Development and evaluation of 200 novel SNP assays for population genetic studies of westslope cutthroat trout and genetic identification of related taxa.

DNA sequence data were collected and screened for single nucleotide polymorphisms (SNPs) in westslope cutthroat trout (Oncorhynchus clarki lewisi) and also for substitutions that could be used to genetically discriminate rainbow trout (O. mykiss) and cutthroat trout, as well as several cutthroat trout subspecies. In total, 260 expressed sequence tag-derived loci were sequenced and allelic discrimination genotyping assays developed from 217 of the variable sites. Another 50 putative SNPs in westslope cutthroat trout were identified by restriction-site-associated DNA sequencing, and seven of these were developed into assays. Twelve O. mykiss SNP assays that were variable within westslope cutthroat trout and 12 previously published SNP assays were also included in downstream testing. A total of 241 assays were tested on six westslope cutthroat trout populations (N = 32 per population), as well as collections of four other cutthroat trout subspecies and a population of rainbow trout. All assays were evaluated for reliability and deviation from Hardy-Weinberg and linkage equilibria. Poorly performing and duplicate assays were removed from the data set, and the remaining 200 assays were used in tests of population differentiation. The remaining markers easily distinguished the various subspecies tested, as evidenced by mean G(ST) of 0.74. A smaller subset of the markers (N = 86; average G(ST) = 0.40) was useful for distinguishing the six populations of westslope cutthroat trout. This study increases by an order of magnitude the number of genetic markers available for the study of westslope cutthroat trout and closely related taxa and includes many markers in genes (developed from ESTs).

Comparison of SNPs and microsatellites for fine-scale application of genetic stock identification of Chinook salmon in the Columbia River Basin.

Genetic stock identification (GSI) is an important tool in fisheries management. Microsatellites (µSATs) have been the dominant genetic marker for GSI; however, increasing availability and numerous advantages of single-nucleotide polymorphism (SNP) markers make them an appealing alternative. We tested performance of 13 µSAT vs. 92 SNP loci in a fine-scale application of GSI, using a new baseline for Chinook salmon consisting of 49 collections (n = 4014) distributed across the Columbia River Basin. In GSI, baseline genotypes for both marker sets were used independently to analyse a real fishery mixture (n = 2731) representing the total run of Chinook salmon passing Bonneville Dam in the Columbia River. Marker sets were evaluated using three criteria: (i) ability to differentiate reporting groups, (ii) proportion of correct assignment in mixture simulation tests and baseline leave-one-out analyses and (iii) individual assignment and confidence intervals around estimated stock proportions of a real fishery mixture. The µSATs outperformed the SNPs in resolving fine-scale relationships, but all 105 markers combined provided greatest power for GSI. SNPs were ranked by relative information content based on both an iterative procedure that optimized correct assignment to the baseline and ranking by minor allele frequency. For both methods, we identified a subset of the top 50 ranked loci, which were similar in assignment accuracy, and both reached maximum available power of the total 92 SNP loci (correct assignment = 73%). Our estimates indicate that between 100 and 200 highly informative SNP loci are required to meet management standards (correct assignment > 90%) for resolving stocks in finer-scale GSI applications.

Relationships between growth and disease resistance in rainbow trout, Oncorhynchus mykiss (Walbaum).

Rainbow trout from 23 families were evaluated for growth and resistance to the bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum and infectious haematopoietic necrosis (IHN) caused by IHN virus. Average family weights were between 161 and 263 g with an average of 225 g at 213 days post-fertilization with specific growth rates ranging from 2.37 to 2.88. Per cent survival of fish challenged with F. psychrophilum was between 18% and 100%, while for those challenged with IHNV, the range was between 12% and 93%. Significant positive correlations were found for end body weight and resistance to IHN (P < 0.05) and for early body weight and resistance to BCWD (P < 0.1). However, no significant correlations were detected between resistance to both pathogens or disease resistance and overall genetic diversity or diversity within the major histocompatibility locus.

Differentiating salmon populations at broad and fine geographical scales with microsatellites and single nucleotide polymorphisms.

Single nucleotide polymorphisms (SNPs) are appealing genetic markers due to several beneficial attributes, but uncertainty remains about how many of these bi-allelic markers are necessary to have sufficient power to differentiate populations, a task now generally accomplished with highly polymorphic microsatellite markers. In this study, we tested the utility of 37 SNPs and 13 microsatellites for differentiating 29 broadly distributed populations of Chinook salmon (n = 2783). Information content of all loci was determined by In and G'(ST), and the top 12 markers ranked by In were microsatellites, but the 6 highest, and 7 of the top 10 G'(ST) ranked markers, were SNPs. The mean ratio of random SNPs to random microsatellites ranged from 3.9 to 4.1, but this ratio was consistently reduced when only the most informative loci were included. Individual assignment test accuracy was higher for microsatellites (73.1%) than SNPs (66.6%), and pooling all 50 markers provided the highest accuracy (83.2%). When marker types were combined, as few as 15 of the top ranked loci provided higher assignment accuracy than either microsatellites or SNPs alone. Neighbour-joining dendrograms revealed similar clustering patterns and pairwise tests of population differentiation had nearly identical results with each suite of markers. Statistical tests and simulations indicated that closely related populations were better differentiated by microsatellites than SNPs. Our results indicate that both types of markers are likely to be useful in population genetics studies and that, in some cases, a combination of SNPs and microsatellites may be the most effective suite of loci.

