Blood transcriptome analysis in a buck-ewe hybrid and its parents.

Examples of living sheep-goat hybrids are rare, mainly due to incorrect chromosome pairing, which is thought to be the main cause for species incompatibility. This case represents the first report of a buck-ewe hybrid and the first mammalian hybrid to be analyzed with next generation sequencing. The buck-ewe hybrid had an intermediate karyotype to the parental species, with 57 chromosomes. Analysis of the blood transcriptomes of the hybrid and both parents revealed that gene expression levels differed between the hybrid and its parents. This could be explained in part by age-dependent differences in gene expression. Contribution to the geep transcriptome was larger from the paternal, compared to the maternal, genome. Furthermore, imprinting patterns deviated considerably from what is known from other mammals. Potentially deleterious variants appeared to be compensated for by monoallelic expression of transcripts. Hence, the data imply that the buck-ewe hybrid compensated for the phylogenetic distance between the parental species by several mechanisms: adjustment of gene expression levels, adaptation to imprinting incompatibilities, and selective monoallelic expression of advantageous transcripts. This study offers a unique opportunity to gain insights into the transcriptome biology and regulation of a hybrid mammal.

Genetic diversity analysis of Thai indigenous pig population using microsatellite markers

Objective: European pigs have been imported to improve the economically important traits of Thai pigs by crossbreeding and was finally completely replaced. Currently Thai indigenous pigs are particularly kept in a small population. Therefore, indigenous pigs risk losing their genetic diversity and identity. Thus, this study was conducted to perform large-scale genetic diversity and phylogenetic analyses on the many pig breeds available in Thailand. Methods: Genetic diversity and phylogenetics analyses of 222 pigs belonging to Thai native pigs (TNP), Thai wild boars (TWB), European commercial pigs, commercial crossbred pigs, and Chinese indigenous pigs were investigated by genotyping using 26 microsatellite markers. Results: The results showed that Thai pig populations had a high genetic diversity with mean total (TNA) and effective (Ne) number of alleles of 14.59 and 3.71, respectively, and expected heterozygosity (He) across loci (0.710). The polymorphic information content (PIC) per locus ranged between 0.651 and 0.914 leading to an average value above all loci of 0.789, and private alleles were found in six populations. The higher He compared to Ho in TNP, TWB, and the commercial pigs indicated some inbreeding within a population. The Nei's genetic distance, mean FST estimates, neighbour-joining tree of populations and individual, as well as multidimensional analysis indicated close genetic relationship between Thai indigenous pigs and some Chinese pigs, and they are distinctly different from European pigs. Conclusion: Our study reveals a close genetic relationship between Thai native pigs and Chinese pigs. The genetic introgression from European breeds is found in some Thai native pig populations, and signs of genetic erosion are shown. Private alleles found in this study should be taken into consideration for the breeding program. The genetic information from this study will be a benefit for both conservation and utilization of Thai pig genetic resources.

mRNA expression profiling in cotyledons reveals significant up-regulation of the two bovine pregnancy-associated glycoprotein genes boPAG-8 and boPAG-11 in early gestation.

The multigene family of pregnancy-associated glycoproteins (PAGs) belongs to a group of aspartic proteases that are exclusively expressed by trophoblast cells in the placenta of even-toed ungulates. In Bovidae, 22 different PAG genes (boPAGs) with a wide range of temporal and spatial expression- and glycosylation patterns have been reported to date. In this study we describe the mRNA expression patterns using real-time quantitative PCR (qPCR) for selected modern (boPAG-1, -9, -21) and ancient bovine PAGs (boPAG-2, -8, -10, -11, -12) in cotyledonary tissue. The highest mean expression was detected in boPAG-8 and lowest in boPAG-10 (P < 0.05). Furthermore, boPAG-8 and -11 were significantly greater expressed in early gestation compared with later pregnancy stages. The characterization of boPAG mRNA-expression levels gives important insights for further protein analyses which will be valuable information for the development of new pregnancy detection systems.

ddRADseq reveals determinants for temperature-dependent sex reversal in Nile tilapia on LG23.

