Genomewide single nucleotide polymorphism discovery in Atlantic salmon (Salmo salar): validation in wild and farmed American and European populations.

A considerable number of single nucleotide polymorphisms (SNPs) are required to elucidate genotype-phenotype associations and determine the molecular basis of important traits. In this work, we carried out de novo SNP discovery accounting for both genome duplication and genetic variation from American and European salmon populations. A total of 9 736 473 nonredundant SNPs were identified across a set of 20 fish by whole-genome sequencing. After applying six bioinformatic filtering steps, 200 K SNPs were selected to develop an Affymetrix Axiom(®) myDesign Custom Array. This array was used to genotype 480 fish representing wild and farmed salmon from Europe, North America and Chile. A total of 159 099 (79.6%) SNPs were validated as high quality based on clustering properties. A total of 151 509 validated SNPs showed a unique position in the genome. When comparing these SNPs against 238 572 markers currently available in two other Atlantic salmon arrays, only 4.6% of the SNP overlapped with the panel developed in this study. This novel high-density SNP panel will be very useful for the dissection of economically and ecologically relevant traits, enhancing breeding programmes through genomic selection as well as supporting genetic studies in both wild and farmed populations of Atlantic salmon using high-resolution genomewide information.

An association and haplotype analysis of porcine maternal infanticide: a model for human puerperal psychosis?

An association analysis using the Illumina porcine SNP60 beadchip was performed to identify SNPs significantly associated with porcine maternal infanticide. We previously hypothesised that this was a good animal model for human puerperal psychosis, an extreme form of postnatal mood disorder. Animals were selected from carefully phenotyped unrelated infanticide and control groups (representing extremes of the phenotypic spectrum), from four different lines. Permutation and sliding window analyses and an analysis to see which haplotypes were in linkage disequilibrium (LD) were compared to identify concordant regions. Across all analyses, intervals on SSCs 1, 3, 4, 10, and 13 were constant, contained genes associated with psychiatric or neurological disorders and were significant in multiple lines. The strongest (near GWS) consistent candidate region across all analyses and all breeds was the one located on SSC3 with one peak at 23.4 Mb, syntenic to a candidate region for bipolar disorder and another at 31.9 Mb, syntenic to a candidate region for human puerperal psychosis (16p13). From the haplotype/LD analysis, two regions reached genome wide significance (GWS): the first on SSC4 (KHDRBS3 to FAM135B), which was significant (-logP 5.57) in one Duroc based breed and is syntenic to a region in humans associated with cognition and neurotism; the second on SSC15, which was significant (-log10P 5.68) in two breeds and contained PAX3, which is expressed in the brain.

Association analyses of porcine SERPINE1 reveal sex-specific effects on muscling, growth, fat accretion and meat quality.

The serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1) gene encodes plasminogen activator inhibitor type 1 (PAI), which is the major physiological inhibitor of tissue-type and urokinase-type plasminogen activators and plays a role in obesity and insulin resistance in women but not in men. We detected SNP FN396538:g.566G>A in intron 3 and a non-synonymous substitution NM_213910:c.612A>G in exon 3 (p.Ile159Val) and mapped the gene to position 8.4 cM on the linkage map of chromosome 3. Association analyses were conducted on the 12th-15th generation of the Meishan × Large White (MLW) cross (n = 565), with records for weight at the end of test, lifetime daily gain, test time daily gain, loin depth and backfat depth, as well as on a European wild boar × Meishan (W × M) F(2) population (n = 333) with 47 traits recorded for carcass composition and meat quality. Analyses performed across the entire MLW population or in the male animals did not show any trait significantly associated with the loci studied. In female animals, both SNPs were associated with loin depth at nominal P < 0.05 with adjusted P values equal to 0.051 (g.566) and 0.057 (c.612). Differences between homozygotes were up to 0.65 SD. In the entire W × M population and female animals, SERPINE1 was significantly associated at adjusted P < 0.05 in descending order with muscling, growth and fat accretion and in male animals with meat quality (R-value). In the studied populations, allele effects were in opposite directions, which implies that the SNPs are markers that are in linkage disequilibrium with a causative mutation.

A gene-based SNP linkage map for pacific white shrimp, Litopenaeus vannamei.

