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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

An extensible vector toolkit and parts library for advanced engineering of plant genomes

Abstract Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T‐DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site‐specific recombinases. Finally, we use a series of dual‐luciferase assays to measure contributions to expression from promoters, terminators, and from cross‐cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

42 ENGINEERING↗
DOE OSTI2014

JGI Plant Genomics Gene Annotation Pipeline

Plant genomes vary in size and are highly complex with a high amount of repeats, genome duplication and tandem duplication. Gene encodes a wealth of information useful in studying organism and it is critical to have high quality and stable gene annotation. Thanks to advancement of sequencing technology, many plant species genomes have been sequenced and transcriptomes are also sequenced. To use these vastly large amounts of sequence data to make gene annotation or re-annotation in a timely fashion, an automatic pipeline is needed. JGI plant genomics gene annotation pipeline, called integrated gene call (IGC), is our effort toward this aim with aid of a RNA-seq transcriptome assembly pipeline. It utilizes several gene predictors based on homolog peptides and transcript ORFs. See Methods for detail. Here we present genome annotation of JGI flagship green plants produced by this pipeline plus Arabidopsis and rice except for chlamy which is done by a third party. The genome annotations of these species and others are used in our gene family build pipeline and accessible via JGI Phytozome portal whose URL and front page snapshot are shown below.

99 GENERAL AND MISCELLANEOUS↗
DOE OSTI2021

Impact of short-read sequencing on the misassembly of a plant genome

Abstract Background Availability of plant genome sequences has led to significant advances. However, with few exceptions, the great majority of existing genome assemblies are derived from short read sequencing technologies with highly uneven read coverages indicative of sequencing and assembly issues that could significantly impact any downstream analysis of plant genomes. In tomato for example, 0.6% (5.1 Mb) and 9.7% (79.6 Mb) of short-read based assembly had significantly higher and lower coverage compared to background, respectively. Results To understand what the causes may be for such uneven coverage, we first established machine learning models capable of predicting genomic regions with variable coverages and found that high coverage regions tend to have higher simple sequence repeat and tandem gene densities compared to background regions. To determine if the high coverage regions were misassembled, we examined a recently available tomato long-read based assembly and found that 27.8% (1.41 Mb) of high coverage regions were potentially misassembled of duplicate sequences, compared to 1.4% in background regions. In addition, using a predictive model that can distinguish correctly and incorrectly assembled high coverage regions, we found that misassembled, high coverage regions tend to be flanked by simple sequence repeats, pseudogenes, and transposon elements. Conclusions Our study provides insights on the causes of variable coverage regions and a quantitative assessment of factors contributing to plant genome misassembly when using short reads and the generality of these causes and factors should be tested further in other species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Architecture and evolution of a minute plant genome

It has been argued that the evolution of plant genome size is principally unidirectional and increasing owing to the varied action of whole-genome duplications (WGDs) and mobile element proliferation. However, extreme genome size reductions have been reported in the angiosperm family tree. Here we report the sequence of the 82-megabase genome of the carnivorous bladderwort plant Utricularia gibba. Despite its tiny size, the U. gibba genome accommodates a typical number of genes for a plant, with the main difference from other plant genomes arising from a drastic reduction in non-genic DNA. Unexpectedly, we identified at least three rounds of WGD in U. gibba since common ancestry with tomato (Solanum) and grape (Vitis). The compressed architecture of the U. gibba genome indicates that a small fraction of intergenic DNA, with few or no active retrotransposons, is sufficient to regulate and integrate all the processes required for the development and reproduction of a complex organism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Editorial: Artificial Intelligence and Machine Learning Applications in Plant Genomics and Genetics

In plants, as in other species, genotypic variation in combination with environmental variation leads to differences in the biochemical makeup of cells, measurable as molecular phenotypes. These in turn influence physiological and developmental traits such as organ formation and plant growth, and eventually traits relevant in agriculture, such as yield and stress tolerance. Thus, relating genotypes to phenotypes yields fundamental insights into the regulation of important processes in plant development and physiology, but also provides the ability to predict yield and quality traits in specific environments, which is essential in basic plant science and molecular breeding of resilient plants in changing environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

The Planteome database: an integrated resource for reference ontologies, plant genomics and phenomics

The Planteome project (http://www.planteome.org) provides a suite of reference and species-specific ontologies for plants and annotations to genes and phenotypes. Ontologies serve as common standards for semantic integration of a large and growing corpus of plant genomics, phenomics and genetics data. The reference ontologies include the Plant Ontology, Plant Trait Ontology and the Plant Experimental Conditions Ontology developed by the Planteome project, along with the Gene Ontology, Chemical Entities of Biological Interest, Phenotype and Attribute Ontology, and others. The project also provides access to species-specific Crop Ontologies developed by various plant breeding and research communities from around the world. We provide integrated data on plant traits, phenotypes, and gene function and expression from 95 plant taxa, annotated with reference ontology terms. The Planteome project is developing a plant gene annotation platform; Planteome Noctua, to facilitate community engagement. All the Planteome ontologies are publicly available and are maintained at the Planteome GitHub site (https://github.com/Planteome) for sharing, tracking revisions and new requests. The annotated data are freely accessible from the ontology browser (http://browser.planteome.org/amigo) and our data repository.

