Whole-genome analysis reveals phylogenetic and demographic history of Eurasian perch.

The contemporary diversity and distribution of species are shaped by their evolutionary and ecological history. This can be deciphered with the help of phylogenetic and demographic analysis methods, ideally combining and supplementing information from mitochondrial and nuclear genomes. In this study, we investigated the demographic history of Eurasian perch (Perca fluviatilis), a highly adaptable teleost with a distribution range across Eurasia. We combined whole-genome resequencing data with available genomic resources to analyse the phylogeny, phylogeography, and demographic history of P. fluviatilis populations from Europe and Siberia. We identified five highly diverged evolutionary mtDNA lineages, three of which show a strong signal of admixture in the Baltic Sea region. The estimated mean divergence time between these lineages ranged from 0.24 to 1.42 million years. Based on nuclear genomes, two distinct demographic trajectories were observed in European and Siberian samples reflecting contrasting demographic histories ca. 30,000-100,000 years before the present. A comparison of mtDNA and nuclear DNA evolutionary trees and AMOVA revealed concordances, as well as incongruences, between the two types of data, most likely reflecting recent postglacial colonization and hybridization events. Overall, our findings demonstrate the power and usefulness of genome-wide information for delineating historical processes that have shaped the genome of P. fluviatilis. We also highlight the added value of data-mining existing transcriptomic resources to complement novel sequence data, helping to shed light on putative glacial refugia and postglacial recolonization routes.

Spatial and intra-host distribution of myxozoan parasite Tetracapsuloides bryosalmonae among Baltic sea trout (Salmo trutta).

Proliferative kidney disease caused by the myxozoan parasite Tetracapsuloides bryosalmonae has been actively studied in juvenile salmonids for decades. However, very little is known about parasite prevalence and its geographical and intra-host distribution at older life stages. We screened T. bryosalmonae among adult sea trout (Salmo trutta) (n = 295) collected along the Estonian Baltic Sea coastline together with juvenile trout from 33 coastal rivers (n = 1752) to assess spatial infection patterns of the adult and juvenile fish. The parasite was detected among 38.6% of adult sea trout with the prevalence increasing from west to east, and south to north, along the coastline. A similar pattern was observed in juvenile trout. Infected sea trout were also older than uninfected fish and the parasite was detected in sea trout up to the age of 6 years. Analysis of intra-host distribution of the parasite and strontium to calcium ratios from the otoliths revealed that (re)infection through freshwater migration may occur among adult sea trout. The results of this study indicate that T. bryosalmonae can persist in a brackish water environment for several years and that returning sea trout spawners most likely contribute to the parasite life cycle by transmitting infective spores.

Whole-genome sequencing illuminates multifaceted targets of selection to humic substances in Eurasian perch.

Extreme environments are inhospitable to the majority of species, but some organisms are able to survive in such hostile conditions due to evolutionary adaptations. For example, modern bony fishes have colonized various aquatic environments, including perpetually dark, hypoxic, hypersaline and toxic habitats. Eurasian perch (Perca fluviatilis) is among the few fish species of northern latitudes that is able to live in very acidic humic lakes. Such lakes represent almost "nocturnal" environments; they contain high levels of dissolved organic matter, which in addition to creating a challenging visual environment, also affects a large number of other habitat parameters and biotic interactions. To reveal the genomic targets of humic-associated selection, we performed whole-genome sequencing of perch originating from 16 humic and 16 clear-water lakes in northern Europe. We identified over 800,000 single nucleotide polymorphisms, of which >10,000 were identified as potential candidates under selection (associated with >3000 genes) using multiple outlier approaches. Our findings suggest that adaptation to the humic environment may involve hundreds of regions scattered across the genome. Putative signals of adaptation were detected in genes and gene families with diverse functions, including organism development and ion transportation. The observed excess of variants under selection in regulatory regions highlights the importance of adaptive evolution via regulatory elements, rather than via protein sequence modification. Our study demonstrates the power of whole-genome analysis to illuminate the multifaceted nature of humic adaptation and provides the foundation for further investigation of causal mutations underlying phenotypic traits of ecological and evolutionary importance.

