A set of 37 microsatellite DNA markers for genetic diversity and structure analysis of Atlantic salmon Salmo salar populations.

Atlantic salmon Salmo salar microsatellite markers from a large database were analysed and selected with technical, economic and genetic criteria to provide an optimized set of polymorphic DNA markers for the analysis of the genetic diversity and the structure of anadromous Atlantic salmon populations. A set of 37 microsatellite markers was identified that are easy to use and provide a high level of differentiation power.

A description of the origins, design and performance of the TRAITS-SGP Atlantic salmon Salmo salar L. cDNA microarray.

The origins, design, fabrication and performance of an Atlantic salmon microarray are described. The microarray comprises 16 950 Atlantic salmon-derived cDNA features, printed in duplicate and mostly sourced from pre-existing expressed sequence tag (EST) collections [SALGENE and salmon genome project (SGP)] but also supplemented with cDNAs from suppression subtractive hybridization libraries and candidate genes involved in immune response, protein catabolism, lipid metabolism and the parr-smolt transformation. A preliminary analysis of a dietary lipid experiment identified a number of genes known to be involved in lipid metabolism. Significant fold change differences (as low as 1.2x) were apparent from the microarray analysis and were confirmed by quantitative real-time polymerase chain reaction analysis. The study also highlighted the potential for obtaining artefactual expression patterns as a result of cross-hybridization of similar transcripts. Examination of the robustness and sensitivity of the experimental design employed demonstrated the greater importance of biological replication over technical (dye flip) replication for identification of a limited number of key genes in the studied system. The TRAITS (TRanscriptome Analysis of Important Traits of Salmon)-salmon genome project microarray has been proven, in a number of studies, to be a powerful tool for the study of key traits of Atlantic salmon biology. It is now available for use by researchers in the wider scientific community.

Interferon type I and type II responses in an Atlantic salmon (Salmo salar) SHK-1 cell line by the salmon TRAITS/SGP microarray.

Interferons (IFNs) are cytokines that have proinflammatory, antiviral, and immunomodulatory effects and play a central role during a host response to pathogens. The IFN family contains both type I and type II molecules. While there are a number of type I IFNs, there is only one type II IFN. Recently both type I and type II IFN genes have been cloned in salmonid fish and recombinant proteins produced showing IFN activity. We have stimulated an Atlantic salmon cell line (SHK-1) with both type I and type II recombinant salmonid IFNs and analyzed the transcriptional response by microarray analysis. Cells were exposed to recombinant IFNs for 6 or 24 h or left unexposed as controls. RNA was hybridized to an Atlantic salmon cDNA microarray (salmon 17K feature TRAITS/SGP array) in order to assess differential gene expression in response to IFN exposure. For IFN I and II, 47 and 72 genes were stimulated, respectively; most genes were stimulated by a single IFN type, but some were affected by both IFNs, indicating coregulation of the IFN response in fish. Real-time PCR analysis was employed to confirm the microarray results for selected differentially expressed genes in both a cell line and primary leukocyte cultures.

Identification of the sex-determining locus of Atlantic salmon (Salmo salar) on chromosome 2.

We have integrated data from linkage mapping, physical mapping and karyotyping to gain a better understanding of the sex-determining locus, SEX, in Atlantic salmon (Salmo salar). SEX has been mapped to Atlantic salmon linkage group 1 (ASL1) and is associated with several microsatellite markers. We have used probes designed from the flanking regions of these sex-linked microsatellite markers to screen a bacterial artificial chromosome (BAC) library, representing an 11.7x coverage of the Atlantic salmon genome, which has been HindIII fingerprinted and assembled into contigs. BACs containing sex-linked microsatellites and their related contigs have been identified and representative BACs have been placed on the Atlantic salmon chromosomes by fluorescent in situ hybridization (FISH). This identified chromosome 2, a large metacentric, as the sex chromosome. By positioning several BACs on this chromosome by FISH, it was possible to orient ASL1 with respect to chromosome 2. The region containing SEX appears to lie on the long arm between marker Ssa202DU and a region of heterochromatin identified by DAPI staining. BAC end-sequencing of clones within sex-linked contigs revealed five hitherto unmapped genes along the sex chromosome. We are using an in silico approach coupled with physical probing of the BAC library to extend the BAC contigs to provide a physical map of ASL1, with a view to sequencing chromosome 2 and, in the process, identifying the sex-determining gene.

