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September 22, 2019  |  

Lentinula edodes genome survey and postharvest transcriptome analysis.

Lentinula edodes is a popular, cultivated edible and medicinal mushroom. Lentinula edodes is susceptible to postharvest problems, such as gill browning, fruiting body softening, and lentinan degradation. We constructed a de novo assembly draft genome sequence and performed gene prediction for Lentinula edodesDe novo assembly was carried out using short reads from paired-end and mate-paired libraries and by using long reads by PacBio, resulting in a contig number of 1,951 and an N50 of 1 Mb. Furthermore, we predicted genes by Augustus using transcriptome sequencing (RNA-seq) data from the whole life cycle of Lentinula edodes, resulting in 12,959 predicted genes. This analysis revealed that Lentinula edodes lacks lignin peroxidase. To reveal genes involved in the loss of quality of Lentinula edodes postharvest fruiting bodies, transcriptome analysis was carried out using serial analysis of gene expression (SuperSAGE). This analysis revealed that many cell wall-related enzymes are upregulated after harvest, such as ß-1,3-1,6-glucan-degrading enzymes in glycoside hydrolase (GH) families GH5, GH16, GH30, GH55, and GH128, and thaumatin-like proteins. In addition, we found that several chitin-related genes are upregulated, such as putative chitinases in GH family 18, exochitinases in GH20, and a putative chitosanase in GH family 75. The results suggest that cell wall-degrading enzymes synergistically cooperate for rapid fruiting body autolysis. Many putative transcription factor genes were upregulated postharvest, such as genes containing high-mobility-group (HMG) domains and zinc finger domains. Several cell death-related proteins were also upregulated postharvest.IMPORTANCE Our data collectively suggest that there is a rapid fruiting body autolysis system in Lentinula edodes The genes for the loss of postharvest quality newly found in this research will be targets for the future breeding of strains that keep fresh longer than present strains. De novoLentinula edodes genome assembly data will be used for the construction of a complete Lentinula edodes chromosome map for future breeding. Copyright © 2017 American Society for Microbiology.

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September 22, 2019  |  

Soil microbial communities are shaped by plant-driven changes in resource availability during secondary succession.

Although we understand the ecological processes eliciting changes in plant community composition during secondary succession, we do not understand whether co-occurring changes in plant detritus shape saprotrophic microbial communities in soil. In this study, we investigated soil microbial composition and function across an old-field chronosequence ranging from 16 to 86 years following agricultural abandonment, as well as three forests representing potential late-successional ecosystems. Fungal and bacterial community composition was quantified from ribosomal DNA, and insight into the functional potential of the microbial community to decay plant litter was gained from shotgun metagenomics and extracellular enzyme assays. Accumulation of soil organic matter across the chronosequence exerted a positive and significant effect on fungal phylogenetic ß-diversity and the activity of extracellular enzymes with lignocellulolytic activity. In addition, the increasing abundance of lignin-rich C4 grasses was positively related to the composition of fungal genes with lignocellulolytic function, thereby linking plant community composition, litter biochemistry, and microbial community function. However, edaphic properties were the primary agent shaping bacterial communities, as bacterial ß-diversity and variation in functional gene composition displayed a significant and positive relationship to soil pH across the chronosequence. The late-successional forests were compositionally distinct from the oldest old fields, indicating that substantial changes occur in soil microbial communities as old fields give way to forests. Taken together, our observations demonstrate that plants govern the turnover of soil fungal communities and functional characteristics during secondary succession, due to the continual input of detritus and differences in litter biochemistry among plant species.

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September 22, 2019  |  

Computational analysis of alternative splicing in plant genomes.

Computational analyses play crucial roles in characterizing splicing isoforms in plant genomes. In this review, we provide a survey of computational tools used in recently published, genome-scale splicing analyses in plants. We summarize the commonly used software and pipelines for read mapping, isoform reconstruction, isoform quantification, and differential expression analysis. We also discuss methods for analyzing long reads and the strategies to combine long and short reads in identifying splicing isoforms. We review several tools for characterizing local splicing events, splicing graphs, coding potential, and visualizing splicing isoforms. We further discuss the procedures for identifying conserved splicing isoforms across plant species. Finally, we discuss the outlook of integrating other genomic data with splicing analyses to identify regulatory mechanisms of AS on genome-wide scale. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019  |  

Initial colonization, community assembly and ecosystem function: fungal colonist traits and litter biochemistry mediate decay rate.

