conditions improved prediction accuracy and reduced computational requirements, providing a scalable, parsimonious approach to multi-environmental trials and GS in early testing stages. We further demonstrate that complementing the full-sib calibration set with optimized historical data results in improved prediction accuracy for the cross-validation schemes.

Maximizing efficiency of genomic selection in CIMMYT's tropical maize breeding program.

KEY MESSAGE: Historical data from breeding programs can be efficiently used to improve genomic selection accuracy, especially when the training set is optimized to subset individuals most informative of the target testing set. The current strategy for large-scale implementation of genomic selection (GS) at the International Maize and Wheat Improvement Center (CIMMYT) global maize breeding program has been to train models using information from full-sibs in a "test-half-predict-half approach." Although effective, this approach has limitations, as it requires large full-sib populations and limits the ability to shorten variety testing and breeding cycle times. The primary objective of this study was to identify optimal experimental and training set designs to maximize prediction accuracy of GS in CIMMYT's maize breeding programs. Training set (TS) design strategies were evaluated to determine the most efficient use of phenotypic data collected on relatives for genomic prediction (GP) using datasets containing 849 (DS1) and 1389 (DS2) DH-lines evaluated as testcrosses in 2017 and 2018, respectively. Our results show there is merit in the use of multiple bi-parental populations as TS when selected using algorithms to maximize relatedness between the training and prediction sets. In a breeding program where relevant past breeding information is not readily available, the phenotyping expenditure can be spread across connected bi-parental populations by phenotyping only a small number of lines from each population. This significantly improves prediction accuracy compared to within-population prediction, especially when the TS for within full-sib prediction is small. Finally, we demonstrate that prediction accuracy in either sparse testing or "test-half-predict-half" can further be improved by optimizing which lines are planted for phenotyping and which lines are to be only genotyped for advancement based on GP.

Diversity analysis of 80,000 wheat accessions reveals consequences and opportunities of selection footprints.

Undomesticated wild species, crop wild relatives, and landraces represent sources of variation for wheat improvement to address challenges from climate change and the growing human population. Here, we study 56,342 domesticated hexaploid, 18,946 domesticated tetraploid and 3,903 crop wild relatives in a massive-scale genotyping and diversity analysis. Using DArTseqTM technology, we identify more than 300,000 high-quality SNPs and SilicoDArT markers and align them to three reference maps: the IWGSC RefSeq v1.0 genome assembly, the durum wheat genome assembly (cv. Svevo), and the DArT genetic map. On average, 72% of the markers are uniquely placed on these maps and 50% are linked to genes. The analysis reveals landraces with unexplored diversity and genetic footprints defined by regions under selection. This provides fertile ground to develop wheat varieties of the future by exploring specific gene or chromosome regions and identifying germplasm conserving allelic diversity missing in current breeding programs.

Strategies for Effective Use of Genomic Information in Crop Breeding Programs Serving Africa and South Asia.

Much of the world's population growth will occur in regions where food insecurity is prevalent, with large increases in food demand projected in regions of Africa and South Asia. While improving food security in these regions will require a multi-faceted approach, improved performance of crop varieties in these regions will play a critical role. Current rates of genetic gain in breeding programs serving Africa and South Asia fall below rates achieved in other regions of the world. Given resource constraints, increased genetic gain in these regions cannot be achieved by simply expanding the size of breeding programs. New approaches to breeding are required. The Genomic Open-source Breeding informatics initiative (GOBii) and Excellence in Breeding Platform (EiB) are working with public sector breeding programs to build capacity, develop breeding strategies, and build breeding informatics capabilities to enable routine use of new technologies that can improve the efficiency of breeding programs and increase genetic gains. Simulations evaluating breeding strategies indicate cost-effective implementations of genomic selection (GS) are feasible using relatively small training sets, and proof-of-concept implementations have been validated in the International Maize and Wheat Improvement Center (CIMMYT) maize breeding program. Progress on GOBii, EiB, and implementation of GS in CIMMYT and International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) breeding programs are discussed, as well as strategies for routine implementation of GS in breeding programs serving Africa and South Asia.

Empirical Comparison of Tropical Maize Hybrids Selected Through Genomic and Phenotypic Selections.