A century of genetic homogenization in Baltic salmon-evidence from archival DNA.

Intra-species genetic homogenization arising from anthropogenic impacts is a major threat to biodiversity. However, few taxa have sufficient historical material to systematically quantify long-term genetic changes. Using archival DNA collected over approximately 100 years, we assessed spatio-temporal genetic change in Atlantic salmon populations across the Baltic Sea, an area heavily impacted by hydropower exploitation and associated with large-scale mitigation stocking. Analysis was carried out by screening 82 SNPs in 1680 individuals from 13 Swedish rivers. We found an overall decrease in genetic divergence and diminished isolation by distance among populations, strongly indicating genetic homogenization over the past century. We further observed an increase in genetic diversity within populations consistent with increased gene flow. The temporal genetic change was lower in larger wild populations than in smaller wild and hatchery-reared ones, indicating that larger populations have been able to support a high number of native spawners in relation to immigrants. Our results demonstrate that stocking practices of salmon in the Baltic Sea have led to the homogenization of populations over the last century, potentially compromising their ability to adapt to environmental change. Stocking of reared fish is common worldwide, and our study is a cautionary example of the potentially long-term negative effects of such activities.

The strength and form of natural selection on transcript abundance in the wild.

Gene transcription variation is known to contribute to disease susceptibility and adaptation, but we currently know very little about how contemporary natural selection shapes transcript abundance. Here, we propose a novel analytical framework to quantify the strength and form of ongoing natural selection at the transcriptome level in a wild vertebrate. We estimated selection on transcript abundance in a cohort of a wild salmonid fish (Salmo trutta) affected by an extracellular myxozoan parasite (Tetracapsuloides bryosalmonae) through mark-recapture field sampling and the integration of RNA-sequencing with classical regression-based selection analysis. We show, based on fin transcriptomes of the host, that infection by the parasite and subsequent host survival is linked to upregulation of mitotic cell cycle process. We also detect a widespread signal of disruptive selection on transcripts linked to host immune defence, host-pathogen interactions, cellular repair and maintenance. Our results provide insights into how selection can be measured at the transcriptome level to dissect the molecular mechanisms of contemporary evolution driven by climate change and emerging anthropogenic threats. We anticipate that the approach described here will enable critical information on the molecular processes and targets of natural selection to be obtained in real time.

Know your enemy - transcriptome of myxozoan Tetracapsuloides bryosalmonae reveals potential drug targets against proliferative kidney disease in salmonids.

The myxozoan Tetracapsuloides bryosalmonae is a widely spread endoparasite that causes proliferative kidney disease (PKD) in salmonid fish. We developed an in silico pipeline to separate transcripts of T. bryosalmonae from the kidney tissue of its natural vertebrate host, brown trout (Salmo trutta). After stringent filtering, we constructed a partial transcriptome assembly T. bryosalmonae, comprising 3427 transcripts. Based on homology-restricted searches of the assembled parasite transcriptome and Atlantic salmon (Salmo salar) proteome, we identified four protein targets (Endoglycoceramidase, Legumain-like protease, Carbonic anhydrase 2, Pancreatic lipase-related protein 2) for the development of anti-parasitic drugs against T. bryosalmonae. Earlier work of these proteins on parasitic protists and helminths suggests that the identified anti-parasitic drug targets represent promising chemotherapeutic candidates also against T. bryosalmonae, and strengthen the view that the known inhibitors can be effective in evolutionarily distant organisms. In addition, we identified differentially expressed T. bryosalmonae genes between moderately and severely infected fish, indicating an increased abundance of T. bryosalmonae sporogonic stages in fish with low parasite load. In conclusion, this study paves the way for future genomic research in T. bryosalmonae and represents an important step towards the development of effective drugs against PKD.

