A GT-seq panel for walleye (Sander vitreus) provides important insights for efficient development and implementation of amplicon panels in non-model organisms.

Targeted amplicon sequencing methods, such as genotyping-in-thousands by sequencing (GT-seq), facilitate rapid, accurate, and cost-effective analysis of hundreds of genetic loci in thousands of individuals. Development of GT-seq panels is nontrivial, but studies describing trade-offs associated with different steps of GT-seq panel development are rare. Here, we construct a dual-purpose GT-seq panel for walleye (Sander vitreus), discuss trade-offs associated with different development and genotyping approaches, and provide suggestions for researchers constructing their own GT-seq panels. Our GT-seq panel was developed using an ascertainment set consisting of restriction site-associated DNA data from 954 individuals sampled from 23 populations in Minnesota and Wisconsin, USA. We conducted simulations to test the utility of all loci for parentage analysis and genetic stock identification and designed 600 primer pairs to maximize joint accuracy for these analyses. We then performed three rounds of primer optimization to remove loci that overamplified and our final panel consisted of 436 loci. We also explored different approaches for DNA extraction, multiplexed polymerase chain reaction (PCR) amplification, and cleanup steps during the GT-seq process and discovered the following: (i) inexpensive Chelex extractions performed well for genotyping; (ii) the exonuclease I and shrimp alkaline phosphatase (ExoSAP) procedure included in some current protocols did not improve results substantially and was probably unnecessary; and (iii) it was possible to PCR amplify panels separately and combine them prior to adapter ligation. Well-optimized GT-seq panels are valuable resources for conservation genetics and our findings and suggestions should aid in their construction in myriad taxa.

Genetic evidence of a northward range expansion in the eastern Bering Sea stock of Pacific cod.

Poleward species range shifts have been predicted to result from climate change, and many observations have confirmed such movement. Poleward shifts may represent a homogeneous shift in distribution, seasonal northward movement of specific populations, or colonization processes at the poleward edge of the distribution. The ecosystem of the Bering Sea has been changing along with the climate, moving from an arctic to a subarctic system. Several fish species have been observed farther north than previously reported and in increasing abundances. We examined one of these fish species, Pacific cod, in the northern Bering Sea (NBS) to assess whether they migrated from another stock in the eastern Bering Sea (EBS), Gulf of Alaska, or Aleutian Islands, or whether they represent a separate population. Genetic analyses using 3,599 single nucleotide polymorphism markers indicated that nonspawning cod collected in August 2017 in the NBS were similar to spawning stocks of cod in the EBS. This result suggests escalating northward movement of the large EBS stock during summer months. Whether the cod observed in the NBS migrate south during winter to spawn or remain in the NBS as a sink population is unknown.

Population assignment and local adaptation along an isolation-by-distance gradient in Pacific cod (Gadus macrocephalus).

The discernment of populations as management units is a fundamental prerequisite for sustainable exploitation of species. A lack of clear stock boundaries complicates not only the identification of spatial management units, but also the assessment of mixed fisheries by population assignment and mixed stock analysis. Many marine species, such as Pacific cod, are characterized by isolation by distance, showing significant differentiation but no clear stock boundaries. Here, we used restriction-site-associated DNA (RAD) sequencing to investigate population structure and assess power to genetically assign Pacific cod to putative populations of origin. Samples were collected across the species range in the eastern Pacific Ocean, from the Salish Sea to the Aleutian Islands. A total of 6,425 putative biallelic single nucleotide polymorphisms were identified from 276 individuals. We found a strong isolation-by-distance signal along coastlines that mirrored previous microsatellite results and pronounced genetic differentiation between coastal samples and those from the inland waters of the Salish Sea, with no evidence for hybridization between these two populations. Individual assignment success based on two methods was high overall (≥84%) but decreased from south to north. Assignment to geographic location of origin also was successful, with average distance between capture and assignment location of 220 km. Outlier analyses identified more loci potentially under selection along the coast than between Salish Sea and coastal samples, suggesting more diverse adaptation to latitudinal environmental factors than inshore vs. offshore environments. Our results confirm previous observations of sharp genetic differentiation of the Salish Sea population and isolation by distance along the coast, but also highlight the feasibility of using modern genomic techniques to inform stock boundaries and fisheries management in a low FST marine species.

Toward responsible stock enhancement: broadcast spawning dynamics and adaptive genetic management in white seabass aquaculture.

The evolutionary effects captive-bred individuals that can have on wild conspecifics are necessary considerations for stock enhancement programs, but breeding protocols are often developed without the knowledge of realized reproductive behavior. To help fill that gap, parentage was assigned to offspring produced by a freely mating group of 50 white seabass (Atractoscion nobilis), a representative broadcast spawning marine finfish cultured for conservation. Similar to the well-known and closely related red drum (Sciaenops ocellatus), A. nobilis exhibited large variation in reproductive success. More males contributed and contributed more equally than females within and among spawns in a mating system best described as lottery polygyny. Two females produced 27% of the seasonal offspring pool and female breeding effective size averaged 1.85 per spawn and 12.38 seasonally, whereas male breeding effective size was higher (6.42 and 20.87, respectively), with every male contributing 1-7% of offspring. Further, females batch spawned every 1-5 weeks, while males displayed continuous reproductive readiness. Sex-specific mating strategies resulted in multiple successful mate pairings and a breeding effective to census size ratio of ≥0.62. Understanding a depleted species' mating system allowed management to more effectively utilize parental genetic variability for culture, but the fitness consequences of long-term stocking can be difficult to address.