Pacific white shrimp (Litopenaeus vannamei) are of particular economic importance to the global shrimp aquaculture industry. However, limited genomics information is available for the penaeid species. We utilized the limited public information available, mainly single nucleotide polymorphisms (SNPs) and expressed sequence tags, to discover markers for the construction of the first SNP genetic map for Pacific white shrimp. In total, 1344 putative SNPs were discovered, and out of 825 SNPs genotyped, 418 SNP markers from 347 contigs were mapped onto 45 sex-averaged linkage groups, with approximate coverage of 2071 and 2130 cm for the female and male maps, respectively. The average-squared correlation coefficient (r(2)), a measure of linkage disequilibrium, for markers located more than 50 cm apart on the same linkage group, was 0.15. Levels of r(2) increased with decreasing inter-marker distance from approximately 80 cm, and increased more rapidly from approximately 30 cm. A QTL for shrimp gender was mapped on linkage group 13. Comparative mapping to model organisms, Daphnia pulex and Drosophila melanogaster, revealed extensive rearrangement of genome architecture for L. vannamei, and that L. vannamei was more related to Daphnia pulex. This SNP genetic map lays the foundation for future shrimp genomics studies, especially the identification of genetic markers or regions for economically important traits.

A major SNP resource for dissection of phenotypic and genetic variation in Pacific white shrimp (Litopenaeus vannamei).

Bioinformatics and re-sequencing approaches were used for the discovery of sequence polymorphisms in Litopenaeus vannamei. A total of 1221 putative single nucleotide polymorphisms (SNPs) were identified in a pool of individuals from various commercial populations. A set of 211 SNPs were selected for further molecular validation and 88% showed variation in 637 samples representing three commercial breeding lines. An association analysis was performed between these markers and several traits of economic importance for shrimp producers including resistance to three major viral diseases. A small number of SNPs showed associations with test weekly gain, grow-out survival and resistance to Taura Syndrome Virus. Very low levels of linkage disequilibrium were revealed between most SNP pairs, with only 11% of SNPs showing an r(2)-value above 0.10 with at least one other SNP. Comparison of allele frequencies showed small changes over three generations of the breeding programme in one of the commercial breeding populations. This unique SNP resource has the potential to catalyse future studies of genetic dissection of complex traits, tracing relationships in breeding programmes, and monitoring genetic diversity in commercial and wild populations of L. vannamei.

Investigation of QTL regions on Chromosome 17 for genes associated with meat color in the pig.

Previous studies have uncovered several significant quantitative trait loci (QTL) relevant to meat colour traits mapped at the end of SSC17 in the pig. Furthermore, results released from the porcine genome sequencing project have identified genes underlying the entire QTL regions and can further contribute to mining the region for likely causative genes. Ten protein coding genes or novel transcripts located within the QTL regions were screened for single nucleotide polymorphisms (SNPs). Linkage mapping and association studies were carried out in the ISU Berkshire x Yorkshire (B x Y) pig resource family. The total length of the new SSC17 linkage map was 126.6 cM and additional markers including endothelin 3 (EDN3) and phosphatase and actin regulator 3 (PHACTR3) genes were assigned at positions 119.4 cM and 122.9 cM, respectively. A new QTL peak was noted at approximately 120 cM, close to the EDN3 gene, and for some colour traits QTL exceeded the 5% chromosome-wise significance threshold. The association analyses in the B x Y family showed that the EDN3 BslI and PHACTR3 PstI polymorphisms were strongly associated with the subjective colour score and objective colour reflectance measures in the loin, as well as average drip loss percentage and pH value. The RNPC1 DpnII and CTCFL HpyCH4III polymorphisms were associated with some meat colour traits. No significant association between CBLN4, TFAP2C, and four novel transcripts and meat colour traits were detected. The association analyses conducted in one commercial pig line found that both EDN3 BslI and PHACTR3 PstI polymorphisms were associated with meat colour reflectance traits such as centre loin hue angle and Minolta Lightness score. The present findings suggested that the EDN3 and PHACTR3 genes might have potential effects on meat colour in pigs, and molecular mechanisms of their functions are worth exploring.

Chromosomal mapping, sequence and transcription analysis of the porcine fertilin beta gene (ADAM2).

Fertilin beta (ADAM2) forms a part of the heterodimeric surface protein fertilin, found on the plasma membrane of mammalian sperm, and has been implicated in the process of sperm-egg fusion. Analysis of cDNA products obtained from adult porcine testis mRNA has presented a sequence corresponding to 2620 bp of the ADAM2 gene. This sequence contained an open reading frame encoding a 735-amino acid protein and homologous to ADAM2 genes known in other mammalian species. Polymerase chain reaction (PCR) analysis of genomic DNA showed that the 2620 bp of cDNA sequence comprises at least 21 exons and spans approximately 76 kb of genomic DNA, with its size and structure being relatively conserved between mouse, human and pig. Fluorescence in situ hybridization was used to map ADAM2 to chromosome 15 of the pig, using a bacterial artificial chromosome clone from the PigE BAC library. This finding is consistent with comparative mapping experiments performed between pig and human chromosomes. Analysis of nine mRNA samples, by reverse transcriptase-PCR, from different porcine tissues has also suggested that expression of ADAM2 is limited to the testis, a finding that is consistent with other mammalian species.

Characterization of the porcine sperm adhesion molecule gene SPAM1- expression analysis, genomic structure, and chromosomal mapping.

