Natural variation in gene expression and viral susceptibility revealed by neural progenitor cell villages.

Human genome variation contributes to diversity in neurodevelopmental outcomes and vulnerabilities; recognizing the underlying molecular and cellular mechanisms will require scalable approaches. Here, we describe a "cell village" experimental platform we used to analyze genetic, molecular, and phenotypic heterogeneity across neural progenitor cells from 44 human donors cultured in a shared in vitro environment using algorithms (Dropulation and Census-seq) to assign cells and phenotypes to individual donors. Through rapid induction of human stem cell-derived neural progenitor cells, measurements of natural genetic variation, and CRISPR-Cas9 genetic perturbations, we identified a common variant that regulates antiviral IFITM3 expression and explains most inter-individual variation in susceptibility to the Zika virus. We also detected expression QTLs corresponding to GWAS loci for brain traits and discovered novel disease-relevant regulators of progenitor proliferation and differentiation such as CACHD1. This approach provides scalable ways to elucidate the effects of genes and genetic variation on cellular phenotypes.

Efficient generation of lower induced motor neurons by coupling Ngn2 expression with developmental cues.

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modeling of hard-to-access tissues (such as the brain). Current protocols either direct neuronal differentiation with small molecules or use transcription-factor-mediated programming. In this study, we couple overexpression of transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced motor neurons (liMoNes/liMNs). This approach induces canonical MN markers including MN-specific Hb9/MNX1 in more than 95% of cells. liMNs resemble bona fide hPSC-derived MN, exhibit spontaneous electrical activity, express synaptic markers, and can contact muscle cells in vitro. Pooled, multiplexed single-cell RNA sequencing on 50 hPSC lines reveals reproducible populations of distinct subtypes of cervical and brachial MNs that resemble their in vivo, embryonic counterparts. Combining small molecule patterning with Ngn2 overexpression facilitates high-yield, reproducible production of disease-relevant MN subtypes, which is fundamental in propelling our knowledge of MN biology and its disruption in disease.

Evolution of population structure in an estuarine-dependent marine fish.

Restriction site-associated DNA (RAD) sequencing was used to characterize neutral and adaptive genetic variation among geographic samples of red drum, Sciaenops ocellatus, an estuarine-dependent fish found in coastal waters along the southeastern coast of the United States (Atlantic) and the northern Gulf of Mexico (Gulf). Analyses of neutral and outlier loci revealed three genetically distinct regional clusters: one in the Atlantic and two in the northern Gulf. Divergence in neutral loci indicated gradual genetic change and followed a linear pattern of isolation by distance. Divergence in outlier loci was at least an order of magnitude greater than divergence in neutral loci, and divergence between the regions in the Gulf was twice that of divergence between other regions. Discordance in patterns of genetic divergence between outlier and neutral loci is consistent with the hypothesis that the former reflects adaptive responses to environmental factors that vary on regional scales, while the latter largely reflects drift processes. Differences in basic habitat, initiated by glacial retreat and perpetuated by contemporary oceanic and atmospheric forces interacting with the geomorphology of the northern Gulf, followed by selection, appear to have led to reduced gene flow among red drum across the northern Gulf, reinforcing differences accrued during isolation and resulting in continued divergence across the genome. This same dynamic also may pertain to other coastal or nearshore fishes (18 species in 14 families) where genetically or morphologically defined sister taxa occur in the three regions.

Genetic mapping and comparative genomics to inform restoration enhancement and culture of southern flounder, Paralichthys lethostigma.

BACKGROUND: Southern flounder, Paralichthys lethostigma, historically support a substantial fishery along the Atlantic and Gulf coasts of the southern United States. Low year-class strengths over the past few years in the western Gulf of Mexico have raised concern that spawning stocks may be overfished. Current management of the resource includes releasing hatchery-raised juveniles to restock bays and estuaries; additionally, there is a growing interest in the potential for commercial aquaculture of the species. Currently, genomic resources for southern flounder do not exist. Here, we used two hatchery-reared families and double-digest, restriction-site-associated DNA (ddRAD) sequencing to create a reduced-representation genomic library consisting of several thousand single nucleotide polymorphisms (SNPs) located throughout the genome. RESULTS: The relative position of each SNP-containing locus was determined to create a high-density genetic map spanning the 24 linkage groups of the southern flounder genome. The consensus map was used to identify regions of shared synteny between southern flounder and seven other fish species for which genome assemblies are available. Finally, syntenic blocks were used to localize genes identified from transcripts in European flounder as potentially being involved in ecotoxicological and osmoregulatory responses, as well as QTLs associated with growth and disease resistance in Japanese flounder, on the southern flounder linkage map. CONCLUSIONS: The information provided by the linkage map will enrich restoration efforts by providing a foundation for interpreting spatial genetic variation within the species, ultimately furthering an understanding of the adaptive potential and resilience of southern flounder to future changes in local environmental conditions. Further, the map will facilitate the use of genetic markers to enhance restoration and commercial aquaculture.

