Schizosaccharomyces versatilis represents a distinct evolutionary lineage of fission yeast.

The fission yeast species Schizosaccharomyces japonicus is currently divided into two varieties-S. japonicus var. japonicus and S. japonicus var. versatilis. Here we examine the var. versatilis isolate CBS5679. The CBS5679 genome shows 88% identity to the reference genome of S. japonicus var. japonicus at the coding sequence level, with phylogenetic analyses suggesting that it has split from the S. japonicus lineage 25 million years ago. The CBS5679 genome contains a reciprocal translocation between chromosomes 1 and 2, together with several large inversions. The products of genes linked to the major translocation are associated with 'metabolism' and 'cellular assembly' ontology terms. We further show that CBS5679 does not generate viable progeny with the reference strain of S. japonicus. Although CBS5679 shares closer similarity to the 'type' strain of var. versatilis as compared to S. japonicus, it is not identical to the type strain, suggesting population structure within var. versatilis. We recommend that the taxonomic status of S. japonicus var. versatilis is raised, with it being treated as a separate species, Schizosaccharomyces versatilis.

Telomere-to-telomere Schizosaccharomyces japonicus genome assembly reveals hitherto unknown genome features.

Schizosaccharomyces japonicus belongs to the single-genus class Schizosaccharomycetes, otherwise known as "fission yeasts." As part of a composite model system with its widely studied S. pombe sister species, S. japonicus has provided critical insights into the workings and the evolution of cell biological mechanisms. Furthermore, its divergent biology makes S. japonicus a valuable model organism in its own right. However, the currently available genome assembly contains gaps and has been unable to resolve centromeres and other repeat-rich chromosomal regions. Here we present a telomere-to-telomere long-read genome assembly of the S. japonicus genome. This includes the three megabase-length chromosomes, with centromeres hundreds of kilobases long, rich in 5S ribosomal RNA genes, transfer RNA genes, long terminal repeats, and short repeats. We identify a gene-sparse region on chromosome 2 that resembles a 331 kb centromeric duplication. We revise the genome size of S. japonicus to at least 16.6 Mb and possibly up to 18.12 Mb, at least 30% larger than previous estimates. Our whole genome assembly will support the growing S. japonicus research community and facilitate research in new directions, including centromere and DNA repeat evolution, and yeast comparative genomics.

Extensive genome introgression between domestic ferret and European polecat during population recovery in Great Britain.

The European polecat (Mustela putorius) is a mammalian predator which occurs across much of Europe east to the Ural Mountains. In Great Britain, following years of persecution the range of the European polecat contracted and by the early 1900s was restricted to unmanaged forests of central Wales. The European polecat has recently undergone a population increase due to legal protection and its range now overlaps that of feral domestic ferrets (Mustela putorius furo). During this range expansion, European polecats hybridized with feral domestic ferrets producing viable offspring. Here, we carry out population-level whole-genome sequencing on 8 domestic ferrets, 19 British European polecats, and 15 European polecats from the European mainland. We used a range of population genomics methods to examine the data, including phylogenetics, phylogenetic graphs, model-based clustering, phylogenetic invariants, ABBA-BABA tests, topology weighting, and Fst. We found high degrees of genome introgression in British polecats outside their previous stronghold, even in those individuals phenotyped as "pure" polecats. These polecats ranged from presumed F1 hybrids (gamma = 0.53) to individuals that were much less introgressed (gamma = 0.2). We quantify this introgression and find introgressed genes containing Fst outliers associated with cognitive function and sight.

Whole genome resequencing data enables a targeted SNP panel for conservation and aquaculture of Oreochromis cichlid fishes.

Cichlid fish of the genus Oreochromis form the basis of the global tilapia aquaculture and fisheries industries. Broodstocks for aquaculture are often collected from wild populations, which in Africa may be from locations containing multiple Oreochromis species. However, many species are difficult to distinguish morphologically, hampering efforts to maintain good quality farmed strains. Additionally, non-native farmed tilapia populations are known to be widely distributed across Africa and to hybridize with native Oreochromis species, which themselves are important for capture fisheries. The morphological identification of these hybrids is particularly unreliable. Here, we describe the development of a single nucleotide polymorphism (SNP) genotyping panel from whole-genome resequencing data that enables targeted species identification in Tanzania. We demonstrate that an optimized panel of 96 genome-wide SNPs based on FST outliers performs comparably to whole genome resequencing in distinguishing species and identifying hybrids. We also show this panel outperforms microsatellite-based and phenotype-based classification methods. Case studies indicate several locations where introduced aquaculture species have become established in the wild, threatening native Oreochromis species. The novel SNP markers identified here represent an important resource for assessing broodstock purity in hatcheries and helping to conserve unique endemic biodiversity.

