The genome and transcriptome of the snail Biomphalaria sudanica s.l.: immune gene diversification and highly polymorphic genomic regions in an important African vector of Schistosoma mansoni.

BACKGROUND: Control and elimination of schistosomiasis is an arduous task, with current strategies proving inadequate to break transmission. Exploration of genetic approaches to interrupt Schistosoma mansoni transmission, the causative agent for human intestinal schistosomiasis in sub-Saharan Africa and South America, has led to genomic research of the snail vector hosts of the genus Biomphalaria. Few complete genomic resources exist, with African Biomphalaria species being particularly underrepresented despite this being where the majority of S. mansoni infections occur. Here we generate and annotate the first genome assembly of Biomphalaria sudanica sensu lato, a species responsible for S. mansoni transmission in lake and marsh habitats of the African Rift Valley. Supported by whole-genome diversity data among five inbred lines, we describe orthologs of immune-relevant gene regions in the South American vector B. glabrata and present a bioinformatic pipeline to identify candidate novel pathogen recognition receptors (PRRs). RESULTS: De novo genome and transcriptome assembly of inbred B. sudanica originating from the shoreline of Lake Victoria (Kisumu, Kenya) resulted in a haploid genome size of ~ 944.2 Mb (6,728 fragments, N50 = 1.067 Mb), comprising 23,598 genes (BUSCO = 93.6% complete). The B. sudanica genome contains orthologues to all described immune genes/regions tied to protection against S. mansoni in B. glabrata, including the polymorphic transmembrane clusters (PTC1 and PTC2), RADres, and other loci. The B. sudanica PTC2 candidate immune genomic region contained many PRR-like genes across a much wider genomic region than has been shown in B. glabrata, as well as a large inversion between species. High levels of intra-species nucleotide diversity were seen in PTC2, as well as in regions linked to PTC1 and RADres orthologues. Immune related and putative PRR gene families were significantly over-represented in the sub-set of B. sudanica genes determined as hyperdiverse, including high extracellular diversity in transmembrane genes, which could be under pathogen-mediated balancing selection. However, no overall expansion in immunity related genes was seen in African compared to South American lineages. CONCLUSIONS: The B. sudanica genome and analyses presented here will facilitate future research in vector immune defense mechanisms against pathogens. This genomic/transcriptomic resource provides necessary data for the future development of molecular snail vector control/surveillance tools, facilitating schistosome transmission interruption mechanisms in Africa.

Comparative chemical genomics in Babesia species identifies the alkaline phosphatase PhoD as a determinant of antiparasitic resistance.

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites and the lack of specific drugs necessitate the discovery of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of Babesia spp. (B. bovis and B. divergens). We identified a potent antibabesial, MMV019266, from the Malaria Box, and selected for resistance in two species of Babesia. After sequencing of multiple independently derived lines in the two species, we identified mutations in a membrane-bound metallodependent phosphatase (phoD). In both species, the mutations were found in the phoD-like phosphatase domain. Using reverse genetics, we validated that mutations in bdphoD confer resistance to MMV019266 in B. divergens. We have also demonstrated that BdPhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of BdPhoD alter the sensitivity to MMV019266 in the parasite. Overexpression of BdPhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting BdPhoD is a pro-susceptibility factor. Together, we have generated a robust pipeline for identification of resistance loci and identified BdPhoD as a resistance mechanism in Babesia species.

The genome and transcriptome of the snail Biomphalaria sudanica s.l.: Immune gene diversification and highly polymorphic genomic regions in an important African vector of Schistosoma mansoni.