Haplotyping RAD loci: an efficient method to filter paralogs and account for physical linkage.

Next-generation sequencing of reduced-representation genomic libraries provides a powerful methodology for genotyping thousands of single-nucleotide polymorphisms (SNPs) among individuals of nonmodel species. Utilizing genotype data in the absence of a reference genome, however, presents a number of challenges. One major challenge is the trade-off between splitting alleles at a single locus into separate clusters (loci), creating inflated homozygosity, and lumping multiple loci into a single contig (locus), creating artefacts and inflated heterozygosity. This issue has been addressed primarily through the use of similarity cut-offs in sequence clustering. Here, two commonly employed, postclustering filtering methods (read depth and excess heterozygosity) used to identify incorrectly assembled loci are compared with haplotyping, another postclustering filtering approach. Simulated and empirical data sets were used to demonstrate that each of the three methods separately identified incorrectly assembled loci; more optimal results were achieved when the three methods were applied in combination. The results confirmed that including incorrectly assembled loci in population-genetic data sets inflates estimates of heterozygosity and deflates estimates of population divergence. Additionally, at low levels of population divergence, physical linkage between SNPs within a locus created artificial clustering in analyses that assume markers are independent. Haplotyping SNPs within a locus effectively neutralized the physical linkage issue without having to thin data to a single SNP per locus. We introduce a Perl script that haplotypes polymorphisms, using data from single or paired-end reads, and identifies potentially problematic loci.

Linkage Mapping and Comparative Genomics of Red Drum (Sciaenops ocellatus) Using Next-Generation Sequencing.

Developments in next-generation sequencing allow genotyping of thousands of genetic markers across hundreds of individuals in a cost-effective manner. Because of this, it is now possible to rapidly produce dense genetic linkage maps for nonmodel species. Here, we report a dense genetic linkage map for red drum, a marine fish species of considerable economic importance in the southeastern United States and elsewhere. We used a prior microsatellite-based linkage map as a framework and incorporated 1794 haplotyped contigs derived from high-throughput, reduced representation DNA sequencing to produce a linkage map containing 1794 haplotyped restriction-site associated DNA (RAD) contigs, 437 anonymous microsatellites, and 44 expressed sequence-tag-linked microsatellites (EST-SSRs). A total of 274 candidate genes, identified from transcripts from a preliminary hydrocarbon exposure study, were localized to specific chromosomes, using a shared synteny approach. The linkage map will be a useful resource for red drum commercial and restoration aquaculture, and for better understanding and managing populations of red drum in the wild.

Molecular phylogenetics of New World searobins (Triglidae; Prionotinae).

Phylogenetic relationships among members of the New World searobin genera Bellator and Prionotus (Family Triglidae, Subfamily Prionotinae) and among other searobins in the families Triglidae and Peristediidae were investigated using both mitochondrial and nuclear DNA sequences. Phylogenetic hypotheses derived from maximum likelihood and Bayesian methodologies supported a monophyletic Prionotinae that included four well resolved clades of uncertain relationship; three contained species in the genus Prionotus and one contained species in the genus Bellator. Bellator was always recovered within the genus Prionotus, a result supported by post hoc model testing. Two nominal species of Prionotus (P. alatus and P. paralatus) were not recovered as exclusive lineages, suggesting the two may comprise a single species. Phylogenetic hypotheses also supported a monophyletic Triglidae but only if armored searobins (Family Peristediidae) were included. A robust morphological assessment is needed to further characterize relationships and suggest classification of clades within Prionotinae; for the time being we recommend that Bellator be considered a synonym of Prionotus. Relationships between armored searobins (Family Peristediidae) and searobins (Family Triglidae) and relationships within Triglidae also warrant further study.

Fine-scale partitioning of genomic variation among recruits in an exploited fishery: causes and consequences.