Priority effects are an important ecological force shaping biotic communities and ecosystem processes, in which the establishment of early colonists alters the colonization success of later-arriving organisms via competitive exclusion and habitat modification. However, we do not understand which biotic and abiotic conditions lead to strong priority effects and lasting historical contingencies. Using saprotrophic fungi in a model leaf decomposition system, we investigated whether compositional and functional consequences of initial colonization were dependent on initial colonizer traits, resource availability or a combination thereof. To test these ideas, we factorially manipulated leaf litter biochemistry and initial fungal colonist identity, quantifying subsequent community composition, using neutral genetic markers, and community functional characteristics, including enzyme potential and leaf decay rates. During the first 3 months, initial colonist respiration rate and physiological capacity to degrade plant detritus were significant determinants of fungal community composition and leaf decay, indicating that rapid growth and lignolytic potential of early colonists contributed to altered trajectories of community assembly. Further, initial colonization on oak leaves generated increasingly divergent trajectories of fungal community composition and enzyme potential, indicating stronger initial colonizer effects on energy-poor substrates. Together, these observations provide evidence that initial colonization effects, and subsequent consequences on litter decay, are dependent upon substrate biochemistry and physiological traits within a regional species pool. Because microbial decay of plant detritus is important to global C storage, our results demonstrate that understanding the mechanisms by which initial conditions alter priority effects during community assembly may be key to understanding the drivers of ecosystem-level processes. © 2015 John Wiley & Sons Ltd.

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September 22, 2019  |  

Laboratory colonization stabilizes the naturally dynamic microbiome composition of field collected Dermacentor andersoni ticks.

Nearly a quarter of emerging infectious diseases identified in the last century are arthropod-borne. Although ticks and insects can carry pathogenic microorganisms, non-pathogenic microbes make up the majority of their microbial communities. The majority of tick microbiome research has had a focus on discovery and description; very few studies have analyzed the ecological context and functional responses of the bacterial microbiome of ticks. The goal of this analysis was to characterize the stability of the bacterial microbiome of Dermacentor andersoni ticks between generations and two populations within a species.The bacterial microbiome of D. andersoni midguts and salivary glands was analyzed from populations collected at two different ecologically distinct sites by comparing field (F1) and lab-reared populations (F1-F3) over three generations. The microbiome composition of pooled and individual samples was analyzed by sequencing nearly full-length 16S rRNA gene amplicons using a Pacific Biosciences CCS platform that allows identification of bacteria to the species level.In this study, we found that the D. andersoni microbiome was distinct in different geographic populations and was tissue specific, differing between the midgut and the salivary gland, over multiple generations. Additionally, our study showed that the microbiomes of laboratory-reared populations were not necessarily representative of their respective field populations. Furthermore, we demonstrated that the microbiome of a few individual ticks does not represent the microbiome composition at the population level.We demonstrated that the bacterial microbiome of D. andersoni was complex over three generations and specific to tick tissue (midgut vs. salivary glands) as well as geographic location (Burns, Oregon vs. Lake Como, Montana vs. laboratory setting). These results provide evidence that habitat of the tick population is a vital component of the complexity of the bacterial microbiome of ticks, and that the microbiome of lab colonies may not allow for comparative analyses with field populations. A broader understanding of microbiome variation will be required if we are to employ manipulation of the microbiome as a method for interfering with acquisition and transmission of tick-borne pathogens.

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September 22, 2019  |  

Assessing the gene content of the megagenome: sugar pine (Pinus lambertiana).