Background: Control and elimination of schistosomiasis is an arduous task, with current strategies proving inadequate to break transmission. Exploration of genetic approaches to interrupt Schistosoma mansoni transmission, the causative agent for human intestinal schistosomiasis in sub-Saharan Africa and South America, has led to genomic research of the snail vector hosts of the genus Biomphalaria. Few complete genomic resources exist, with African Biomphalaria species being particularly underrepresented despite this being where the majority of S. mansoni infections occur. Here we generate and annotate the first genome assembly of Biomphalaria sudanica sensu lato, a species responsible for S. mansoni transmission in lake and marsh habitats of the African Rift Valley. Supported by whole-genome diversity data among five inbred lines, we describe orthologs of immune-relevant gene regions in the South American vector B. glabrata and present a bioinformatic pipeline to identify candidate novel pathogen recognition receptors (PRRs). Results: De novo genome and transcriptome assembly of inbred B. sudanica originating from the shoreline of Lake Victoria (Kisumu, Kenya) resulted in a haploid genome size of ~944.2 Mb (6732 fragments, N50=1.067 Mb), comprising 23,598 genes (BUSCO=93.6% complete). The B. sudanica genome contains orthologues to all described immune genes/regions tied to protection against S. mansoni in B. glabrata. The B. sudanica PTC2 candidate immune genomic region contained many PRR-like genes across a much wider genomic region than has been shown in B. glabrata, as well as a large inversion between species. High levels of intra-species nucleotide diversity were seen in PTC2, as well as in regions linked to PTC1 and RADres orthologues. Immune related and putative PRR gene families were significantly over-represented in the sub-set of B. sudanica genes determined as hyperdiverse, including high extracellular diversity in transmembrane genes, which could be under pathogen-mediated balancing selection. However, no overall expansion in immunity related genes were seen in African compared to South American lineages. Conclusions: The B. sudanica genome and analyses presented here will facilitate future research in vector immune defense mechanisms against pathogens. This genomic/transcriptomic resource provides necessary data for the future development of molecular snail vector control/surveillance tools, facilitating schistosome transmission interruption mechanisms in Africa.

Comparative chemical genomics in Babesia species identifies the alkaline phosphatase phoD as a novel determinant of resistance.

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites, coupled with the lack of potent inhibitors necessitates the discovery of novel conserved druggable targets for the generation of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of novel and conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of evolutionarily-related Babesia spp. ( B. bovis and B. divergens ). We identified a potent antibabesial inhibitor from the Malaria Box, MMV019266. We were able to select for resistance to this compound in two species of Babesia, achieving 10-fold or greater resistance after ten weeks of intermittent selection. After sequencing of multiple independently derived lines in the two species, we identified mutations in a single conserved gene in both species: a membrane-bound metallodependent phosphatase (putatively named PhoD). In both species, the mutations were found in the phoD-like phosphatase domain, proximal to the predicted ligand binding site. Using reverse genetics, we validated that mutations in PhoD confer resistance to MMV019266. We have also demonstrated that PhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of PhoD alter the sensitivity to MMV019266 in the parasite: overexpression of PhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting PhoD is a resistance mechanism. Together, we have generated a robust pipeline for identification of resistance loci, and identified PhoD as a novel determinant of resistance in Babesia species. Highlights: Use of two species for in vitro evolution identifies a high confidence locus associated with resistance Resistance mutation in phoD was validated using reverse genetics in B. divergens Perturbation of phoD using function genetics results in changes in the level of resistance to MMV019266Epitope tagging reveals localization to the ER/apicoplast, a conserved localization with a similar protein in diatoms Together, phoD is a novel resistance determinant in multiple Babesia spp .

PTC2 region genotypes counteract Biomphalaria glabrata population differences between M-line and BS90 in resistance to infection by Schistosoma mansoni.

Background: Biomphalaria glabrata is a snail intermediate host for Schistosoma mansoni, a trematode responsible for human schistosomiasis. BS90 is one of the most well studied strains of B. glabrata owing to its high resistance to infection by most strains of S. mansoni. An F2 mapping study from 1999 identified two RAPD markers that associated with what appeared to be single-locus, dominant resistance by the BS90 population relative to the susceptible M-line population. One marker cannot be mapped, but the other, OPM-04, maps to within 5 Mb of PTC2, a region we recently showed has a very large effect on resistance within another snail population challenged by the same strain of parasite (PR1). Here we tested the hypothesis that the PTC2 region contains the causal gene/s that explain the iconic resistance of BS90 snails. Methods: We used marker-assisted backcrossing to drive the BS90 version of the PTC2 region (+/-~1 Mb on either side) into an M-line (susceptible strain) genetic background, and the M-line version into a BS90 genetic background. We challenged the offspring with PR1-strain schistosomes and tested for effects of allelic variation in the PTC2 region in a common genetic background. Results: Relative to M-line haplotypes, the BS90 haplotype actually confers enhanced susceptibility. So we reject our original hypothesis. One possible explanation for our result was that the causal gene linked to OPM-04 is near, but not in the PTC2 block that we introgressed into each line. So we used an F2 cross to independently test the effects of the PTC2 and OPM-04 regions in a randomized genetic background. We confirmed that the BS90 haplotype confers increased susceptibility, and we see a similar, although non-significant effect at OPM-04. We discuss possible reasons why our results differed so dramatically from those of the 1999 study. We also present Pacbio assemblies of the PTC2 and flanking region in BS90 and M-line, compare with previously published PTC2 haplotypes, and discuss candidate genes that might be behind the enhanced susceptibility of the BS90 haplotype.

