Comparative genomic analysis of Atlantic salmon, Salmo salar, from Europe and North America

Background: Several lines of evidence including allozyme analysis, restriction digest patterns and sequencing ofmtDNA as well as mini- and micro-satellite allele frequencies indicate that Atlantic salmon (Salmo salar) from NorthAmerica and Europe are genetically distinct. These observations are supported by karyotype analysis, whichrevealed that North American Atlantic salmon have 27 pairs of chromosomes whereas European salmon have 29pairs. We set out to construct a linkage map for a North American Atlantic salmon family and to compare this mapwith the well developed map for European Atlantic salmon.Results: We used microsatellite markers, which had previously been mapped in the two Atlantic salmon SALMAPmapping families from the River Tay, Scotland, to carry out linkage analysis in an Atlantic salmon family (NB1)whose parents were derived from the Saint John River stock in New Brunswick, Canada. As large differences inrecombination rates between female and male Atlantic salmon have been noted, separate genetic maps wereconstructed for each sex. The female linkage map comprises 218 markers in 37 linkage groups while the male maphas 226 markers in 28 linkage groups. We combined 280 markers from the female and male maps into 27composite linkage groups, which correspond to the haploid number of chromosomes in Atlantic salmon from theWestern Atlantic.Conclusions: A comparison of the composite NB1 and SALMAP linkage maps revealed the reason for thedifference in the chromosome numbers between European and North American Atlantic salmon: Linkage groupsAS-4 and AS-32 in the Scottish salmon, which correspond to chromosomes Ssa-6 and Ssa-22, are combined into asingle NB1 linkage group as are linkage groups AS-21 and AS-33 (corresponding to chromosomes Ssa-26 and Ssa-28). The comparison of the linkage maps also suggested some additional chromosomal rearrangements, but it willrequire finer mapping, potentially using SNPs, to test these predictions. Our results provide the first comparison ofthe genomic architecture of Atlantic salmon from North America and Europe with respect to chromosomeorganization

Developing Successful Breeding Programs for New Zealand Aquaculture: A Perspective on Progress and Future Genomic Opportunities

Over the past 40 years New Zealand (NZ) aquaculture has grown into a significant primary industry. Tonnage is small on a global scale, but the industry has built an international reputation for the supply of high quality seafood to many overseas markets. Since the early 1990s the industry has recognized the potential gains from selective breeding and the challenge has been to develop programs that can overcome biological obstacles (such as larval rearing and mortality) and operate cost-effectively on a relatively small scale while still providing significant gains in multiple traits of economic value. This paper provides an overview of the current status, and a perspective on genomic technology implementation, for the family based genetic improvement programs established for the two main species farmed in NZ: Chinook (king) salmon (Oncorhynchus tshawytscha) and GreenshellTM mussel (Perna canaliculus). These programs have provided significant benefit to the industry in which we are now developing genomic resources based on genotyping-by-sequencing to complement the breeding programs, enable evaluation of the genetic diversity and identify the potential benefits of genomic selection. This represents an opportunity to increase genetic gain and more effectively utilize the potential for within family selection

