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Development and characterisation of microsatellite and single nucleotide polymorphism markers in Atlantic cod (Gadus morhua L.)
S250
Abstracts / Aquaculture 272S1 (2007) S238–S321
studies are needed. One such option is to build a dual expression (Eukaryotic and Prokaryotic) vector capable of providing the desired outcomes. Most commercially available delivery vectors employ the CMV promoter to drive expression of the foreign protein. However, given that ELI relies on the simultaneous expression of many foreign proteins (sometimes thousands) at once then it may be prudent to incorporate a promoter that has increased activity within fish. To this end we have isolated and characterized the β-actin promoter from Atlantic salmon. Expression activity of the β-actin promoter was compared to a CMV promoter via a CAT assay and was shown to be far more efficient in transient assays, involving intramuscular injections. Finally we constructed a new dual expression vector, pbS-Sfi-CAT, incorporating the β-actin promoter, a prokaryotic promoter (T5/lacO), and SfiI restriction sites for ease of cloning. The efficacy of this new vector for Atlantic salmon vaccination studies will be discussed in the context of expression library immunization.
alignments of approximately 2300 EST sequences using an SNP detection pipeline. The approach resulted in 140 putative SNPs, 70% of which were shown by experimental validation to be true SNPs and segregating in the breeding population. More studies are currently under way in which more ESTs will be searched towards our goal: the establishment of 1000 functional microsatellite and SNP markers and construction of genetic linkage map for Atlantic cod. A genetic linkage map enables for instance the possibilities of finding markers linked to quantitative trait loci (QTLs) for different traits. Such QTLs together with phenotypic data can form a basis for marker-assisted selection (MAS) in a selective breeding programme.
doi:10.1016/j.aquaculture.2007.07.044
A. Derayat c, R.D. Houston a, D.R. Guy b, A. Hamilton b, J. Ralph b, N. Spreckley b, J.B. Taggart c, B.J. McAndrew c, C.S. Haley a a Division of Genetics and Genomics, Roslin Institute, Roslin, Midlothian EH25 9PS, UK b Landcatch Natural Selection, Cooperage Way, Alloa, Clackmannanshire FK10 3LP, UK c Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK
Development and characterisation of microsatellite and single nucleotide polymorphism markers in Atlantic cod (Gadus morhua L.) M. Delghandi a, J. Stenvik a , T. Moen b, M.S. Wesmajervi a, J.I. Westgård d, K.T. Fjalestad a, A. Kettunen a, F. Nilsen c a Norwegian Institute of Fisheries and Aquaculture Research, Norway b AKVAFORSK, Norway c Institute of Marine Research, Norway d Norwegian College of Fishery Science, Norway The first phase of constructing a genetic map for Atlantic cod (Gadus morhua L.) is the development of genetic markers, such as microsatellites and single nucleotide polymorphisms (SNPs). Approximately 5000 expressed sequence tags (ESTs) for type I and type II microsatellites and SNPs and 400 cloned inserts enriched for DNA sequences containing tandem di-, triand tetra nucleotide repeats for type II microsatellites were screened. As a result, a total of 133 microsatellite loci were selected for evaluation. Among those loci which were successfully amplified 40 revealed a reliable pattern of genetic variation. The number of alleles varied between 2 and 19 with an observed heterozygosity of 0.02 to 0.89. SNP markers were detected from
doi:10.1016/j.aquaculture.2007.07.045
Mapping QTL affecting body lipid percentage in Atlantic salmon (Salmo salar)
A medium genetic component to body lipid percentage within commercial lines has previously been shown (h2 = 0.17–0.24) To investigate whether this genetic component includes loci of major effect, a genome-wide QTL scan was performed within commercially bred families (1999 strip) that were analysed for a range of commercially important harvest traits. Eleven large fullsib families were chosen for analysis. To utilise the large difference between male and female recombination rate, a two-stage genotyping and analysis strategy was employed. Initially, the parents and offspring were genotyped for between two and four markers per linkage group, and a sire-based QTL analysis was used to detect linkage groups with significant effects on commercially important traits. To confirm the QTL and to provide an improved estimate of position, a dam-based analysis was then employed. One major QTL was located at the genome-wide significance level for percentage body lipid. Other microsatellites loci within this linkage group are being mapped and marker density is being increased
Abstracts / Aquaculture 272S1 (2007) S238–S321
studies are needed. One such option is to build a dual expression (Eukaryotic and Prokaryotic) vector capable of providing the desired outcomes. Most commercially available delivery vectors employ the CMV promoter to drive expression of the foreign protein. However, given that ELI relies on the simultaneous expression of many foreign proteins (sometimes thousands) at once then it may be prudent to incorporate a promoter that has increased activity within fish. To this end we have isolated and characterized the β-actin promoter from Atlantic salmon. Expression activity of the β-actin promoter was compared to a CMV promoter via a CAT assay and was shown to be far more efficient in transient assays, involving intramuscular injections. Finally we constructed a new dual expression vector, pbS-Sfi-CAT, incorporating the β-actin promoter, a prokaryotic promoter (T5/lacO), and SfiI restriction sites for ease of cloning. The efficacy of this new vector for Atlantic salmon vaccination studies will be discussed in the context of expression library immunization.