Integration of whole genome sequencing and transcriptomics reveals a complex picture of the reestablishment of insecticide resistance in the major malaria vector Anopheles coluzzii.

Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids, and a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance.

A population genomic unveiling of a new cryptic mosquito taxon within the malaria-transmitting Anopheles gambiae complex.

The Anopheles gambiae complex consists of multiple morphologically indistinguishable mosquito species including the most important vectors of the malaria parasite Plasmodium falciparum in sub-Saharan Africa. Nine cryptic species have been described so far within the complex. The ecological, immunological and reproductive differences among these species will critically impact population responses to disease control strategies and environmental changes. Here, we examine whole-genome sequencing data from a longitudinal study of putative A. coluzzii in western Burkina Faso. Surprisingly, many specimens are genetically divergent from A. coluzzii and all other Anopheles species and represent a new taxon, here designated Anopheles TENGRELA (AT). Population genetic analysis suggests that the cryptic GOUNDRY subgroup, previously collected as larvae in central Burkina Faso, represents an admixed population descended from both A. coluzzii and AT. AT harbours low nucleotide diversity except for the 2La inversion polymorphism which is maintained by overdominance. It shows numerous fixed differences with A. coluzzii concentrated in several regions reflecting selective sweeps, but the two taxa are identical at standard diagnostic loci used for taxon identification, and thus, AT may often go unnoticed. We present an amplicon-based genotyping assay for identifying AT which could be usefully applied to numerous existing samples. Misidentified cryptic taxa could seriously confound ongoing studies of Anopheles ecology and evolution in western Africa, including phenotypic and genotypic surveys of insecticide resistance. Reproductive barriers between cryptic species may also complicate novel vector control efforts, for example gene drives, and hinder predictions about evolutionary dynamics of Anopheles and Plasmodium.

Three Signatures of Adaptive Polymorphism Exemplified by Malaria-Associated Genes.

Malaria has been one of the strongest selective pressures on our species. Many of the best-characterized cases of adaptive evolution in humans are in genes tied to malaria resistance. However, the complex evolutionary patterns at these genes are poorly captured by standard scans for nonneutral evolution. Here, we present three new statistical tests for selection based on population genetic patterns that are observed more than once among key malaria resistance loci. We assess these tests using forward-time evolutionary simulations and apply them to global whole-genome sequencing data from humans, and thus we show that they are effective at distinguishing selection from neutrality. Each test captures a distinct evolutionary pattern, here called Divergent Haplotypes, Repeated Shifts, and Arrested Sweeps, associated with a particular period of human prehistory. We clarify the selective signatures at known malaria-relevant genes and identify additional genes showing similar adaptive evolutionary patterns. Among our top outliers, we see a particular enrichment for genes involved in erythropoiesis and for genes previously associated with malaria resistance, consistent with a major role for malaria in shaping these patterns of genetic diversity. Polymorphisms at these genes are likely to impact resistance to malaria infection and contribute to ongoing host-parasite coevolutionary dynamics.

Clusters of polymorphic transmembrane genes control resistance to schistosomes in snail vectors.

