Six diagnostic single nucleotide polymorphism markers for detecting introgression between cutthroat and rainbow trouts

Ten primer pairs were screened to develop single nucleotide polymorphism (SNP) TaqMan assays that will distinguish California golden trout and some rainbow trouts (Oncorhynchus mykiss sspp., O. m. aguabonita) from the Paiute and Lahontan cutthroat trouts (Oncorhynchus clarkii seleniris, O. c. henshawi). From these 10 primer pairs, one mitochondrial and five nuclear fixed SNP differences were discovered and developed into TaqMan assays. These six assays will be useful for characterizing and monitoring hybridization between these groups. Additional Oncorhynchus clarkii sspp. and Oncorhynchus mykiss sspp. were assayed to determine if these assays are useful in closely related species.     

A suite of twelve single nucleotide polymorphism markers for detecting introgression between cutthroat and rainbow trout

A suite of 12 subspecies and species-specific single nucleotide polymorphism (species-specific SNP) markers was developed to distinguish rainbow trout (RT) Oncorhynchus mykiss from the four major subspecies of cutthroat trout: westslope cutthroat trout (WCT) Oncorhynchus clarki lewisi, Yellowstone cutthroat trout (YCT) Oncorhynchus clarki bouvieri, coastal cutthroat trout (CCT) Oncorhynchus clarki clarki, Lahontan cutthroat trout (LCT) Oncorhynchus clarki henshawi, and their hybrids. Several of the markers were linked to help strengthen hybrid determinations, and sex-specific species-specific SNP assays were also developed.     

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.     

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.     

Relationship between stress, feeding and plasma ghrelin levels in rainbow trout, Oncorhynchus mykiss

Sexually immature rainbow trout were acclimated to small-volume (1 m³) holding tanks and then exposed to short-term stress to examine the relationship between feeding, stress, plasma ghrelin levels and other plasma stress parameters. Plasma ghrelin levels showed an increase 24 h after a single feed, plasma lactate and glucose levels decreased over the same period and plasma cortisol levels were low and constant. One hour of confinement stress resulted in elevations of plasma cortisol, glucose and lactate and depression of plasma ghrelin levels. In a separate experiment, 2 h of confinement stress also depressed feeding immediately after stress, concomitant with increases in plasma cortisol, lactate and glucose; however, in this case there was no change in plasma ghrelin concentrations. A repeat of the 2-h confinement experiment using fish that had not been acclimated to small-volume holding tanks produced a more marked elevation in plasma cortisol and a stronger suppression of feeding post-stress but in this case also, there was no change in plasma ghrelin levels. The results of this study confirm that feeding in rainbow trout is suppressed by confinement stress although the effect is transitory in this domesticated stock. Similar to that in other fishes, plasma ghrelin levels appear to be modulated by feeding status and may be influenced by stress, suggesting an orexigenic role for ghrelin in rainbow trout.     

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).     

A molecular analysis of hybridization between native westslope cutthroat trout and introduced rainbow trout in southeastern British Columbia, Canada

Restriction site variation in the Ikaros gene intron was used to assess the incidence of westslope cutthroat trout ( Oncorhynchus clarki lewisi ), rainbow trout ( O. mykiss ) and interspecific hybrids at 11 localities among eight streams tributary to the upper Kootenay River system in south-eastern British Columbia, Canada. Out of 356 fish assayed by this technique, hybrids ( n =16) were found at seven of the 11 sites across five different streams. Rainbow trout ( n =6) were found at two of the 11 sites. Analysis of hybrids with a second genetic marker (heat shock 71 intron) indicated that most represented either backcrosses to both westslope cutthroat and rainbow trout, or post F₁ hybrids. Mitochondrial DNA analysis indicated that hybrid matings occur between male rainbow trout and female westslope cutthroat trout and vice versa. Comparison of present hybridization in five tributaries relative to an allozyme-based analysis in the mid-1980s, that documented hybrids in only a single tributary of seven that were common to the two studies, suggests that hybridization and introgression has increased in upper Kootenay River tributaries. The present analysis is a conservative estimate of genetic interaction between the species because introgression was not tested in the majority of samples. Identification of genetically pure westslope cutthroat trout populations, and why they might be resistant to introgression from rainbow trout, are crucial conservation priorities for this unique subspecies of cutthroat trout.     