Genomic selection predicts the genomic estimated breeding values (GEBVs) of individuals not previously phenotyped. Several studies have investigated the accuracy of genomic predictions in maize but there is little empirical evidence on the practical performance of lines selected based on phenotype in comparison with those selected solely on GEBVs in advanced testcross yield trials. The main objectives of this study were to (1) empirically compare the performance of tropical maize hybrids selected through phenotypic selection (PS) and genomic selection (GS) under well-watered (WW) and managed drought stress (WS) conditions in Kenya, and (2) compare the cost-benefit analysis of GS and PS. For this study, we used two experimental maize data sets (stage I and stage II yield trials). The stage I data set consisted of 1492 doubled haploid (DH) lines genotyped with rAmpSeq SNPs. A subset of these lines (855) representing various DH populations within the stage I cohort was crossed with an individual single-cross tester chosen to complement each population. These testcross hybrids were evaluated in replicated trials under WW and WS conditions for grain yield and other agronomic traits, while the remaining 637 DH lines were predicted using the 855 lines as a training set. The second data set (stage II) consists of 348 DH lines from the first data set. Among these 348 best DH lines, 172 lines selected were solely based on GEBVs, and 176 lines were selected based on phenotypic performance. Each of the 348 DH lines were crossed with three common testers from complementary heterotic groups, and the resulting 1042 testcross hybrids and six commercial checks were evaluated in four to five WW locations and one WS condition in Kenya. For stage I trials, the cross-validated prediction accuracy for grain yield was 0.67 and 0.65 under WW and WS conditions, respectively. We found similar responses to selection using PS and GS for grain yield other agronomic traits under WW and WS conditions. The top 15% of hybrids advanced through GS and PS gave 21%-23% higher grain yield under WW and 51%-52% more grain yield under WS than the mean of the checks. The GS reduced the cost by 32% over the PS with similar selection gains. We concluded that the use of GS for yield under WW and WS conditions in maize can produce selection candidates with similar performance as those generated from conventional PS, but at a lower cost, and therefore, should be incorporated into maize breeding pipelines to increase breeding program efficiency.

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants.

Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can be used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters.

Patterns of metabolite changes identified from large-scale gene perturbations in Arabidopsis using a genome-scale metabolic network.

Metabolomics enables quantitative evaluation of metabolic changes caused by genetic or environmental perturbations. However, little is known about how perturbing a single gene changes the metabolic system as a whole and which network and functional properties are involved in this response. To answer this question, we investigated the metabolite profiles from 136 mutants with single gene perturbations of functionally diverse Arabidopsis (Arabidopsis thaliana) genes. Fewer than 10 metabolites were changed significantly relative to the wild type in most of the mutants, indicating that the metabolic network was robust to perturbations of single metabolic genes. These changed metabolites were closer to each other in a genome-scale metabolic network than expected by chance, supporting the notion that the genetic perturbations changed the network more locally than globally. Surprisingly, the changed metabolites were close to the perturbed reactions in only 30% of the mutants of the well-characterized genes. To determine the factors that contributed to the distance between the observed metabolic changes and the perturbation site in the network, we examined nine network and functional properties of the perturbed genes. Only the isozyme number affected the distance between the perturbed reactions and changed metabolites. This study revealed patterns of metabolic changes from large-scale gene perturbations and relationships between characteristics of the perturbed genes and metabolic changes.

The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of Pathway/Genome Databases.

The MetaCyc database (MetaCyc.org) is a comprehensive and freely accessible database describing metabolic pathways and enzymes from all domains of life. MetaCyc pathways are experimentally determined, mostly small-molecule metabolic pathways and are curated from the primary scientific literature. MetaCyc contains >2100 pathways derived from >37,000 publications, and is the largest curated collection of metabolic pathways currently available. BioCyc (BioCyc.org) is a collection of >3000 organism-specific Pathway/Genome Databases (PGDBs), each containing the full genome and predicted metabolic network of one organism, including metabolites, enzymes, reactions, metabolic pathways, predicted operons, transport systems and pathway-hole fillers. Additions to BioCyc over the past 2 years include YeastCyc, a PGDB for Saccharomyces cerevisiae, and 891 new genomes from the Human Microbiome Project. The BioCyc Web site offers a variety of tools for querying and analysis of PGDBs, including Omics Viewers and tools for comparative analysis. New developments include atom mappings in reactions, a new representation of glycan degradation pathways, improved compound structure display, better coverage of enzyme kinetic data, enhancements of the Web Groups functionality, improvements to the Omics viewers, a new representation of the Enzyme Commission system and, for the desktop version of the software, the ability to save display states.