Schistosomiasis is a debilitating parasitic disease infecting hundreds of millions of people. Schistosomes use aquatic snails as intermediate hosts. A promising avenue for disease control involves leveraging innate host mechanisms to reduce snail vectorial capacity. In a genome-wide association study of Biomphalaria glabrata snails, we identify genomic region PTC2 which exhibits the largest known correlation with susceptibility to parasite infection (>15 fold effect). Using new genome assemblies with substantially higher contiguity than the Biomphalaria reference genome, we show that PTC2 haplotypes are exceptionally divergent in structure and sequence. This variation includes multi-kilobase indels containing entire genes, and orthologs for which most amino acid residues are polymorphic. RNA-Seq annotation reveals that most of these genes encode single-pass transmembrane proteins, as seen in another resistance region in the same species. Such groups of hyperdiverse snail proteins may mediate host-parasite interaction at the cell surface, offering promising targets for blocking the transmission of schistosomiasis.

Schistosomiasis is a widespread parasitic disease, affecting over 200 million people in tropical countries. It is caused by schistosome worms, which are carried by freshwater snails. These snails release worm larvae into the water, where they can infect humans ­ for example, after bathing or swimming. Treatment options for schistosomiasis are limited. Eliminating the freshwater snails is one way to control the disease, but this is not always effective in the long term and the chemicals used can also harm other animals in the water. Another way to manage schistosomiasis could be to stop the worms from infecting their snail host by breaking the parasites' life cycle without killing the snails. It is already known that some snails are naturally resistant to infection by some strains of schistosomes. Since this immunity is also inherited by the offspring of resistant snails, there is likely a genetic mechanism behind it. However, very little else is known about any genes that might be involved. Tennessen et al. therefore set out to identify what genes were responsible for schistosome resistance and how they worked. The experiments used a large laboratory colony of snails, whose susceptibility to schistosome infection varied among individual animals. To determine the genes behind this variation, Tennessen et al. first searched for areas of DNA that also differed between the immune and infected snails. Comparing genetic sequences across over 1,000 snails revealed a distinct region of DNA that had a large effect on how likely they were to be infected. This section of DNA turned out to be highly diverse, with different snails carrying varying numbers and different forms of the genes within this region. Many of these genes appear to encode proteins found on the surface of snail cells, which could affect whether snails and worms can recognize each other when they come into contact. This in turn could determine whether or not the worms can infect their hosts. These results shed new light on how the snails that carry schistosomes may be able to resist infections. In the future, this knowledge could be key to controlling schistosomiasis, either by releasing genetically engineered, immune snails into the wild (thus making it harder for the parasites to reproduce) or by using the snails' mechanism of resistance to design better drug therapies.

Allelic variation in a single genomic region alters the hemolymph proteome in the snail Biomphalaria glabrata.

Freshwater snails are obligate intermediate hosts for numerous parasitic trematodes, most notably schistosomes. Schistosomiasis is a devastating human and veterinary illness, which is primarily controlled by limiting the transmission of these parasites from their intermediate snail hosts. Understanding how this transmission occurs, as well as the basic immunobiology of these snails may be important for controlling this disease in the future. Allelic variation in the Guadeloupe resistance complex (GRC) of Biomphalaria glabrata partially determines their susceptibility to parasitic infection, and can influence the microbiome diversity and microbial defenses in the hemolymph of these snails. In the present study, we examine the most abundant proteins present in the hemolymph of snails that are resistant or susceptible to schistosomes, as determined by their GRC genotype. Using proteomic analysis, we found that snails with different GRC genotypes have differentially abundant hemolymph proteins that are not explained by differences in transcription. There are 13 revealed hemolymph proteins that differ significantly between resistant and susceptible genotypes, nearly 40% of which are involved in immune responses. These findings build on the mounting evidence that genes in the GRC region have multiple physiological roles, and likely contribute more extensively to the general immune response than previously believed. These data also raise the intriguing possibility that the GRC region controls resistance to schistosomes, not directly, but indirectly via its effects on the snail's proteome and potentially its microbiome.

Repeated translocation of a gene cassette drives sex-chromosome turnover in strawberries.