Characterization of tetranucleotide microsatellites for Rio Grande cutthroat trout and rainbow trout,and their cross‐amplification in other cutthroat trout subspecies

We describe the isolation and characterization of 12 tetranucleotide microsatellites for Rio Grande cutthroat trout (Oncorhynchus clarkii virginalis) and rainbow trout (Oncorhynchus mykiss), and subsequently investigate their performance in Colorado River cutthroat trout (Oncorhynchus clarkii pleuriticus), greenback cutthroat trout (Oncorhynchus clarkii stomias) and Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri). All 12 loci are polymorphic in all subspecies of O. clarkii examined.     

Estimation of the number of SNP genetic markers required for parentage verification

Formulae were developed to compute exclusion probabilities for parentage confirmation for any number of diallelic markers under the assumption that the minor allele frequency (MAF) varied among markers, but has a uniform distribution. Three scenarios were analysed: a progeny with (1) a single putative parent; (2) two putative parents; and (3) one actual parent and one putative parent. Exclusion probabilities were computed for minimum values for the MAFs of 0.1, 0.2 and 0.3, and required either one or at least two conflicts for exclusion. The numbers of markers required to obtain 99% exclusion probabilities based on a single conflict for the three minimum MAFs were 54, 45 and 39 for scenario 1; 17, 16 and 15 for scenario 2; and 28, 25 and 24 for scenario 3. The requirement of at least two conflicts for exclusion increased the number of markers required by approximately 45% for all three scenarios and all three minimum MAFs. The results obtained by the analytical formulae were very close to results obtained by simulation and to values in the literature for specific marker sets.     

Modelling competition and hybridization between native cutthroat trout and nonnative rainbow and hybrid trout

Native salmonid fish have been displaced worldwide by nonnatives through hybridization, competition, and predation, but the dynamics of these factors are poorly understood. We apply stochastic Lotka–Volterra models to the displacement of cutthroat trout by rainbow/hybrid trout in the Snake River, Idaho, USA. Cutthroat trout are susceptible to hybridization in the river but are reproductively isolated in tributaries via removal of migratory rainbow/hybrid spawners at weirs. Based on information-theoretic analysis, population data provide evidence that hybridization was the primary mechanism for cutthroat trout displacement in the first 17 years of the invasion. However, under some parameter values, the data provide evidence for a model in which interaction occurs among fish from both river and tributary subpopulations. This situation is likely to occur when tributary-spawned cutthroat trout out-migrate to the river as fry. The resulting competition with rainbow/hybrid trout can result in the extinction of cutthroat trout even when reproductive segregation is maintained.     

Characterization of 18 SNP markers for sperm whale (Physeter macrocephalus)

We report the characterization of 18 new single nucleotide polymorphism (SNP) markers for an endangered species, the sperm whale (Physeter macrocephalus), developed using a targeted gene approach. SNP markers were derived from autosomal regions of the genome using primers originally characterized for genome mapping in other mammals. These SNP markers are the first to be designed for genotyping sperm whale populations and will provide a necessary addition to the genetic tools employed for understanding population structure on a global scale and for developing a conservation management strategy for this endangered species.     

Repeated unidirectional introgression towards Populus balsamifera in contact zones of exotic and native poplars

As the evolutionary significance of hybridization is largely dictated by its extent beyond the first generation, we broadly surveyed patterns of introgression across a sympatric zone of two native poplars ( Populus balsamifera , Populus deltoides ) in Quebec, Canada within which European exotic Populus nigra and its hybrids have been extensively planted since the 1800s. Single nucleotide polymorphisms (SNPs) that appeared fixed within each species were characterized by DNA-sequencing pools of pure individuals. Thirty-five of these diagnostic SNPs were employed in a high-throughput assay that genotyped 635 trees of different age classes, sampled from 15 sites with various degrees of anthropogenic disturbance. The degree of admixture within sampled trees was then assessed through Bayesian clustering of genotypes. Hybrids were present in seven of the populations, with 2.4% of all sampled trees showing spontaneous admixture. Sites with hybrids were significantly more disturbed than pure stands, while hybrids comprised both immature juveniles and trees of reproductive age. All three possible F1s were detected. Advanced-generation hybrids were consistently biased towards P. balsamifera regardless of whether hybridization had occurred with P. deltoides or P. nigra. Gene exchange between P. deltoides and P. nigra was not detected beyond the F1 generation; however, detection of a trihybrid demonstrates that even this apparent reproductive isolation does not necessarily result in an evolutionary dead end. Collectively, results demonstrate the natural fertility of hybrid poplars and suggest that introduced genes could potentially affect the genetic integrity of native trees, similar to that arising from introgression between natives.     