Newly discovered cichlid fish biodiversity threatened by hybridization with non-native species.

Invasive freshwater fishes are known to readily hybridize with indigenous congeneric species, driving loss of unique and irreplaceable genetic resources. Here we reveal that newly discovered (2013-2016) evolutionarily significant populations of Korogwe tilapia (Oreochromis korogwe) from southern Tanzania are threatened by hybridization with the larger invasive Nile tilapia (Oreochromis niloticus). We use a combination of morphology, microsatellite allele frequencies and whole genome sequences to show that O. korogwe from southern lakes (Nambawala, Rutamba and Mitupa) are distinct from geographically disjunct populations in northern Tanzania (Zigi River and Mlingano Dam). We also provide genetic evidence of O. korogwe × niloticus hybrids in three southern lakes and demonstrate heterogeneity in the extent of admixture across the genome. Finally, using the least admixed genomic regions we estimate that the northern and southern O. korogwe populations most plausibly diverged ~140,000 years ago, suggesting that the geographical separation of the northern and southern groups is not a result of a recent translocation, and instead these populations represent independent evolutionarily significant units. We conclude that these newly discovered and phenotypically unique cichlid populations are already threatened by hybridization with an invasive species, and propose that these irreplaceable genetic resources would benefit from conservation interventions.

Whole-genome sequencing of European autochthonous and commercial pig breeds allows the detection of signatures of selection for adaptation of genetic resources to different breeding and production systems.

BACKGROUND: Natural and artificial directional selection in cosmopolitan and autochthonous pig breeds and wild boars have shaped their genomes and resulted in a reservoir of animal genetic diversity. Signatures of selection are the result of these selection events that have contributed to the adaptation of breeds to different environments and production systems. In this study, we analysed the genome variability of 19 European autochthonous pig breeds (Alentejana, Bísara, Majorcan Black, Basque, Gascon, Apulo-Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano, Sarda, Krskopolje pig, Black Slavonian, Turopolje, Moravka, Swallow-Bellied Mangalitsa, Schwäbisch-Hällisches Schwein, Lithuanian indigenous wattle and Lithuanian White old type) from nine countries, three European commercial breeds (Italian Large White, Italian Landrace and Italian Duroc), and European wild boars, by mining whole-genome sequencing data obtained by using a DNA-pool sequencing approach. Signatures of selection were identified by using a single-breed approach with two statistics [within-breed pooled heterozygosity (HP) and fixation index (FST)] and group-based FST approaches, which compare groups of breeds defined according to external traits and use/specialization/type. RESULTS: We detected more than 22 million single nucleotide polymorphisms (SNPs) across the 23 compared populations and identified 359 chromosome regions showing signatures of selection. These regions harbour genes that are already known or new genes that are under selection and relevant for the domestication process in this species, and that affect several morphological and physiological traits (e.g. coat colours and patterns, body size, number of vertebrae and teats, ear size and conformation, reproductive traits, growth and fat deposition traits). Wild boar related signatures of selection were detected across all the genome of several autochthonous breeds, which suggests that crossbreeding (accidental or deliberate) occurred with wild boars. CONCLUSIONS: Our findings provide a catalogue of genetic variants of many European pig populations and identify genome regions that can explain, at least in part, the phenotypic diversity of these genetic resources.

Arabidopsis late blight: infection of a nonhost plant by Albugo laibachii enables full colonization by Phytophthora infestans.

The oomycete pathogen Phytophthora infestans causes potato late blight, and as a potato and tomato specialist pathogen, is seemingly poorly adapted to infect plants outside the Solanaceae. Here, we report the unexpected finding that P. infestans can infect Arabidopsis thaliana when another oomycete pathogen, Albugo laibachii, has colonized the host plant. The behaviour and speed of P. infestans infection in Arabidopsis pre-infected with A. laibachii resemble P. infestans infection of susceptible potato plants. Transcriptional profiling of P. infestans genes during infection revealed a significant overlap in the sets of secreted-protein genes that are induced in P. infestans upon colonization of potato and susceptible Arabidopsis, suggesting major similarities in P. infestans gene expression dynamics on the two plant species. Furthermore, we found haustoria of A. laibachii and P. infestans within the same Arabidopsis cells. This Arabidopsis-A. laibachii-P. infestans tripartite interaction opens up various possibilities to dissect the molecular mechanisms of P. infestans infection and the processes occurring in co-infected Arabidopsis cells.