Evaluation of salmon sperm electroporation for gene transfer

The aim of this thesis was to evaluate the potential of using sperm cells as a vector for gene transfer in chinook salmon (Oncorhynchus tshawytscha). Preliminary studies indicated that while salmon sperm were inefficient at picking up DNA during simple incubation, electroporation of the sperm significantly enhanced the sperm/DNA association. Therefore, a number of factors were examined to identify the optimal conditions for DNA uptake by salmon sperm and the subsequent transfer of foreign DNA into salmon embryos through fertilisation. The effect of electroporation on salmon sperm/DNA association was examined using bacterial transformation, the polymerase chain reaction, dot blot and Southern analysis. Sperm were electroporated in the presence of circular or linear pRSV–lacZ DNA at 20 µg/ml and 100 µg/ml, with field strengths of 625 V/cm or 1000 V/cm, a pulse length of 27.4 ms, and with either a single or double pulse. Electroporation enhanced sperm retention of circular and linear pRSV-lacZ DNA, and retention was greatest at the higher field strength with two pulses, and a DNA concentration of 100 µg/ml. The sperm/DNA association was strong as it resisted repeated washing and digestion by DNase 1, suggesting that some of the foreign DNA was internalised into the sperm. It was estimated that, following washing and DNase 1 digestion, on average, 0.12 fg (16 copies), of DNA were associated with each sperm cell. The ability of electroporated sperm to transfer plasmid DNA into embryos was investigated using a number of combinations of field strength, pulse length, pulse number and DNA concentration. Optimal conditions were found to be two pulses of 27.4 ms at 1000 V/cm, using 0.5 x HBS, with 200 µg/ml of plasmid DNA The transferred DNA was detected by PCR in up to 85% of the resulting 12 week old fry. Gene transfer efficiencies of up to 47% were repeatedly achieved over two separate spawning seasons. Southern analysis failed to detect any plasmid DNA in these fry, indicating that although electroporated sperm are capable of transferring the plasmid DNA, the levels of this DNA in the fry are low and the resulting fry are mosaic. PCR and Southern analysis of DNA extracted from embryos of five developmental stages was also carried out. Introduced plasmid DNA was detected in over 90% of the 0.8 day old embryos, and up to 107 copies of the plasmid DNA were observed in 1.2 day old embryos. The results indicated that the DNA introduced by electroporated sperm is amplified during the early stages of embryo development, remains predominantly extrachromosomal in an unmodified form, and is gradually lost during development. Expression of the introduced pRSV-lacZ DNA was detected in only a small number of embryos despite the presence of high levels of plasmid DNA in most of the embryos analysed. The methods of sperm electroporation and egg microinjection for gene transfer were directly compared. In a parallel study, a growth hormone gene construct, OnMTGH1, was transferred into chinook and coho salmon using both these methods. While the microinjected DNA was converted into high molecular weight concatemers, the sperm transferred DNA remained unmodified after introduction into salmon embryos. A greater proportion of 11 day old embryos resulting from electroporated sperm (5/5) were found to contain OnMTGH1 DNA than those resulting from microinjected eggs (4/8). However, no introduced DNA, or growth enhanced individuals, were detected in the 10 month old salmon developing from sperm electroporated with the GH construct. In contrast, introduced DNA was detected in 12% of the microinjected group, and growth enhanced individuals were obtained. It is clear from these results that electroporated salmon sperm can successfully take up DNA, and are efficient vectors for transferring genes into embryos. However, the introduced DNA does not persist and the levels decrease as the fish develop. Nevertheless, sperm electroporation has the potential to be further developed as an efficient mass gene transfer method in salmon

Advantages of selective medium for surveillance of Tenacibaculum species in marine fish aquaculture

We evaluated the suitability of three diagnostic techniques to detect Tenacibaculum maritimum in farmed Chinook salmon (or ‘king’ salmon), Oncorhynchus tshawytscha, exhibiting clinical signs of tenacibaculosis with skin spots and ulcers. A selective bacterial culture technique using Marine Shieh's Selective Medium (MSSM) found 100% detection of T. maritimum in sampled farmed O. tshawytscha. In comparison, pathogen detection using a non-selective culture medium (Marine Agar) revealed 33.3% detection of T. maritimum, while a droplet digital polymerase chain reaction (ddPCR) specific assay provided 81.5% positive detection. Skin abnormality type was a significant predictor of ddPCR detection, with spots less likely to be found positive compared to ulcers (p = 0.01). This study also reports the first isolation of co-occurring Tenacibaculum soleae and Tenacibaculum dicentrarchi from skin abnormalities of farmed O. tshawytscha in New Zealand. The 16S ribosomal RNA sequences amplified from representative isolates shared 100% and 99% identities with T. soleae and T. dicentrarchi 16S rRNA sequences, respectively. T. soleae and T. dicentrarchi colonies were morphologically distinct from T. maritimum. The MSSM culture technique facilitated rapid recovery of all three Tenacibaculum species within 48 h when incubated at room temperature (20–23 °C). In this study we discuss the advantages of a selective medium for surveillance of pathogenic Tenacibaculum species that infect fish when used in combination with confirmatory identification by molecular techniques, in contrast to relying only on PCR based detection methods. We suggest that culture dependent diagnostic techniques provide sufficiently sensitive, rapid, and cost-effective on-farm screening as this method detects only viable organisms which pose immediate risk to fish. The rapid identification and management of emerging fish-pathogenic species of Tenacibaculum in New Zealand is essential for the diagnosis of tenacibaculosis outbreaks which can impact farm production and animal welfare

Vertebral abnormalities in free-living Chinook salmon (Oncorhynchus tshawytscha, Walbaum) in New Zealand