Turnovers of sex-determining systems represent important diversifying forces across eukaryotes. Shifts in sex chromosomes-but conservation of the master sex-determining genes-characterize distantly related animal lineages. Yet in plants, in which separate sexes have evolved repeatedly and sex chromosomes are typically homomorphic, we do not know whether such translocations drive sex-chromosome turnovers within closely related taxonomic groups. This phenomenon can only be demonstrated by identifying sex-associated nucleotide sequences, still largely unknown in plants. The wild North American octoploid strawberries (Fragaria) exhibit separate sexes (dioecy) with homomorphic, female heterogametic (ZW) inheritance, yet sex maps to three different chromosomes in different taxa. To characterize these turnovers, we identified sequences unique to females and assembled their reads into contigs. For most octoploid Fragaria taxa, a short (13 kb) sequence was observed in all females and never in males, implicating it as the sex-determining region (SDR). This female-specific "SDR cassette" contains both a gene with a known role in fruit and pollen production and a novel retrogene absent on Z and autosomal chromosomes. Phylogenetic comparison of SDR cassettes revealed three clades and a history of repeated translocation. Remarkably, the translocations can be ordered temporally due to the capture of adjacent sequence with each successive move. The accumulation of the "souvenir" sequence-and the resultant expansion of the hemizygous SDR over time-could have been adaptive by locking genes into linkage with sex. Terminal inverted repeats at the insertion borders suggest a means of movement. To our knowledge, this is the first plant SDR shown to be translocated, and it suggests a new mechanism ("move-lock-grow") for expansion and diversification of incipient sex chromosomes.

Gene buddies: linked balanced polymorphisms reinforce each other even in the absence of epistasis.

The fates of genetic polymorphisms maintained by balancing selection depend on evolutionary dynamics at linked sites. While coevolution across linked, epigenetically-interacting loci has been extensively explored, such supergenes may be relatively rare. However, genes harboring adaptive variation can occur in close physical proximity while generating independent effects on fitness. Here, I present a model in which two linked loci without epistasis are both under balancing selection for unrelated reasons. Using forward-time simulations, I show that recombination rate strongly influences the retention of adaptive polymorphism, especially for intermediate selection coefficients. A locus is more likely to retain adaptive variation if it is closely linked to another locus under balancing selection, even if the two loci have no interaction. Thus, two linked polymorphisms can both be retained indefinitely even when they would both be lost to drift if unlinked. While these results may be intuitive, they have important implications for genetic architecture: clusters of mutually reinforcing genes may underlie phenotypic variation in natural populations, and such genes cannot be assumed to be functionally associated. Future studies that measure selection coefficients and recombination rates among closely linked genes will be fruitful for characterizing the extent of this phenomenon.

Plastid genomes reveal recurrent formation of allopolyploid Fragaria.

PREMISE OF THE STUDY: Recurrent formation of polyploid taxa is a common observation in many plant groups. Haploid, cytoplasmic genomes like the plastid genome can be used to overcome the problem of homeologous genes and recombination in polyploid taxa. Fragaria (Rosaceae) contains several octo- and decaploid species. We use plastome sequences to infer the plastid ancestry of these taxa with special focus on the decaploid Fragaria cascadensis. METHODS: We used genome skimming of 96 polyploid Fragaria samples on a single Illumina HiSeq 3000 lane to obtain whole plastome sequences. These sequences were used for phylogenetic reconstructions and dating analyses. Ploidy of all samples was inferred with flow cytometry, and plastid inheritance was examined in a controlled cross of F. cascadensis. KEY RESULTS: The plastid genome phylogeny shows that only the octoploid F. chiloensis is monophyletic, all other polyploid taxa were supported to be para- or polyphyletic. The decaploid Fragaria cascadensis has biparental plastid inheritance and four different plastid donors. Diversification of the F. cascadensis clades occurred in the last 230,000 years. The southern part of its distribution range harbors considerably higher genetic diversity, suggestive of a potential refugium. CONCLUSIONS: Fragaria cascadensis had at least four independent origins from parents with different plastomes. In contrast, para- and polyphyletic taxa of the octoploid Fragaria species are best explained by incomplete lineage sorting and/or hybridization. Biogeographic patterns in F. cascadensis are probably a result of range shift during the last glacial maximum.

