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The post-glacial colonization of Gander Lake in Newfoundland, Canada, by Arctic Charr (Salvelinus alpinus) provides the opportunity to study the genomic basis of adaptation to extreme deep-water environments. Colonization of deep-water (>50 m) habitats often requires extensive adaptation to cope with novel environmental challenges from high hydrostatic pressure, low temperature, and low light, but the genomic mechanisms underlying evolution in these environments are rarely known. Here, we compare genomic divergence between a deep-water morph adapted to depths of up to 288 m and a larger, piscivorous pelagic morph occupying shallower depths. Using both a SNP array and resequencing of whole nuclear and mitochondrial genomes, we find clear genetic divergence (FST = 0.11-0.15) between deep and shallow water morphs, despite an absence of morph divergence across the mitochondrial genome. Outlier analyses identified many diverged genomic regions containing genes enriched for processes such as gene expression and DNA repair, cardiac function, and membrane transport. Detection of putative copy number variants (CNVs) uncovered 385 genes with CNVs distinct to piscivorous morphs, and 275 genes with CNVs distinct to deep-water morphs, enriched for processes associated with synapse assembly. Demographic analyses identified evidence for recent and local morph divergence, and ongoing reductions in diversity consistent with postglacial colonization. Together, these results show that Arctic Charr morph divergence has occurred through genome-wide differentiation and elevated divergence of genes underlying multiple cellular and physiological processes, providing insight into the genomic basis of adaptation in a deep-water habitat following postglacial recolonization.
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Trucha , Agua , Adaptación Fisiológica/genética , Animales , Genoma , Genómica , Trucha/genéticaRESUMEN
A salmon louse (Lepeophtheirus salmonis salmonis) genetic linkage map was constructed to serve as a genomic resource for future investigations into the biology of this important marine parasitic copepod species, and to provide insights into the inheritance patterns of genetic markers in this species. SNP genotyping of 8 families confirmed the presence of 15 linkage groups based upon the assignment of 93,773 markers. Progeny sample size weight adjusted map sizes in males (with the exception of SL12 and SL15) ranged in size from 96.50 cM (SL11) to 134.61 cM (SL06), and total combined map steps or bins ranged from 143 (SL09) to 203 (SL13). The SL12 male map was the smallest linkage group with a weight-averaged size of 3.05 cM with 6 recombination bins. Male:female specific recombination rate differences are 10.49:1 and represent one of the largest reported sex-specific differences for any animal species. Recombination ratio differences (M:F) ranged from 1.0 (SL12) to 29:1 (SL15). The number of markers exhibiting normal Mendelian segregation within the sex linkage group SL15 was extremely low (N = 80) in comparison to other linkage groups genotyped [range: 1459 (SL12)-10206 markers (SL05)]. Re-evaluation of Mendelian inheritance patterns of markers unassigned to any mapping parent according to hemizygous segregation patterns (models presented) identified matches for many of these markers to hemizygous patterns. The greatest proportion of these markers assigned to SL15 (N increased to 574). Inclusion of the hemizygous markers revised SL15 sex-specific recombination rate differences to 28:1. Recombination hot- and coldspots were identified across all linkage groups with all linkage groups possessing multiple peaks. Nine of 13 linkage groups evaluated possessed adjacent domains with hot-coldspot transitional zones. The most common pattern was for one end of the linkage to show elevated recombination in addition to internal regions. For SL01 and SL06, however, a terminal region with high recombination was not evident while a central domain possessing extremely high-recombination levels was present. High levels of recombination were weakly coupled to higher levels of SNP variation within domains, but this association was very strong for the central domains of SL01 and SL06. From the pooled paternal half-sib lots (several virgin females placed with 1 male), only 1 or two surviving family lots were obtained. Surviving families possessed parents where both the male and female possessed either inherently low or high recombination rates. This study provides insight into the organization of the sea louse genome, and describes large differences in recombination rate that exist among individuals of the same sex, and between the sexes. These differences in recombination rate may be coupled to the capabilities of this species to adapt to environmental and pharmaceutical treatments, given that family survivorship appears to be enhanced when parents have similar recombination levels.