Loss of a Conserved tRNA Anticodon Modification Perturbs Plant Immunity.

tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined. Despite their vastly different chemical structures and their presence in different tRNAs, occurring in different locations in tRNA, the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent discoveries have revealed unprecedented complexity in the modification patterns of tRNA, their regulation and function, suggesting that each modified nucleoside in tRNA may have its own specific function. However, in plants, our knowledge on the role of individual tRNA modifications and how they are regulated is very limited. In a genetic screen designed to identify factors regulating disease resistance and activation of defenses in Arabidopsis, we identified SUPPRESSOR OF CSB3 9 (SCS9). Our results reveal SCS9 encodes a tRNA methyltransferase that mediates the 2´-O-ribose methylation of selected tRNA species in the anticodon loop. These SCS9-mediated tRNA modifications enhance during the course of infection with the bacterial pathogen Pseudomonas syringae DC3000, and lack of such tRNA modification, as observed in scs9 mutants, severely compromise plant immunity against the same pathogen without affecting the salicylic acid (SA) signaling pathway which regulates plant immune responses. Our results support a model that gives importance to the control of certain tRNA modifications for mounting an effective immune response in Arabidopsis, and therefore expands the repertoire of molecular components essential for an efficient disease resistance response.

bio-samtools 2: a package for analysis and visualization of sequence and alignment data with SAMtools in Ruby.

MOTIVATION: bio-samtools is a Ruby language interface to SAMtools, the highly popular library that provides utilities for manipulating high-throughput sequence alignments in the Sequence Alignment/Map format. Advances in Ruby, now allow us to improve the analysis capabilities and increase bio-samtools utility, allowing users to accomplish a large amount of analysis using a very small amount of code. bio-samtools can also be easily developed to include additional SAMtools methods and hence stay current with the latest SAMtools releases. RESULTS: We have added new Ruby classes for the MPileup and Variant Call Format (VCF) data formats emitted by SAMtools and introduced more analysis methods for variant analysis, including alternative allele calculation and allele frequency calling for SNPs. Our new implementation of bio-samtools also ensures that all the functionality of the SAMtools library is now supported and that bio-samtools can be easily extended to include future changes in SAMtools. bio-samtools 2 also provides methods that allow the user to directly produce visualization of alignment data.

Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations.

RenSeq is a NB-LRR (nucleotide binding-site leucine-rich repeat) gene-targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB-LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB-LRRs and can be accessed through a genome browser that we provide. We compared these NB-LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum 'Heinz 1706' extended the NB-LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co-segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi-ber2) and S. ruiz-ceballosii (Rpi-rzc1), we were able to apply RenSeq successfully to identify markers that co-segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy-to-adapt Galaxy pipelines.

EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics.

BACKGROUND: Next Generation Sequencing technologies have facilitated differential gene expression analysis through RNA-seq and Tag-seq methods. RNA-seq has biases associated with transcript lengths, lacks uniform coverage of regions in mRNA and requires 10-20 times more reads than a typical Tag-seq. Most existing Tag-seq methods either have biases or not high throughput due to use of restriction enzymes or enzymatic manipulation of 5' ends of mRNA or use of RNA ligations. RESULTS: We have developed EXpression Profiling through Randomly Sheared cDNA tag Sequencing (EXPRSS) that employs acoustic waves to randomly shear cDNA and generate sequence tags at a relatively defined position (~150-200 bp) from the 3' end of each mRNA. Implementation of the method was verified through comparative analysis of expression data generated from EXPRSS, NlaIII-DGE and Affymetrix microarray and through qPCR quantification of selected genes. EXPRSS is a strand specific and restriction enzyme independent tag sequencing method that does not require cDNA length-based data transformations. EXPRSS is highly reproducible, is high-throughput and it also reveals alternative polyadenylation and polyadenylated antisense transcripts. It is cost-effective using barcoded multiplexing, avoids the biases of existing SAGE and derivative methods and can reveal polyadenylation position from paired-end sequencing. CONCLUSIONS: EXPRSS Tag-seq provides sensitive and reliable gene expression data and enables high-throughput expression profiling with relatively simple downstream analysis.