BACKGROUND: In Nile tilapia sex determination is governed by a male heterogametic system XX/XY either on LG1 or LG23. The latter carries a Y-specific duplicate of the amh gene, which is a testis-determining factor. Allelic variants in the amh gene demonstrated to be major triggers for autosomal and temperature-dependent sex reversal. Further, QTL on LG23 and LG20 show a temperature-responsiveness with influence on the phenotypic sex relative to the sex chromosomes. Here we present a ddRADseq based approach to identify genomic regions that show unusual large differentiation in terms of fixation index (FST) between temperature-treated pseudomales and non-masculinized females using a comparative genome-scan. Genome-wide associations were identified for the temperature-dependent sex using a genetically all-female population devoid of amh-ΔY. RESULTS: Twenty-two thousand three hundred ninety-two SNPs were interrogated for the comparison of temperature-treated pseudomales and females, which revealed the largest differentiation on LG23. Outlier FST-values (0.35-0.44) were determined for six SNPs in the genomic interval (9,190,077-11,065,693) harbouring the amh gene (9,602,693-9,605,808), exceeding the genome-wide low FST of 0.013. Association analysis with a set of 9104 selected SNPs confirmed that the same genomic region on LG23 exerts a significant effect on the temperature-dependent sex. CONCLUSIONS: This study highlights the role of LG23 in sex determination, harbouring major determinants for temperature-dependent sex reversal in Nile tilapia. Furthermore FST outlier detection proves a powerful tool for detection of sex-determining regions in fish genomes.

Validation of blood vitamin A concentrations in cattle: comparison of a new cow-side test (iCheck™ FLUORO) with high-performance liquid chromatography (HPLC).

BACKGROUND: Plasma concentration of retinol is an accepted indicator to assess the vitamin A (retinol) status in cattle. However, the determination of vitamin A requires a time consuming multi-step procedure, which needs specific equipment to perform extraction, centrifugation or saponification prior to high-performance liquid chromatography (HPLC). METHODS: The concentrations of retinol in whole blood (n = 10), plasma (n = 132) and serum (n = 61) were measured by a new rapid cow-side test (iCheck™ FLUORO) and compared with those by HPLC in two independent laboratories in Germany (DE) and Japan (JP). RESULTS: Retinol concentrations in plasma ranged from 0.033 to 0.532 mg/L, and in serum from 0.043 to 0.360 mg/L (HPLC method). No significant differences in retinol levels were observed between the new rapid cow-side test and HPLC performed in different laboratories (HPLC vs. iCheck™ FLUORO: 0.320 ± 0.047 mg/L vs. 0.333 ± 0.044 mg/L, and 0.240 ± 0.096 mg/L vs. 0.241 ± 0.069 mg/L, lab DE and lab JP, respectively). A similar comparability was observed when whole blood was used (HPLC vs. iCheck™ FLUORO: 0.353 ± 0.084 mg/L vs. 0.341 ± 0.064 mg/L). Results showed a good agreement between both methods based on correlation coefficients of r2 = 0.87 (P < 0.001) and Bland-Altman blots revealed no significant bias for all comparison. CONCLUSIONS: With the new rapid cow-side test (iCheck™ FLUORO) retinol concentrations in cattle can be reliably assessed within a few minutes and directly in the barn using even whole blood without the necessity of prior centrifugation. The ease of the application of the new rapid cow-side test and its portability can improve the diagnostic of vitamin A status and will help to control vitamin A supplementation in specific vitamin A feeding regimes such as used to optimize health status in calves or meat marbling in Japanese Black cattle.

Characterization of a very rare case of living ewe-buck hybrid using classical and molecular cytogenetics.

The natural occurrence of live hybrid offsprings between sheep and goats has been documented in literature, however all the studies have reported the mating of goats with rams, whereas the reciprocal cross was never documented. This study reports on a very rare case of interspecies hybridization occurred between a ewe (2n = 54, XX) and a buck (2n = 60, XY). The hybrid, born in a German flock under natural conditions, is characterised by an intermediate karyotype (2n = 57, XX). The CBA-banding has shown 3 metacentric and 54 acrocentric chromosomes, whereas the GTG- and RBA-banding have revealed that the autosomes involved in the hybrid combination were CHI1, 3; CHI2, 8 and CHI5, 11 corresponding to the metacentric chromosomes OAR1, OAR2 and OAR3. A tri-colour FISH using chromosome paintings and BAC probes has validated this arrangement. A further FISH analysis has been carried out to analyse the telomeres, which showed a normal structure. Nucleolus organiser-bearing chromosomes were identified as pairs OAR1p(CHI3), OAR2q(CHI2), OAR3q(CHI5), OAR4(CHI4) and OAR25(CHI28), and nuclear associations were found. Sex chromosomes were correctly arranged. The odd number of the karyotype might be responsible for a reduced fertility as consequence of the incorrect chromosomal pairing and/or segregation during the meiosis.