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Mapeo Cromosómico , Copépodos/genética , Genómica , Recombinación Genética , Animales , Femenino , Ligamiento Genético/genética , Marcadores Genéticos/genética , Genoma/genética , Genotipo , Masculino , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Caracteres SexualesRESUMEN
BACKGROUND: The sablefish (order: Scorpaeniformes) is an economically important species in commercial fisheries of the North Pacific and an emerging species in aquaculture. Aside from a handful of sequences in NCBI and a few published microsatellite markers, little is known about the genetics of this species. The development of genetic tools, including polymorphic markers and a linkage map will allow for the successful development of future broodstock and mapping of phenotypes of interest. The significant sexual dimorphism between females and males makes a genetic test for early identification of sex desirable. RESULTS: A full mitochondrial genome is presented and the resulting phylogenetic analysis verifies the placement of the sablefish within the Scorpaeniformes. Nearly 35,000 assembled transcript sequences are used to identify genes and obtain polymorphic SNP and microsatellite markers. 360 transcribed polymorphic loci from two sablefish families produce a map of 24 linkage groups. The sex phenotype maps to sablefish LG14 of the male map. We show significant conserved synteny and conservation of gene-order between the threespine stickleback Gasterosteus aculeatus and sablefish. An additional 1843 polymorphic SNP markers are identified through next-generation sequencing techniques. Sex-specific markers and sequence insertions are identified immediately upstream of the gene gonadal-soma derived factor (gsdf), the master sex determinant locus in the medaka species Oryzias luzonensis. CONCLUSIONS: The first genomic resources for sablefish provide a foundation for further studies. Over 35,000 transcripts are presented, and the genetic map represents, as far as we can determine, the first linkage map for a member of the Scorpaeniformes. The observed level of conserved synteny and comparative mapping will allow the use of the stickleback genome in future genetic studies on sablefish and other related fish, particularly as a guide to whole-genome assembly. The identification of sex-specific insertions immediately upstream of a known master sex determinant implicates gsdf as an excellent candidate for the master sex determinant for sablefish.
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Mapeo Cromosómico , Peces/genética , Genómica , Mitocondrias/genética , Filogenia , Caracteres Sexuales , Procesos de Determinación del Sexo/genética , Animales , Femenino , Peces/fisiología , Marcadores Genéticos/genética , Genoma Mitocondrial/genética , Técnicas de Genotipaje , Masculino , Fenotipo , Smegmamorpha/genética , Sintenía/genéticaRESUMEN
Gene flow between wild and domestic populations has been repeatedly demonstrated across a diverse range of taxa. Ultimately, the genetic impacts of gene flow from domestic into wild populations depend both on the degree of domestication and the original source of the domesticated population. Atlantic salmon, Salmo salar, used in North American aquaculture are ostensibly of North American origin. However, evidence of European introgression into North American aquaculture salmon has accumulated in recent decades, even though the use of diploid European salmon has never been approved in Canada. The full extent of such introgression as well as the potential impacts on wild salmon in the Northwest Atlantic remains uncertain. Here, we extend previous work comparing North American and European wild salmon (n = 5799) using a 220 K SNP array to quantify levels of recent European introgression into samples of domestic salmon, aquaculture escapees, and wild salmon collected throughout Atlantic Canada. Analysis of North American farmed salmon (n = 403) and escapees (n = 289) displayed significantly elevated levels of European ancestry by comparison with wild individuals (p < 0.001). Of North American farmed salmon sampled between 2011 and 2018, ~17% had more than 10% European ancestry and several individuals exceeded 40% European ancestry. Samples of escaped farmed salmon similarly displayed elevated levels of European ancestry, with two individuals classified as 100% European. Analysis of juvenile salmon collected in rivers proximate to aquaculture locations also revealed evidence of elevated European ancestry and larger admixture tract in comparison to individuals collected at distance from aquaculture. Overall, our results demonstrate that even though diploid European salmon have never been approved for use in Canada, individuals of full and partial European ancestry have been in use over the last decade, and that some of these individuals have escaped and hybridized in the wild.
