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April 21, 2020  |  

Sequencing a Juglans regia?×?J. microcarpa hybrid yields high-quality genome assemblies of parental species.

Members of the genus Juglans are monecious wind-pollinated trees in the family Juglandaceae with highly heterozygous genomes, which greatly complicates genome sequence assembly. The genomes of interspecific hybrids are usually comprised of haploid genomes of parental species. We exploited this attribute of interspecific hybrids to avoid heterozygosity and sequenced an interspecific hybrid Juglans microcarpa?×?J. regia using a novel combination of single-molecule sequencing and optical genome mapping technologies. The resulting assemblies of both genomes were remarkably complete including chromosome termini and centromere regions. Chromosome termini consisted of arrays of telomeric repeats about 8?kb long and heterochromatic subtelomeric regions about 10?kb long. The centromeres consisted of arrays of a centromere-specific Gypsy retrotransposon and most contained genes, many of them transcribed. Juglans genomes evolved by a whole-genome-duplication dating back to the Cretaceous-Paleogene boundary and consist of two subgenomes, which were fractionated by numerous short gene deletions evenly distributed along the length of the chromosomes. Fractionation was shown to be asymmetric with one subgenome exhibiting greater gene loss than the other. The asymmetry of the process is ongoing and mirrors an asymmetry in gene expression between the subgenomes. Given the importance of J. microcarpa?×?J. regia hybrids as potential walnut rootstocks, we catalogued disease resistance genes in the parental genomes and studied their chromosomal distribution. We also estimated the molecular clock rates for woody perennials and deployed them in estimating divergence times of Juglans genomes and those of other woody perennials.

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April 21, 2020  |  

The complexity of the Fragaria x ananassa (octoploid) transcriptome by single-molecule long-read sequencing.

Strawberry (Fragaria x ananassa) is an allopolyploid species with diverse and complex transcripts. The regulatory mechanisms of fruit development and maturation have been extensively studied; however, little is known about the signaling mechanisms that direct this process in octoploid strawberry (Fragaria x ananassa). Here, we used long-read sequencing (LRS) technology and RNA-seq analysis to investigate the diversity and complexity of the polyploid transcriptome and differentially expressed transcripts along four successive fruit developmental stages of cultivated strawberry. We obtained a reference transcriptome with 119,897 unique full-length isoforms, including 2017 new isoforms and 2510 long noncoding RNAs. Based on the genome of the plausible progenitor (Fragaria vesca), 20,229 alternative splicing (AS) events were identified. Using this transcriptome, we found 17,485 differentially expressed transcripts during strawberry fruit development, including 527 transcription factors (TFs) belonging to 41 families. The expression profiles of all members of the auxin, ABA pathway, and anthocyanin biosynthesis gene families were also examined, and many of them were highly expressed at the ripe fruit stage, strongly indicating that the role of those genes is in the regulation of fruit ripening. We produce a high-quality reference transcriptome for octoploid strawberry, including much of the full-length transcript diversity, to help understand the regulatory mechanisms of fruit development and maturation of polyploid species, particularly via elucidation of the biochemical pathways involved in auxin, ABA, and anthocyanin biosynthesis.

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April 21, 2020  |  

Comparative genomic and phylogenetic analyses of Populus section Leuce using complete chloroplast genome sequences

Species of Populus section Leuce are distributed throughout most parts of the Northern Hemisphere and have important economic and ecological significance. However, due to frequent hybridization within Leuce, the phylogenetic relationship between species has not been clarified. The chloroplast (cp) genome is characterized by maternal inheritance and relatively conservative mutation rates; thus, it is a powerful tool for building phylogenetic trees. In this study, we used the PacBio SEQUEL software to determine that the cp genome of Populus tomentosa has a length of 156,558 bp including a long single-copy region (84,717 bp), a small single-copy region (16,555 bp), and a pair of inverted repeat regions (27,643 bp). The cp genome contains 131 unique genes, including 37 transfer RNAs, 8 ribosomal RNAs, and 86 protein-coding genes. We compared the cp genomes of seven species of section Leuce and identified five cp DNA markers with >?1% variable sites. Phylogenetic analyses revealed two evolutionary branches for section Leuce. The species with the closest relationship with P. tomenstosa was P. adenopoda, followed by P. alba. These cp genome data will help to determine the cp evolution of section Leuce and further elucidate the origin of P. tomentosa.

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April 21, 2020  |  

Full-length transcript sequencing and comparative transcriptomic analysis to evaluate the contribution of osmotic and ionic stress components towards salinity tolerance in the roots of cultivated alfalfa (Medicago sativa L.).

