Subspecies-informative SNP assays for evaluating introgression between native golden trout and introduced rainbow trout

We characterize 20 single nucleotide polymorphism assays for evaluating hybridization between native golden trout subspecies (Oncorhynchus mykiss aguabonita and O. m. whitei) and introduced rainbow trout strains. These assays utilize the 5′‐nuclease reaction, facilitating high‐throughput genotyping of many individuals and making them useful in quantifying and monitoring introgression and potentially applicable to studies of other O. mykiss groups. Minor allele frequency differentials (δq) among native and introduced rainbow groups ranged from 0 to 1, with an average differential of 0.75 for both subspecies aguabonita and whitei relative to the hatchery rainbow trout strain.     

Somatostatin inhibits growth of rainbow trout

Implantation of rainbow trout Oncorhynchus mykiss with somatostatin-14 (SS-14) for 20 days resulted in reduced food conversion as well as significant growth retardation compared to controls. Relative growth as mass was reduced by 20%, whereas relative growth by length was reduced by 45%. A single intraperitoneal injection of SS-14 reduced plasma levels of growth hormone (GH), insulin-like growth factor-1 (IGF-I) and insulin. SS-14 injection also reduced [³⁵S]-sulphate incorporation into gill cartilage compared to saline-injected fish. In addition, in vitro incubation of gill cartilage with SS-14 reduced [³⁵S]-sulphate incorporation in a doserelated manner. These results indicate that SS-14 inhibits growth of rainbow trout and suggests that SS-14, in addition to influencing GH, may play an extra-pituitary role in the modulation of the GH-IGF-I axis.     

Use of sequence data from rainbow trout and Atlantic salmon for SNP detection in Pacific salmon

Single nucleotide polymorphisms (SNPs) are a class of genetic markers that are well suited to a broad range of research and management applications. Although advances in genotyping chemistries and analysis methods continue to increase the potential advantages of using SNPs to address molecular ecological questions, the scarcity of available DNA sequence data for most species has limited marker development. As the number and diversity of species being targeted for large-scale sequencing has increased, so has the potential for using sequence from sister taxa for marker development in species of interest. We evaluated the use of Oncorhynchus mykiss and Salmo salar sequence data to identify SNPs in three other species (Oncorhynchus tshawytscha, Oncorhynchus nerka and Oncorhynchus keta). Primers designed based on O. mykiss and S. salar alignments were more successful than primers designed based on Oncorhynchus-only alignments for sequencing target species, presumably due to the much larger number of potential targets available from the former alignments and possibly greater sequence conservation in those targets. In sequencing approximately 89 kb we observed a frequency of 4.30 x 10(-3) SNPs per base pair. Approximately half (53/101) of the subsequently designed validation assays resulted in high-throughput SNP genotyping markers. We speculate that this relatively low conversion rate may reflect the duplicated nature of the salmon genome. Our results suggest that a large number of SNPs could be developed for Pacific salmon using sequence data from other species. While the costs of DNA sequencing are still significant, these must be compared to the costs of using other marker classes for a given application.     

Characterization of 38 polymorphic microsatellite markers for rainbow trout (Oncorhynchus mykiss)

Thirty‐eight new microsatellite markers were developed for genome mapping and population genetics studies in rainbow trout (Oncorhynchus mykiss). The amount of polymorphism, percentage of heterozygosity and ability of each marker to amplify genomic DNA from other salmonids were recorded. Five markers were observed to be duplicated in the rainbow trout genome by containing more than one allele in homozygous (clonal) fish.     