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Teleosts exhibit extensive diversity of sex determination (SD) systems and mechanisms, providing the opportunity to study the evolution of SD and sex chromosomes. Here we sequenced the genome of the common lumpfish (Cyclopterus lumpus Linnaeus), a species of increasing importance to aquaculture, and identified the SD region and master SD locus using a 70 K single nucleotide polymorphism array and tissue-specific expression data. The chromosome-level assembly identified 25 diploid chromosomes with a total size of 572.89 Mb, a scaffold N50 of 23.86 Mb and genome annotation-predicted 21,480 protein-coding genes. Genome-wide association analysis located a highly sex-associated region on chromosome 13, suggesting that anti-Müllerian hormone (AMH) is the putative SD factor. Linkage disequilibrium and heterozygosity across chromosome 13 support a proto-XX/XY system, with an absence of widespread chromosome divergence between sexes. We identified three copies of AMH in the lumpfish primary and alternate haplotype assemblies localized in the SD region. Comparison to sequences from other teleosts suggested a monophyletic relationship and conservation within the Cottioidei. One AMH copy showed similarity to AMH/AMHY in a related species and was also the only copy with expression in testis tissue, suggesting this copy may be the functional copy of AMH in lumpfish. The two other copies arranged in tandem inverted duplication were highly similar, suggesting a recent duplication event. This study provides a resource for the study of early sex chromosome evolution and novel genomic resources that benefits lumpfish conservation management and aquaculture.
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Hormona Antimülleriana , Perciformes , Animales , Hormona Antimülleriana/genética , Acuicultura , Estudio de Asociación del Genoma Completo , Masculino , Perciformes/genética , Cromosomas SexualesRESUMEN
BACKGROUND: Salmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. This duplication greatly impacts species diversification, functional specialization, and adaptation. Extensive new genomic resources have recently become available for Atlantic salmon (Salmo salar), but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution. RESULTS: From existing expressed sequence tag (EST) resources and three new full-length cDNA libraries, 9,057 reference quality full-length gene insert clones were identified for Atlantic salmon. A further 1,365 reference full-length clones were annotated from 29,221 northern pike (Esox lucius) ESTs. Pairwise dN/dS comparisons within each of 408 sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates. CONCLUSIONS: 9,057 full-length reference genes were characterized in S. salar and can be used to identify alleles and gene family members. Comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. In addition, there is evidence that evolution has acted asymmetrically on paralogs, allowing one of the pair to diverge at a faster rate.
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ADN Complementario/genética , Esocidae/genética , Evolución Molecular , Genoma/genética , Poliploidía , Salmo salar/genética , Animales , Secuencia de Bases , Clonación Molecular , Mapeo Contig , Etiquetas de Secuencia Expresada , Duplicación de Gen , Perfilación de la Expresión Génica , Alineación de SecuenciaRESUMEN
The resiliency of populations and species to environmental change is dependent on the maintenance of genetic diversity, and as such, quantifying diversity is central to combating ongoing widespread reductions in biodiversity. With the advent of next-generation sequencing, several methods now exist for resolving fine-scale population structure, but the comparative performance of these methods for genetic assignment has rarely been tested. Here, we evaluate the performance of sequenced microsatellites and a single nucleotide polymorphism (SNP) array to resolve fine-scale population structure in a critically important salmonid in north eastern Canada, Arctic Charr (Salvelinus alpinus). We also assess the utility of sequenced microsatellites for fisheries applications by quantifying the spatial scales of movement and exploitation through genetic assignment of fishery samples to rivers of origin and comparing these results with a 29-year tagging dataset. Self-assignment and simulation-based analyses of 111 genome-wide microsatellite loci and 500 informative SNPs from 28 populations of Arctic Charr in north-eastern Canada identified largely river-specific genetic structure. Despite large differences (~4X) in the number of loci surveyed between panels, mean self-assignment accuracy was similar with the microsatellite loci and the SNP panel (>90%). Subsequent analysis of 996 fishery-collected samples using the microsatellite panel revealed that larger rivers contribute greater numbers of individuals to the fishery and that coastal fisheries largely exploit individuals originating from nearby rivers, corroborating results from traditional tagging experiments. Our results demonstrate the efficacy of sequence-based microsatellite genotyping to advance understanding of fine-scale population structure and harvest composition in northern and understudied species.