Defining the full tomato NB-LRR resistance gene repertoire using genomic and cDNA RenSeq.

BACKGROUND: The availability of draft crop plant genomes allows the prediction of the full complement of genes that encode NB-LRR resistance gene homologs, enabling a more targeted breeding for disease resistance. Recently, we developed the RenSeq method to reannotate the full NB-LRR gene complement in potato and to identify novel sequences that were not picked up by the automated gene prediction software. Here, we established RenSeq on the reference genome of tomato (Solanum lycopersicum) Heinz 1706, using 260 previously identified NB-LRR genes in an updated Solanaceae RenSeq bait library. RESULT: Using 250-bp MiSeq reads after RenSeq on genomic DNA of Heinz 1706, we identified 105 novel NB-LRR sequences. Reannotation included the splitting of gene models, combination of partial genes to a longer sequence and closing of assembly gaps. Within the draft S. pimpinellifolium LA1589 genome, RenSeq enabled the annotation of 355 NB-LRR genes. The majority of these are however fragmented, with 5'- and 3'-end located on the edges of separate contigs. Phylogenetic analyses show a high conservation of all NB-LRR classes between Heinz 1706, LA1589 and the potato clone DM, suggesting that all sub-families were already present in the last common ancestor. A phylogenetic comparison to the Arabidopsis thaliana NB-LRR complement verifies the high conservation of the more ancient CCRPW8-type NB-LRRs. Use of RenSeq on cDNA from uninfected and late blight-infected tomato leaves allows the avoidance of sequence analysis of non-expressed paralogues. CONCLUSION: RenSeq is a promising method to facilitate analysis of plant resistance gene complements. The reannotated tomato NB-LRR complements, phylogenetic relationships and chromosomal locations provided in this paper will provide breeders and scientists with a useful tool to identify novel disease resistance traits. cDNA RenSeq enables for the first time next-gen sequencing approaches targeted to this very low-expressed gene family without the need for normalization.

SVGenes: a library for rendering genomic features in scalable vector graphic format.

MOTIVATION: Drawing genomic features in attractive and informative ways is a key task in visualization of genomics data. Scalable Vector Graphics (SVG) format is a modern and flexible open standard that provides advanced features including modular graphic design, advanced web interactivity and animation within a suitable client. SVGs do not suffer from loss of image quality on re-scaling and provide the ability to edit individual elements of a graphic on the whole object level independent of the whole image. These features make SVG a potentially useful format for the preparation of publication quality figures including genomic objects such as genes or sequencing coverage and for web applications that require rich user-interaction with the graphical elements. RESULTS: SVGenes is a Ruby-language library that uses SVG primitives to render typical genomic glyphs through a simple and flexible Ruby interface. The library implements a simple Page object that spaces and contains horizontal Track objects that in turn style, colour and positions features within them. Tracks are the level at which visual information is supplied providing the full styling capability of the SVG standard. Genomic entities like genes, transcripts and histograms are modelled in Glyph objects that are attached to a track and take advantage of SVG primitives to render the genomic features in a track as any of a selection of defined glyphs. The feature model within SVGenes is simple but flexible and not dependent on particular existing gene feature formats meaning graphics for any existing datasets can easily be created without need for conversion. AVAILABILITY: The library is provided as a Ruby Gem from https://rubygems.org/gems/bio-svgenes under the MIT license, and open source code is available at https://github.com/danmaclean/bioruby-svgenes also under the MIT License. CONTACT: dan.maclean@tsl.ac.uk.

Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen.

Pest and pathogen losses jeopardise global food security and ever since the 19(th) century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.

Transcriptome analysis of the sulfate deficiency response in the marine microalga Emiliania huxleyi.