RAD sequencing yields a high success rate for westslope cutthroat and rainbow trout species-diagnostic SNP assays

Hybridization with introduced rainbow trout threatens most native westslope cutthroat trout populations. Understanding the genetic effects of hybridization and introgression requires a large set of high-throughput, diagnostic genetic markers to inform conservation and management. Recently, we identified several thousand candidate single-nucleotide polymorphism (SNP) markers based on RAD sequencing of 11 westslope cutthroat trout and 13 rainbow trout individuals. Here, we used flanking sequence for 56 of these candidate SNP markers to design high-throughput genotyping assays. We validated the assays on a total of 92 individuals from 22 populations and seven hatchery strains. Forty-six assays (82%) amplified consistently and allowed easy identification of westslope cutthroat and rainbow trout alleles as well as heterozygote controls. The 46 SNPs will provide high power for early detection of population admixture and improved identification of hybrid and nonhybridized individuals. This technique shows promise as a very low-cost, reliable and relatively rapid method for developing and testing SNP markers for nonmodel organisms with limited genomic resources.     

Does cyclicity of growth rate in rainbow trout exist?

Ninety rainbow trout Oncorhynchus mykiss were reared in three tanks ( n= 30), and their growth rate was monitored for 25 weeks. During the experiment, fish were fed ad libitum , and kept under simulated natural photoperiod and constant temperature (14 ± 1°C). Throughout the experiment, water quality (pH, O₂, N-NH₃, N-NH₄, NO₂) was considered optimum. Two growth rate periods were observed on the population: first an acclimation period (weeks 1–5), followed by a period of general decrease in growth rate (weeks 5–25). This trend was modelled with a third-order polynomial function. The autocorrelation analysis of the fluctuations revealed a cyclicity of the growth rate with a 4–5-week period. This cyclicity was independent of the tanks and of the growth rate trend type (long- v . short-term acclimation period). However, at the individual level, trends as well as short-term fluctuations were heterogeneous and no clear cyclicity could be detected. The biological relevance of the population growth rate cyclicity and the apparent absence of similar individual fluctuations are discussed.     

Muscle fibre growth dynamics in diploid and triploid rainbow trout

The effect of triploidy on muscle fibre growth was determined by comparing hyperplasia and hypertrophy of white muscle fibres in all-female, diploid and triploid rainbow trout Oncorhynchus mykiss (100–400 mm total length). Conventional morphometry and protein and DNA concentrations were used to assess muscle fibre hyperplasia and hypertrophy in white muscle samples derived from an anterio-dorsal location. Muscle fibre distributions were significantly different between triploids and diploids in trout <300 mm. The proportion of fibres <20 μm was higher in diploids than in triploids and the proportion of fibres in the 20–40 μm category was higher in triploids than in diploids. This indicates that the hyperplastic fibres of triploids are larger than those of diploids. Larger hyperplastic fibres in triploids are probably due to the combined effect of increased nuclear size in triploids and the relatively high nucleus: cell ratio observed in small muscle fibres. These larger fibres may be less favourable to cellular metabolic exchange because of their smaller surface area to volume ratios, and perhaps account for reduced viability and growth observed in triploids during early life stages. On the other hand, the lack of difference in the distribution of fibres <20 μm between diploids and triploids at larger body size ranges (301–400 mm) imply that triploid trout may have higher rates of new fibre recruitment and growth capacity at these sizes. There was no difference between diploid and triploid trout in the mean size of muscle fibres; however, the number of fibres per unit area was reduced by 10% in triploids. No differences were observed in protein or DNA concentrations in muscle tissues between the two genetic groups. Since triploid nuclei have 1·5 times more DNA than diploid nuclei, this deviation from the expected muscle DNA concentration (1·3–1·4 times more DNA in triploids when the 10% reduction in fibre density is considered) suggests that the number of nuclei per muscle fibre is reduced. In both diploids and triploids, mean fibre size increased with body length while fibre density decreased. Similarly, protein concentration in the muscle tissue increased and DNA concentration declined with increasing body length. Protein/DNA ratio was strongly and positively correlated with fibre size. These results demonstrate that changes in DNA and protein concentrations can be used to assess hyperplasia and hypertrophy in muscle tissues. However, the morphometric procedure provides better insight into muscle fibre growth as it enables the direct visualization and analysis of muscle fibre distribution patterns.     