Conservation and management of exploited species depends on accurate knowledge of how genetic variation is partitioned across a fishery, especially as it relates to recruitment. Using double-digest restriction-site associated DNA sequencing, we surveyed variation in 7,382 single nucleotide polymorphisms (SNPs) in red snapper (Lutjanus campechanus) young-of-the-year (YOY) sampled at six localities and in adults sampled at two localities in the northern Gulf of Mexico. Significant genetic heterogeneity was detected between the two adult samples, separated by ~600 km, and at spatial scales less than five kilometers among samples of YOY. Genetic differences between YOY samples and between YOY samples and adult samples were not associated with geographic distance, and a genome scan revealed no evidence of loci under selection. Estimates of the effective number of breeders, allelic richness, and relatedness within YOY samples were not consistent with sweepstakes recruitment. Instead, the data demonstrate, at least within one recruitment season, that multiple pulses of recruits originate from distinct groups of spawning adults, even at small spatial scales. For exploited species with this type of recruitment pattern, protection of spawning adults over wide geographic areas may be critical for ensuring productivity and stability of the fishery by maintaining larval supply and connectivity.

dDocent: a RADseq, variant-calling pipeline designed for population genomics of non-model organisms.

Restriction-site associated DNA sequencing (RADseq) has become a powerful and useful approach for population genomics. Currently, no software exists that utilizes both paired-end reads from RADseq data to efficiently produce population-informative variant calls, especially for non-model organisms with large effective population sizes and high levels of genetic polymorphism. dDocent is an analysis pipeline with a user-friendly, command-line interface designed to process individually barcoded RADseq data (with double cut sites) into informative SNPs/Indels for population-level analyses. The pipeline, written in BASH, uses data reduction techniques and other stand-alone software packages to perform quality trimming and adapter removal, de novo assembly of RAD loci, read mapping, SNP and Indel calling, and baseline data filtering. Double-digest RAD data from population pairings of three different marine fishes were used to compare dDocent with Stacks, the first generally available, widely used pipeline for analysis of RADseq data. dDocent consistently identified more SNPs shared across greater numbers of individuals and with higher levels of coverage. This is due to the fact that dDocent quality trims instead of filtering, incorporates both forward and reverse reads (including reads with INDEL polymorphisms) in assembly, mapping, and SNP calling. The pipeline and a comprehensive user guide can be found at http://dDocent.wordpress.com.

A comparison of neutral and immune genetic variation in Atlantic salmon, Salmo salar L. in Chilean aquaculture facilities.

Genetic diversity was assessed in samples of cultured Atlantic salmon, Salmo salar L., obtained from facilities in Chile between 2005 and 2010, a period of time during which the infectious pathogens Infectious Salmon Anemia (ISA) virus, Caligus rogercresseyi (sea lice), and Piscirickettsia salmonis (salmon rickettsial syndrome) were common. Two panels of microsatellite markers were utilized: one with microsatellites with no known gene associations (neutral) and one featuring microsatellites linked to putative immune-related genes (immune-related). Allelic richness and gene diversity across samples were significantly greater in neutral loci as compared to immune-related loci. Both diversity measures were homogeneous among samples for immune-related loci and heterogeneous among samples for neutral loci. Immune-related loci were identified as F(ST) outliers in pairwise comparisons of samples at a 10-fold higher frequency than neutral loci. These results indicate that neutral and immune-related portions of the Atlantic salmon genome may have differed in response to the gauntlet of pathogens and that monitoring of specific, well characterized immune-related loci as well as neutral loci in cultured species could be useful when disease control and prevention is a goal.

Use of comparative genomics to develop EST-SSRs for red drum (Sciaenops ocellatus).

Microsatellites physically linked to expressed sequence tags (EST-SSRs) are an important resource for linkage mapping and comparative genomics, and data mining in publicly available EST databases is a common strategy for EST-SSR discovery. At present, many species lack species-specific EST sequence data needed for the efficient characterization of EST-SSRs. This paper describes the discovery and development of EST-SSRs for red drum (Sciaenops ocellatus), an estuarine-dependent sciaenid species of economic importance in the USA and elsewhere, using a phylogenetically informed, comparative genomics approach to primer design. The approach entailed comparing existing genomic resources from species closely allied phylogenetically to red drum, with resources from more distantly related outgroup species. By taking into account the degree to which flanking regions are conserved across taxa, the efficiency of PCR primer design was increased greatly. The amplification success rate for primers designed for red drum was 100 % when using EST libraries from confamilial species and 92 % when using an EST library from a species in the same suborder. The primers developed also amplified EST-SSRs in a wide range of perciform fishes, suggesting potential use in comparative genomics. This study demonstrates that EST-SSRs can be efficiently developed for an organism when limited species-specific data are available by exploiting genomic resources from well-studied species, even those at extended phylogenetic distances.

