Efficient population representation with more genetic markers increases performance of a steelhead (Oncorhynchus mykiss) genetic stock identification baseline.

Genetic stock identification (GSI) is an important fisheries management tool to identify the origin of fish harvested in mixed stock fisheries. Periodic updates of genetic baselines can improve performance via the addition of unsampled or under-sampled populations and the inclusion of more informative markers. We used a combination of baselines to evaluate how population representation, marker number, and marker type affected the performance and accuracy of genetic stock assignments (self-assignment, bias, and holdout group tests) for steelhead (Oncorhynchus mykiss) in the Snake River basin. First, we compared the performance of an existing genetic baseline with a newly developed one which had a reduced number of individuals from more populations using the same set of markers. Self-assignment rates were significantly higher (p < 0.001; +5.4%) for the older, larger baseline, bias did not differ significantly between the two, but there was a significant improvement in performance for the new baseline in holdout results (p < 0.001; mean increase of 25.0%). Second, we compared the performance of the new baseline with increased numbers of genetic markers (~2x increase of single-nucleotide polymorphisms; SNPs) for the same set of baseline individuals. In this comparison, results produced significantly higher rates of self-assignment (p < 0.001; +9.7%) but neither bias nor leave-one-out were significantly affected. Third, we compared 334 SNPs versus opportunistically discovered microhaplotypes from the same amplicons for the new baseline, and showed the latter produced significantly higher rates of self-assignment (p < 0.01; +2.6%), similar bias, but slightly lower holdout performance (-0.1%). Combined, we show the performance of genetic baselines can be improved via representative and efficient sampling, that increased marker number consistently improved performance over the original baseline, and that opportunistic discovery of microhaplotypes can lead to small improvements in GSI performance.

Estimating microhaplotype allele frequencies from low-coverage or pooled sequencing data.

BACKGROUND: Microhaplotypes have the potential to be more cost-effective than SNPs for applications that require genetic panels of highly variable loci. However, development of microhaplotype panels is hindered by a lack of methods for estimating microhaplotype allele frequency from low-coverage whole genome sequencing or pooled sequencing (pool-seq) data. RESULTS: We developed new methods for estimating microhaplotype allele frequency from low-coverage whole genome sequence and pool-seq data. We validated these methods using datasets from three non-model organisms. These methods allowed estimation of allele frequency and expected heterozygosity at depths routinely achieved from pooled sequencing. CONCLUSIONS: These new methods will allow microhaplotype panels to be designed using low-coverage WGS and pool-seq data to discover and evaluate candidate loci. The python script implementing the two methods and documentation are available at https://www.github.com/delomast/mhFromLowDepSeq .

Single-parentage assignments reveal negative-assortative mating in an endangered salmonid.

Understanding reproductive patterns in endangered species is critical for supporting their recovery efforts. In this study we use a combination of paired-parent and single-parent assignments to examine the reproductive patterns in an endangered population of sockeye salmon (Oncorhynchus nerka) that uses Redfish Lake in central Idaho as a spawning and nursery lake. Recovery efforts include the release of maturing adults into the lake for volitional spawning. The lake is also inhabited by a population of resident O. nerka that is genetically indistinguishable, but phenotypically smaller, to the maturing adults released into the lake. The resident population is difficult to sample and the reproductive patterns between the two groups are unknown. We used results of paired- and single-parentage assignments to specifically examine the reproductive patterns of male fish released into the lake under an equal sex ratio and a male-biased sex ratio. Assignment results of offspring leaving the lake indicated a reproductive shift by males under the two scenarios. Males displayed an assortative mating pattern under an equal sex ratio and spawned almost exclusively with the released females. Under a male-biased sex ratio most males shifted to a negative-assortative mating pattern and spawned with smaller females from the resident population. These males were younger and smaller than males that spawned with released females suggesting they were unable to compete with larger males for spawning opportunities with the larger, released females. The results provided insights into the reproductive behavior of this endangered population and has implications for recovery efforts.

Sex-specific markers undetected in green sunfish Lepomis cyanellus using restriction-site associated DNA sequencing.