Genetic relationships between Japanese native and commercial breeds using 70 chicken autosomal SNP genotypes by the DigiTag2 assay

Recently, single nucleotide polymorphisms (SNPs) have been used to identify genes or genomic regions responsible for economic traits, including genetic diseases in domestic animals, and to examine genetic diversity of populations. In this study, we genotyped 70 chicken autosomal SNPs using DigiTag2 assay to understand the genetic structure of the Japanese native chicken breeds Satsumadori and Ingie, and the relationship of these breeds with other established breeds, Rhode Island Red (RIR), commercial broiler and layer. Five breeds, each consisting of approximately 20 chickens, were subjected to the assay, revealing the following: Average expected heterozygosities of broiler, Satsumadori, RIR, layer and Ingie were 0.265, 0.254, 0.244, 0.179 and 0.176, respectively. Phylogenetic analysis using the concatenated 70 autosomal SNP genotypes distinguished all chickens and formed clusters of chickens belonging to the respective breeds. In addition, the 2-D scatter plot of the first two principal components was consistent with the phylogenic tree. Taken together with the pairwise F(st) distances, broiler and RIR were closely positioned near each other, while Ingie was positioned far from the other breeds. Structure analysis revealed that the probable number of genetic clusters (K) was six and four with maximum likelihood and ΔK values, respectively. The clustering with maximum likelihood revealed that, in addition to the clustering of the other five breeds, the Satsumadori was subdivided into two genetic clusters. The clustering with ΔK value indicated that the broiler and Rhode Island Red were assigned to the same genetic cluster.     

Primary and secondary indices of stress in the progeny of rainbow trout (Oncorhynchus mykiss) selected for high and low responsiveness to stress

A series of pooled gamete matings was carried out employing eggs and milt from mature male and female rainbow trout selected for a consistently high- or low-responsiveness to stress, as indicated by post-stress plasma cortisol elevation. Development of the progeny was closely monitored and the responsiveness to stress of the progeny of high-responding parents and the progeny of low-responding parents was assessed by two methods. For a period of 14 months, at approximately monthly intervals, the plasma cortisol elevation evoked by a standardized confinement stress was determined in fish from each group, and secondly, on one occasion, the time-course of the plasma cortisol response to a 24-h period of confinement was monitored. Progeny of high-responding parents snowed a significantly greater cortisol response to stress than the progeny of low-responding parents during both testing procedures. However, when the effect of a 14-day confinement stress was examined, high-responding progeny showed a more rapid recovery of plasma cortisol levels, while levels in the low-responding progeny, although initially lower, showed a more sustained elevation. To assess the possible functional implications of these observations, circulating lymphocyte numbers, an immunologically important cortisol-sensitive component of the blood cell complement, were determined. The duration of the lymphocytopenia observed following the onset of confinement was found to be related to the initial, not the sustained, cortisol response. These data suggest that manipulation of the sensitivity to stress of fish is feasible by selective breeding, but that careful.choice of the indices employed to identify traits considered desirable is necessary.     

Construction and characterization of BAC libraries for three fish species; rainbow trout, carp and tilapia

Bacterial artificial chromosome (BAC) libraries are important tools for genomic research. We have constructed seven genomic BAC libraries from three fish species, rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio) and tilapia (Oreochromis niloticus). The two rainbow trout BAC libraries have average insert sizes of 58 and 110 kb. The average size of inserts in the carp BAC library is 160 kb. The average insert sizes of the four tilapia BAC libraries are 65, 105, 145 and 194 kb, respectively. These libraries represent good coverage of each genome (2-64 x coverage). The libraries can be screened by conventional colony hybridization and provide a starting point for the construction of high-density filtres or polymerase chain reaction (PCR) screening approaches. These BAC libraries will facilitate the positional cloning of quantitative trait loci (QTLs) for a variety of economically important traits in these species.     