Rapid genetic adaptation to a novel environment despite a genome-wide reduction in genetic diversity.

Introduced species often colonize regions that have vastly different ecological and environmental conditions than those found in their native range. As such, species introductions can provide a deeper understanding into the process of adaptive evolution. In the 1880s, steelhead trout (Oncorhynchus mykiss) from California were introduced into Lake Michigan (Laurentian Great Lakes, North America) where they established naturally reproducing populations. In their native range, steelhead hatch in rivers, migrate to the ocean and return to freshwater to spawn. Steelhead in Lake Michigan continue to swim up rivers to spawn, but now treat the freshwater environment of the Great Lakes as a surrogate ocean. To examine the effects of this introduction, we sequenced the genomes of 264 fish. By comparing steelhead from Lake Michigan to steelhead from their ancestral range, we determined that the introduction led to consistent reductions in genetic diversity across all 29 chromosomes. Despite this reduction in genetic diversity, three chromosomal regions were associated with rapid genetic adaptation to the novel environment. The first region contained functional changes to ceramide kinase, which likely altered metabolic and wound-healing rates in Lake Michigan steelhead. The second and third regions encoded carbonic anhydrases and a solute carrier protein, both of which are critical for osmoregulation, and demonstrate how steelhead physiologically adapted to freshwater. Furthermore, the contemporary release of diverse hatchery strains into the lake increased genetic diversity but reduced the signature of genetic adaptation. This study illustrates that species can rapidly adapt to novel environments despite genome-wide reductions in genetic diversity.

Allelic Variation in a Single Genomic Region Alters the Microbiome of the Snail Biomphalaria glabrata.

Freshwater snails are the intermediate hosts for numerous parasitic worms which can have negative consequences for human health and agriculture. Understanding the transmission of these diseases requires a more complete characterization of the immunobiology of snail hosts. This includes the characterization of its microbiome and genetic factors which may interact with this important commensal community. Allelic variation in the Guadeloupe resistance complex (GRC) genomic region of Guadeloupean Biomphalaria glabrata influences their susceptibility to schistosome infection and may have other roles in the snail immune response. In the present study, we examined whether a snail's GRC genotype has a role in shaping the bacterial diversity and composition present on or in whole snails. We show that the GRC haplotype, including the resistant genotype, has a significant effect on the diversity of bacterial species present in or on whole snails, including the relative abundances of Gemmatimonas aurantiaca and Micavibrio aeruginosavorus. These findings support the hypothesis that the GRC region is likely involved in pathways that can modify the microbial community of these snails and may have more immune roles in B. glabrata than originally believed. This is also one of few examples in which allelic variation at a particular locus has been shown to affect the microbiome in any species.

Genetic Mapping and Phylogenetic Analysis Reveal Intraspecific Variation in Sex Chromosomes of the Virginian Strawberry.

With their extraordinary diversity in sexual systems, flowering plants offer unparalleled opportunities to understand sex determination and to reveal generalities in the evolution of sex chromosomes. Comparative genetic mapping of related taxa with good phylogenetic resolution can delineate the extent of sex chromosome diversity within plant groups, and lead the way to understanding the evolutionary drivers of such diversity. The North American octoploid wild strawberries provide such an opportunity. We performed linkage mapping using targeted sequence capture for the subdioecious western Fragaria virginiana ssp. platypetala and compared the location of its sex-determining region (SDR) to those of 2 other (sub)dioecious species, the eastern subspecies, F. virginiana ssp. virginiana (whose SDR is at 0-5.5 Mb on chromosome VI of the B2 subgenome), and the sister species F. chiloensis (whose SDR is at 37 Mb on chromosome VI of the Av subgenome). Male sterility was dominant in F. virginiana ssp. platypetala and mapped to a chromosome also in homeologous group VI. Likewise, one major quantitative trait locus (QTL) for female fertility overlapped the male sterility region. However, the SDR mapped to yet another subgenome (B1), and to a different location (13 Mb), but similar to the location inferred in one population of the naturally occurring hybrid between F. chiloensis and F. virginiana (F. ×ananassa ssp. cuneifolia). Phylogenetic analysis of chromosomes across the octoploid taxa showed consistent subgenomic composition reflecting shared evolutionary history but also reinforced within-species variation in the SDR-carrying chromosome, suggesting either repeated evolution, or recent turnovers in SDR.

