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September 22, 2019  |  

Comparative Annotation Toolkit (CAT)-simultaneous clade and personal genome annotation.

The recent introductions of low-cost, long-read, and read-cloud sequencing technologies coupled with intense efforts to develop efficient algorithms have made affordable, high-quality de novo sequence assembly a realistic proposition. The result is an explosion of new, ultracontiguous genome assemblies. To compare these genomes, we need robust methods for genome annotation. We describe the fully open source Comparative Annotation Toolkit (CAT), which provides a flexible way to simultaneously annotate entire clades and identify orthology relationships. We show that CAT can be used to improve annotations on the rat genome, annotate the great apes, annotate a diverse set of mammals, and annotate personal, diploid human genomes. We demonstrate the resulting discovery of novel genes, isoforms, and structural variants-even in genomes as well studied as rat and the great apes-and how these annotations improve cross-species RNA expression experiments.© 2018 Fiddes et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019  |  

Full-length transcriptome sequences of ephemeral plant Arabidopsis pumila provides insight into gene expression dynamics during continuous salt stress.

Arabidopsis pumila is native to the desert region of northwest China and it is extraordinarily well adapted to the local semi-desert saline soil, thus providing a candidate plant system for environmental adaptation and salt-tolerance gene mining. However, understanding of the salt-adaptation mechanism of this species is limited because of genomic sequences scarcity. In the present study, the transcriptome profiles of A. pumila leaf tissues treated with 250 mM NaCl for 0, 0.5, 3, 6, 12, 24 and 48 h were analyzed using a combination of second-generation sequencing (SGS) and third-generation single-molecule real-time (SMRT) sequencing.Correction of SMRT long reads by SGS short reads resulted in 59,328 transcripts. We found 8075 differentially expressed genes (DEGs) between salt-stressed tissues and controls, of which 483 were transcription factors and 1157 were transport proteins. Most DEGs were activated within 6 h of salt stress and their expression stabilized after 48 h; the number of DEGs was greatest within 12 h of salt stress. Gene annotation and functional analyses revealed that expression of genes associated with the osmotic and ionic phases rapidly and coordinately changed during the continuous salt stress in this species, and salt stress-related categories were highly enriched among these DEGs, including oxidation-reduction, transmembrane transport, transcription factor activity and ion channel activity. Orphan, MYB, HB, bHLH, C3H, PHD, bZIP, ARF and NAC TFs were most enriched in DEGs; ABCB1, CLC-A, CPK30, KEA2, KUP9, NHX1, SOS1, VHA-A and VP1 TPs were extensively up-regulated in salt-stressed samples, suggesting that they play important roles in slat tolerance. Importantly, further experimental studies identified a mitogen-activated protein kinase (MAPK) gene MAPKKK18 as continuously up-regulated throughout salt stress, suggesting its crucial role in salt tolerance. The expression patterns of the salt-responsive 24 genes resulted from quantitative real-time PCR were basically consistent with their transcript abundance changes identified by RNA-Seq.The full-length transcripts generated in this study provide a more accurate depiction of gene transcription of A. pumila. We identified potential genes involved in salt tolerance of A. pumila. These data present a genetic resource and facilitate better understanding of salt-adaptation mechanism for ephemeral plants.

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September 22, 2019  |  

The microbiome of the leaf surface of Arabidopsis protects against a fungal pathogen.

We have explored the importance of the phyllosphere microbiome in plant resistance in the cuticle mutants bdg (BODYGUARD) or lacs2.3 (LONG CHAIN FATTY ACID SYNTHASE 2) that are strongly resistant to the fungal pathogen Botrytis cinerea. The study includes infection of plants under sterile conditions, 16S ribosomal DNA sequencing of the phyllosphere microbiome, and isolation and high coverage sequencing of bacteria from the phyllosphere. When inoculated under sterile conditions bdg became as susceptible as wild-type (WT) plants whereas lacs2.3 mutants retained the resistance. Adding washes of its phyllosphere microbiome could restore the resistance of bdg mutants, whereas the resistance of lacs2.3 results from endogenous mechanisms. The phyllosphere microbiome showed distinct populations in WT plants compared to cuticle mutants. One species identified as Pseudomonas sp isolated from the microbiome of bdg provided resistance to B. cinerea on Arabidopsis thaliana as well as on apple fruits. No direct activity was observed against B. cinerea and the action of the bacterium required the plant. Thus, microbes present on the plant surface contribute to the resistance to B. cinerea. These results open new perspectives on the function of the leaf microbiome in the protection of plants.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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September 22, 2019  |  