Climate change-driven disease in sympatric hosts: Temporal dynamics of parasite burden and proliferative kidney disease in wild brown trout and Atlantic salmon.

Global climate change is altering the abundance and spread of various parasites, which has important consequences not only for host-parasite interactions but also for the relationships between different host species. Here, we focus on the myxozoan endoparasite Tetracapsuloides bryosalmonae that causes temperature-dependent proliferative kidney disease (PKD) in salmonids. We characterized the temporal changes in the parasite load and the severity of PKD signs (renal hyperplasia, haematocrit) in two sympatric populations of wild brown trout (Salmo trutta) and Atlantic salmon (Salmo salar). We found that both the parasite load and disease signs vary considerably between individuals, species, rivers and sampling periods. We showed that Atlantic salmon was able to slow down the initial parasite proliferation rate and subsequently tolerate high parasite burden without obvious disease signs. In contrast, the initial parasite proliferation rate was much higher in brown trout, which was followed by the development of severe PKD signs. Thus, the speed of parasite proliferation, rather than the absolute number of the parasites in the host kidney, may play an important role in interspecific variation in PKD susceptibility. To conclude, this study illustrates the usefulness of temporal perspective for understanding host defence mechanisms and climate change-mediated impacts in the wild.

Mapping of quantitative trait loci for life history traits segregating within common frog populations.

The evolution of complex traits is often shaped by adaptive divergence. However, very little is known about the number, effect size, and location of the genomic regions influencing the variation of these traits in natural populations. Based on a dense linkage map of the common frog, Rana temporaria, we have localized, for the first time in amphibians, three significant and nine suggestive quantitative trait loci (QTLs) for metabolic rate, growth rate, development time, and weight at metamorphosis, explaining 5.6-18.9% of the overall phenotypic variation in each trait. We also found a potential pleiotropic QTL between development time and size at metamorphosis that, if confirmed, might underlie the previously reported genetic correlation between these traits. Furthermore, we demonstrate that the genetic variation linked to fitness-related larval traits segregates within Rana temporaria populations. This study provides the first insight into the genomic regions that affect larval life history traits in anurans, providing a valuable resource to delve further into the genomic basis of evolutionary change in amphibians.

Quantitative Genetic Variation in, and Environmental Effects on, Pathogen Resistance and Temperature-Dependent Disease Severity in a Wild Trout.

Health after pathogen contact varies among individuals because of differences in pathogen load (which is limited by resistance) and disease severity in response to pathogen load (which is limited by tolerance). To understand pathogen-induced host evolution, it is critical to know not only the relative contributions of nongenetic and genetic variation to resistance and tolerance but also how they change environmentally. We quantified nongenetic and genetic variation in parasite load and the associated temperature-dependent disease among trout siblings from two rivers. We detected a genetic variance for parasite load 6.6 times as large in the colder river. By contrast, genetic variance for disease traits tended to be larger in the warmer river, where the disease was manifested more severely. The relationships between disease severity and pathogen load (tolerance) exhibited plateaus at low pathogen load and stronger steepening slopes at high pathogen load in the warmer river. Our study demonstrates the environmental influence on disease severity, nongenetic and genetic variance for health-damage-limiting host abilities, and the shape of tolerance curves. Environmental variability is predicted to govern the presence and intensity of selection, change the relative contributions of nongenetic and genetic variance, and therefore hamper evolution toward more resistant and tolerant hosts.

Telomere length and antioxidant defense associate with parasite-induced retarded growth in wild brown trout.