Multiple marker mapping of quantitative trait loci in a cross between outbred wild boar and large white pigs.

A quantitative trait locus (QTL) analysis of growth and fatness data from a three generation pig experiment is presented. The population of 199 F2 animals was derived from a cross between wild boar and Large White pigs. Animals were typed for 240 markers spanning 23 Morgans of 18 autosomes and the X chromosome. A series of analyses are presented within a least squares framework. First, these identify chromosomes containing loci controlling trait variation and subsequently attempt to map QTLs to locations within chromosomes. This population gives evidence for a large QTL affecting back fat and another for abdominal fat segregating on chromosome 4. The best locations for these QTLs are within 4 cM of each other and, hence, this is likely to be a single QTL affecting both traits. The allele inherited from the wild boar causes an increase in fat deposition. A QTL for intestinal length was also located in the same region on chromosome 4 and could be the same QTL with pleiotropic effects. Significant effects, owing to multiple QTLs, for intestinal length were identified on chromosomes 3 and 5. A single QTL affecting growth rate to 30 kg was located on chromosome 13 such that the Large White allele increased early growth rate, another QTL on chromosome 10 affected growth rate from 30 to 70 kg and another on chromosome 4 affected growth rate to 70 kg.

A microsatellite linkage map of rainbow trout (Oncorhynchus mykiss) characterized by large sex-specific differences in recombination rates.

We constructed a genetic linkage map for a tetraploid derivative species, the rainbow trout (Oncorhynchus mykiss), using 191 microsatellite, 3 RAPD, 7 ESMP, and 7 allozyme markers in three backcross families. The linkage map consists of 29 linkage groups with potential arm displacements in the female map due to male-specific pseudolinkage arrangements. Synteny of duplicated microsatellite markers was used to identify and confirm some previously reported pseudolinkage arrangements based upon allozyme markers. Fifteen centromeric regions (20 chromosome arms) were identified with a half-tetrad analysis using gynogenetic diploids. Female map length is approximately 10 M, but this is a large underestimate as many genotyped segments remain unassigned at a LOD threshold of 3.0. Extreme differences in female:male map distances were observed (ratio F:M, 3.25:1). Females had much lower recombination rates (0.14:1) in telomeric regions than males, while recombination rates were much higher in females within regions proximal to the centromere (F:M, 10:1). Quadrivalent formations that appear almost exclusively in males are postulated to account for the observed differences.

Genetic alterations in a malignant schwannoma from a patient with neurofibromatosis (NF1).

In a patient with neurofibromatosis (von Recklinghausen disease; NF1), normal lymphocytes, five cutaneous neurofibromas, and tumour tissue from a recurrence of a malignant schwannoma were analysed for genetic alterations. Eleven DNA markers located on chromosome 17 and nine randomly chosen markers representing chromosomes 1, 2, 3, 4, 5, 6, and 11, were analysed. High resolution Giemsa banding of lymphocytes revealed no chromosomal rearrangement. The DNA from the neurofibromas were all found to have the same restricted fragment length polymorphism pattern as the constitutional DNA from the patient. In the malignant schwannoma a complete loss of one allele was found at polymorphic loci on chromosome arm 17p. One gene copy of the TP53 gene (17p13.1) and the NF1 gene (17q11.2) was lost, as was one copy of the PGA gene (11q13). No mutations were detected in the mutational hotspots of the TP53 gene. Partial losses were detected at three loci on chromosomes 1, 2 and 6, indicating a clonal variation within the tumour since histological evaluation disclosed no normal tissue in the analysed specimen. Our data indicate that the NF1 gene may function as a tumour suppressor gene, and that, either by effect of dose reduction or complete inactivation, both the NF1 gene and the TP53 gene may be critical for the progression of a neurofibroma to a malignant schwannoma. The observations made are consistent with the concept of stepwise multigenetic changes in tumour progression.

Alignment of two genetic linkage maps on porcine chromosome 13.

A set of four microsatellite markers from the USDA genetic linkage map of porcine chromosome 13 were mapped in the European Pig Gene Mapping Project (PiGMaP) reference pedigrees. A two-point linkage analysis was performed between these markers and a set of markers known to map to chromosome 13. Pairs of markers that had a lod score greater than three were used to construct a multi-point linkage map, permitting alignment of the United States Department of Agriculture (USDA) map to the PiGMaP.