Alfalfa is the most extensively cultivated forage legume. Salinity is a major environmental factor that impacts on alfalfa’s productivity. However, little is known about the molecular mechanisms underlying alfalfa responses to salinity, especially the relative contribution of the two important components of osmotic and ionic stress.In this study, we constructed the first full-length transcriptome database for alfalfa root tips under continuous NaCl and mannitol treatments for 1, 3, 6, 12, and 24?h (three biological replicates for each time points, including the control group) via PacBio Iso-Seq. This resulted in the identification of 52,787 full-length transcripts, with an average length of 2551?bp. Global transcriptional changes in the same 33 stressed samples were then analyzed via BGISEQ-500 RNA-Seq. Totals of 8861 NaCl-regulated and 8016 mannitol-regulated differentially expressed genes (DEGs) were identified. Metabolic analyses revealed that these DEGs overlapped or diverged in the cascades of molecular networks involved in signal perception, signal transduction, transcriptional regulation, and antioxidative defense. Notably, several well characterized signalling pathways, such as CDPK, MAPK, CIPK, and PYL-PP2C-SnRK2, were shown to be involved in osmotic stress, while the SOS core pathway was activated by ionic stress. Moreover, the physiological shifts of catalase and peroxidase activity, glutathione and proline content were in accordance with dynamic transcript profiles of the relevant genes, indicating that antioxidative defense system plays critical roles in response to salinity stress.Overall, our study provides evidence that the response to salinity stress in alfalfa includes both osmotic and ionic components. The key osmotic and ionic stress-related genes are candidates for future studies as potential targets to improve resistance to salinity stress via genetic engineering.

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April 21, 2020  |  

Differential retention of transposable element-derived sequences in outcrossing Arabidopsis genomes.

Transposable elements (TEs) are genomic parasites with major impacts on host genome architecture and host adaptation. A proper evaluation of their evolutionary significance has been hampered by the paucity of short scale phylogenetic comparisons between closely related species. Here, we characterized the dynamics of TE accumulation at the micro-evolutionary scale by comparing two closely related plant species, Arabidopsis lyrata and A. halleri.Joint genome annotation in these two outcrossing species confirmed that both contain two distinct populations of TEs with either ‘recent’ or ‘old’ insertion histories. Identification of rare segregating insertions suggests that diverse TE families contribute to the ongoing dynamics of TE accumulation in the two species. Orthologous TE fragments (i.e. those that have been maintained in both species), tend to be located closer to genes than those that are retained in one species only. Compared to non-orthologous TE insertions, those that are orthologous tend to produce fewer short interfering RNAs, are less heavily methylated when found within or adjacent to genes and these tend to have lower expression levels. These findings suggest that long-term retention of TE insertions reflects their frequent acquisition of adaptive roles and/or the deleterious effects of removing nearly neutral TE insertions when they are close to genes.Our results indicate a rapid evolutionary dynamics of the TE landscape in these two outcrossing species, with an important input of a diverse set of new insertions with variable propensity to resist deletion.

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April 21, 2020  |  

Retrotranspositional landscape of Asian rice revealed by 3000 genomes.

The recent release of genomic sequences for 3000 rice varieties provides access to the genetic diversity at species level for this crop. We take advantage of this resource to unravel some features of the retrotranspositional landscape of rice. We develop software TRACKPOSON specifically for the detection of transposable elements insertion polymorphisms (TIPs) from large datasets. We apply this tool to 32 families of retrotransposons and identify more than 50,000 TIPs in the 3000 rice genomes. Most polymorphisms are found at very low frequency, suggesting that they may have occurred recently in agro. A genome-wide association study shows that these activations in rice may be triggered by external stimuli, rather than by the alteration of genetic factors involved in transposable element silencing pathways. Finally, the TIPs dataset is used to trace the origin of rice domestication. Our results suggest that rice originated from three distinct domestication events.

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April 21, 2020  |  

CAMISIM: simulating metagenomes and microbial communities.