Effects of clodronate on cortical and trabecular bone in ovariectomized rats on a low calcium diet.

The aim of this study was to evaluate the contribution of a low calcium diet to the cortical and trabecular osteoporosis seen in ovariectomized rats after 7 weeks on a low calcium diet and to investigate the effects of the bisphosphonate clodronate on this development of osteoporosis. Thirty-six mature, female Wistar rats were randomized into four groups: Ovx-B (bisphosphonate) and Ovx-C (control) were ovariectomized, and Sham-Ca (low calcium) and Sham+Ca (normal calcium) were sham operated. The first three groups were fed a low calcium diet (0.01%) and Sham+Ca normal rat chow (Ca 1.1%). The Ovx-B received 10 mg/kg s.c. clodronate daily for nine weeks, and Ovx-C, Sham-Ca, and Sham+Ca received the same volumes of saline. Bone mineral turnover measured as 85Sr-uptake was increased in all low calcium groups compared to Sham+Ca. The Sham+Ca femora had higher dry weight and ash weight than the other groups, and Ovx-C had higher dry weight compared with Ovx-B and Sham-Ca. Calcium content was lower in both Ovx groups compared to both Sham groups. Magnesium was lower in all groups compared to Sham+Ca and higher in Ovx-B compared with Ovx-C. In the femoral shaft, Sham+Ca had significantly higher ultimate bending moment, energy absorption, and deflection compared to the other three groups. Ultimate bending moment was higher in Sham-Ca than in Ovx-C. Stiffness was increased in both Sham+Ca and Ovx-B compared to Ovx-C. The maximum stress in the femoral midshaft was higher in Sham+Ca than in the other groups, and higher in Ovx-B than in Ovx-C. Histomorphometry showed increased medullary area in all low calcium groups compared to Sham+Ca and larger cortical area in Sham+Ca and Ovx-B compared to Ovx-C. Compared to Sham+Ca the trabecular bone volume was decreased to 30% in Sham-Ca and to 9% in Ovx-C, but was unchanged in Ovx-B. The low calcium diet generally increased bone mineral turnover and reduced the tibial bone volume. Femoral changes led to a reduction of cortical fracture strength and maximal stress. Ovariectomy in addition to a low calcium diet reduced femoral strength even more. Daily injections of clodronate to ovariectomized rats on a low calcium diet increased femoral shaft stiffness and maximum stress, and clodronate preserved both trabecular and cortical tibial bone volume completely.

Effect of intensive training on lower leg structural strength: an in vivo study in ovariectomized rats.

The aim of this study was to investigate the effect of training on the in vivo tibial structural strength during the development of post-ovariectomy osteoporosis. Seventeen mature Wistar rats (215 g) were ovariectomized and randomized into two groups. The sedentary control group was kept cage confined, while 3 days postoperatively the trained group started treadmill running with high intensity for 1 h 5 days a week. All were given a low calcium diet (Ca 0.01%). After 8 weeks the animals were anaesthetized and the right lower legs fractured during muscle contraction in three-point ventral bending. The left legs were fractured at the same level after removal of all soft tissues. Histomorphometry of the meta- and diaphysis of the distal tibiae was performed. Weight-gain was higher in sedentary (108 g) than in trained (61 g) rats (P<0.0001). There were no significant differences in mechanical results between the groups at in vivo or in vitro fracture. Correcting for weight-gain differences did not change these results. Histomorphometry showed no differences between the groups. Corticosterone was higher in trained than in sedentary rats (P<0.02), and corticosterone may have had a negative influence both on muscle and bone. The study could not show an effect of high intensity training in the early phase after ovariectomy on in vivo or in vitro fracture strength.

Training increases the in vivo fracture strength in osteoporotic bone. Protection by muscle contraction examined in rat tibiae.

The effect of high-intensity training on the in vivo lower leg fracture strength during muscle contraction was investigated in osteoporotic rats. 20 Wistar rats were ovariectomized and given a low calcium (0.01%) diet. 7 weeks after ovariectomy they were randomized into training (T) and sedentary (S). The S group was kept cage-confined without any intervention. The T group ran on a treadmill with 10 degrees inclination 5/7 days for 8 weeks. A maximum intensity of 27 m/min was reached after 4 weeks. After 8 weeks, the right lower legs of the anesthetized animals were loaded in three-point ventral bending until fracture occurred during electrically-induced muscle contraction. The left tibiae were excised and fractured at the same level as the right tibiae. Weight gain was equal in the two groups. Energy absorption and deflection at fracture were significantly higher in the T group than in the S group in vivo during muscle contraction. In vitro, there were no significant differences in mechanical results. The mediolateral outer diameter was larger in the T group, and the maximal stress that the tibia could withstand was lower than in the S group. We conclude that 8 weeks of high-intensity training of osteoporotic rats increased the structural lower leg strength during muscle contraction. The reduced maximal stress in the training animals indicates a reduction in bone material quality. The increase of in vivo structural strength must reflect an increased protective effect of muscle contraction due to training.