Spinal abnormalities are common in farmed Chinook Salmon in New Zealand. We report spinal abnormalities in adult Chinook salmon which were predominantly hatchery reared and released as smolts and which we term free-living. We compare these to rates seen in farmed New Zealand salmon. 101 free-living adult salmon were radiographically assessed for spinal curvatures (lordosis, kyphosis, scoliosis; LKS) and other vertebral abnormalities. Severity of abnormality was assessed on a three-point scale. Abnormal vertebral bodies were detected in 88.1% of free-living salmon. Spinal curvatures were the most common abnormality type with 83.2% of fish showing this abnormality but only one free-living Chinook had LKS of severity greater than 1. Farmed Chinook salmon are reported to have LKS rates of 29% with 18% of LKS abnormalities of severity greater than 1. These results suggest that free-living Chinook salmon frequently develop spinal abnormalities, but these abnormalities are less severe than those observed in farmed salmon

Effects of commercial diets and temperature on the growth performance and stress response of hapuku (Polyprion oxygeneios)

There is a growing interest in the development of hapuku (Polyprion oxygeneios) for aquaculture in New Zealand and Australia. This is driven by the high value of this species prized for its excellent flesh quality, texture and its rapid growth capability. As a relatively new aquaculture candidate, little is currently known about their thermal tolerance and stress response. Juveniles inhabit surface waters, have a high rate of growth and move into a demersal habitat at an age between 3 and 4 years, where water temperature is cooler (7-15. °C) and more stable. The sea surface temperature in New Zealand can reach 22. °C during the summer months in more northerly locations, and captive rearing has indicated that during periods of high temperature, growth is reduced and it is possible that the physiological response is compromised. We examined the effects of two rearing temperatures (18. °C and 22. °C) and three commercial diets on the growth of P. oxygeneios during a 14 week trial. At the end of this trial, fish were exposed to a crowding stressor, and their stress response (plasma cortisol, glucose and cholesterol levels) determined. In addition, we examined the temporal stress response of P. oxygeneios acclimated to 18. °C and 22. °C subjected to a single acute handling stress. Specific growth rate and condition factor significantly increased over time in fish reared at 18. °C, but not at 22. °C. Plasma cortisol levels in hapuku prior to and after application of the stressors were within the range observed in other teleost species and the magnitude of the cortisol response was higher in hapuku subjected to crowding than handling stress. In summary, the results indicated that rearing P. oxygeneios at temperatures of 22. °C compromised their growth and that all three diets tested promoted growth in hapuku reared at 18. °C but not at 22. °C.Statement of relevanceHapuku over 1 kg had better growth rates at 18. °C than 22. °C

Type I collagen differences in farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand

The integrity and function of Type I collagen (Col-I), a fundamental structural molecule, is central to fish movement. Farmed Chinook salmon in New Zealand are reported to develop a late onset curvature syndrome, lordosis, kyphosis and scoliosis (LKS), associated with inflammation and fibrosis, which affects movement and product quality. To investigate if type I collagen integrity is associated with LKS, salmon from a farm with high LKS (Farm 1) were compared with a farm with low LKS (Farm 2). Representative salmon from Farm 1 and Farm 2 were harvested at 25 months of age and their physical metrics measured. Condition factor (K) was derived. White muscle samples from the abdominal and caudal regions were sampled and analysed. The properties of Col-I were determined using liquid chromatography-electrospray ionization mass spectrometry. The amount of Col-I in white muscle, inferred from hydroxy-proline [Hyp], was 0.071 and 0.130 ([Hyp (mg) / Dry sample (mg)]%) for Farm 1 and Farm 2 respectively. There was a significant (p 4-fold hydroxylysinonorleucine (HLNL) higher in Farm 1 salmon. Mature pyridinoline (PYR) crosslinks were readily detectable in salmon from Farm 2 but below the threshold for reliable detection in those from Farm 1. The mature crosslink of elastin, desmosine (DES), was ~1.5-fold higher in Farm 2 salmon. We have quantified Col-I in the white muscle compartment of farmed Chinook salmon and established methods to compare the crosslink profile. PYR and DHLNL crosslinks associated with myosepta were significantly different between the populations. Salmon from Farm 2 had both a higher proportion of mature PYR crosslinks and higher [Hyp], additive differences that may be of functional significance. Higher levels of crosslinks (HLNL, HHMD) associated with loose connective tissue and the extracellular matrix were seen in salmon from Farm 1 and also associated with condition factor. These results demonstrated differences in the amount of Col-I and crosslink profile of farmed Chinook salmon which could be linked with a population-based susceptibility toward LKS. The association between LKS and genetic and/or husbandry differences requires additional controlled experiments to determine these relationships more precisely