The response to sulfate deficiency of plants and freshwater green algae has been extensively analysed by system biology approaches. By contrast, seawater sulfate concentration is high and very little is known about the sulfur metabolism of marine organisms. Here, we used a combination of metabolite analysis and transcriptomics to analyse the response of the marine microalga Emiliania huxleyi as it acclimated to sulfate limitation. Lowering sulfate availability in artificial seawater from 25 to 5 mM resulted in significant reduction in growth and intracellular concentrations of dimethylsulfoniopropionate and glutathione. Sulfate-limited E. huxleyi cells showed increased sulfate uptake but sulfate reduction to sulfite did not seem to be regulated. Sulfate limitation in E. huxleyi affected expression of 1718 genes. The vast majority of these genes were upregulated, including genes involved in carbohydrate and lipid metabolism, and genes involved in the general stress response. The acclimation response of E. huxleyi to sulfate deficiency shows several similarities to the well-described responses of Arabidopsis and Chlamydomonas, but also has many unique features. This dataset shows that even though E. huxleyi is adapted to constitutively high sulfate concentration, it retains the ability to re-program its gene expression in response to reduced sulfate availability.

Panhematopoietic RNA barcoding enables kinetic measurements of nucleate and anucleate lineages and the activation of myeloid clones following acute platelet depletion.

BACKGROUND: Platelets and erythrocytes constitute over 95% of all hematopoietic stem cell output. However, the clonal dynamics of HSC contribution to these lineages remains largely unexplored. RESULTS: We use lentiviral genetic labeling of mouse hematopoietic stem cells to quantify output from all lineages, nucleate, and anucleate, simultaneously linking these with stem and progenitor cell transcriptomic phenotypes using single-cell RNA-sequencing. We observe dynamic shifts of clonal behaviors through time in same-animal peripheral blood and demonstrate that acute platelet depletion shifts the output of multipotent hematopoietic stem cells to the exclusive production of platelets. Additionally, we observe the emergence of new myeloid-biased clones, which support short- and long-term production of blood cells. CONCLUSIONS: Our approach enables kinetic studies of multi-lineage output in the peripheral blood and transcriptional heterogeneity of individual hematopoietic stem cells. Our results give a unique insight into hematopoietic stem cell reactivation upon platelet depletion and of clonal dynamics in both steady state and under stress.

Identification and localisation of the NB-LRR gene family within the potato genome.

BACKGROUND: The potato genome sequence derived from the Solanum tuberosum Group Phureja clone DM1-3 516 R44 provides unparalleled insight into the genome composition and organisation of this important crop. A key class of genes that comprises the vast majority of plant resistance (R) genes contains a nucleotide-binding and leucine-rich repeat domain, and is collectively known as NB-LRRs. RESULTS: As part of an effort to accelerate the process of functional R gene isolation, we performed an amino acid motif based search of the annotated potato genome and identified 438 NB-LRR type genes among the ~39,000 potato gene models. Of the predicted genes, 77 contain an N-terminal toll/interleukin 1 receptor (TIR)-like domain, and 107 of the remaining 361 non-TIR genes contain an N-terminal coiled-coil (CC) domain. Physical map positions were established for 370 predicted NB-LRR genes across all 12 potato chromosomes. The majority of NB-LRRs are physically organised within 63 identified clusters, of which 50 are homogeneous in that they contain NB-LRRs derived from a recent common ancestor. CONCLUSIONS: By establishing the phylogenetic and positional relationship of potato NB-LRRs, our analysis offers significant insight into the evolution of potato R genes. Furthermore, the data provide a blueprint for future efforts to identify and more rapidly clone functional NB-LRR genes from Solanum species.

Investigation of the potential for triterpene synthesis in rice through genome mining and metabolic engineering.

The first committed step in sterol biosynthesis in plants involves the cyclization of 2,3-oxidosqualene by the oxidosqualene cyclase (OSC) enzyme cycloartenol synthase. 2,3-Oxidosqualene is also a precursor for triterpene synthesis. Antimicrobial triterpenes are common in dicots, but seldom found in monocots, with the notable exception of oat. Here, through genome mining and metabolic engineering, we investigate the potential for triterpene synthesis in rice. The first two steps in the oat triterpene pathway are catalysed by a divergent OSC (AsbAS1) and a cytochrome P450 (CYP51). The genes for these enzymes form part of a metabolic gene cluster. To investigate the origins of triterpene synthesis in monocots, we analysed systematically the OSC and CYP51 gene families in rice. We also engineered rice for elevated triterpene content. We discovered a total of 12 OSC and 12 CYP51 genes in rice and uncovered key events in the evolution of triterpene synthesis. We further showed that the expression of AsbAS1 in rice leads to the accumulation of the simple triterpene, ß-amyrin. These findings provide new insights into the evolution of triterpene synthesis in monocots and open up opportunities for metabolic engineering for disease resistance in rice and other cereals.