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September 22, 2019  |  

Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides.

The basidiomycete yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded Agrobacterium tumefaciens T-DNA insertions. We identified 1,337 putative essential genes with low T-DNA insertion rates. We functionally profiled genes required for fatty acid catabolism and lipid accumulation, validating results with 35 targeted deletion strains. We identified a high-confidence set of 150 genes affecting lipid accumulation, including genes with predicted function in signaling cascades, gene expression, protein modification and vesicular trafficking, autophagy, amino acid synthesis and tRNA modification, and genes of unknown function. These results greatly advance our understanding of lipid metabolism in this oleaginous species and demonstrate a general approach for barcoded mutagenesis that should enable functional genomics in diverse fungi.

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September 22, 2019  |  

Genomics of habitat choice and adaptive evolution in a deep-sea fish.

Intraspecific diversity promotes evolutionary change, and when partitioned among geographic regions or habitats can form the basis for speciation. Marine species live in an environment that can provide as much scope for diversification in the vertical as in the horizontal dimension. Understanding the relevant mechanisms will contribute significantly to our understanding of eco-evolutionary processes and effective biodiversity conservation. Here, we provide an annotated genome assembly for the deep-sea fish Coryphaenoides rupestris and re-sequencing data to show that differentiation at non-synonymous sites in functional loci distinguishes individuals living at different depths, independent of horizontal spatial distance. Our data indicate disruptive selection at these loci; however, we find no clear evidence for differentiation at neutral loci that may indicate assortative mating. We propose that individuals with distinct genotypes at relevant loci segregate by depth as they mature (supported by survey data), which may be associated with ecotype differentiation linked to distinct phenotypic requirements at different depths.

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September 22, 2019  |  

The genome of the marine medaka Oryzias melastigma.

Marine medaka (Oryzias melastigma) is considered to be a useful fish model for marine and estuarine ecotoxicology studies and has good potential for field-based population genomics because of its geographical distribution in Asian estuarine and coastal areas. In this study, we present the first whole-genome draft of O. melastigma. The genome assembly consists of 8,602 scaffolds (N50 = 23.737 Mb) and a total genome length of 779.4 Mb. A total of 23,528 genes were predicted, and 12,670 gene families shared with three teleost species (Japanese medaka, mangrove killifish and zebrafish) were identified. Genome analyses revealed that the O. melastigma genome is highly heterozygous and contains a large number of repeat sequences. This assembly represents a useful genomic resource for fish scientists.© 2018 John Wiley & Sons Ltd.

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September 22, 2019  |  

A transposable element annotation pipeline and expression analysis reveal potentially active elements in the microalga Tisochrysis lutea.

Transposable elements (TEs) are mobile DNA sequences known as drivers of genome evolution. Their impacts have been widely studied in animals, plants and insects, but little is known about them in microalgae. In a previous study, we compared the genetic polymorphisms between strains of the haptophyte microalga Tisochrysis lutea and suggested the involvement of active autonomous TEs in their genome evolution.To identify potentially autonomous TEs, we designed a pipeline named PiRATE (Pipeline to Retrieve and Annotate Transposable Elements, download: https://doi.org/10.17882/51795 ), and conducted an accurate TE annotation on a new genome assembly of T. lutea. PiRATE is composed of detection, classification and annotation steps. Its detection step combines multiple, existing analysis packages representing all major approaches for TE detection and its classification step was optimized for microalgal genomes. The efficiency of the detection and classification steps was evaluated with data on the model species Arabidopsis thaliana. PiRATE detected 81% of the TE families of A. thaliana and correctly classified 75% of them. We applied PiRATE to T. lutea genomic data and established that its genome contains 15.89% Class I and 4.95% Class II TEs. In these, 3.79 and 17.05% correspond to potentially autonomous and non-autonomous TEs, respectively. Annotation data was combined with transcriptomic and proteomic data to identify potentially active autonomous TEs. We identified 17 expressed TE families and, among these, a TIR/Mariner and a TIR/hAT family were able to synthesize their transposase. Both these TE families were among the three highest expressed genes in a previous transcriptomic study and are composed of highly similar copies throughout the genome of T. lutea. This sum of evidence reveals that both these TE families could be capable of transposing or triggering the transposition of potential related MITE elements.This manuscript provides an example of a de novo transposable element annotation of a non-model organism characterized by a fragmented genome assembly and belonging to a poorly studied phylum at genomic level. Integration of multi-omics data enabled the discovery of potential mobile TEs and opens the way for new discoveries on the role of these repeated elements in genomic evolution of microalgae.