Influence of growth rate on white muscle dynamics in rainbow trout and brook trout

In trout, fast growth stimulated white muscle fibre hypertrophy ( P 0·001) and hyperplasia ( P <0·01) in outer fibres but not in deep fibres. Glycogen was most prevalent in outer fibres ( P <0·01) and in brook trout ( P <0·01) that on average had three to four times larger fibres than rainbow trout.     

Compensatory growth in the rainbow trout, Salmo gairdneri Richardson

The effect of a period of starvation and subsequent refeeding on the weight and length of rainbow trout at different times of the year has been investigated. Fish that have been starved for 3 weeks and then fed for 3 weeks show a weight gain equivalent to or greater than that of fish fed normally for the 6 week period, in four out of the five periods studied. The study provided evidence of the adaptation of the fish to starvation followed by what may be termed compensatory growth once feeding was resumed. The length changes of the fish indicate that the weight gains were due to growth rather than increases in gut fat deposits or increased water uptake.     

Next-generation RAD sequencing identifies thousands of SNPs for assessing hybridization between rainbow and westslope cutthroat trout

The increased numbers of genetic markers produced by genomic techniques have the potential to both identify hybrid individuals and localize chromosomal regions responding to selection and contributing to introgression. We used restriction-site-associated DNA sequencing to identify a dense set of candidate SNP loci with fixed allelic differences between introduced rainbow trout (Oncorhynchus mykiss) and native westslope cutthroat trout (Oncorhynchus clarkii lewisi). We distinguished candidate SNPs from homeologs (paralogs resulting from whole-genome duplication) by detecting excessively high observed heterozygosity and deviations from Hardy-Weinberg proportions. We identified 2923 candidate species-specific SNPs from a single Illumina sequencing lane containing 24 barcode-labelled individuals. Published sequence data and ongoing genome sequencing of rainbow trout will allow physical mapping of SNP loci for genome-wide scans and will also provide flanking sequence for design of qPCR-based TaqMan(?) assays for high-throughput, low-cost hybrid identification using a subset of 50-100 loci. This study demonstrates that it is now feasible to identify thousands of informative SNPs in nonmodel species quickly and at reasonable cost, even if no prior genomic information is available.     

Characterization of 22 novel single nucleotide polymorphism markers in steelhead and rainbow trout

Thirty‐two individuals representing coastal and inland populations of steelhead and rainbow trout (Oncorhynchus mykiss) were sequenced at 18 expressed sequence tags and nine microsatellite loci to identify single nucleotide polymorphisms. A total of 98 polymorphisms were discovered during the screen and 22 were developed into 5′ exonuclease assays (Taqman assays). Genotypes from TaqMan assays were compared to sequence data from individuals in the ascertainment panel to confirm proper allele designations. A larger number of samples (n = 192) from six regions were tested with the validated assays. Per‐locus F_ST values ranged from 0.001 to 0.414.     

Characterization of twenty‐four microsatellite markers for rainbow trout (Oncorhynchus mykiss)

Twenty‐four new microsatellite markers were developed for genome mapping and population genetics studies in rainbow trout (Oncorhynchus mykiss). The amount of polymorphism, percentage heterozygosity and ability of each marker to amplify genomic DNA from other salmonids were recorded. Seven markers were observed to be duplicated in the rainbow trout genome by containing more than one allele in homozygous (clonal) fish.     

Overwinter feeding in rainbow trout

The contents of the stomachs of 38 rainbow trout stocked in Llyn Alaw, Anglesey, in August 1969 and caught between October 1969 and February 1970 were analysed. The fish were actively feeding on the bottom fauna throughout the winter and 21 of the stomachs were full or distended. The mean volume of the contents of the stomachs was 2–8 times greater than that of the contents of stomachs of similarly sized brown trout caught at the same time.