We used restriction-site associated DNA sequencing for SNP discovery and genotyping of known-sex green sunfish Lepomis cyanellus DNA samples to search for sex-diagnostic single nucleotide polymorphisms (SNPs) and restriction-site associated sequences present in one sex and absent in the other. The bioinformatic analyses discovered candidate SNPs and sex-specific restriction-site associated sequences that fit patterns of male or female heterogametic sex determination systems. However, when primers were developed and tested, no candidates reliably identified phenotypic sex. The top performing SNP candidate (ZW_218) correlated with phenotypic sex 63.0% of the time and the presence-absence loci universally amplified in both sexes. We recommend further investigations that interrogate a larger fraction of the L. cyanellus genome. Additionally, studies on the effect of temperature and rearing density on sex determination, as well as breeding of sex-reversed individuals, could provide more insights into the sex determination system of L. cyanellus.

Asymmetric viability in reciprocal crosses of zebrafish Danio rerio and pearl danio Danio albolineatus.

Interspecies hybrids have long been studied to further understanding of speciation. Reciprocal crosses sometimes have asymmetric viability, a phenomenon termed 'Darwin's corollary to Haldane's rule'. It has been proposed that this asymmetry is caused by Dobzhansky-Muller incompatibilities between nuclear genes and cytoplasmic factors (e.g., maternal transcripts, mitochondrial genome). The molecular basis of this hypothesis has received little empirical investigation, presumably due to the lack of an appropriate model system. We report a case of extreme asymmetry in viability between reciprocal hybrids of zebrafish Danio rerio and pearl danio Danio albolineatus. Hybrids from D. rerio females × D. albolineatus males (n = 4 crosses) were viable, with 83.2 ± 9.6% surviving from fertilization to 5 days post-fertilization (dpf) and 80.1 ± 14.4% surviving from 5 to 21 dpf. Hybrids from D. albolineatus females × D. rerio males (n = 6 crosses) were inviable after embryonic development. These hybrids developed pericardial oedema at 1 dpf and only 37.2 ± 18.0% survived from fertilization to 5 dpf. Of the 595 larvae alive at 5 dpf, only one juvenile with stunted growth survived to 21 dpf. We propose that given the resources available for the D. rerio model system and the strong asymmetry in viability between reciprocal crosses, these hybrids will allow investigation of the molecular basis for Darwin's corollary to Haldane's rule.

Genotyping single nucleotide polymorphisms and inferring ploidy by amplicon sequencing for polyploid, ploidy-variable organisms.

Whole genome duplication is hypothesized to have played a critical role in the evolution of several major taxa, including vertebrates, and while many lineages have rediploidized, some retain polyploid genomes. Additionally, variation in ploidy can occur naturally or be artificially induced within select plant and animal species. Modern genetic techniques have not been widely applied to polyploid or ploidy-variable species, in part due to the difficulty of obtaining genotype data from polyploids. In this study, we demonstrate a strategy for developing an amplicon sequencing panel of single nucleotide polymorphisms for high-throughput genotyping of polyploid organisms. We then develop a method to infer ploidy of individuals from amplicon sequencing data that is generalized to apply to any ploidy and does not require prior identification of heterozygous genotypes. Combining these two techniques will allow researchers to both infer ploidy and generate ploidy-aware genotypes with the same amplicon sequencing panel. We demonstrate this approach with white sturgeon Acipenser transmontanus, a ploidy-variable (octoploid, decaploid and dodecaploid) imperiled species under conservation management in the Pacific Northwest and obtained a panel of 325 loci. These loci were validated by examining inheritance in known-cross families, and the ploidy inference method was validated with known ploidy samples. We provide scripts that adapt existing pipelines to genotype polyploids and an R package for application of the ploidy inference method. We expect that these techniques will empower studies of genetic variation and inheritance in polyploid organisms that vary in ploidy level, either naturally or as a result of artificial propagation practices.

Distribution of genetic variation underlying adult migration timing in steelhead of the Columbia River basin.