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September 22, 2019  |  

Gene duplication and evolution dynamics in the homeologous regions harboring multiple prolamin and resistance gene families in hexaploid wheat.

Improving end-use quality and disease resistance are important goals in wheat breeding. The genetic loci controlling these traits are highly complex, consisting of large families of prolamin and resistance genes with members present in all three homeologous A, B, and D genomes in hexaploid bread wheat. Here, orthologous regions harboring both prolamin and resistance gene loci were reconstructed and compared to understand gene duplication and evolution in different wheat genomes. Comparison of the two orthologous D regions from the hexaploid wheat Chinese Spring and the diploid progenitor Aegilops tauschii revealed their considerable difference due to the presence of five large structural variations with sizes ranging from 100 kb to 2 Mb. As a result, 44% of the Ae. tauschii and 71% of the Chinese Spring sequences in the analyzed regions, including 79 genes, are not shared. Gene rearrangement events, including differential gene duplication and deletion in the A, B, and D regions, have resulted in considerable erosion of gene collinearity in the analyzed regions, suggesting rapid evolution of prolamin and resistance gene families after the separation of the three wheat genomes. We hypothesize that this fast evolution is attributed to the co-evolution of the two gene families dispersed within a high recombination region. The identification of a full set of prolamin genes facilitated transcriptome profiling and revealed that the A genome contributes the least to prolamin expression because of its smaller number of expressed intact genes and their low expression levels, while the B and D genomes contribute similarly.

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September 22, 2019  |  

The complete chloroplast genome sequence of Actinidia arguta using the PacBio RS II platform.

Actinidia arguta is the most basal species in a phylogenetically and economically important genus in the family Actinidiaceae. To better understand the molecular basis of the Actinidia arguta chloroplast (cp), we sequenced the complete cp genome from A. arguta using Illumina and PacBio RS II sequencing technologies. The cp genome from A. arguta was 157,611 bp in length and composed of a pair of 24,232 bp inverted repeats (IRs) separated by a 20,463 bp small single copy region (SSC) and an 88,684 bp large single copy region (LSC). Overall, the cp genome contained 113 unique genes. The cp genomes from A. arguta and three other Actinidia species from GenBank were subjected to a comparative analysis. Indel mutation events and high frequencies of base substitution were identified, and the accD and ycf2 genes showed a high degree of variation within Actinidia. Forty-seven simple sequence repeats (SSRs) and 155 repetitive structures were identified, further demonstrating the rapid evolution in Actinidia. The cp genome analysis and the identification of variable loci provide vital information for understanding the evolution and function of the chloroplast and for characterizing Actinidia population genetics.

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September 22, 2019  |  

The genome of Rhizophagus clarus HR1 reveals a common genetic basis for auxotrophy among arbuscular mycorrhizal fungi.