Ecological genomics of tropical trees: how local population size and allelic diversity of resistance genes relate to immune responses, cosusceptibility to pathogens, and negative density dependence

In tropical forests, rarer species show increased sensitivity to species-specific soil pathogens and more negative effects of conspecific density on seedling survival (NDD). These patterns suggest a connection between ecology and immunity, perhaps because small population size disproportionately reduces genetic diversity of hyperdiverse loci such as immunity genes. In an experiment examining seedling roots from six species in one tropical tree community, we found that smaller populations have reduced amino acid diversity in pathogen resistance (R) genes but not the transcriptome in general. Normalized R gene amino acid diversity varied with local abundance and prior measures of differences in sensitivity to conspecific soil and NDD. After exposure to live soil, species with lower R gene diversity had reduced defence gene induction, more cosusceptibility of maternal cohorts to colonization by potentially pathogenic fungi, reduced root growth arrest (an R gene-mediated response) and their root-associated fungi showed lower induction of self-defence (antioxidants). Local abundance was not related to the ability to induce immune responses when pathogen recognition was bypassed by application of salicylic acid, a phytohormone that activates defence responses downstream of R gene signalling. These initial results support the hypothesis that smaller local tree populations have reduced R gene diversity and recognition-dependent immune responses, along with greater cosusceptibility to species-specific pathogens that may facilitate disease transmission and NDD. Locally rare species may be less able to increase their equilibrium abundance without genetic boosts to defence via immigration of novel R gene alleles from a larger and more diverse regional population.

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September 22, 2019  |  

Circular RNA architecture and differentiation during leaf bud to young leaf development in tea (Camellia sinensis).

Circular RNA (circRNA) discovery, expression patterns and experimental validation in developing tea leaves indicates its correlation with circRNA-parental genes and potential roles in ceRNA interaction network. Circular RNAs (circRNAs) have recently emerged as a novel class of abundant endogenous stable RNAs produced by circularization with regulatory potential. However, identification of circRNAs in plants, especially in non-model plants with large genomes, is challenging. In this study, we undertook a systematic identification of circRNAs from different stage tissues of tea plant (Camellia sinensis) leaf development using rRNA-depleted circular RNA-seq. By combining two state-of-the-art detecting tools, we characterized 3174 circRNAs, of which 342 were shared by each approach, and thus considered high-confidence circRNAs. A few predicted circRNAs were randomly chosen, and 20 out of 24 were experimental confirmed by PCR and Sanger sequencing. Similar in other plants, tissue-specific expression was also observed for many C. sinensis circRNAs. In addition, we found that circRNA abundances were positively correlated with the mRNA transcript abundances of their parental genes. qRT-PCR validated the differential expression patterns of circRNAs between leaf bud and young leaf, which also indicated the low expression abundance of circRNAs compared to the standard mRNAs from the parental genes. We predicted the circRNA-microRNA interaction networks, and 54 of the differentially expressed circRNAs were found to have potential tea plant miRNA binding sites. The gene sets encoding circRNAs were significantly enriched in chloroplasts related GO terms and photosynthesis/metabolites biosynthesis related KEGG pathways, suggesting the candidate roles of circRNAs in photosynthetic machinery and metabolites biosynthesis during leaf development.

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September 22, 2019  |  

Identification and analysis of glutathione S-transferase gene family in sweet potato reveal divergent GST-mediated networks in aboveground and underground tissues in response to abiotic stresses.

Sweet potato, a hexaploid species lacking a reference genome, is one of the most important crops in many developing countries, where abiotic stresses are a primary cause of reduction of crop yield. Glutathione S-transferases (GSTs) are multifunctional enzymes that play important roles in oxidative stress tolerance and cellular detoxification.A total of 42 putative full-length GST genes were identified from two local transcriptome databases and validated by molecular cloning and Sanger sequencing. Sequence and intraspecific phylogenetic analyses revealed extensive differentiation in their coding sequences and divided them into eight subfamilies. Interspecific phylogenetic and comparative analyses indicated that most examined GST paralogs might originate and diverge before the speciation of sweet potato. Results from large-scale RNA-seq and quantitative real-time PCR experiments exhibited extensive variation in gene-expression profiles across different tissues and varieties, which implied strong evolutionary divergence in their gene-expression regulation. Moreover, we performed five manipulated stress experiments and uncovered highly divergent stress-response patterns of sweet potato GST genes in aboveground and underground tissues.Our study identified a large number of sweet potato GST genes, systematically investigated their evolutionary diversification, and provides new insights into the GST-mediated stress-response mechanisms in this worldwide crop.