Sugar pine (Pinus lambertiana Douglas) is within the subgenus Strobus with an estimated genome size of 31 Gbp. Transcriptomic resources are of particular interest in conifers due to the challenges presented in their megagenomes for gene identification. In this study, we present the first comprehensive survey of the P. lambertiana transcriptome through deep sequencing of a variety of tissue types to generate more than 2.5 billion short reads. Third generation, long reads generated through PacBio Iso-Seq has been included for the first time in conifers to combat the challenges associated with de novo transcriptome assembly. A technology comparison is provided here contribute to the otherwise scarce comparisons of 2nd and 3rd generation transcriptome sequencing approaches in plant species. In addition, the transcriptome reference was essential for gene model identification and quality assessment in the parallel project responsible for sequencing and assembly of the entire genome. In this study, the transcriptomic data was also used to address some of the questions surrounding lineage-specific Dicer-like proteins in conifers. These proteins play a role in the control of transposable element proliferation and the related genome expansion in conifers. Copyright © 2016 Author et al.

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September 22, 2019  |  

A comparative transcriptional landscape of maize and sorghum obtained by single-molecule sequencing.

Maize and sorghum are both important crops with similar overall plant architectures, but they have key differences, especially in regard to their inflorescences. To better understand these two organisms at the molecular level, we compared expression profiles of both protein-coding and noncoding transcripts in 11 matched tissues using single-molecule, long-read, deep RNA sequencing. This comparative analysis revealed large numbers of novel isoforms in both species. Evolutionarily young genes were likely to be generated in reproductive tissues and usually had fewer isoforms than old genes. We also observed similarities and differences in alternative splicing patterns and activities, both among tissues and between species. The maize subgenomes exhibited no bias in isoform generation; however, genes in the B genome were more highly expressed in pollen tissue, whereas genes in the A genome were more highly expressed in endosperm. We also identified a number of splicing events conserved between maize and sorghum. In addition, we generated comprehensive and high-resolution maps of poly(A) sites, revealing similarities and differences in mRNA cleavage between the two species. Overall, our results reveal considerable splicing and expression diversity between sorghum and maize, well beyond what was reported in previous studies, likely reflecting the differences in architecture between these two species.© 2018 Wang et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019  |  

Application of circular consensus sequencing and network analysis to characterize the bovine IgG repertoire.

Vertebrate immune systems generate diverse repertoires of antibodies capable of mediating response to a variety of antigens. Next generation sequencing methods provide unique approaches to a number of immuno-based research areas including antibody discovery and engineering, disease surveillance, and host immune response to vaccines. In particular, single-molecule circular consensus sequencing permits the sequencing of antibody repertoires at previously unattainable depths of coverage and accuracy. We approached the bovine immunoglobulin G (IgG) repertoire with the objective of characterizing diversity of expressed IgG transcripts. Here we present single-molecule real-time sequencing data of expressed IgG heavy-chain repertoires of four individual cattle. We describe the diversity observed within antigen binding regions and visualize this diversity using a network-based approach.We generated 49,945 high quality cDNA sequences, each spanning the entire IgG variable region from four Bos taurus calves. From these sequences we identified 49,521 antigen binding regions using the automated Paratome web server. Approximately 9% of all unique complementarity determining 2 (CDR2) sequences were of variable lengths. A bimodal distribution of unique CDR3 sequence lengths was observed, with common lengths of 5-6 and 21-25 amino acids. The average number of cysteine residues in CDR3s increased with CDR3 length and we observed that cysteine residues were centrally located in CDR3s. We identified 19 extremely long CDR3 sequences (up to 62 amino acids in length) within IgG transcripts. Network analyses revealed distinct patterns among the expressed IgG antigen binding repertoires of the examined individuals.We utilized circular consensus sequencing technology to provide baseline data of the expressed bovine IgG repertoire that can be used for future studies important to livestock research. Somatic mutation resulting in base insertions and deletions in CDR2 further diversifies the bovine antibody repertoire. In contrast to previous studies, our data indicate that unusually long CDR3 sequences are not unique to IgM antibodies in cattle. Centrally located cysteine residues in bovine CDR3s provide further evidence that disulfide bond formation is likely of structural importance. We hypothesize that network or cluster-based analyses of expressed antibody repertoires from controlled challenge experiments will help identify novel natural antigen binding solutions to specific pathogens of interest.