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Drug resistance in the salmon louse Lepeophtheirus salmonis is a global issue for Atlantic salmon aquaculture. Multiple resistance has been described across most available compound classes with the exception of the benzoylureas. To target this gap in effective management of L. salmonis and other species of sea lice (e.g. Caligus spp.), Elanco Animal Health is developing an in-feed treatment containing lufenuron (a benzoylurea) to be administered prior to seawater transfer of salmon smolts and to provide long-term protection of salmon against sea lice infestations. Benzoylureas disrupt chitin synthesis, formation, and deposition during all moulting events. However, the mechanism(s) of action are not yet fully understood and most research completed to date has focused on insects. We exposed the first parasitic stage of L. salmonis to 700â¯ppb lufenuron for three hours and observed over 90% reduction in survival to the chalimus II life stage on the host, as compared to vehicle controls. This agrees with a follow up in vivo administration study on the host, which showed >95% reduction by the chalimus I stage. Transcriptomic responses of salmon lice exposed to lufenuron included genes related to moulting, epithelial differentiation, solute transport, and general developmental processes. Global metabolite profiles also suggest that membrane stability and fluidity is impacted in treated lice. These molecular signals are likely the underpinnings of an abnormal moulting process and cuticle formation observed ultrastructurally using transmission electron microscopy. Treated nauplii-staged lice exhibited multiple abnormalities in the integument, suggesting that the coordinated assembly of the epi- and procuticle is impaired. In all cases, treatment with lufenuron had rapid impacts on L. salmonis development. We describe multiple experiments to characterize the efficacy of lufenuron on eggs, larvae, and parasitic stages of L. salmonis, and provide the most comprehensive assessment of the physiological responses of a marine arthropod to a benzoylurea chemical.
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Benzamidas/farmacología , Muda/efectos de los fármacos , Phthiraptera/efectos de los fármacos , Salmo salar/parasitología , Animales , Acuicultura , Benzamidas/administración & dosificación , Enfermedades de los Peces/parasitología , Infestaciones por Piojos/tratamiento farmacológico , Infestaciones por Piojos/prevención & control , Estadios del Ciclo de Vida/efectos de los fármacos , Estadios del Ciclo de Vida/genética , Metabolómica , Muda/genética , Phthiraptera/genética , Phthiraptera/fisiología , Salmo salar/crecimiento & desarrollo , Agua de Mar , TranscriptomaRESUMEN
Antiparasitic drugs such as emamectin benzoate (EMB) are relied upon to reduce the parasite load, particularly of the sea louse Lepeophtheirus salmonis, on farmed salmon. The decline in EMB treatment efficacy for this purpose is an important issue for salmon producers around the world, and particularly for those in the Atlantic Ocean where widespread EMB tolerance in sea lice is recognized as a significant problem. Salmon farms in the Northeast Pacific Ocean have not historically experienced the same issues with treatment efficacy, possibly due to the relatively large population of endemic salmonid hosts that serve to both redistribute surviving lice and dilute populations potentially under selection by introducing naïve lice to farms. Frequent migration of lice among farmed and wild hosts should limit the effect of farm-specific selection pressures on changes to the overall allele frequencies of sea lice in the Pacific Ocean. A previous study using microsatellites examined L. salmonis oncorhynchi from 10 Pacific locations from wild and farmed hosts and found no population structure. Recently however, a farm population of sea lice was detected where EMB bioassay exposure tolerance was abnormally elevated. In response, we have developed a Pacific louse draft genome that complements the previously-released Atlantic louse sequence. These genomes were combined with whole-genome re-sequencing data to design a highly sensitive 201,279 marker SNP array applicable for both subspecies (90,827 validated Pacific loci; 153,569 