First-generation SNP/InDel markers tagging loci for pathogen resistance in the potato genome

A panel of 17 tetraploid and 11 diploid potato genotypes was screened by comparative sequence analysis of polymerase chain reaction (PCR) products for single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (InDels), in regions of the potato genome where genes for qualitative and/or quantitative resistance to different pathogens have been localized. Most SNP and InDel markers were derived from bacterial artificial chromosome (BAC) insertions that contain sequences similar to the family of plant genes for pathogen resistance having nucleotide-binding-site and leucine-rich-repeat domains (NBS-LRR-type genes). Forty-four such NBS-LRR-type genes containing BAC-insertions were mapped to 14 loci, which tag most known resistance quantitative trait loci (QTL) in potato. Resistance QTL not linked to known resistance-gene-like (RGL) sequences were tagged with other markers. In total, 78 genomic DNA fragments with an overall length of 31 kb were comparatively sequenced in the panel of 28 genotypes. 1498 SNPs and 127 InDels were identified, which corresponded, on average, to one SNP every 21 base pairs and one InDel every 243 base pairs. The nucleotide diversity of the tetraploid genotypes (pi = 0.72 x 10(-3)) was lower when compared with diploid genotypes (pi = 2.31 x 10(-3)). RGL sequences showed higher nucleotide diversity when compared with other sequences, suggesting evolution by divergent selection. Information on sequences, sequence similarities, SNPs and InDels is provided in a database that can be queried via the Internet.     

A suite of molecular markers for identifying species, detecting introgression and describing population structure in spadefoot toads (Spea spp.)

Two congeneric species of spadefoot toad, Spea multiplicata and Spea bombifrons, have been the focus of hybridization studies since the 1970s. Because complex hybrids are not readily distinguished phenotypically, genetic markers are needed to identify introgressed individuals. We therefore developed a set of molecular markers (amplified fragment length polymorphism, polymerase chain reaction-restriction fragment length polymorphism and single nucleotide polymorphism) for identifying pure-species, F1 hybrids and more complex introgressed types. To do so, we tested a series of markers across both species and known hybrids using populations in both allopatry and sympatry. We retained those markers that differentiated the two pure-species and also consistently identified known species hybrids. These markers are well suited for identifying hybrids between these species. Moreover, those markers that show variation within each species can be used in conjunction with existing molecular markers in studies of population structure and gene flow.     

Relationship between single nucleotide polymorphism of interleukin-18 and susceptibility to pulmonary tuberculosis in the Chinese Han population

Interleukin-18 (IL-18) is a multi-functional cytokine capable of inducing either Th1 or Th2 polarization depending on the immunologic milieu. IL-18 may influence the host response to Mycobacterium tuberculosis (M.tb) infection. To investigate the relationship between single nucleotide polymorphisms of the IL-18 and susceptibility to pulmonary tuberculosis in the Chinese Han population, the IL-18 gene was sequenced to detect polymorphisms and to examine the genotype frequencies in 300 patients and 702 healthy controls. DNA sequencing revealed three IL-18 variants: rs1946518, rs5744247, and rs549908. It also revealed that allele A of rs1946518 confers a 1.47-fold increased risk of developing tuberculosis (TB) (P = 0.0001, OR [95%CI] = 1.47 [1.21-1.78]), and that the C allele of rs5744247 confers a 0.77-fold decreased risk of disease (P = 0.01, R [95%CI] = 0.77 [0.632-0.937]). The genotypes rs1946518, rs5744247 and rs549908 were found to be significantly associated with TB. Estimation of the frequencies of haplotypes revealed a potential risk haplotype AGA (P = 0.01, OR [95%CI] = 1.41 [1.15-1.72]) and a protective haplotype CCA (P = 0.01, OR [95%CI] = 0.70 [0.57-0.85]) for TB. The present findings suggest that polymorphisms in the IL-18 gene may affect susceptibility to TB and increase the risk of developing the disease in the Chinese Han population.