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September 22, 2019  |  

PacBio sequencing and its applications.

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Additionally, PacBio’s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

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September 22, 2019  |  

Global identification of the full-length transcripts and alternative splicing related to phenolic acid biosynthetic genes in Salvia miltiorrhiza.

Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing) of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and four alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that six candidate cytochrome P450s and five candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza.

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September 22, 2019  |  

Generation and comparative analysis of full-length transcriptomes in sweetpotato and its putative wild ancestor I. trifida.

Sweetpotato [Ipomoea batatas (L.) Lam.] is one of the most important crops in many developing countries and provides a candidate source of bioenergy. However, neither high-quality reference genome nor large-scale full-length cDNA sequences for this outcrossing hexaploid are still lacking, which in turn impedes progress in research studies in sweetpotato functional genomics and molecular breeding. In this study, we apply a combination of second- and third-generation sequencing technologies to sequence full-length transcriptomes in sweetpotato and its putative ancestor I. trifida. In total, we obtained 53,861/51,184 high-quality transcripts, which includes 34,963/33,637 putative full-length cDNA sequences, from sweetpotato/I. trifida. Amongst, we identified 104,540/94,174 open reading frames, 1476/1475 transcription factors, 25,315/27,090 simple sequence repeats, 417/531 long non-coding RNAs out of the sweetpotato/I. trifida dataset. By utilizing public available genomic contigs, we analyzed the gene features (including exon number, exon size, intron number, intron size, exon-intron structure) of 33,119 and 32,793 full-length transcripts in sweetpotato and I. trifida, respectively. Furthermore, comparative analysis between our transcript datasets and other large-scale cDNA datasets from different plant species enables us assessing the quality of public datasets, estimating the genetic similarity across relative species, and surveyed the evolutionary pattern of genes. Overall, our study provided fundamental resources of large-scale full-length transcripts in sweetpotato and its putative ancestor, for the first time, and would facilitate structural, functional and comparative genomics studies in this important crop.

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September 22, 2019  |  

MUMmer4: A fast and versatile genome alignment system.

The MUMmer system and the genome sequence aligner nucmer included within it are among the most widely used alignment packages in genomics. Since the last major release of MUMmer version 3 in 2004, it has been applied to many types of problems including aligning whole genome sequences, aligning reads to a reference genome, and comparing different assemblies of the same genome. Despite its broad utility, MUMmer3 has limitations that can make it difficult to use for large genomes and for the very large sequence data sets that are common today. In this paper we describe MUMmer4, a substantially improved version of MUMmer that addresses genome size constraints by changing the 32-bit suffix tree data structure at the core of MUMmer to a 48-bit suffix array, and that offers improved speed through parallel processing of input query sequences. With a theoretical limit on the input size of 141Tbp, MUMmer4 can now work with input sequences of any biologically realistic length. We show that as a result of these enhancements, the nucmer program in MUMmer4 is easily able to handle alignments of large genomes; we illustrate this with an alignment of the human and chimpanzee genomes, which allows us to compute that the two species are 98% identical across 96% of their length. With the enhancements described here, MUMmer4 can also be used to efficiently align reads to reference genomes, although it is less sensitive and accurate than the dedicated read aligners. The nucmer aligner in MUMmer4 can now be called from scripting languages such as Perl, Python and Ruby. These improvements make MUMer4 one the most versatile genome alignment packages available.

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September 22, 2019  |  

Aberration or analogy? The atypical plastomes of Geraniaceae

A number of plant groups have been proposed as ideal systems to explore plastid inheritance, plastome evolution and plastome-nuclear genome coevolution. Quick generation times and a compact nuclear genome in Arabidopsis thaliana, the relative ease of plastid isolation from Spinacia oleracea and the tractability of plastid transformation in Nicotiana tabacum are all desirable attributes in a model system; however, these and most other groups all lack novelty in terms of plastome structure and nucleotide sequence evolution. Contemporary sequencing and assembly technologies have facilitated analyses of atypical plastomes and, as predicted by early investigations, Geraniaceae plastomes have experienced unprecedented rearrangements relative to the canonical structure and exhibit remarkably high rates of synonymous and nonsynonymous nucleotide substitutions. While not the only lineage with unusual plastome features, likely no other group represents the array of aberrant phenomena recorded for the family. In this chapter, Geraniaceae plastomes will be discussed and, where possible, compared with other taxa.