Permanent genetic resources added to Molecular Ecology Resources Database 1 December 2011-31 January 2012.

This article documents the addition of 473 microsatellite marker loci and 71 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Barteria fistulosa, Bombus morio, Galaxias platei, Hematodinium perezi, Macrocentrus cingulum Brischke (a.k.a. M. abdominalis Fab., M. grandii Goidanich or M. gifuensis Ashmead), Micropogonias furnieri, Nerita melanotragus, Nilaparvata lugens Stål, Sciaenops ocellatus, Scomber scombrus, Spodoptera frugiperda and Turdus lherminieri. These loci were cross-tested on the following species: Barteria dewevrei, Barteria nigritana, Barteria solida, Cynoscion acoupa, Cynoscion jamaicensis, Cynoscion leiarchus, Cynoscion nebulosus, Cynoscion striatus, Cynoscion virescens, Macrodon ancylodon, Menticirrhus americanus, Nilaparvata muiri and Umbrina canosai. This article also documents the addition of 116 sequencing primer pairs for Dicentrarchus labrax.

Isolation and characterization of microsatellite markers in the Serra Spanish mackerel, Scomberomorus brasiliensis.

Thirteen nuclear-encoded microsatellites from a genomic DNA library of Serra Spanish mackerel, Scomberomorus brasiliensis, were isolated and characterized. The microsatellites include 10 perfect repeats (eight tetranucleotide and two dinucleotide) and three imperfect repeats (two tetranucleotide and one dinucleotide). An additional five microsatellites, isolated originally from two congeneric species (S. cavalla and S. niphonius), were characterized in S. brasiliensis. Serra Spanish mackerel support artisanal fisheries along the Caribbean and Atlantic coasts of Central and South America, from Belize to Brazil.

PERMANENT GENETIC RESOURCES: PCR primers for 100 microsatellites in red drum (Sciaenops ocellatus).

One hundred nuclear-encoded microsatellites from a genomic library of red drum (Sciaenops ocellatus) were isolated and characterized. Eight microsatellites had tetranucleotide motifs; 92 had dinucleotide motifs. The average number of alleles per microsatellite (sample of 22-24 fish) was 17.7 (range = 2-30); gene diversity averaged 0.796 (range = 0.227-1.000). Following Bonferroni correction, genotype frequencies at 90 microsatellites did not deviate significantly from Hardy-Weinberg equilibrium expectations. Occurrence of null alleles was inferred at 15 microsatellites; alleles differing by only a single base were observed at 11 microsatellites. The microsatellites developed should prove useful for population-genetic studies of 'wild' red drum and in construction of a genetic map.

Access for all: the rise of the Paralympic Games.

The Paralympic, or Parallel, Games for athletes with disabilities have played a major role over the past half century in changing attitudes towards disability and accelerating the agenda for inclusion. This article charts their development from small beginnings as a competition for disabled ex-servicemen and women in England founded shortly after the Second World War to the present day ambulatory international festival of Summer and Winter Games organized in conjunction with the Olympic Games. The Paralympic Games trace their origins to the work of Dr (later Sir) Ludwig Guttmann at the National Spinal Injuries Unit at Stoke Mandeville Hospital in Buckinghamshire who used sport as an integral part of the treatment of paraplegic patients. A sports competition was held at the hospital to coincide with the Opening Ceremony of the London Games in July 1948. This became an annual event attracting the first international participation in 1952, after which it became the International Stoke Mandeville Games. From 1960 onwards attempts were made to hold every fourth Games in the Olympic host city. Despite initial success in staging the 1960 Games in Rome and the 1964 Games in Tokyo, subsequent host cities refused to host the competitions and alternative locations were found where a package of official support, finance and suitable venues could be assembled. In 1976, the scope of the Games was widened to accept other disabilities. From 1988 onwards, a process of convergence took place that saw the Paralympics brought into the central arena of the Olympics, both literally and figuratively. In the process they have embraced new sports, have encompassed a wider range of disabilities, and helped give credence to the belief that access to sport is available to all. The Paralympics also underline the change from sport as therapeutic competition to that of elite events that carry intrinsic prestige, with growing rivalry over medal tables. For the future, however, questions remain as to whether the current arrangements of separate but supposedly equal festivals assist the continuing development of the Paralympics or perpetuate difference.

Factors contributing to the regulation of thermoinhibition in Tagetes minuta L.