Porcine EEF1A1 and EEF1A2 genes: genomic structure, polymorphism, mapping and expression.

Eukaryotic translation elongation factor 1 alpha (EEF1A) plays a key role in protein synthesis. In higher vertebrates EEF1A occurs in two isoforms, EEF1A1 and EEF1A2, encoded by distinct genes. The purpose of this study was to compare the two porcine genes as for the genomic sequence, gene organization and mRNA expression in different tissues, as well as to search for polymorphism and chromosomal assignment. Standard methods of DNA and mRNA analysis were used. We determined the complete genomic sequence of the porcine EEF1A1 and EEF1A2 genes. The two genes differ in the lengths of transcription units (3102 and 8588 bp, respectively), but have similar genomic organization and their coding sequences are highly similar (78% identity of coding sequences and 92.4% identity of amino acid sequences). Several polymorphisms in the two genes were detected. EEF1A1 and EEF1A2 were mapped to SSC1p11.1 and SSC17q23.3, respectively. mRNA of EEF1A1 was expressed in all studied tissues (the highest expression was in 44-day fetal muscle and low expression in adult liver and brain), while EEF1A2 was expressed only in skeletal-muscle, tongue, heart, diaphragm and brain tissues. EEF1A2 was not expressed in fetal muscle tissue (44 days). In this paper results are provided on genomic sequences, genomic organization, polymorphism, chromosomal assignment and spatial and temporal expressions of the porcine EEF1A1 and EEF1A2 genes. Novel polymorphisms were described in both genes. Porcine EEF1A2 was studied for the first time.

Allelic variant in the anti-Müllerian hormone gene leads to autosomal and temperature-dependent sex reversal in a selected Nile tilapia line.

Owing to the demand for sustainable sex-control protocols in aquaculture, research in tilapia sex determination is gaining momentum. The mutual influence of environmental and genetic factors hampers disentangling the complex sex determination mechanism in Nile tilapia (Oreochromis niloticus). Previous linkage analyses have demonstrated quantitative trait loci for the phenotypic sex on linkage groups 1, 3, and 23. Quantitative trait loci for temperature-dependent sex reversal similarly reside on linkage group 23. The anti-Müllerian hormone gene (amh), located in this genomic region, is important for sexual fate in higher vertebrates, and shows sexually dimorphic expression in Nile tilapia. Therefore this study aimed at detecting allelic variants and marker-sex associations in the amh gene. Sequencing identified six allelic variants. A significant effect on the phenotypic sex for SNP ss831884014 (p<0.0017) was found by stepwise logistic regression. The remaining variants were not significantly associated. Functional annotation of SNP ss831884014 revealed a non-synonymous amino acid substitution in the amh protein. Consequently, a fluorescence resonance energy transfer (FRET) based genotyping assay was developed and validated with a representative sample of fish. A logistic linear model confirmed a highly significant effect of the treatment and genotype on the phenotypic sex, but not for the interaction term (treatment: p<0.0001; genotype: p<0.0025). An additive genetic model proved a linear allele substitution effect of 12% in individuals from controls and groups treated at high temperature, respectively. Moreover, the effect of the genotype on the male proportion was significantly higher in groups treated at high temperature, giving 31% more males on average of the three genotypes. In addition, the groups treated at high temperature showed a positive dominance deviation (+11.4% males). In summary, marker-assisted selection for amh variant ss831884014 seems to be highly beneficial to increase the male proportion in Nile tilapia, especially when applying temperature-induced sex reversal.

Learning to smell: repeated exposure increases sensitivity to androstenone, a major component of boar taint.

This study aimed to assess whether repeated exposure affects subjects' ability to detect androstenone. Olfactory acuity of 77 female and 44 male subjects (age 37.5 ± 11.7 years) was assessed three times during six weeks. Replicate triangle tests using various dilutions of androstenone (0.5, 5.0, and 50.0 µg/g) on filter paper smell strips were applied. Subjects were either assigned to a test group (TRAIN) using androstenone for daily training, or to a control group (CONTR) using a placebo. For the low, the intermediate, and the high level of androstenone presented, detection rate increased from 14.1 to 30.6%, 40.5 to 62.8%, and 65.3 to 78.5% respectively within 6 weeks from the initial assessment. Results suggest that mere exposure during repeated olfactory testing increased subjects' ability to correctly discriminate androstenone. The olfactory improvement was, however, more pronounced in the TRAIN group. Androstenone detection appears to be associated with its individual appreciation.