Arabidopsis database and stock resources.

The volume of Arabidopsis information has increased enormously in recent years as a result of the sequencing of the reference genome and other large-scale functional genomics projects. Much of the data is stored in public databases, where data are organized, analyzed, and made freely accessible to the research community. These databases are resources that researchers can utilize for making predictions and developing testable hypotheses. The methods in this chapter describe ways to access and utilize Arabidopsis data and genomic resources found in databases and stock centers.

Putting The Plant Metabolic Network pathway databases to work: going offline to gain new capabilities.

Metabolic databases such as The Plant Metabolic Network/MetaCyc and KEGG PATHWAY are publicly accessible resources providing organism-specific information on reactions and metabolites. KEGG PATHWAY depicts metabolic networks as wired, electronic circuit-like maps, whereas the MetaCyc family of databases uses a canonical textbook-like representation. The first MetaCyc-based database for a plant species was AraCyc, which describes metabolism in the model plant Arabidopsis. This database was created over 10 years ago and has since then undergone extensive manual curation to reflect updated information on enzymes and pathways in Arabidopsis. This chapter describes accessing and using AraCyc and its underlying Pathway Tools software. Specifically, methods for (1) navigating Pathway Tools, (2) visualizing omics data and superimposing the data on a metabolic pathway map, and (3) creating pathways and pathway components are discussed.

The challenge of constructing, classifying, and representing metabolic pathways.

Scientists, educators, and students benefit from having free and centralized access to the wealth of metabolic information that has been gathered over the decades. Curators of the MetaCyc database work to present this information in an easily understandable pathway-based framework. MetaCyc is used not only as an encyclopedic resource for metabolic information but also as a template for the pathway prediction software that generates pathway/genome databases for thousands of organisms with sequenced genomes (available at www.biocyc.org). Curators need to define pathway boundaries and classify pathways within a broader pathway ontology to maximize the utility of the pathways to both users and the pathway prediction software. These seemingly simple tasks pose several challenges. This review describes these challenges as well as the criteria that need to be considered, and the rules that have been developed by MetaCyc curators as they make decisions regarding the representation and classification of metabolic pathway information in MetaCyc. The functional consequences of these decisions in regard to pathway prediction in new species are also discussed.

The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases.

The MetaCyc database (http://metacyc.org/) provides a comprehensive and freely accessible resource for metabolic pathways and enzymes from all domains of life. The pathways in MetaCyc are experimentally determined, small-molecule metabolic pathways and are curated from the primary scientific literature. MetaCyc contains more than 1800 pathways derived from more than 30,000 publications, and is the largest curated collection of metabolic pathways currently available. Most reactions in MetaCyc pathways are linked to one or more well-characterized enzymes, and both pathways and enzymes are annotated with reviews, evidence codes and literature citations. BioCyc (http://biocyc.org/) is a collection of more than 1700 organism-specific Pathway/Genome Databases (PGDBs). Each BioCyc PGDB contains the full genome and predicted metabolic network of one organism. The network, which is predicted by the Pathway Tools software using MetaCyc as a reference database, consists of metabolites, enzymes, reactions and metabolic pathways. BioCyc PGDBs contain additional features, including predicted operons, transport systems and pathway-hole fillers. The BioCyc website and Pathway Tools software offer many tools for querying and analysis of PGDBs, including Omics Viewers and comparative analysis. New developments include a zoomable web interface for diagrams; flux-balance analysis model generation from PGDBs; web services; and a new tool called Web Groups.

The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools.