Thermoinhibition in Tagetes minuta achenes is tightly and rapidly regulated with regard to its imposition and release, with both processes occurring within 2-3h. Germination at high temperatures is almost exclusively regulated by the embryo, while the pericarp appears to play only a minor role. Thermoinhibition in T. minuta could not be alleviated by any single plant growth regulator application, but a combination of treatments that both reduced ABA levels and increased ethylene levels were able to restore germination at supraoptimal temperatures. This suggests a role for both ethylene and ABA in the imposition of thermoinhibition in this species.

Microsatellite and mitochondrial DNA analyses of Atlantic bluefin tuna (Thunnus thynnus thynnus) population structure in the Mediterranean Sea.

Genetic variation was surveyed at nine microsatellite loci and the mitochondrial control region (868 bp) to test for the presence of genetic stock structure in young-of-the-year Atlantic bluefin tuna (Thunnus thynnus thynnus) from the Mediterranean Sea. Bluefin tuna were sampled over a period of 5 years from the Balearic and Tyrrhenian seas in the western basin of the Mediterranean Sea, and from the southern Ionian Sea in the eastern basin of the Mediterranean Sea. Analyses of multilocus microsatellite genotypes and mitochondrial control region sequences revealed no significant heterogeneity among collections taken from the same location in different years; however, significant spatial genetic heterogeneity was observed across all samples for both microsatellite markers and mitochondrial control region sequences (FST=0.0023, P=0.038 and PhiST=0.0233, P=0.000, respectively). Significant genetic differentiation between the Tyrrhenian and Ionian collections was found for both microsatellite and mitochondrial markers (FST=0.0087, P=0.015 and PhiST=0.0367, P=0.030, respectively). These results suggest the possibility of a genetically discrete population in the eastern basin of the Mediterranean Sea.

Genetic effective size is three orders of magnitude smaller than adult census size in an abundant, Estuarine-dependent marine fish (Sciaenops ocellatus).

Using eight microsatellite loci and a variety of analytical methods, we estimated genetic effective size (N(e)) of an abundant and long-lived marine fish species, the red drum (Sciaenops ocellatus), in the northern Gulf of Mexico (Gulf). The ratio N(e)/N, where short-term variance N(e) was estimated via the temporal method from shifts in allele-frequency data over four cohorts and where N reflected a current estimate of adult census size in the northern Gulf, was approximately 0.001. In an idealized population, this ratio should approximate unity. The extraordinarily low value of N(e)/N appears to arise from high variance in individual reproductive success and perhaps more importantly from variance in productivity of critical spawning and nursery habitats located in spatially discrete bays and estuaries throughout the northern Gulf. An estimate of N(e) based on a coalescent approach, which measures long-term, inbreeding effective size, was four orders of magnitude lower than the estimate of current census size, suggesting that factors presently driving N(e)/N to low values among red drum in the northern Gulf may have operated similarly in the past. Models that predict N(e)/N exclusively from demographic and life-history features will seriously overestimate N(e) if variance in reproductive success and variance in productivity among spatially discrete demes is underestimated. Our results indicate that these variances, especially variance in productivity among demes, must be large for red drum. Moreover, our study indicates that vertebrate populations with enormous adult census numbers may still be at risk relative to decline and extinction from genetic factors.

Mutation patterns of mitochondrial H- and L-strand DNA in closely related Cyprinid fishes.

Mitochondrial genome replication is asymmetric. Replication starts from the origin of heavy (H)-strand replication, displacing the parental H-strand as it proceeds along the molecule. The H-strand remains single stranded until light (L)-strand replication is initiated from a second origin of replication. It has been suggested that single-stranded H-strand DNA is more sensitive to mutational damage, giving rise to substitutional rate differences between the two strands and among genes in mammalian mitochondrial DNA. In this study, we analyzed sequences of the cytochrome b, ND4, ND4L, and COI genes of cyprinid fishes to investigate rates and patterns of nucleotide substitution in the mitochondrial genome. To test for strand-asymmetric mutation pressure, a likelihood-ratio test was developed and applied to the cyprinid sequences. Patterns of substitution and levels of strand-asymmetric mutation pressure were largely consistent with a mutation gradient between the H- and L-strand origins of replication. Significant strand bias was observed among rates of transitional substitution. However, biological interpretation of the direction and strength of strand asymmetry for specific classes of substitutions is problematic. The problem occurs because the rate of any single class of substitution inferred from one strand is actually a sum of rates on two strands. The validity of the likelihood-ratio test is not affected by this problem.