Early growth conditions can have profound impacts on individuals' development, growth and physiology, with subsequent long-term consequences for individuals' fitness and life expectancy. Telomere length (TL) has been suggested to indicate both individual fitness and life expectancy in wide range of species, as the telomere attrition rate at early age can be accelerated due to exposure to various stressors, including parasites and inflammatory diseases, which increase production of reactive oxygen species (ROS) and influence antioxidant (AO) levels. We investigated impacts of Tetracapsuloides bryosalmonae infection, a causative agent of proliferative kidney disease (PKD), on AO status and TL in a natural population of juvenile brown trout (Salmo trutta). The fish with higher parasite load showed more severe kidney hyperplasia, anemia and smaller body size compared to less parasitized fish. Furthermore, fish with severe PKD symptoms had lower SOD-, CAT- and GST activity than fish with milder kidney hyperplasia. However, parasite load was not directly correlated either with AOs or with TL. Smaller fish showed shorter TLs, potentially reflecting lower individual quality. The fish, which were less sensitive to parasite-induced impaired growth, quantified as parasite load-adjusted fork length, showed also longer TLs, lower GR- and GST activity and less GSHtot compared to more sensitive fish. These results provide novel knowledge about the impacts of the PKD in brown trout at the molecular level and support the idea that TL may reflect individual quality and ability to cope with parasitic infections.

First record of proliferative kidney disease agent Tetracapsuloides bryosalmonae in wild brown trout and European grayling in Finland.

The myxozoan endoparasite Tetracapsuloides bryosalmonae causes temperature-driven proliferative kidney disease (PKD) in salmonid fishes. Despite the economic and ecological importance of PKD, information about the distribution of the parasite is still scarce. Here, we report for the first time the occurrence of T. bryosalmonae in wild brown trout Salmo trutta and European grayling Thymallus thymallus populations in Finland. We detected T. bryosalmonae at high prevalence in both brown trout and European grayling from the transboundary Finnish-Russian River Koutajoki system (Rivers Oulankajoki, Kuusinkijoki, Kitkajoki, Maaninkajoki, and Juumajoki) in north-eastern Finland. In southern Finland, T. bryosalmonae was detected in River Siuntionjoki young-of-the-year brown trout collected both in 2015 and 2016 (100% prevalence), while the parasite was not observed in fish from 3 other rivers (Ingarskila, Mustajoki, and Vantaanjoki) flowing to the Gulf of Finland. Our results, together with those from recent studies of Atlantic salmon, indicate that T. bryosalmonae is distributed over much higher latitudes in northern Europe than previously appreciated. We expect that increasing water temperatures will likely cause new PKD outbreaks in these more northerly regions in the future.

Is telomere length a molecular marker of past thermal stress in wild fish?

Telomeres protect eukaryotic chromosomes; variation in telomere length has been linked (primarily in homoeothermic animals) to variation in stress, cellular ageing and disease risk. Moreover, telomeres have been suggested to function as biomarker for quantifying past environmental stress, but studies in wild animals remain rare. Environmental stress, such as extreme environmental temperatures in poikilothermic animals, may result in oxidative stress that accelerates telomere attrition. However, growth, which may depend on temperature, can also contribute to telomere attrition. To test for associations between multitissue telomere length and past water temperature while accounting for the previous individual growth, we used quantitative PCR to analyse samples from 112 young-of-the-year brown trout from 10 natural rivers with average water temperature differences of up to 6°C (and an absolute maximum of 23°C). We found negative associations between relative telomere length (RTL) and both average river temperature and individual body size. We found no indication of RTL-temperature association differences among six tissues, but we did find indications for differences among the tissues for associations between RTL and body size; size trends, albeit nonsignificant in their differences, were strongest in muscle and weakest in fin. Although causal relationships among temperature, growth, oxidative stress, and cross-sectional telomere length remain largely unknown, our results indicate that telomere-length variation in a poikilothermic wild animal is associated with both past temperature and growth.

Genetic variation facilitates seedling establishment but not population growth rate of a perennial invader.