Shotgun metagenome data sets of microbial communities are highly diverse, not only due to the natural variation of the underlying biological systems, but also due to differences in laboratory protocols, replicate numbers, and sequencing technologies. Accordingly, to effectively assess the performance of metagenomic analysis software, a wide range of benchmark data sets are required.We describe the CAMISIM microbial community and metagenome simulator. The software can model different microbial abundance profiles, multi-sample time series, and differential abundance studies, includes real and simulated strain-level diversity, and generates second- and third-generation sequencing data from taxonomic profiles or de novo. Gold standards are created for sequence assembly, genome binning, taxonomic binning, and taxonomic profiling. CAMSIM generated the benchmark data sets of the first CAMI challenge. For two simulated multi-sample data sets of the human and mouse gut microbiomes, we observed high functional congruence to the real data. As further applications, we investigated the effect of varying evolutionary genome divergence, sequencing depth, and read error profiles on two popular metagenome assemblers, MEGAHIT, and metaSPAdes, on several thousand small data sets generated with CAMISIM.CAMISIM can simulate a wide variety of microbial communities and metagenome data sets together with standards of truth for method evaluation. All data sets and the software are freely available at https://github.com/CAMI-challenge/CAMISIM.

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April 21, 2020  |  

Genome sequence and transcriptomic profiles of a marine bacterium, Pseudoalteromonas agarivorans Hao 2018.

Members of the marine genus Pseudoalteromonas have attracted great interest because of their ability to produce a large number of biologically active substances. Here, we report the complete genome sequence of Pseudoalteromonas agarivorans Hao 2018, a strain isolated from an abalone breeding environment, using second-generation Illumina and third-generation PacBio sequencing technologies. Illumina sequencing offers high quality and short reads, while PacBio technology generates long reads. The scaffolds of the two platforms were assembled to yield a complete genome sequence that included two circular chromosomes and one circular plasmid. Transcriptomic data for Pseudoalteromonas were not available. We therefore collected comprehensive RNA-seq data using Illumina sequencing technology from a fermentation culture of P. agarivorans Hao 2018. Researchers studying the evolution, environmental adaptations and biotechnological applications of Pseudoalteromonas may benefit from our genomic and transcriptomic data to analyze the function and expression of genes of interest.

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April 21, 2020  |  

Comprehensive analysis of full genome sequence and Bd-milRNA/target mRNAs to discover the mechanism of hypovirulence in Botryosphaeria dothidea strains on pear infection with BdCV1 and BdPV1

Pear ring rot disease, mainly caused by Botryosphaeria dothidea, is widespread in most pear and apple-growing regions. Mycoviruses are used for biocontrol, especially in fruit tree disease. BdCV1 (Botryosphaeria dothidea chrysovirus 1) and BdPV1 (Botryosphaeria dothidea partitivirus 1) influence the biological characteristics of B. dothidea strains. BdCV1 is a potential candidate for the control of fungal disease. Therefore, it is vital to explore interactions between B. dothidea and mycovirus to clarify the pathogenic mechanisms of B. dothidea and hypovirulence of B. dothidea in pear. A high-quality full-length genome sequence of the B. dothidea LW-Hubei isolate was obtained using Single Molecule Real-Time sequencing. It has high repeat sequence with 9.3% and DNA methylation existence in the genome. The 46.34?Mb genomes contained 14,091 predicted genes, which of 13,135 were annotated. B. dothidea was predicted to express 3833 secreted proteins. In bioinformatics analysis, 351 CAZy members, 552 transporters, 128 kinases, and 1096 proteins associated with plant-host interaction (PHI) were identified. RNA-silencing components including two endoribonuclease Dicer, four argonaute (Ago) and three RNA-dependent RNA polymerase (RdRp) molecules were identified and expressed in response to mycovirus infection. Horizontal transfer of the LW-C and LW-P strains indicated that BdCV1 induced host gene silencing in LW-C to suppress BdPV1 transmission. To investigate the role of RNA-silencing in B. dothidea defense, we constructed four small RNA libraries and sequenced B. dothidea micro-like RNAs (Bd-milRNAs) produced in response to BdCV1 and BdPV1 infection. Among these, 167 conserved and 68 candidate novel Bd-milRNAs were identified, of which 161 conserved and 20 novel Bd-milRNA were differentially expressed. WEGO analysis revealed involvement of the differentially expressed Bd-milRNA-targeted genes in metabolic process, catalytic activity, cell process and response to stress or stimulus. BdCV1 had a greater effect on the phenotype, virulence, conidiomata, vertical and horizontal transmission ability, and mycelia cellular structure biological characteristics of B. dothidea strains than BdPV1 and virus-free strains. The results obtained in this study indicate that mycovirus regulates biological processes in B. dothidea through the combined interaction of antiviral defense mediated by RNA-silencing and milRNA-mediated regulation of target gene mRNA expression.

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January 23, 2017  |  

Tutorial: Iso-Seq analysis application

This tutorial provides an overview of the Isoform Sequencing (Iso-Seq) analysis application. The Iso-Seq application provides reads that span entire transcript isoforms, from the 5′ end to the 3′ polyA-tail….

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