A linkage map of Atlantic salmon (Salmo salar) reveals an uncommonly large difference in recombination rate between the sexes.

A genetic linkage map of the Atlantic salmon (Salmo salar) was constructed, using 54 microsatellites and 473 amplified fragment length polymorphism (AFLP) markers. The mapping population consisted of two full-sib families within one paternal half-sib family from the Norwegian breeding population. A mapping strategy was developed that facilitated the construction of separate male and female maps, while retaining all the information contributed by the dominant AFLP markers. By using this strategy, we were able to map a significant number of the AFLP markers for which all informative offspring had two heterozygous parents; these markers then served as bridges between the male and female maps. The female map spanned 901 cM and had 33 linkage groups, while the male spanned 103 cM and had 31 linkage groups. Twenty-five linkage groups were common between the two maps. The construction of the genetic map revealed a large difference in recombination rate between females and males. The ratio of female recombination rate vs. male recombination rate was 8.26, the highest ratio reported for any vertebrate. This map constitutes the first linkage map of Atlantic salmon, one of the most important aquaculture species worldwide.

Recent fusion events during evolution of pig chromosomes 3 and 6 identified by comparison with the babirusa karyotype.

The chromosomes of the babirusa, a species considered to have diverged from an ancestor of the pig during the Miocene epoch, about 12-26 million years ago, were studied to determine the sites of recent rearrangements during evolution of the domestic pig. It is shown that there is a pericentric inversion of the entire short arm on pig chromosome 1, compared to its counterpart in the babirusa (chromosome 15). We also present evidence suggesting that pig chromosome 3 was derived by a telomere-centromere fusion of two ancestral chromosomes homoelogous to babirusa chromosomes 12 and 17. Likewise, we conclude that pig chromosome 6 was most likely derived by a telomere-telomere fusion of ancestral chromosomes homoelogous to babirusa chromosomes 6 and 14. The detection of interstitial hybridization signals from presumptive subteloemeric repeats in the same chromosome region as the evolutionary fusion points on pig chromosomes 3 and 6 indicates that the fusion sites may still contain elements that are otherwise restricted to the telomere regions of pig chromosomes.

Characterization of microsatellites from flow-sorted porcine chromosome 13.

Porcine flow-sorted Chromosome (Chr) 13 was PCR amplified with primers based on porcine short interspersed element (SINE) sequences. The product was cloned, gridded in microtiter plates, and screened with a [GT]10 oligonucleotide which gave 45 positive clones. Sequencing of these clones showed that 36 were unique, and 26 [GT]n microsatellites were characterized. Six other simple repeat sequences, the majority of which were associated with the 3' end of the SINE sequence, were also detected. Twenty-one primers sets were selected, and 13 of these detected useful polymorphisms in the grandparents (n = 26) of the European porcine mapping collaboration (PiGMaP) reference families. These 13 markers were mapped in the "PiGMaP" reference families, and a two-point linkage analysis was performed. The Lod scores indicated that three of the markers were not linked and the remaining 11 formed two linkage groups of two and nine markers respectively. The larger linkage group was also linked to the transferrin locus, permitting assignment of nine markers to porcine Chr 13.

The gene encoding human transmembrane secretory component (locus PIGR) is linked to D1S58 on chromosome 1.

The human transmembrane secretory component (SC or poly-Ig receptor, PIGR) is expressed basolaterally on glandular epithelial cells and is responsible for the external translocation of polymeric IgA and IgM. SC is hence a key molecule in antibody protection of mucosal surfaces. The human SC gene (locus PIGR) is located on chromosome 1 (1q31-q41). Here we present the first genetic linkage study of PIGR versus syntenic markers, including D1S58 and F13B, which have been previously regionalized to 1q31-q32 and 1q31-q32.1, respectively. We found that PIGR is closely linked to D1S58 (lods + 5.06 at theta max = 0.06, without sex difference). PIGR versus F13B showed + 1.46 at theta max = 0.25 for both sexes combined. A recombination of 0.06 between F13B and D1S58 (lods + 2.24) was in contrast to a previously published study giving theta max = 0.22 (lods + 3.9), the combined lods being 5.6 at theta max = 0.20. The progeny of a triply heterozygotic female indicated that PIGR is the flanking locus, therefore suggesting a cen-F13B-D1S58-PIGR-qter gene sequence on human chromosome 1. Only negative lod scores to RH, C8@, and PGM1 on 1p, and FY on proximal 1q, were found. Current combined Norwegian allele frequencies were estimated for PIGR to be A1 = 0.63, A2 = 0.37 (370 chromosomes), and for D1S58 to be A1 = 0.44, A2 = 0.56 (218 chromosomes).