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September 22, 2019  |  

Complete genome sequences of two genotype A2 small ruminant lentiviruses isolated from infected U.S. sheep.

Two distinct subgroups of genotype A2 small ruminant lentiviruses (SRLVs) have been identified in the United States that infect sheep with specific host transmembrane protein 154 (TMEM154) diplotypes. Here, we report the first two complete genome sequences of SRLV strains infecting U.S. sheep belonging to genotype A2, subgroups 1 and 2. Copyright © 2017 Workman et al.

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September 22, 2019  |  

Sequence of the sugar pine megagenome.

Until very recently, complete characterization of the megagenomes of conifers has remained elusive. The diploid genome of sugar pine (Pinus lambertiana Dougl.) has a highly repetitive, 31 billion bp genome. It is the largest genome sequenced and assembled to date, and the first from the subgenus Strobus, or white pines, a group that is notable for having the largest genomes among the pines. The genome represents a unique opportunity to investigate genome “obesity” in conifers and white pines. Comparative analysis of P. lambertiana and P. taeda L. reveals new insights on the conservation, age, and diversity of the highly abundant transposable elements, the primary factor determining genome size. Like most North American white pines, the principal pathogen of P. lambertiana is white pine blister rust (Cronartium ribicola J.C. Fischer ex Raben.). Identification of candidate genes for resistance to this pathogen is of great ecological importance. The genome sequence afforded us the opportunity to make substantial progress on locating the major dominant gene for simple resistance hypersensitive response, Cr1 We describe new markers and gene annotation that are both tightly linked to Cr1 in a mapping population, and associated with Cr1 in unrelated sugar pine individuals sampled throughout the species’ range, creating a solid foundation for future mapping. This genomic variation and annotated candidate genes characterized in our study of the Cr1 region are resources for future marker-assisted breeding efforts as well as for investigations of fundamental mechanisms of invasive disease and evolutionary response. Copyright © 2016 by the Genetics Society of America.

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September 22, 2019  |  

Metagenomic and near full-length 16S rRNA sequence data in support of the phylogenetic analysis of the rumen bacterial community in steers.

Amplicon sequencing utilizing next-generation platforms has significantly transformed how research is conducted, specifically microbial ecology. However, primer and sequencing platform biases can confound or change the way scientists interpret these data. The Pacific Biosciences RSII instrument may also preferentially load smaller fragments, which may also be a function of PCR product exhaustion during sequencing. To further examine theses biases, data is provided from 16S rRNA rumen community analyses. Specifically, data from the relative phylum-level abundances for the ruminal bacterial community are provided to determine between-sample variability. Direct sequencing of metagenomic DNA was conducted to circumvent primer-associated biases in 16S rRNA reads and rarefaction curves were generated to demonstrate adequate coverage of each amplicon. PCR products were also subjected to reduced amplification and pooling to reduce the likelihood of PCR product exhaustion during sequencing on the Pacific Biosciences platform. The taxonomic profiles for the relative phylum-level and genus-level abundance of rumen microbiota as a function of PCR pooling for sequencing on the Pacific Biosciences RSII platform were provided. For more information, see “Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers” P.R. Myer, M. Kim, H.C. Freetly, T.P.L. Smith (2016) [1].

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September 22, 2019  |  

PacBio for haplotyping in gene families.

The throughput and read length provided by Pacific Bioscience (PacBio) Single Molecule Real Time (SMRT) sequencing platform makes it feasible to construct contiguous, non-chimeric sequences. This is especially useful for genes with repetitive sequences in their gene bodies in gene families. We illustrate the use of PacBio to sequence and assemble hundreds of transcripts of gluten gene families from different cultivars of wheat using sequence from a single SMRT cell. To this end, we barcoded amplicons from different cultivars, then pooled these into one library for sequencing. Sequencing reads were later separated by the barcodes and further sorted into different gene groups by blast. The reads from each gene are then assembled by SeqmanNGen software. Given the length of 1 kb for each sequence derived from an initial molecule, the phase of the polymorphisms is not lost and can be used to infer also haplotype differences between different cultivars.