Fish migrations are energetically costly, especially when moving between freshwater and saltwater, but are a viable strategy for Pacific salmon and trout (Oncorhynchus spp.) due to the advantageous resources available at various life stages. Anadromous steelhead (O. mykiss) migrate vast distances and exhibit variation for adult migration phenotypes that have a genetic basis at candidate genes known as greb1L and rock1. We examined the distribution of genetic variation at 13 candidate markers spanning greb1L, intergenic, and rock1 regions versus 226 neutral markers for 113 populations (n = 9,471) of steelhead from inland and coastal lineages in the Columbia River. Patterns of population structure with neutral markers reflected genetic similarity by geographic region as demonstrated in previous studies, but candidate markers clustered populations by genetic variation associated with adult migration timing. Mature alleles for late migration had the highest frequency overall in steelhead populations throughout the Columbia River, with only 9 of 113 populations that had a higher frequency of premature alleles for early migration. While a single haplotype block was evident for the coastal lineage, we identified multiple haplotype blocks for the inland lineage. The inland lineage had one haplotype block that corresponded to candidate markers within the greb1L gene and immediately upstream in the intergenic region, and the second block only contained candidate markers from the intergenic region. Haplotype frequencies had similar patterns of geographic distribution as single markers, but there were distinct differences in frequency between the two haplotype blocks for the inland lineage. This may represent multiple recombination events that differed between lineages where phenotypic differences exist between freshwater entry versus arrival timing as indicated by Micheletti et al. (2018a). Redundancy analyses were used to model environmental effects on allelic frequencies of candidate markers, and significant variables were migration distance, temperature, isothermality, and annual precipitation. This study improves our understanding of the spatial distribution of genetic variation underlying adult migration timing in steelhead as well as associated environmental factors and has direct conservation and management implications.

Differentiating diploid and triploid individuals using single nucleotide polymorphisms genotyped by amplicon sequencing.

Triploidy can occur naturally or be induced in fish and shellfish during artificial propagation in order to produce sterile individuals. Fisheries managers often stock these sterile triploids as a means of improving angling opportunities without risking unwanted reproduction of the stocked fish. Additionally, the rearing of all-triploid individuals has been suggested as a means to reduce the possibility of escaped aquaculture fish interbreeding with wild populations. Efficient means of determining if an individual is triploid or diploid are therefore needed both to monitor the efficacy of triploidy-inducing treatments and, when sampling fish from a body of water that has a mixture of diploids and triploids, to determine the ploidy of a fish prior to further analyses. Currently, ploidy is regularly measured through flow cytometry, but this technique typically utilizes a fresh blood sample. This study presents an alternative, cost-effective method of determining ploidy by analysing amplicon-sequencing data for biallelic single-nucleotide polymorphisms (SNPs). For each sample, heterozygous genotypes are identified and the likelihoods of diploidy and triploidy are calculated based on the read counts for each allele. The accuracy of this method is demonstrated using triploid and diploid brook trout (Salvelinus fontinalis) genotyped with a panel of 234 SNPs and Chinook salmon (Oncorhynchus tshawytscha) genotyped with a panel of 298 SNPs following the GT-seq methodology of amplicon sequencing.

Effects of homozygosity on sex determination in zebrafish Danio rerio.

Gynogenetic zebrafish Danio rerio were obtained by activating D. rerio oocytes with UV irradiated common carp Cyprinus carpio sperm and then applying one of four different shocks [two (early) meiotic and two (late) mitotic shocks]. Gynogens produced by three of the shocks survived to maturity. All adult gynogens (n = 52) except one were found to be male. There was no difference in growth rate between the biparental controls and gynogens produced through the most effective shock, thereby eliminating growth rate as a possible cause of the skewed sex ratio. Gynogen males had reduced fertility compared to biparental controls, with about half of gynogens being unable to reproduce through natural spawning (all controls reproduced successfully). Gynogen males that did reproduce gave lower fertilization rates compared with controls. This demonstrates the negative effects of increased homozygosity on male reproductive function. Families sired by meiotic gynogen males were more likely to be female biased (33% of families) compared with families sired by biparental control males (11%). In addition to confirming the polygenic nature of sex determination in D. rerio, these observations suggest that recessive or over-dominant male-determining alleles are present in domesticated D. rerio populations.

Why are triploid zebrafish all male?