Mycorrhizal symbiosis is one of the most fundamental types of mutualistic plant-microbe interaction. Among the many classes of mycorrhizae, the arbuscular mycorrhizae have the most general symbiotic style and the longest history. However, the genomes of arbuscular mycorrhizal (AM) fungi are not well characterized due to difficulties in cultivation and genetic analysis. In this study, we sequenced the genome of the AM fungus Rhizophagus clarus HR1, compared the sequence with the genome sequence of the model species R. irregularis, and checked for missing genes that encode enzymes in metabolic pathways related to their obligate biotrophy.In the genome of R. clarus, we confirmed the absence of cytosolic fatty acid synthase (FAS), whereas all mitochondrial FAS components were present. A KEGG pathway map identified the absence of genes encoding enzymes for several other metabolic pathways in the two AM fungi, including thiamine biosynthesis and the conversion of vitamin B6 derivatives. We also found that a large proportion of the genes encoding glucose-producing polysaccharide hydrolases, that are present even in ectomycorrhizal fungi, also appear to be absent in AM fungi.In this study, we found several new genes that are absent from the genomes of AM fungi in addition to the genes previously identified as missing. Missing genes for enzymes in primary metabolic pathways imply that AM fungi may have a higher dependency on host plants than other biotrophic fungi. These missing metabolic pathways provide a genetic basis to explore the physiological characteristics and auxotrophy of AM fungi.

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September 22, 2019  |  

RAD sequencing and a hybrid Antarctic fur seal genome assembly reveal rapidly decaying linkage disequilibrium, global population structure and evidence for inbreeding.

Recent advances in high throughput sequencing have transformed the study of wild organisms by facilitating the generation of high quality genome assemblies and dense genetic marker datasets. These resources have the potential to significantly advance our understanding of diverse phenomena at the level of species, populations and individuals, ranging from patterns of synteny through rates of linkage disequilibrium (LD) decay and population structure to individual inbreeding. Consequently, we used PacBio sequencing to refine an existing Antarctic fur seal (Arctocephalus gazella) genome assembly and genotyped 83 individuals from six populations using restriction site associated DNA (RAD) sequencing. The resulting hybrid genome comprised 6,169 scaffolds with an N50 of 6.21 Mb and provided clear evidence for the conservation of large chromosomal segments between the fur seal and dog (Canis lupus familiaris). Focusing on the most extensively sampled population of South Georgia, we found that LD decayed rapidly, reaching the background level by around 400 kb, consistent with other vertebrates but at odds with the notion that fur seals experienced a strong historical bottleneck. We also found evidence for population structuring, with four main Antarctic island groups being resolved. Finally, appreciable variance in individual inbreeding could be detected, reflecting the strong polygyny and site fidelity of the species. Overall, our study contributes important resources for future genomic studies of fur seals and other pinnipeds while also providing a clear example of how high throughput sequencing can generate diverse biological insights at multiple levels of organization. Copyright © 2018 Humble et al.

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September 22, 2019  |  

A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits.

Rose is the world’s most important ornamental plant, with economic, cultural and symbolic value. Roses are cultivated worldwide and sold as garden roses, cut flowers and potted plants. Roses are outbred and can have various ploidy levels. Our objectives were to develop a high-quality reference genome sequence for the genus Rosa by sequencing a doubled haploid, combining long and short reads, and anchoring to a high-density genetic map, and to study the genome structure and genetic basis of major ornamental traits. We produced a doubled haploid rose line (‘HapOB’) from Rosa chinensis ‘Old Blush’ and generated a rose genome assembly anchored to seven pseudo-chromosomes (512?Mb with N50 of 3.4?Mb and 564 contigs). The length of 512?Mb represents 90.1-96.1% of the estimated haploid genome size of rose. Of the assembly, 95% is contained in only 196 contigs. The anchoring was validated using high-density diploid and tetraploid genetic maps. We delineated hallmark chromosomal features, including the pericentromeric regions, through annotation of transposable element families and positioned centromeric repeats using fluorescent in situ hybridization. The rose genome displays extensive synteny with the Fragaria vesca genome, and we delineated only two major rearrangements. Genetic diversity was analysed using resequencing data of seven diploid and one tetraploid Rosa species selected from various sections of the genus. Combining genetic and genomic approaches, we identified potential genetic regulators of key ornamental traits, including prickle density and the number of flower petals. A rose APETALA2/TOE homologue is proposed to be the major regulator of petal number in rose. This reference sequence is an important resource for studying polyploidization, meiosis and developmental processes, as we demonstrated for flower and prickle development. It will also accelerate breeding through the development of molecular markers linked to traits, the identification of the genes underlying them and the exploitation of synteny across Rosaceae.