Biochemistry & Molecular Biology↗
DOE OSTI2016

Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research

Genome editing with site-specific nucleases has become a powerful tool for functional characterization of plant genes and genetic improvement of agricultural crops. Among the various site-specific nuclease-based technologies available for genome editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems have shown the greatest potential for rapid and efficient editing of genomes in plant species. Here, this article reviews the current status of application of CRISPR/Cas9 to plant genomics research, with a focus on loss-of-function and gain-of-function analysis of individual genes in the context of perennial plants and the potential application of CRISPR/Cas9 to perturbation of gene expression, as well as identification and analysis of gene modules as part of an accelerated domestication and synthetic biology effort.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Phytozome Comparative Plant Genomics Portal

The Dept. of Energy Joint Genome Institute is a genomics user facility supporting DOE mission science in the areas of Bioenergy, Carbon Cycling, and Biogeochemistry. The Plant Program at the JGI applies genomic, analytical, computational and informatics platforms and methods to: 1. Understand and accelerate the improvement (domestication) of bioenergy crops 2. Characterize and moderate plant response to climate change 3. Use comparative genomics to identify constrained elements and infer gene function 4. Build high quality genomic resource platforms of JGI Plant Flagship genomes for functional and experimental work 5. Expand functional genomic resources for Plant Flagship genomes

09 BIOMASS FUELS↗
DOE OSTI2015

Evolution of plant virus movement proteins from the 30K superfamily and of their homologs integrated in plant genomes

Homologs of Tobacco mosaic virus 30K cell-to-cell movement protein are encoded by diverse plant viruses. Mechanisms of action and evolutionary origins of these proteins remain obscure. We expand the picture of conservation and evolution of the 30K proteins, producing sequence alignment of the 30K superfamily with the broadest phylogenetic coverage thus far and illuminating structural features of the core all-beta fold of these proteins. Integrated copies of pararetrovirus 30K movement genes are prevalent in euphyllophytes, with at least one copy intact in nearly every examined species, and mRNAs detected for most of them. Sequence analysis suggests repeated integrations, pseudogenizations, and positive selection in those provirus genes. An unannotated 30K-superfamily gene in Arabidopsis thaliana genome is likely expressed as a fusion with the At1g37113 transcript. This molecular background of endopararetrovirus gene products in plants may change our view of virus infection and pathogenesis, and perhaps of cellular homeostasis in the hosts. - Highlights: • Sequence region shared by plant virus “30K” movement proteins has an all-beta fold. • Most euphyllophyte genomes contain integrated copies of pararetroviruses. • These integrated virus genomes often include intact movement protein genes. • Molecular evidence suggests that these “30K” genes may be selected for function.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

A host plant genome ( Zizania latifolia ) after a century-long endophyte infection

In spite of the importance of host–microbe interactions in natural ecosystems, agriculture and medicine, the impact of long-term (especially decades or longer) microbial colonization on the dynamics of host genomes is not well understood. Moreover, the vegetable crop ‘Jiaobai’ with enlarged edible stems was domesticated from wild Zizania latifolia (Oryzeae) approximately 2000 years ago as a result of persistent infection by a fungal endophyte, Ustilago esculenta. Asexual propagation via infected rhizomes is the only means of Jiaobai production, and the Z. latifolia–endophyte complex has been maintained continuously for two centuries. Here, genomic analysis revealed that cultivated Z. latifolia has a significantly smaller repertoire of immune receptors compared with wild Z. latifolia. There are widespread gene losses/mutations and expression changes in the plant–pathogen interaction pathway in Jiaobai. Finally, these results show that continuous long-standing endophyte association can have a major effect on the evolution of the structural and transcriptomic components of the host genome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genome editing in plants using the compact editor CasΦ

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasΦ guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele, we show that CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wild-type CasΦ enzyme. Consistently, vCasΦ and nCasΦ yielded offspring plants with inherited edits at much higher rates compared to WTCasΦ. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, T-rich minimal protospacer adjacent motif (PAM), and wide range of working temperatures make CasΦ an excellent supplement to existing plant genome editing systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Precision genome editing in plants using gene targeting and prime editing: existing and emerging strategies

Precise modification of plant genomes, such as seamless insertion, deletion, or replacement of DNA sequences at a predefined site, is a challenging task. Gene targeting (GT) and prime editing are currently the best approaches for this purpose. However, these techniques are inefficient in plants, which limits their applications for crop breeding programs. Recently, substantial developments have been made to improve the efficiency of these techniques in plants. Several strategies, such as RNA donor templating, chemically modified donor DNA template, and tandem-repeat homology-directed repair, are aimed at improving GT. Additionally, improved prime editing gRNA design, use of engineered reverse transcriptase enzymes, and splitting prime editing components have improved the efficacy of prime editing in plants. These emerging strategies and existing technologies are reviewed along with various perspectives on their future improvement and the development of robust precision genome editing technologies for plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants