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September 22, 2019  |  

An ancient integration in a plant NLR is maintained as a trans-species polymorphism

Plant immune receptors are under constant selective pressure to maintain resistance to plant pathogens. Nucleotide-binding leucine-rich repeat (NLR) proteins are one class of cytoplasmic immune receptors whose genes commonly show signatures of adaptive evolution. While it is known that balancing selection contributes to maintaining high intraspecific allelic diversity, the evolutionary mechanism that influences the transmission of alleles during speciation remains unclear. The barley Mla locus has over 30 described alleles conferring isolate-specific resistance to barley powdery mildew and contains three NLR families (RGH1, RGH2, and RGH3). We discovered (using sequence capture and RNAseq) the presence of a novel integrated Exo70 domain in RGH2 in the Mla3 haplotype. Allelic variation across barley accessions includes presence/absence of the integrated domain in RGH2. Expanding our search to several Poaceae species, we found shared interspecific conservation in the RGH2-Exo70 integration. We hypothesise that balancing selection has maintained allelic variation at Mla as a trans-species polymorphism over 24 My, thus contributing to and preserving interspecific allelic diversity during speciation.

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September 22, 2019  |  

Identification of candidate genes at the Dp-fl locus conferring resistance against the rosy apple aphid Dysaphis plantaginea

The cultivated apple is susceptible to several pests including the rosy apple aphid (RAA; Dysaphis plantaginea Passerini), control of which is mainly based on chemical treatments. A few cases of resistance to aphids have been described in apple germplasm resources, laying the basis for the development of new resistant cultivars by breeding. The cultivar ‘Florina’ is resistant to RAA, and recently, the Dp-fl locus responsible for its resistance was mapped on linkage group 8 of the apple genome. In this paper, a chromosome walking approach was performed by using a ‘Florina’ bacterial artificial chromosome (BAC) library. The walking started from the available tightly linked molecular markers flanking the resistance region. Various walking steps were performed in order to identify the minimum tiling path of BAC clones covering the Dp-fl region from both the “resistant” and “susceptible” chromosomes of ‘Florina’. A genomic region of about 279 Kb encompassing the Dp-fl resistance locus was fully sequenced by the PacBio technology. Through the development of new polymorphic markers, the mapping interval around the resistance locus was narrowed down to a physical region of 95 Kb. The annotation of this sequence resulted in the identification of four candidate genes putatively involved in the RAA resistance response.

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September 22, 2019  |  

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.

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September 22, 2019  |  

Genome sequences of Chlorella sorokiniana UTEX 1602 and Micractinium conductrix SAG 241.80: implications to maltose excretion by a green alga.

Green algae represent a key segment of the global species capable of photoautotrophic-driven biological carbon fixation. Algae partition fixed-carbon into chemical compounds required for biomass, while diverting excess carbon into internal storage compounds such as starch and lipids or, in certain cases, into targeted extracellular compounds. Two green algae were selected to probe for critical components associated with sugar production and release in a model alga. Chlorella sorokiniana UTEX 1602 – which does not release significant quantities of sugars to the extracellular space – was selected as a control to compare with the maltose-releasing Micractinium conductrix SAG 241.80 – which was originally isolated from an endosymbiotic association with the ciliate Paramecium bursaria. Both strains were subjected to three sequencing approaches to assemble their genomes and annotate their genes. This analysis was further complemented with transcriptional studies during maltose release by M. conductrix SAG 241.80 versus conditions where sugar release is minimal. The annotation revealed that both strains contain homologs for the key components of a putative pathway leading to cytosolic maltose accumulation, while transcriptional studies found few changes in mRNA levels for the genes associated with these established intracellular sugar pathways. A further analysis of potential sugar transporters found multiple homologs for SWEETs and tonoplast sugar transporters. The analysis of transcriptional differences revealed a lesser and more measured global response for M. conductrix SAG 241.80 versus C. sorokiniana UTEX 1602 during conditions resulting in sugar release, providing a catalog of genes that might play a role in extracellular sugar transport.© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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