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September 22, 2019  |  

Conservation genomics of the declining North American bumblebee Bombus terricola reveals inbreeding and selection on immune genes.

The yellow-banded bumblebee Bombus terricola was common in North America but has recently declined and is now on the IUCN Red List of threatened species. The causes of B. terricola’s decline are not well understood. Our objectives were to create a partial genome and then use this to estimate population data of conservation interest, and to determine whether genes showing signs of recent selection suggest a specific cause of decline. First, we generated a draft partial genome (contig set) for B. terricola, sequenced using Pacific Biosciences RS II at an average depth of 35×. Second, we sequenced the individual genomes of 22 bumblebee gynes from Ontario and Quebec using Illumina HiSeq 2500, each at an average depth of 20×, which were used to improve the PacBio genome calls and for population genetic analyses. The latter revealed that several samples had long runs of homozygosity, and individuals had high inbreeding coefficient F, consistent with low effective population size. Our data suggest that B. terricola’s effective population size has decreased orders of magnitude from pre-Holocene levels. We carried out tests of selection to identify genes that may have played a role in ameliorating environmental stressors underlying B. terricola’s decline. Several immune-related genes have signatures of recent positive selection, which is consistent with the pathogen-spillover hypothesis for B. terricola’s decline. The new B. terricola contig set can help solve the mystery of bumblebee decline by enabling functional genomics research to directly assess the health of pollinators and identify the stressors causing declines.

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September 22, 2019  |  

A miR172 target-deficient AP2-like gene correlates with the double flower phenotype in roses.

One of the well-known floral abnormalities in flowering plants is the double-flower phenotype, which corresponds to flowers that develop extra petals, sometimes even containing entire flowers within flowers. Because of their highly priced ornamental value, spontaneous double-flower variants have been found and selected for in a wide range of ornamental species. Previously, double flower formation in roses was associated with a restriction of AGAMOUS expression domain toward the centre of the meristem, leading to extra petals. Here, we characterized the genomic region containing the mutation associated with the switch from simple to double flowers in the rose. An APETALA2-like gene (RcAP2L), a member of the Target Of EAT-type (TOE-type) subfamily, lies within this interval. In the double flower rose, two alleles of RcAP2L are present, one of which harbours a transposable element inserted into intron 8. This insertion leads to the creation of a miR172 resistant RcAP2L variant. Analyses of the presence of this variant in a set of simple and double flower roses demonstrate a correlation between the presence of this allele and the double flower phenotype. These data suggest a role of this miR172 resistant RcAP2L variant in regulating RcAGAMOUS expression and double flower formation in Rosa sp.

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September 22, 2019  |  

Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing.

The recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or “orphan” crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6-$9/sample) for consumables.We modified a non-organic solvent protocol to extract microgram quantities (1-13 µg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384-576 samples/day), and is inexpensive at US$0.62/sample for consumables.This versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.

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September 22, 2019  |  

Whole-genome resequencing and pan-transcriptome reconstruction highlight the impact of genomic structural Variation on secondary metabolite gene clusters in the grapevine Esca pathogen Phaeoacremonium minimum.