alignments of approximately 2300 EST sequences using an SNP detection pipeline. The approach resulted in 140 putative SNPs, 70% of which were shown by experimental validation to be true SNPs and segregating in the breeding population. More studies are currently under way in which more ESTs will be searched towards our goal: the establishment of 1000 functional microsatellite and SNP markers and construction of genetic linkage map for Atlantic cod. A genetic linkage map enables for instance the possibilities of finding markers linked to quantitative trait loci (QTLs) for different traits. Such QTLs together with phenotypic data can form a basis for marker-assisted selection (MAS) in a selective breeding programme.
doi:10.1016/j.aquaculture.2007.07.044
A. Derayat c, R.D. Houston a, D.R. Guy b, A. Hamilton b, J. Ralph b, N. Spreckley b, J.B. Taggart c, B.J. McAndrew c, C.S. Haley a a Division of Genetics and Genomics, Roslin Institute, Roslin, Midlothian EH25 9PS, UK b Landcatch Natural Selection, Cooperage Way, Alloa, Clackmannanshire FK10 3LP, UK c Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK
Development and characterisation of microsatellite and single nucleotide polymorphism markers in Atlantic cod (Gadus morhua L.) M. Delghandi a, J. Stenvik a , T. Moen b, M.S. Wesmajervi a, J.I. Westgård d, K.T. Fjalestad a, A. Kettunen a, F. Nilsen c a Norwegian Institute of Fisheries and Aquaculture Research, Norway b AKVAFORSK, Norway c Institute of Marine Research, Norway d Norwegian College of Fishery Science, Norway The first phase of constructing a genetic map for Atlantic cod (Gadus morhua L.) is the development of genetic markers, such as microsatellites and single nucleotide polymorphisms (SNPs). Approximately 5000 expressed sequence tags (ESTs) for type I and type II microsatellites and SNPs and 400 cloned inserts enriched for DNA sequences containing tandem di-, triand tetra nucleotide repeats for type II microsatellites were screened. As a result, a total of 133 microsatellite loci were selected for evaluation. Among those loci which were successfully amplified 40 revealed a reliable pattern of genetic variation. The number of alleles varied between 2 and 19 with an observed heterozygosity of 0.02 to 0.89. SNP markers were detected from
doi:10.1016/j.aquaculture.2007.07.045
Mapping QTL affecting body lipid percentage in Atlantic salmon (Salmo salar)
A medium genetic component to body lipid percentage within commercial lines has previously been shown (h2 = 0.17–0.24) To investigate whether this genetic component includes loci of major effect, a genome-wide QTL scan was performed within commercially bred families (1999 strip) that were analysed for a range of commercially important harvest traits. Eleven large fullsib families were chosen for analysis. To utilise the large difference between male and female recombination rate, a two-stage genotyping and analysis strategy was employed. Initially, the parents and offspring were genotyped for between two and four markers per linkage group, and a sire-based QTL analysis was used to detect linkage groups with significant effects on commercially important traits. To confirm the QTL and to provide an improved estimate of position, a dam-based analysis was then employed. One major QTL was located at the genome-wide significance level for percentage body lipid. Other microsatellites loci within this linkage group are being mapped and marker density is being increased