BACKGROUND AND AIMS: Assessing the demographic consequences of genetic variation is fundamental to invasion biology. However, genetic and demographic approaches are rarely combined to explore the effects of genetic variation on invasive populations in natural environments. This study combined population genetics, demographic data and a greenhouse experiment to investigate the consequences of genetic variation for the population fitness of the perennial, invasive herb Lupinus polyphyllus. METHODS: Genetic and demographic data were collected from 37 L. polyphyllus populations representing different latitudes in Finland, and genetic variation was characterized based on 13 microsatellite loci. Associations between genetic variation and population size, population density, latitude and habitat were investigated. Genetic variation was then explored in relation to four fitness components (establishment, survival, growth, fecundity) measured at the population level, and the long-term population growth rate (λ). For a subset of populations genetic variation was also examined in relation to the temporal variability of λ. A further assessment was made of the role of natural selection in the observed variation of certain fitness components among populations under greenhouse conditions. KEY RESULTS: It was found that genetic variation correlated positively with population size, particularly at higher latitudes, and differed among habitat types. Average seedling establishment per population increased with genetic variation in the field, but not under greenhouse conditions. Quantitative genetic divergence (Q(ST)) based on seedling establishment in the greenhouse was smaller than allelic genetic divergence (F'(ST)), indicating that unifying selection has a prominent role in this fitness component. Genetic variation was not associated with average survival, growth or fecundity measured at the population level, λ or its variability. CONCLUSIONS: The study suggests that although genetic variation may facilitate plant invasions by increasing seedling establishment, it may not necessarily affect the long-term population growth rate. Therefore, established invasions may be able to grow equally well regardless of their genetic diversity.

Use of sibling relationship reconstruction to complement traditional monitoring in fisheries management and conservation of brown trout.

Declining trends in the abundance of many fish urgently call for more efficient and informative monitoring methods that would provide necessary demographic data for the evaluation of existing conservation, restoration, and management actions. We investigated how genetic sibship reconstruction from young-of-the-year brown trout (Salmo trutta L.) juveniles provides valuable, complementary demographic information that allowed us to disentangle the effects of habitat quality and number of breeders on juvenile density. We studied restored (n = 15) and control (n = 15) spawning and nursery habitats in 16 brown trout rivers and streams over 2 consecutive years to evaluate the effectiveness of habitat restoration activities. Similar juvenile densities both in restored and control spawning and nursery grounds were observed. Similarly, no differences in the effective number of breeders, Nb(SA) , were detected between habitats, indicating that brown trout readily used recently restored spawning grounds. Only a weak relationship between the Nb(SA) and juvenile density was observed, suggesting that multiple factors affect juvenile abundance. In some areas, very low estimates of Nb(SA) were found at sites with high juvenile density, indicating that a small number of breeders can produce a high number of progeny in favorable conditions. In other sites, high Nb(SA) estimates were associated with low juvenile density, suggesting low habitat quality or lack of suitable spawning substrate in relation to available breeders. Based on these results, we recommend the incorporation of genetic sibship reconstruction to ongoing and future fish evaluation and monitoring programs to gain novel insights into local demographic and evolutionary processes relevant for fisheries management, habitat restoration, and conservation.

El Uso de la Reconstrucción de Relaciones Fraternales para Complementar el Monitoreo Tradicional en el Manejo de Pesquerías y en la Conservación de la Trucha Café Resumen Las tendencias de declinación en la abundancia de muchos peces son un llamado urgente para métodos de monitoreo más eficientes e informativos que proporcionarían los datos demográficos necesarios para la evaluación de la conservación actual, la restauración y las acciones de manejo. Investigamos cómo la reconstrucción genética del número de crías producidas por una pareja de padres de juveniles de ese mismo año de trucha café (Salmo trutta L.) proporciona información demográfica valiosa y complementaria, la cual nos permitió revelar los efectos de la calidad de hábitat y el número de reproductores sobre la densidad juvenil. Estudiamos hábitats de reproducción y crianza restaurados (n = 15) y controlados (n = 15) en 16 ríos y arroyos de trucha café a lo largo de dos años consecutivos para evaluar la efectividad de las actividades de restauración de hábitat. Se observaron densidades juveniles similares en ambos tipos de hábitat. De manera similar, no se detectaron diferencias entre los hábitats en el número efectivo de reproductores (Nb(SA) ), lo que indica que la trucha café usó de manera inmediata y reciente los hábitats de reproducción restaurados. Sólo se observó una relación débil entre el Nb(SA) y la densidad juvenil, lo que sugiere que múltiples factores afectan a la abundancia juvenil. En algunas áreas, se encontraron estimados muy bajos de Nb(SA) en sitios con una alta densidad juvenil, lo que indica que un pequeño número de reproductores puede producir un alto número de descendencia en condiciones favorables. En otros sitios, los estimados altos de Nb(SA) estuvieron asociados con una baja densidad juvenil, lo que sugiere que una baja calidad de hábitat o la falta de un sustrato adecuado para la reproducción en relación con los reproductores disponibles. Con base en estos resultados, recomendamos la incorporación de esta reconstrucción genética a la evaluación actual y futura de peces y a los programas de monitoreo para obtener percepciones novedosas de los procesos demográficos y evolutivos relevantes para el manejo de pesqueras, restauración de hábitat y conservación.