Sequence analysis of cDNA products, derived from adult porcine testis mRNA, gave overlapping nucleotide sequence correlating to 1952 bp of the sperm adhesion molecule 1 (SPAM1) gene. This sequence was shown to be homologous to SPAM1 genes known in other mammalian species and contained an open reading frame encoding a 493-amino acid protein. Fluorescence in situ hybridization (FISH), using a bacterial artificial chromosome (BAC) clone from the PigE BAC library, was used to map SPAM1 to chromosome 18 of the pig. This finding is consistent with comparative mapping experiments performed between pig and human chromosomes. Polymerase chain reaction (PCR) analysis of genomic DNA has shown that the 1952 bp of cDNA sequence spans approximately 9 kb of genomic DNA and comprises of at least four exons, with its size and structure being relatively conserved between mouse, human and pig. Reverse transcriptase (RT)-PCR analysis of mRNA from nine porcine tissues has also suggested that expression of SPAM1 is limited to the testis.

Morphologically distinct sperm subpopulations defined by Fourier shape descriptors in fresh ejaculates correlate with variation in boar semen quality following cryopreservation.

This study investigated two hypotheses: 1) that consistent between-boar variation in frozen semen quality exists and is genetically determined, and 2) that morphologically distinct subpopulations of spermatozoa exist within fresh boar ejaculates and that the incidence of these subpopulations is correlated with semen quality following cryopreservation. Five ejaculates were collected from each of 15 boars (5 boars from each of 3 breeds). An objective sperm morphology analyzer used Fourier shape descriptors to describe variation in the morphology of 300 spermatozoa per ejaculate before freezing. Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% glycerol) and 5 straws (0.5 mL) per ejaculate were cryopreserved (to -5 degrees C at 6 degrees C/min, then -5 degrees C to -80 degrees C at 40 degrees C/min). Semen was assessed for percentage of motile cells and motility characteristics (with computer-aided sperm analysis), plasma membrane integrity (SYBR-14 positive), and acrosome integrity (fluorescein-labeled peanut agglutinin positive). Consistent between-boar variability was detected for post-thaw sperm motility (P < .01), membrane integrity (P < .01), acrosome integrity (P < .01), curvilinear velocity (P < .01), straight-line velocity (P < .05), beat cross-frequency (P < .05), and amplitude of lateral head displacement (P < .01). Three morphologically distinct subpopulations of spermatozoa, defined by Fourier descriptors, were detected. The proportion of these subpopulations within the fresh ejaculate correlated with semen quality assessments made following cryopreservation. These findings support the hypothesis that consistent interindividual variation in sperm freezability is genetically determined and may relate to processes that occur during spermatogenesis. Subsequent characterization of these genetic differences between "good" and "poor" freezers may ultimately identify biophysical components of the spermatozoa that are essential for successful cryopreservation.

Identification of microorganisms using random primed PCR.

The polymerase chain reaction has facilitated the use of molecular approaches in microbiology including new strategies for the rapid identification of micro-organisms. Approaches based on the use of random primers and standard conditions, allows characteristic DNA fingerprints to be generated from any micro-organism even in the absence of information about its DNA sequence. Different primers can be used to produce genus-specific, species-specific, or even strain-specific DNA fingerprints. This article covers the background to this strategy, describes three different approaches to generating DNA fingerprints using random primers, and provides experimental detail for one method, RAPD.

Effect of sperm motility on separation of bovine X- and Y-bearing spermatozoa by means of free-flow electrophoresis.

Free-flow electrophoresis was used on bovine spermatozoa to test the hypothesis that there are surface charge differences between X and Y chromosome-bearing spermatozoa. Spermatozoa were deflected towards the anode by means of tromethamine (THAM) buffers of varying concentrations, and were separated into two populations under specific conditions. Experimental temperature and initial sperm motility had a significant effect on sperm distribution in the electric field. The results of DNA hybridization assays indicated an enrichment for Y chromosome-bearing spermatozoa.

Effect of the estrogen receptor locus on reproduction and production traits in four commercial pig lines.

We investigated the effect of the estrogen receptor (ESR) gene on growth and reproductive traits in four Large White-based commercial pig lines. A total of 9,015 litter records from 4,262 sows genotyped at the ESR locus were analyzed to determine whether ESR influenced total number born (TNB) or number born alive (NBA). Teat number (TN), test ADG, ADFI, feed:gain ratio (F/G), and ultrasonic backfat (BF) were also analyzed to determine effects of ESR. The TNB and NBA were increased per favorable allele of ESR (P < .01) with additive effects of .42 (.31) and .39 (.31) pigs/litter in the first parity (later parities), respectively. Dominance effects were near zero in parity one, but they were .16 and .14 pigs for TNB and NBA, respectively, in later parities (P < .05). A favorable additive pleiotropic effect was detected for BF (P < .001; -.11 mm per copy of the favorable litter size allele). There were no detectable effects on ADG or F/G (P > .10), although ADF was reduced 18 g/d per copy of the favorable litter size allele (P < .05). Average TN was 13.1 for pigs carrying the favorable litter size allele vs 13.2 for noncarriers (P < .05). Marker-assisted selection using ESR is warranted to increase litter size in the Large White-based lines considered here and will be of considerable economic value to pork producers.