The Arabidopsis Information Resource (TAIR, http://arabidopsis.org) is a genome database for Arabidopsis thaliana, an important reference organism for many fundamental aspects of biology as well as basic and applied plant biology research. TAIR serves as a central access point for Arabidopsis data, annotates gene function and expression patterns using controlled vocabulary terms, and maintains and updates the A. thaliana genome assembly and annotation. TAIR also provides researchers with an extensive set of visualization and analysis tools. Recent developments include several new genome releases (TAIR8, TAIR9 and TAIR10) in which the A. thaliana assembly was updated, pseudogenes and transposon genes were re-annotated, and new data from proteomics and next generation transcriptome sequencing were incorporated into gene models and splice variants. Other highlights include progress on functional annotation of the genome and the release of several new tools including Textpresso for Arabidopsis which provides the capability to carry out full text searches on a large body of research literature.

Creation of a genome-wide metabolic pathway database for Populus trichocarpa using a new approach for reconstruction and curation of metabolic pathways for plants.

Metabolic networks reconstructed from sequenced genomes or transcriptomes can help visualize and analyze large-scale experimental data, predict metabolic phenotypes, discover enzymes, engineer metabolic pathways, and study metabolic pathway evolution. We developed a general approach for reconstructing metabolic pathway complements of plant genomes. Two new reference databases were created and added to the core of the infrastructure: a comprehensive, all-plant reference pathway database, PlantCyc, and a reference enzyme sequence database, RESD, for annotating metabolic functions of protein sequences. PlantCyc (version 3.0) includes 714 metabolic pathways and 2,619 reactions from over 300 species. RESD (version 1.0) contains 14,187 literature-supported enzyme sequences from across all kingdoms. We used RESD, PlantCyc, and MetaCyc (an all-species reference metabolic pathway database), in conjunction with the pathway prediction software Pathway Tools, to reconstruct a metabolic pathway database, PoplarCyc, from the recently sequenced genome of Populus trichocarpa. PoplarCyc (version 1.0) contains 321 pathways with 1,807 assigned enzymes. Comparing PoplarCyc (version 1.0) with AraCyc (version 6.0, Arabidopsis [Arabidopsis thaliana]) showed comparable numbers of pathways distributed across all domains of metabolism in both databases, except for a higher number of AraCyc pathways in secondary metabolism and a 1.5-fold increase in carbohydrate metabolic enzymes in PoplarCyc. Here, we introduce these new resources and demonstrate the feasibility of using them to identify candidate enzymes for specific pathways and to analyze metabolite profiling data through concrete examples. These resources can be searched by text or BLAST, browsed, and downloaded from our project Web site (http://plantcyc.org).

Using the Arabidopsis information resource (TAIR) to find information about Arabidopsis genes.

The Arabidopsis Information Resource (TAIR; http://arabidopsis.org) is a comprehensive Web resource of Arabidopsis biology for plant scientists. TAIR curates and integrates information about genes, proteins, gene function, gene expression, mutant phenotypes, biological materials such as clones and seed stocks, genetic markers, genetic and physical maps, biochemical pathways, genome organization, images of mutant plants, protein sub-cellular localizations, publications, and the research community. The various data types are extensively interconnected and can be accessed through a variety of Web-based search and display tools. This unit primarily focuses on some basic methods for searching, browsing, visualizing, and analyzing information about Arabidopsis genes and describes several new tools such as a new TAIR genome browser (GBrowse), and the TAIR synteny viewer (GBrowse_syn). We also describe how to use AraCyc for mining plant metabolic pathways.

PlantMetabolomics.org: a web portal for plant metabolomics experiments.

PlantMetabolomics.org (PM) is a web portal and database for exploring, visualizing, and downloading plant metabolomics data. Widespread public access to well-annotated metabolomics datasets is essential for establishing metabolomics as a functional genomics tool. PM integrates metabolomics data generated from different analytical platforms from multiple laboratories along with the key visualization tools such as ratio and error plots. Visualization tools can quickly show how one condition compares to another and which analytical platforms show the largest changes. The database tries to capture a complete annotation of the experiment metadata along with the metabolite abundance databased on the evolving Metabolomics Standards Initiative. PM can be used as a platform for deriving hypotheses by enabling metabolomic comparisons between genetically unique Arabidopsis (Arabidopsis thaliana) populations subjected to different environmental conditions. Each metabolite is linked to relevant experimental data and information from various annotation databases. The portal also provides detailed protocols and tutorials on conducting plant metabolomics experiments to promote metabolomics in the community. PM currently houses Arabidopsis metabolomics data generated by a consortium of laboratories utilizing metabolomics to help elucidate the functions of uncharacterized genes. PM is publicly available at http://www.plantmetabolomics.org.