Genotyping-by-sequencing enables linkage mapping in three octoploid cultivated strawberry families.

Genotyping-by-sequencing (GBS) was used to survey genome-wide single-nucleotide polymorphisms (SNPs) in three biparental strawberry (Fragaria × ananassa) populations with the goal of evaluating this technique in a species with a complex octoploid genome. GBS sequence data were aligned to the F. vesca 'Fvb' reference genome in order to call SNPs. Numbers of polymorphic SNPs per population ranged from 1,163 to 3,190. Linkage maps consisting of 30-65 linkage groups were produced from the SNP sets derived from each parent. The linkage groups covered 99% of the Fvb reference genome, with three to seven linkage groups from a given parent aligned to any particular chromosome. A phylogenetic analysis performed using the POLiMAPS pipeline revealed linkage groups that were most similar to ancestral species F. vesca for each chromosome. Linkage groups that were most similar to a second ancestral species, F. iinumae, were only resolved for Fvb 4. The quantity of missing data and heterogeneity in genome coverage inherent in GBS complicated the analysis, but POLiMAPS resolved F. × ananassa chromosomal regions derived from diploid ancestor F. vesca.

Present-day sympatry belies the evolutionary origin of a high-order polyploid.

Disentangling the evolutionary histories of polyploids, especially those with high ploidies, can reveal fundamental processes in speciation. Despite occurring frequently during evolution, the origins of many extant polyploid plant species remain largely unknown. By integrating linkage mapping, polyploid phylogeny and sex-determining region (SDR) in a unified framework, we statistically evaluated evolutionary hypotheses concerning the origin of a recently recognized decaploid strawberry (Fragaria cascadensis). The maximum-likelihood phylogenies and topology tests across homeologous groups consistently rejected the seemingly parsimonious hypothesis of 'contemporary sympatric speciation' via hybridization between octoploid and diploid congeners. Instead, most chromosomes supported 'ancient hybrid speciation' between a maternal octoploid progenitor ancestral to extant octoploid strawberries and a paternal, extinct Fragaria iinumae-like diploid progenitor, probably in Beringia during the Pleistocene. The absence of a shared SDR between the decaploid and other Fragaria is also consistent with an older origin rather than a recent hybrid origin in situ. Our study reveals a long evolutionary history of the decaploid despite its recent discovery, and highlights the pitfalls of inferring polyploid origins from niche/range alone or combined with morphology. It can serve as an exemplary starting step towards building much-needed model systems of established polyploids that have been, and remain to be, recognized.

A Targeted Capture Linkage Map Anchors the Genome of the Schistosomiasis Vector Snail, Biomphalaria glabrata.

The aquatic planorbid snail Biomphalaria glabrata is one of the most intensively-studied mollusks due to its role in the transmission of schistosomiasis. Its 916 Mb genome has recently been sequenced and annotated, but it remains poorly assembled. Here, we used targeted capture markers to map over 10,000 B. glabrata scaffolds in a linkage cross of 94 F1 offspring, generating 24 linkage groups (LGs). We added additional scaffolds to these LGs based on linkage disequilibrium (LD) analysis of targeted capture and whole-genome sequences of 96 unrelated snails. Our final linkage map consists of 18,613 scaffolds comprising 515 Mb, representing 56% of the genome and 75% of genic and nonrepetitive regions. There are 18 large (> 10 Mb) LGs, likely representing the expected 18 haploid chromosomes, and > 50% of the genome has been assigned to LGs of at least 17 Mb. Comparisons with other gastropod genomes reveal patterns of synteny and chromosomal rearrangements. Linkage relationships of key immune-relevant genes may help clarify snail-schistosome interactions. By focusing on linkage among genic and nonrepetitive regions, we have generated a useful resource for associating snail phenotypes with causal genes, even in the absence of a complete genome assembly. A similar approach could potentially improve numerous poorly-assembled genomes in other taxa. This map will facilitate future work on this host of a serious human parasite.