A comparison of the copper sensitivity of two economically important saltwater mussel species and a review of previously reported copper toxicity data for mussels: important implications for determining future ambient copper saltwater criteria in the USA.

Saltwater bivalves of the genus Mytilus are among the most copper sensitive taxa listed in both the current and recently proposed U.S. EPA ambient saltwater copper criteria documents. The copper saltwater quality criteria are somewhat unique in that the criteria were set specifically to protect Mytilus. However, there is considerable uncertainty in the reported taxonomy of Mytilus species in the criteria database and it has recently been demonstrated the copper toxicity to M. galloprovincialis is dependent on the organic matter content of the test water. A review of the toxicity and biogeography literature was conducted to rationalize the existing criteria database. Elimination of some data is suggested due to the uncertainty of test organism genotype. Moreover, due to the lack of reported dissolved organic matter content of the test waters in tests included in the criteria database, it is impossible to determine if the difference in species mean acute values reported in the criteria documents for Mytilus was due to differences in water chemistry or differences in species sensitivity. Experiments were designed and conducted with M. galloprovincialis and M. edulis (genetically confirmed) to determine if copper toxicity is a function of organic matter content for these two species and if there is a significant difference in species copper sensitivity. Results showed that copper toxicity is a function of organic matter concentration for both species and copper sensitivity of each species was statistically similar. Results support the normalization of the saltwater copper criteria database with respect to dissolved organic matter when developing ambient saltwater copper criteria. The USEPA toxicity database would benefit from future testing of M. trossulus and M. californianus.

Estrogen receptor-alpha variants are associated with lipoprotein size distribution and particle levels in women: the Framingham Heart Study.

Plasma lipid profile is affected by endogenous estrogen levels and hormone replacement therapy (HRT). As plasma lipid concentrations have a significant heritable basis and the effects of both endogenous estrogen and use of HRT are mediated by estrogen receptors, we sought to investigate the relationships between polymorphisms in estrogen receptor-alpha (ESR1) and plasma lipid and lipoprotein concentrations. We analyzed data from 854 women (mean age 52+/-10 years) from the Framingham Heart Study. A TA repeat in the promoter region, c.30T>C in exon 1, c.454-397T>C, and c.454-351A>G in intron 1, all in linkage disequilibrium (LD), were significantly associated with low-density lipoprotein (LDL) particle size and concentration of small LDL particles. Women with the c.454-397C allele had larger LDL particle size (21.09+/-0.02 nm versus 21.01+/-0.03 nm, p=0.021) concurrent with lower small LDL particle concentration (0.47+/-0.02 mmol/L versus 0.58+/-0.03 mmol/L, p=0.008). Moreover, the TA[L]-c.30C-c.454-397C-c.454-351G haplotype (frequency, 32%) was associated with lower small LDL particle concentrations (-0.06+/-0.03 mmol/L change associated with each copy of this haplotype, p=0.011) when compared to the TA[S]-c.30T-c.454-397T-c.454-351A haplotype (frequency, 46%), where L and S are long and short TA repeats. Our results suggest that common ESR1 polymorphisms have a significant effect on lipoprotein metabolism in women.

Estrogen receptor beta polymorphisms are associated with bone mass in women and men: the Framingham Study.

UNLABELLED: ESR2 is expressed in bone cells, yet few studies have tested its variation for association with BMD, an important determinant of osteoporotic fractures. This was investigated in 723 men and 795 women from the Framingham study. Results show association of variation in this gene with BMD in both women and men. INTRODUCTION: Osteoporotic fracture risk is highly dependent on bone density, a quantitative multifactorial trait with a substantial genetic component. In contrast to the growing body of evidence that estrogen receptor alpha (ESR1) plays a role in bone metabolism, few studies have examined the estrogen receptor beta (ESR2) gene for association with BMD. An ESR2 CA repeat polymorphism, D14S1026, was associated with BMD in two small studies, each with <200 women. MATERIALS AND METHODS: The objective of this investigation was to assess whether D14S1026 or four other intronic polymorphisms were associated with BMD in 723 men and 795 women (mean age, 60 years) from the offspring cohort of the population-based Framingham Study. BMD was measured at the femur (neck, trochanter, and Ward's area) and the lumbar spine (L(2)-L(4)). RESULTS: In both women and men, there was significant association of D14S1026 genotype with measures of femoral but not spinal BMD. In addition, genotypes of two common single nucleotide polymorphisms, rs1256031 and rs1256059, in strong linkage disequilibrium with one another but not with D14S1026, were associated with measures of femoral BMD in men. The rs1256031 genotypes had up to a 4.0% difference in mean femoral BMD. An inferred rs1256031-D14S1026-rs1256059 haplotype C-23CA-T was significantly associated with reduced femoral BMD in women (p = 0.03, 0.003, and 0.01 for neck, trochanter, and Ward's area, respectively). Haplotype-based BMD differences ranged from 3.0% to 4.3%. CONCLUSIONS: We have observed significant association of common ESR2 variants with measures of femoral BMD in both men and women.