First microsatellite loci of the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD).

Proliferative kidney disease (PKD) caused by the myxozoan parasite Tetracapsuloides bryosalmonae is a severe parasitic disease of salmonid fish. Estimates of genetic variation in parasite populations across Europe are currently lacking. We developed the first polymorphic microsatellite markers for T. bryosalmonae using Illumina MiSeq sequence data derived from genomic DNA. Twelve polymorphic loci were identified from 24 tested loci. Allelic variation was low at most loci, ranging from 2 to 6 (average 3.0). The markers developed here are expected to be useful in future genetic studies of T. bryosalmonae, aimed at further understanding the dispersal of the parasite, host-parasite relationships and the epidemiology of PKD.

Proliferative kidney disease (PKD) agent Tetracapsuloides bryosalmonae in brown trout populations in Estonia.

Proliferative kidney disease (PKD) caused by the myxozoan parasite Tetracapsuloides bryosalmonae is a serious parasitic disease threatening both farmed and wild salmonid populations, but very little is currently known about the distribution of the parasite in the Baltic Sea region. In this study we (1) report the development of a novel multiplex PCR method for fast and reliable screening of T. bryosalmonae; (2) use this multiplex PCR method to show that the PKD agent T. bryosalmonae is widespread in natural brown trout Salmo trutta L. populations in Estonia; (3) evaluate monthly and yearly variation of T. bryosalmonae prevalence in juvenile trout; (4) assess T. bryosalmonae prevalence in different age-classes of fish (0+ vs. 1+ and older) and report the presence of the PKD agent in the kidneys of returning sea trout spawners; and (5) suggest the freshwater bryozoan Plumatella fungosa as a putative invertebrate host of T. bryosalmonae in Estonia. Our results demonstrate a highly heterogeneous distribution of T. bryosalmonae at the micro-geographic scale, indicating that PKD could have an important negative effect on recruitment in Estonian brown trout populations.

DNA metabarcoding reveals spatial and temporal variation of fish eye fluke communities in lake ecosystems.

Eye flukes (Diplostomidae) are diverse and abundant trematode parasites that form multi-species communities in fish with negative effects on host fitness and survival. However, the environmental factors and host-related characteristics that determine species diversity, composition, and coexistence in such communities remain poorly understood. Here, we developed a cost-effective cox1 region-specific DNA metabarcoding approach to characterize parasitic diplostomid communities in two common fish species (Eurasian perch and common roach) collected from seven temperate lakes in Estonia. We found considerable inter- and intra-lake, as well as inter-host species, variation in diplostomid communities. Sympatric host species characterization revealed that parasite communities were typically more diverse in roach than perch. Additionally, we detected five positive and two negative diplostomid species associations in roach, whereas only a single negative association was observed in perch. These results indicate that diplostomid communities in temperate lakes are complex and dynamic systems exhibiting both spatial and temporal heterogeneity. They are influenced by various environmental factors and by host-parasite and inter-parasite interactions. We expect that the described methodology facilitates ecological and biodiversity research of diplostomid parasites. It is also adaptable to other parasite groups where it could serve to improve current understanding of diversity, distribution, and interspecies interactions of other understudied taxa.