validated Atlantic loci). Notably, kmer spectrum analysis of the re-sequenced samples indicated that Pacific lice exhibit a large within-individual heterozygosity rate (average of 1 in every 72 bases) that is markedly higher than that of Atlantic individuals (1 in every 173 bases). The SNP chip was used to produce a high-density map for Atlantic sea louse linkage group 5 that was previously shown to be associated with EMB tolerance in Atlantic lice. Additionally, 478 Pacific louse samples from farmed and wild hosts obtained between 2005 and 2014 were also genotyped on the array. Clustering analysis allowed us to detect the apparent emergence of an otherwise rare genotype at a high frequency among the lice collected from two farms in 2013 that had reported elevated EMB tolerance. This genotype was not observed in louse samples collected from the same farm in 2010, nor in any lice sampled from other locations prior to 2013. However, this genotype was detected at low frequencies in louse samples from farms in two locations reporting elevated EMB tolerance in 2014. These results suggest that a rare genotype present in Pacific lice may be locally expanded in farms after EMB treatment. Supporting this hypothesis, 437 SNPs associated with this genotype were found to be in a region of linkage group 5 that overlaps the region associated with EMB resistance in Atlantic lice. Finally, five of the top diagnostic SNPs within this region were used to screen lice that had been subjected to an EMB survival assay, revealing a significant association between these SNPs and EMB treatment outcome. To our knowledge this work is the first report to identify a genetic link to altered EMB efficacy in L. salmonis in the Pacific Ocean.
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Antiparasitarios/farmacología , Copépodos/efectos de los fármacos , Copépodos/genética , Ivermectina/análogos & derivados , Polimorfismo de Nucleótido Simple , Animales , Mapeo Cromosómico , Análisis por Conglomerados , Femenino , Enfermedades de los Peces/tratamiento farmacológico , Enfermedades de los Peces/parasitología , Ligamiento Genético , Sitios Genéticos , Marcadores Genéticos , Genética de Población , Genotipo , Ivermectina/farmacología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Océano Pacífico , Salmón/parasitologíaRESUMEN
Cypermethrin has been administered for decades to control salmon lice (Lepeophtheirus salmonis) infestations in Atlantic salmon farming regions globally. However, resistance to cypermethrin and other available therapeutants has threatened the sustainability of this growing industry. To better understand the effects of cypermethrin on L. salmonis, a 38K oligonucleotide microarray and RT-qPCR analyses were applied to pools of copepodid larvae exposed to 1.0ppb cypermethrin or seawater controls for 24h. Phenotypic assessments and global gene expression profiles showed a significant disruption of homeostasis in copepodid L. salmonis exposed to cypermethrin. Multiple degradative enzymes were overexpressed in cypermethrin-treated lice including five trypsin-like serine proteases and three cytochrome p450s CYP3a24 (p=0.03, fold change (FC)=3.8; GenBank accession no. JP326960.1), CYP6w1 (p=0.008, FC=5.3; GenBank accession no. JP317875.1), and CYP6d4 (p=0.01; FC=7.9; GenBank accession no. JP334550.1). These enzymes represent preliminary markers for understanding the physiological response of L. salmonis to cypermethrin exposure. A general stress response was also observed in cypermethrin-treated lice which included differential expression of cell signaling genes involved in the induction of cell growth, solute transport, and metabolism. Lastly, a consensus-based analysis was completed with two previously published L. salmonis transcriptome studies revealing genes that respond to cypermethrin, emamectin benzoate (another delousing agent) and hyposalinity. This included concordant differential expression of heat shock beta-1, ammonium transporter Rh types B, and 72kDa type IV collagenase across different L. salmonis studies. This is currently the most comprehensive transcriptome assessment of chemical exposure on the first infectious stage of L. salmonis, providing novel markers for studying drug resistance and general stress in this important parasite.