Adult triploid zebrafish Danio rerio has previously been reported to be all male. This phenomenon has only been reported in one other gonochoristic fish species, the rosy bitterling Rhodeus ocellatus, despite the fact that triploidy is induced in numerous species. To investigate the mechanism responsible, we first produced triploid zebrafish and observed gonad development. Histological sections of juvenile triploid gonads showed that primary growth oocytes were able to develop in the juvenile ovary, but no cortical alveolus or more advanced oocytes were found. All adult triploids examined were male (n = 160). Male triploids were able to induce oviposition by diploid females during natural spawning trials, but fertilization rates were low (1.0 ± 3.1%) compared with diploid male siblings (67.4 ± 16.6%). The embryos produced by triploid sires were aneuploid with a mean ploidy of 2.4 ± 0.1n, demonstrating that triploid males produce aneuploid spermatozoa. After confirming that adult triploids are all male, we produced an additional batch of triploid zebrafish and exposed them (and a group of diploid siblings) to 100 ng/L estradiol (E2) from 5 to 28 dpf. The E2 treated triploids and nontreated triploids were all male. The nontreated diploids were also all male, but the E2 treated diploids were 89% female. This demonstrates that triploidy acts downstream of estrogen synthesis in the sex differentiation pathway to induce male development. Based on this and the observations of juvenile gonad development in triploids, we suggest that triploidy inhibits development of oocytes past the primary growth stage, and this causes female to male sex reversal.

Larval rearing of zebrafish at suboptimal temperatures.

Temperature-sensitive mutants have been widely utilized in single-cell and invertebrate model systems, particularly to study the function of essential genes. Few temperature-sensitive mutants have been identified in zebrafish, likely due to the difficulty of raising zebrafish at low temperatures. We describe a novel rearing protocol that allows rapid growth of larval and juvenile zebrafish at 23 °C compared to previous data in the literature. Embryos collected from four breeding pairs were maintained at 28.5 ±â€¯0.5 °C until 5 days post-fertilization (dpf) - the onset of exogenous feeding. Larvae were then divided to six tanks and three tanks were cooled to 23 ±â€¯0.2 °C. Fish were fed a live diet (marine rotifers Brachionus plicatilis and Artemia nauplii) and maintained under a set of environmental parameters shown to increase growth rate: continuous light, low salinity (3ppt), and algal turbidity. Mean total length and weight of fish at 21dpf were 12.7 ±â€¯0.3 mm and 20.5 ±â€¯1.5 mg for the 23 °C treatment and 18.5 ±â€¯0.4 mm and 67.3 ±â€¯3.4 mg for the 28.5 °C control. By 35 dpf, the fish raised at 23 °C had reached a mean length and weight of 18.9 ±â€¯0.7 mm and 76.4 ±â€¯6.7 mg, approximately the size control fish reached at 21 dpf. At 35 dpf, water temperature was raised to 28 °C and fish were reared to maturity (75 dpf) under standard conditions (freshwater, 13 L:11D photoperiod, dry diet, no added algal turbidity). Sex ratio and fertility were assessed and compared between temperature groups. There were no significant differences in sex ratio, fertilization rate, embryo viability at 1 dpf, clutch size, or relative fecundity. This rearing protocol will allow for efficient utilization of temperature-sensitive mutations in the zebrafish model system.

Zebrafish embryonic development is induced by carp sperm.

Haploid gynogenetic screens increase the efficiency of forward genetic screens and linkage analysis in fish. Typically, UV-irradiated zebrafish sperm is used to activate zebrafish oocytes for haploid screens. We describe the use of UV-irradiated common carp sperm to activate haploid gynogenetic zebrafish development. Carp × zebrafish hybrids are shown to have a characteristic set of features during embryonic development and exhibit functional development of several tissues (muscle, heart and nervous system). Hybrids become inviable past the embryonic stages. This technique eliminates the possibility of incompletely irradiated zebrafish spermatozoa contaminating haploid progenies. While developing this protocol, one unique zebrafish female was identified which, upon insemination with UV-irradiated carp spermatozoa, repeatedly displayed spontaneous diploidization of the maternal chromosomes in her offspring.