Cost-effective genome-wide estimation of allele frequencies from pooled DNA in Atlantic salmon (Salmo salar L.).

BACKGROUND: New sequencing technologies have tremendously increased the number of known molecular markers (single nucleotide polymorphisms; SNPs) in a variety of species. Concurrently, improvements to genotyping technology have now made it possible to efficiently genotype large numbers of genome-wide distributed SNPs enabling genome wide association studies (GWAS). However, genotyping significant numbers of individuals with large number of SNPs remains prohibitively expensive for many research groups. A possible solution to this problem is to determine allele frequencies from pooled DNA samples, such 'allelotyping' has been presented as a cost-effective alternative to individual genotyping and has become popular in human GWAS. In this article we have tested the effectiveness of DNA pooling to obtain accurate allele frequency estimates for Atlantic salmon (Salmo salar L.) populations using an Illumina SNP-chip. RESULTS: In total, 56 Atlantic salmon DNA pools from 14 populations were analyzed on an Atlantic salmon SNP-chip containing probes for 5568 SNP markers, 3928 of which were bi-allelic. We developed an efficient quality control filter which enables exclusion of loci showing high error rate and minor allele frequency (MAF) close to zero. After applying multiple quality control filters we obtained allele frequency estimates for 3631 bi-allelic loci. We observed high concordance (r > 0.99) between allele frequency estimates derived from individual genotyping and DNA pools. Our results also indicate that even relatively small DNA pools (35 individuals) can provide accurate allele frequency estimates for a given sample. CONCLUSIONS: Despite of higher level of variation associated with array replicates compared to pool construction, we suggest that both sources of variation should be taken into account. This study demonstrates that DNA pooling allows fast and high-throughput determination of allele frequencies in Atlantic salmon enabling cost-efficient identification of informative markers for discrimination of populations at various geographical scales, as well as identification of loci controlling ecologically and economically important traits.

Fish scales and SNP chips: SNP genotyping and allele frequency estimation in individual and pooled DNA from historical samples of Atlantic salmon (Salmo salar).

BACKGROUND: DNA extracted from historical samples is an important resource for understanding genetic consequences of anthropogenic influences and long-term environmental change. However, such samples generally yield DNA of a lower amount and quality, and the extent to which DNA degradation affects SNP genotyping success and allele frequency estimation is not well understood. We conducted high density SNP genotyping and allele frequency estimation in both individual DNA samples and pooled DNA samples extracted from dried Atlantic salmon (Salmo salar) scales stored at room temperature for up to 35 years, and assessed genotyping success, repeatability and accuracy of allele frequency estimation using a high density SNP genotyping array. RESULTS: In individual DNA samples, genotyping success and repeatability was very high (> 0.973 and > 0.998, respectively) in samples stored for up to 35 years; both increased with the proportion of DNA of fragment size > 1000 bp. In pooled DNA samples, allele frequency estimation was highly repeatable (Repeatability = 0.986) and highly correlated with empirical allele frequency measures (Mean Adjusted R2 = 0.991); allele frequency could be accurately estimated in > 95% of pooled DNA samples with a reference group of at least 30 individuals. SNPs located in polyploid regions of the genome were more sensitive to DNA degradation: older samples had lower genotyping success at these loci, and a larger reference panel of individuals was required to accurately estimate allele frequencies. CONCLUSIONS: SNP genotyping was highly successful in degraded DNA samples, paving the way for the use of degraded samples in SNP genotyping projects. DNA pooling provides the potential for large scale population genetic studies with fewer assays, provided enough reference individuals are also genotyped and DNA quality is properly assessed beforehand. We provide recommendations for future studies intending to conduct high-throughput SNP genotyping and allele frequency estimation in historical samples.