The genes for apolipoprotein all (APOA2) and the Duffy blood group (FY) are linked on chromosome 1 in man.

We have cloned a cDNA probe for human apolipoprotein AII and used it to analyze linkage relationships on chromosome 1. We found no recombinations between APOA2 and the gene coding for the Duffy blood group antigens (FY) in the 19 meioses examined. Our maximal lod score is 4.2 at zero recombination rate. K. Berg (1987, Cytogenet. Cell Genet. 46:579) found a maximal score of 2.5 at recombination fraction 0.14 in 54 meioses. When results from both studies are combined, the most likely distance between FY and APOA2 is about 10% recombination with a combined lod score of 5.6 for both sexes.

Pigs with the dominant white coat color phenotype carry a duplication of the KIT gene encoding the mast/stem cell growth factor receptor.

Comparative mapping data suggested that the dominant white coat color in pigs may be due to a mutation in KIT which encodes the mast/stem cell growth factor receptor. We report here that dominant white pigs lack melanocytes in the skin, as would be anticipated for a KIT mutation. We found a complete association between the dominant white mutation and a duplication of the KIT gene, or part of it, in samples of unrelated pigs representing six different breeds. The duplication was revealed by single strand conformation polymorphism (SSCP) analysis and subsequent sequence analysis showing that white pigs transmitted two nonallelic KIT sequences. Quantitative Southern blot and quantitative PCR analysis, as well as fluorescence in situ hybridization (FISH) analysis, confirmed the presence of a gene duplication in white pigs. FISH analyses showed that KIT and the very closely linked gene encoding the platelet-derived growth factor receptor (PDGFRA) are both located on the short arm of Chromosome (Chr) 8 at band 8p12. The result revealed an extremely low rate of recombination in the centromeric region of this chromosome, since the closely linked (0.5 cM) serum albumin (ALB) locus has previously been in situ mapped to the long arm (8q12). Pig Chr 8 shares extensive conserved synteny with human Chr 4, but the gene order is rearranged.

Linkage and association studies with C8A and C8B RFLPs on chromosome 1.

Linkage relations for the C8A and C8B BamHI RFLPs have been investigated. A peak lod score of 4.52 at recombination fraction zero was obtained between the two C8 genes. Combined with our previously obtained linkage data (Rogde et al. 1986) the maximum lod score is 7.53 at recombination fraction zero. The compiled C8-PGM1 linkage data from this and the previous study gave a maximum lod score of 22.02 at recombination fraction 0.11 (0.07-0.16) with no sex difference. A chromosome 1p reference marker, D1S57, has been applied in this linkage study. A maximum lod score of 5.06 between the C8 cluster and D1S57 at theta = 0.18 (0.11-0.28) was recorded. The linkage analyses and triply informative families gave evidence that the C8 loci are situated about halfway between PGM1 and D1S57 on the short arm of chromosome 1. There was no evidence of allelic association between the C8A and C8B BamHI RFLPs in 62 unrelated haplotypes.

The porcine hormone-sensitive lipase gene: sequence, structure, polymorphisms and linkage mapping.

The porcine hormone-sensitive lipase gene and its cDNA have been isolated and sequenced. Several putative regulatory sequences have been detected in the promotor region. The deduced amino acid sequence is 85% identical to the corresponding human, mouse and rat sequence. A search for polymorphisms revealed one intronic and one exonic polymorphism, the latter resulting in a conservative amino acid substitution. Linkage mapping located the LIPE gene close to the calcium release channel (CRC) locus on chromosome 6.

The Kidd (JK) blood group locus assigned to chromosome 18 by close linkage to a DNA-RFLP.