The estrogen receptor locus is associated with a major gene influencing litter size in pigs.

Identification of individual major genes affecting quantitative traits in livestock species has been limited to date. By using a candidate gene approach and a divergent breed cross involving the Chinese Meishan pig, we have shown that a specific allele of the estrogen receptor (ER) locus is associated with increased litter size. Female pigs from synthetic lines with a 50% Meishan background that were homozygous for this beneficial allele produced 2.3 more pigs in first parities and 1.5 more pigs averaged over all parities than females from the same synthetic lines and homozygous for the undesirable allele. This beneficial ER allele was also found in pigs with Large White breed ancestory. Analysis of females with Large White breed background showed an advantage for females homozygous for the beneficial allele as compared to females homozygous for the other allele of more than 1 total pig born. Analyses of growth performance test records detected no significant unfavorable associations of the beneficial allele with growth and developmental traits. Mapping of the ER gene demonstrated that the closest known genes or markers were 3 centimorgans from ER. To our knowledge, one of these, superoxide dismutase gene (SOD2), was mapped for the first time in the pig. Analysis of ER and these linked markers indicated that ER is the best predictor of litter size differences. Introgression of the beneficial allele into commercial pig breeding lines, in which the allele was not present, and marker-assisted selection for the beneficial allele in lines with Meishan and Large White background have begun.

Nucleotide sequence of the luxA gene of Vibrio harveyi and the complete amino acid sequence of the alpha subunit of bacterial luciferase.

The nucleotide sequence of the 1.85-kilobase EcoRI fragment from Vibrio harveyi that was cloned using a mixed-sequence synthetic oligonucleotide probe (Cohn, D. H., Ogden, R. C., Abelson, J. N., Baldwin, T. O., Nealson, K. H., Simon, M. I., and Mileham, A. J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 120-123) has been determined. The alpha subunit-coding region (luxA) was found to begin at base number 707 and end at base number 1771. The alpha subunit has a calculated molecular weight of 40,108 and comprises a total of 355 amino acid residues. There are 34 base pairs separating the start of the alpha subunit structural gene and a 669-base open reading frame extending from the proximal EcoRI site. At the 3' end of the luxA coding region there are 26 bases between the end of the structural gene and the start of the luxB structural gene. Approximately two-thirds of the alpha subunit was sequenced by protein chemical techniques. The amino acid sequence implied by the DNA sequence, with few exceptions, confirmed the chemically determined sequence. Regions of the alpha subunit thought to comprise the active center were found to reside in two discrete and relatively basic regions, one from around residues 100-115 and the second from around residues 280-295.

Nucleotide sequence and transcription analysis of a linear DNA plasmid associated with the killer character of the yeast Kluyveromyces lactis.

In killer strains of the yeast Kluyveromyces lactis, production of a protein toxin which inhibits the growth of sensitive yeast cells is associated with the presence of two linear DNA plasmids, k1 and k2. We have determined the nucleotide sequence of the smaller plasmid k1 (8.9kb) which is thought to carry the structural gene(s) encoding the toxin. The plasmid has a low G + C content (26.8%) and contains four long open reading frames which account for over 95% of the total sequence. The longest open reading frame (1146 amino acids) probably corresponds to a structural gene for the killer toxin. Transcripts from three of the putative genes have been detected in K.lactis by Northern hybridisation.

Transposon mutagenesis of marine Vibrio spp.

Coliphage P1 was used to transduce derivatives of transposons Tn5 and mini-Mu into marine Vibrio spp. Transposon Tn5 encoding tetracycline resistance (Tn5-132) was used to isolate mutants of Vibrio harveyi defective in genes for bioluminescence (lux). Insertion of transposon Tn5-132 into the lux gene region was demonstrated by intraspecific transduction with phage hv-1 and by Southern blot hybridization. Transposon mini-Mu, modified to specify tetracycline resistance, was employed to mutagenize genes for lateral flagella synthesis of Vibrio parahaemolyticus. Mini-Mu contains the lacZ structural gene, and transposition results in transcriptional fusion of Vibrio genes with the transposon lacZ gene. Thus, in these fusions, lacZ expression was proportional to the level of transcription of the target gene. Regulation of lateral flagella gene expression was studied in vivo by measuring beta-galactosidase activity, and conditions which activate transcription of these genes were identified. A method for gene cloning with transposon-induced mutations is discussed.