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September 22, 2019  |  

Genome-wide analysis of complex wheat gliadins, the dominant carriers of celiac disease epitopes.

Gliadins, specified by six compound chromosomal loci (Gli-A1/B1/D1 and Gli-A2/B2/D2) in hexaploid bread wheat, are the dominant carriers of celiac disease (CD) epitopes. Because of their complexity, genome-wide characterization of gliadins is a strong challenge. Here, we approached this challenge by combining transcriptomic, proteomic and bioinformatic investigations. Through third-generation RNA sequencing, full-length transcripts were identified for 52 gliadin genes in the bread wheat cultivar Xiaoyan 81. Of them, 42 were active and predicted to encode 25 a-, 11 ?-, one d- and five ?-gliadins. Comparative proteomic analysis between Xiaoyan 81 and six newly-developed mutants each lacking one Gli locus indicated the accumulation of 38 gliadins in the mature grains. A novel group of a-gliadins (the CSTT group) was recognized to contain very few or no CD epitopes. The d-gliadins identified here or previously did not carry CD epitopes. Finally, the mutant lacking Gli-D2 showed significant reductions in the most celiac-toxic a-gliadins and derivative CD epitopes. The insights and resources generated here should aid further studies on gliadin functions in CD and the breeding of healthier wheat.

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September 22, 2019  |  

Soil microbial communities and elk foraging intensity: implications for soil biogeochemical cycling in the sagebrush steppe.

Foraging intensity of large herbivores may exert an indirect top-down ecological force on soil microbial communities via changes in plant litter inputs. We investigated the responses of the soil microbial community to elk (Cervus elaphus) winter range occupancy across a long-term foraging exclusion experiment in the sagebrush steppe of the North American Rocky Mountains, combining phylogenetic analysis of fungi and bacteria with shotgun metagenomics and extracellular enzyme assays. Winter foraging intensity was associated with reduced bacterial richness and increasingly distinct bacterial communities. Although fungal communities did not respond linearly to foraging intensity, a greater ß-diversity response to winter foraging exclusion was observed. Furthermore, winter foraging exclusion increased soil cellulolytic and hemicellulolytic enzyme potential and higher foraging intensity reduced chitinolytic gene abundance. Thus, future changes in winter range occupancy may shape biogeochemical processes via shifts in microbial communities and subsequent changes to their physiological capacities to cycle soil C and N.© 2017 John Wiley & Sons Ltd/CNRS.

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September 22, 2019  |  

Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers.

Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplification primer selection, and read length, which can affect the apparent microbial community. In this study, we compared short read 16S rRNA variable regions, V1-V3, with that of near-full length 16S regions, V1-V8, using highly diverse steer rumen microbial communities, in order to examine the impact of technology selection on phylogenetic profiles. Short paired-end reads from the Illumina MiSeq platform were used to generate V1-V3 sequence, while long “circular consensus” reads from the Pacific Biosciences RSII instrument were used to generate V1-V8 data. The two platforms revealed similar microbial operational taxonomic units (OTUs), as well as similar species richness, Good’s coverage, and Shannon diversity metrics. However, the V1-V8 amplified ruminal community resulted in significant increases in several orders of taxa, such as phyla Proteobacteria and Verrucomicrobia (P < 0.05). Taxonomic classification accuracy was also greater in the near full-length read. UniFrac distance matrices using jackknifed UPGMA clustering also noted differences between the communities. These data support the consensus that longer reads result in a finer phylogenetic resolution that may not be achieved by shorter 16S rRNA gene fragments. Our work on the cattle rumen bacterial community demonstrates that utilizing near full-length 16S reads may be useful in conducting a more thorough study, or for developing a niche-specific database to use in analyzing data from shorter read technologies when budgetary constraints preclude use of near-full length 16S sequencing. Copyright © 2016 Elsevier B.V. All rights reserved.

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