The close linkage between the PstI-restriction fragment length polymorphism (RFLP) disclosed by the L2.7 genomic DNA probe and the Kidd blood group locus is described. The maximum lod score is +8.53 at recombination fraction theta = 0.03. The upper probability limit of the recombination fraction is theta 1 = 0.11. The L2.7 probe, previously assigned provisionally to chromosome 17, is by the present study assigned to chromosome 18. This also assigns the Kidd blood group locus (JK) to chromosome 18. Accepting previous deletion mapping, the shortest regions of overlap (SRO) for JK is 18q11-12, whereas one of our hybrids assigns L2.7 to 18p11-pter, suggesting centromeric localisation of the linkage group. JK has been assigned previously to chromosome 2 because of its provisional linkage to IGK which in turn has been mapped to 2p12. Our own JK-IGK linkage data do in fact support the previous positive lod scores at high recombination fractions (total lods +4.12 at theta1 = 0.30). No obvious explanation for the conflicting gene mapping data is found.

The extension coat color locus and the loci for blood group O and tyrosine aminotransferase are on pig chromosome 6.

A linkage map of pig chromosome 6 was constructed using a wild pig/Large White intercross pedigree. The map comprises 23 polymorphic loci, and the sex-average map length is approximately 170 cM. The study adds three new genes to the chromosome 6 map: the extension (E) coat color locus, and the blood group O (EAO) and tyrosine aminotransferase (TAT) loci. Segregation at the E locus determined two coat color phenotypes among the F2 animals: wild-type color (El-) and black-spotting (Ep/Ep). The E locus showed close genetic linkage to the most distal marker (S0035) on the short arm of chromosome 6. Comparative coat color genetics as well as comparative mapping strongly suggest that E in pigs encodes the melanocyte-stimulating hormone receptor, as previously shown for the corresponding coat color loci in mouse and cattle. TAT was also mapped to the distal part of 6p, whereas EAO was the most distal marker on 6q. A clear tendency for a higher recombination rate in both terminal regions was observed. A model for the evolution of pig chromosome 6, based on comparative mapping data, is presented.

A physically anchored linkage map of pig chromosome 1 uncovers sex- and position-specific recombination rates.

A linkage map of pig chromosome 1 (SSC1) has been constructed using 15 polymorphic markers. Eleven markers constitute PCR-based microsatellites (three associated with coding sequences), six markers have also been mapped by in situ hybridization, and homologues to four of the markers have been mapped in human. The physical assignments show that almost the entire SSC1 is covered by the linkage map, which measures 164 cM (sex-averaged). In a large region on the q arm, representing about 40% of the chromosome, there is a significant excess of male recombination. In contrast, there is a significantly higher recombination rate in females in both terminal regions. In the penultimate marker intervals on the q arm as well as the p arm, females show a fivefold excess of recombination compared to males. The rate of genetic recombination in relation to the physical DNA content is 1 cM per 2-4 Mb over at least 80% of the chromosome. In the terminal part of the q arm, however, there is a tremendous increase in the recombination rate, with 1 cM equaling about 0.4 Mb. Two homologous chromosome segments in human could be detected, ESR-CGA on human chromosome 6 (HSA6) and IFNA-RLN-GRP78 on human chromosome 9 (HSA9). Since the porcine blood group locus EAA is located close to (or possibly within) the latter conserved segment and the suggested human counterpart, the ABO system, is similarly close to the segment on HSA9, our data provide indirect evidence that these blood group systems are homologous.

Life-long course and molecular characterization of the original Dutch family with epidermolysis bullosa simplex with muscular dystrophy due to a homozygous novel plectin point mutation.

Plectin is one of the largest and most versatile cytolinker proteins known. Cloned and sequenced in 1991, it was later shown to have nonsense mutations in recessive epidermolysis bullosa with muscular dystrophy. A dominant mutation in the gene was found to cause epidermolysis bullosa simplex Ogna without muscular dystrophy. Here we report the DNA sequencing of the plectin gene (PLEC1) in a Dutch family originally described in 1972 as having epidermolysis bullosa with muscular dystrophy. The results revealed homozygosity for a new plectin nonsense mutation at position 13187 and its specific 8q24 marker haplotype profile. Western blotting of cultured fibroblasts and immunofluorescence microscopy of skin biopsy confirm that the plectin protein expression is grossly reduced or absent. A summary of the life-long clinical course of the two affected brothers homozygous for the new E1914X mutation is given.