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Copépodos/genética , Proteínas de Peces/genética , Regulación de la Expresión Génica/efectos de los fármacos , Insecticidas/farmacología , Piretrinas/farmacología , Estrés Fisiológico/genética , Transcriptoma/genética , Animales , Biología Computacional , Copépodos/efectos de los fármacos , Copépodos/crecimiento & desarrollo , Perfilación de la Expresión Génica , Genoma/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcriptoma/efectos de los fármacosRESUMEN
The products of dax1, foxl2a and mis have each been shown to have proliferative and/or differentiative activities during mammalian organogenesis. These factors also play a role in regulating the biosynthesis of estrogen, particularly by modulating the activity of aromatase cyp19a. We demonstrate the transcription and translation of these genes during salmon embryogenesis. We were able to track sex-specific differences in these processes through accurate determination of the sex of each embryo and larva examined from genotyped microsatellites. We detected sex- and stage-specific immunolabeling of the embryonic gut, kidney, gonads, neural cord and skeletal muscle by DAX-1, FOXL2A and MIS. These results indicate the potential of these factors to mediate proliferation and/or differentiation programs during development of these tissues. As well, immunolabeling of skeletal muscle by CYP19B1 throughout the study reveals probable neurogenic activity associated with peripheral radial glial cells and the growing embryonic musculature.
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Aromatasa/genética , Salmo salar/metabolismo , Animales , Aromatasa/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Genotipo , Larva/enzimología , Larva/crecimiento & desarrollo , Masculino , Mesodermo/embriología , Mesodermo/enzimología , Mesodermo/crecimiento & desarrollo , Morfogénesis , Músculo Esquelético/embriología , Músculo Esquelético/enzimología , Salmo salar/embriología , Salmo salar/crecimiento & desarrollo , Factores SexualesRESUMEN
The northern pike is the most frequently studied member of the Esociformes, the closest order to the diverse and economically important Salmoniformes. The ancestor of all salmonids purportedly experienced a whole-genome duplication (WGD) event, making salmonid species ideal for studying the early impacts of genome duplication while complicating their use in wider analyses of teleost evolution. Studies suggest that the Esociformes diverged from the salmonid lineage prior to the WGD, supporting the use of northern pike as a pre-duplication outgroup. Here we present the first genome assembly, reference transcriptome and linkage map for northern pike, and evaluate the suitability of this species to provide a representative pre-duplication genome for future studies of salmonid and teleost evolution. The northern pike genome sequence is composed of 94,267 contigs (N50 = 16,909 bp) contained in 5,688 scaffolds (N50 = 700,535 bp); the total scaffolded genome size is 878 million bases. Multiple lines of evidence suggest that over 96% of the protein-coding genome is present in the genome assembly. The reference transcriptome was constructed from 13 tissues and contains 38,696 transcripts, which are accompanied by normalized expression data in all tissues. Gene-prediction analysis produced a total of 19,601 northern pike-specific gene models. The first-generation linkage map identifies 25 linkage groups, in agreement with northern pike's diploid karyotype of 2N = 50, and facilitates the placement of 46% of assembled bases onto linkage groups. Analyses reveal a high degree of conserved synteny between northern pike and other model teleost genomes. While conservation of gene order is limited to smaller syntenic blocks, the wider conservation of genome organization implies the northern pike exhibits a suitable approximation of a non-duplicated Protacanthopterygiian genome. This dataset will facilitate future studies of esocid biology and empower ongoing examinations of the Atlantic salmon and rainbow trout genomes by facilitating their comparison with other major teleost groups.
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Esocidae/genética , Ligamiento Genético , Genoma , Animales , Esocidae/clasificación , Datos de Secuencia Molecular , Filogenia , TranscriptomaRESUMEN
This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross-tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.