The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases.

The MetaCyc database (MetaCyc.org) is a comprehensive and freely accessible resource for metabolic pathways and enzymes from all domains of life. The pathways in MetaCyc are experimentally determined, small-molecule metabolic pathways and are curated from the primary scientific literature. With more than 1400 pathways, MetaCyc is the largest collection of metabolic pathways currently available. Pathways reactions are linked to one or more well-characterized enzymes, and both pathways and enzymes are annotated with reviews, evidence codes, and literature citations. BioCyc (BioCyc.org) is a collection of more than 500 organism-specific Pathway/Genome Databases (PGDBs). Each BioCyc PGDB contains the full genome and predicted metabolic network of one organism. The network, which is predicted by the Pathway Tools software using MetaCyc as a reference, consists of metabolites, enzymes, reactions and metabolic pathways. BioCyc PGDBs also contain additional features, such as predicted operons, transport systems, and pathway hole-fillers. The BioCyc Web site offers several tools for the analysis of the PGDBs, including Omics Viewers that enable visualization of omics datasets on two different genome-scale diagrams and tools for comparative analysis. The BioCyc PGDBs generated by SRI are offered for adoption by any party interested in curation of metabolic, regulatory, and genome-related information about an organism.

Ubiquitin, hormones and biotic stress in plants.

BACKGROUND: The covalent attachment of ubiquitin to a substrate protein changes its fate. Notably, proteins typically tagged with a lysine48-linked polyubiquitin chain become substrates for degradation by the 26S proteasome. In recent years many experiments have been performed to characterize the proteins involved in the ubiquitylation process and to identify their substrates, in order to understand better the mechanisms that link specific protein degradation events to regulation of plant growth and development. SCOPE: This review focuses on the role that ubiquitin plays in hormone synthesis, hormonal signalling cascades and plant defence mechanisms. Several examples are given of how targeted degradation of proteins affects downstream transcriptional regulation of hormone-responsive genes in the auxin, gibberellin, abscisic acid, ethylene and jasmonate signalling pathways. Additional experiments suggest that ubiquitin-mediated proteolysis may also act upstream of the hormonal signalling cascades by regulating hormone biosynthesis, transport and perception. Moreover, several experiments demonstrate that hormonal cross-talk can occur at the level of proteolysis. The more recently established role of the ubiquitin/proteasome system (UPS) in defence against biotic threats is also reviewed. CONCLUSIONS: The UPS has been implicated in the regulation of almost every developmental process in plants, from embryogenesis to floral organ production probably through its central role in many hormone pathways. More recent evidence provides molecular mechanisms for hormonal cross-talk and links the UPS system to biotic defence responses.

The Arabidopsis Aux/IAA protein family has diversified in degradation and auxin responsiveness.

Rapid, auxin-responsive degradation of multiple auxin/indole-3-acetic acid (Aux/IAA) proteins is essential for plant growth and development. Domain II residues were previously shown to be required for the degradation of several Arabidopsis thaliana Aux/IAA proteins. We examined the degradation of additional full-length family members and the proteolytic importance of N-terminal residues outside domain II using luciferase (LUC) fusions. Elimination of domain I did not affect degradation. However, substituting an Arg for a conserved Lys between domains I and II specifically impaired basal degradation without compromising the auxin-mediated acceleration of degradation. IAA8, IAA9, and IAA28 contain domain II and a conserved Lys, but they were degraded more slowly than previously characterized family members when expressed as LUC fusions, suggesting that sequences outside domain II influence proteolysis. We analyzed the degradation of IAA31, with a region somewhat similar to domain II but without the conserved Lys, and of IAA20, which lacks domain II and the conserved Lys. Both IAA20:LUC and epitope-tagged IAA20 were long-lived, and their longevity was not influenced by auxin. Epitope-tagged IAA31 was long-lived, like IAA20, but by contrast, it showed accelerated degradation in response to auxin. The existence of long-lived and auxin-insensitive Aux/IAA proteins suggeststhat they may play a novel role in auxin signaling.