CRISPR/Cas9 has been widely applied to various plant species accelerating the pace of plant genome editing and precision breeding in crops. Unintended effects beyond off-target nucleotide mutations are still somewhat unexplored. We investigated the degree and patterns of epigenetic changes after gene editing. We examined changes in DNA methylation in genome-edited promoters of naturally hypermethylated genes (AT1G72350 and AT1G09970) and hypomethylated genes (AT3G17320 and AT5G28770) from Arabidopsis. Transgenic plants were developed via Agrobacterium-mediated floral dip transformation. Homozygous edited lines were selected from segregated T2 plants by an in vitro digestion assay using ribonucleoprotein complex. Bisulfite sequencing comparisons were made between paired groups of edited and non-edited plants to identify changes in DNA methylation of the targeted loci. We found that directed mutagenesis via CRISPR/Cas9 resulted in no unintended morphological or epigenetic alterations. Phenotypes of wild-type, transgenic empty vector, and transgenic edited plants were similar. Epigenetic profiles revealed that methylation patterns of promoter regions flanking target sequences were identical among wild-type, transgenic empty vector, and transgenic edited plants. There was no effect of mutation type on epigenetic status. We also evaluated off-target mutagenesis effects in the edited plants. Potential off-target sites containing up to 4-bp mismatch of each target were sequenced. No off-target mutations were detected in candidate sites. Our results showed that CRISPR/Cas9 did not leave an epigenetic footprint on either the immediate gene-edited DNA and flanking DNA or introduce off-target mutations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Detailed analysis of putative genes encoding small proteins in legume genomes

Diverse plant genome sequencing projects coupled with powerful bioinformatics tools have facilitated massive data analysis to construct specialized databases classified according to cellular function. However, there are still a considerable number of genes encoding proteins whose function has not yet been characterized. Included in this category are small proteins (SPs, 30–150 amino acids) encoded by short open reading frames (sORFs). SPs play important roles in plant physiology, growth, and development. Unfortunately, protocols focused on the genome-wide identification and characterization of sORFs are scarce or remain poorly implemented. As a result, these genes are underrepresented in many genome annotations. In this work, we exploited publicly available genome sequences of Phaseolus vulgaris, Medicago truncatula, Glycine max, and Lotus japonicus to analyze the abundance of annotated SPs in plant legumes. Our strategy to uncover bona fide sORFs at the genome level was centered in bioinformatics analysis of characteristics such as evidence of expression (transcription), presence of known protein regions or domains, and identification of orthologous genes in the genomes explored. We collected 6170, 10,461, 30,521, and 23,599 putative sORFs from P. vulgaris, G. max, M. truncatula, and L. japonicus genomes, respectively. Expressed sequence tags (ESTs) available in the DFCI Gene Index database provided evidence that ~one-third of the predicted legume sORFs are expressed. Most potential SPs have a counterpart in a different plant species and counterpart regions or domains in larger proteins. Potential functional sORFs were also classified according to a reduced set of GO categories, and the expression of 13 of them during P. vulgaris nodule ontogeny was confirmed by qPCR. This analysis provides a collection of sORFs that potentially encode for meaningful SPs, and offers the possibility of their further functional evaluation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

The Plant Ontology as a Tool for Comparative Plant Anatomy and Genomic Analyses

The Plant Ontology (PO; http://www.plantontology.org/) is a publicly available, collaborative effort to develop and maintain a controlled, structured vocabulary (‘ontology’) of terms to describe plant anatomy, morphology and the stages of plant development. The goals of the PO are to link (annotate) gene expression and phenotype data to plant structures and stages of plant development, using the data model adopted by the Gene Ontology. From its original design covering only rice, maize and Arabidopsis, the scope of the PO has been expanded to include all green plants. The PO was the first multispecies anatomy ontology developed for the annotation of genes and phenotypes. Also, to our knowledge, it was one of the first biological ontologies that provides translations (via synonyms) in non-English languages such as Japanese and Spanish. As of Release #18 (July 2012), there are about 2.2 million annotations linking PO terms to >110,000 unique data objects representing genes or gene models, proteins, RNAs, germplasm and quantitative trait loci (QTLs) from 22 plant species. In this paper, we focus on the plant anatomical entity branch of the PO, describing the organizing principles, resources available to users and examples of how the PO is integrated into other plant genomics databases and web portals. We also provide two examples of comparative analyses, demonstrating how the ontology structure and PO-annotated data can be used to discover the patterns of expression of the LEAFY (LFY) and terpene synthase (TPS) gene homologs.

59 BASIC BIOLOGICAL SCIENCES↗