The Ascomycete fungus Phaeoacremonium minimum is one of the primary causal agents of Esca, a widespread and damaging grapevine trunk disease. Variation in virulence among Pm. minimum isolates has been reported, but the underlying genetic basis of the phenotypic variability remains unknown. The goal of this study was to characterize intraspecific genetic diversity and explore its potential impact on virulence functions associated with secondary metabolism, cellular transport, and cell wall decomposition. We generated a chromosome-scale genome assembly, using single molecule real-time sequencing, and resequenced the genomes and transcriptomes of multiple isolates to identify sequence and structural polymorphisms. Numerous insertion and deletion events were found for a total of about 1 Mbp in each isolate. Structural variation in this extremely gene dense genome frequently caused presence/absence polymorphisms of multiple adjacent genes, mostly belonging to biosynthetic clusters associated with secondary metabolism. Because of the observed intraspecific diversity in gene content due to structural variation we concluded that a transcriptome reference developed from a single isolate is insufficient to represent the virulence factor repertoire of the species. We therefore compiled a pan-transcriptome reference of Pm. minimum comprising a non-redundant set of 15,245 protein-coding sequences. Using naturally infected field samples expressing Esca symptoms, we demonstrated that mapping of meta-transcriptomics data on a multi-species reference that included the Pm. minimum pan-transcriptome allows the profiling of an expanded set of virulence factors, including variable genes associated with secondary metabolism and cellular transport.

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September 22, 2019  |  

Population genomics of Culiseta melanura, the principal vector of Eastern equine encephalitis virus in the United States.

Eastern Equine Encephalitis (EEE) (Togaviridae, Alphavirus) is a highly pathogenic mosquito-borne arbovirus that circulates in an enzootic cycle involving Culiseta melanura mosquitoes and wild Passeriformes birds in freshwater swamp habitats. Recently, the northeastern United States has experienced an intensification of virus activity with increased human involvement and northward expansion into new regions. In addition to its principal role in enzootic transmission of EEE virus among avian hosts, recent studies on the blood-feeding behavior of Cs. melanura throughout its geographic range suggest that this mosquito may also be involved in epizootic / epidemic transmission to equines and humans in certain locales. Variations in blood feeding behavior may be a function of host availability, environmental factors, and/or underlying genetic differences among regional populations. Despite the importance of Cs. melanura in transmission and maintenance of EEE virus, the genetics of this species remains largely unexplored.To investigate the occurrence of genetic variation in Cs. melanura, the genome of this mosquito vector was sequenced resulting in a draft genome assembly of 1.28 gigabases with a contig N50 of 93.36 kilobases. Populations of Cs. melanura from 10 EEE virus foci in the eastern North America were genotyped with double-digest RAD-seq. Following alignment of reads to the reference genome, variant calling, and filtering, 40,384 SNPs were retained for downstream analyses. Subsequent analyses revealed genetic differentiation between northern and southern populations of this mosquito species. Moreover, limited fine-scale population structure was detected throughout northeastern North America, suggesting local differentiation of populations but also a history of ancestral polymorphism or contemporary gene flow. Additionally, a genetically distinct cluster was identified predominantly at two northern sites.This study elucidates the first evidence of fine-scale population structure in Cs. melanura throughout its eastern range and detects evidence of gene flow between populations in northeastern North America. This investigation provides the groundwork for examining the consequences of genetic variations in the populations of this mosquito species that could influence vector-host interactions and the risk of human and equine infection with EEE virus.

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September 22, 2019  |  

Changes in the genetic requirements for microbial interactions with increasing community complexity.

Microbial community structure and function rely on complex interactions whose underlying molecular mechanisms are poorly understood. To investigate these interactions in a simple microbiome, we introduced E. coli into an experimental community based on a cheese rind and identified the differences in E. coli’s genetic requirements for growth in interactive and non-interactive contexts using Random Barcode Transposon Sequencing (RB-TnSeq) and RNASeq. Genetic requirements varied among pairwise growth conditions and between pairwise and community conditions. Our analysis points to mechanisms by which growth conditions change as a result of increasing community complexity and suggests that growth within a community relies on a combination of pairwise and higher-order interactions. Our work provides a framework for using the model organism E. coli as a readout to investigate microbial interactions regardless of the genetic tractability of members of the studied ecosystem.© 2018, Morin et al.

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