Consumer perception of boar meat as affected by labelling information, malodorous compounds and sensitivity to androstenone.

This study aimed to assess the influence of two label conditions on the acceptance of boar meat. A central location test was conducted with 145 consumers each assessing 4 pieces of pork loin. Samples varied with respect to two factors: actual meat type (boar vs. standard pork) and label information (young boar meat vs. pork). Androstenone and skatole levels in the tested boar meat ranged from 0.51 to 2.72 µg/g and 0.01 to 0.23 µg/g melted fat, respectively. Consumers' sensitivity to and appreciation of androstenone and skatole odour was determined through a smell experiment. The acceptance of taste, tenderness, juiciness, and overall liking was neither influenced by the label information nor by the meat type. Twenty-seven % of all participants were classified as insensitive to androstenone odour, whereas 52% perceived it as positive and 21% as negative. Consumers who disliked the androstenone odour indicated a higher disliking of boar meat.

A single nucleotide polymorphism in the CYP2E1 gene promoter affects skatole content in backfat of boars of two commercial Duroc-sired crossbred populations.

The prevention of unpleasant boar taint is the main reason for castration of male piglets. This study aimed to investigate how the malodorous compound skatole is affected by a single nucleotide polymorphism (g.2412 C>T at -586 ATG) in the porcine cytochrome p450 II E1 (CYP2E1) gene. 119 boars of two commercial Duroc-sired crossbred populations raised at different farms were investigated. Skatole and androstenone in backfat averaged 114±125 ng/g and 1206±895 ng/g melted fat, respectively. The frequency of the genotypes CC, CT, and TT was 25, 52, and 23%, respectively. CC boars had the highest average skatole levels (175 ng/g) compared to CT (92 ng/g) and TT (93 ng/g). Applying suggested sensory threshold levels for skatole (>150 ng/g) and androstenone (>2000 ng/g), 30% of the carcasses may be unacceptably tainted while the proportion of tainted carcasses is significantly higher within genotype CC (56.7%) compared to genotypes CT (24.3%) and TT (14.8%). Effective reduction of tainted carcasses appears feasible applying marker assisted selection.

Polymorphisms in the bovine HSP90AB1 gene are associated with heat tolerance in Thai indigenous cattle.

Heat shock proteins act as molecular chaperones that have preferentially been transcribed in response to severe perturbations of the cellular homeostasis such as heat stress. Here the traits respiration rate (RR), rectal temperature (RT), pack cell volume (PCV) and the individual heat tolerance coefficient (HTC) were recorded as physiological responses on heat stress (environmental temperatures) in Bos taurus (crossbred Holstein Friesian; HF) and B. indicus (Thai native cattle: White Lamphun; WL and Mountain cattle; MT) animals (n = 47) in Thailand. Polymorphisms of the heat shock protein 90-kDa beta gene (HSP90AB1) were evaluated by comparative sequencing. Nine single nucleotide polymorphisms (SNP) were identified, i.e. three in exons 10 and 11, five in introns 8, 9, 10 and 11, and one in the 3'UTR. The exon 11 SNP g.5082C>T led to a missense mutation (alanine to valine). During the period of extreme heat (in the afternoon) RR and RT were elevated in each of the three breeds, whereas the PCV decreased. Mountain cattle and White Lamphun heifers recorded significantly better physiologic parameters (p < 0.05) in all traits considered, including or particularly HTC than Holstein Friesian heifers. The association analysis revealed that the T allele at SNP g.4338T>C within intron 3 improved the heat tolerance (p < 0.05). Allele T was exclusively found in White Lamphun animals and to 84% in Mountain cattle. Holstein Friesian heifers revealed an allele frequency of only 18%. Polymorphisms within HSP90AB1 were not causative for the physiological responses; however, we propose that they should at least be used as genetic markers to select appropriate breeds for hot climates.

Genome-wide QTL mapping for three traits related to teat number in a White Duroc x Erhualian pig resource population.

BACKGROUND: Teat number is an important fertility trait for pig production, reflecting the mothering ability of sows. It is also a discrete and often canalized trait presenting bilateral symmetry with minor differences between the two sides, providing a potential power to evaluate fluctuating asymmetry and developmental instability. The knowledge of its genetic control is still limited. In this study, a genome-wide scan was performed with 183 microsatellites covering the pig genome to identify quantitative trait loci (QTL) for three traits related to teat number including the total teat number (TTN), the teat number at the left (LTN) and right (RTN) sides in a large scale White Duroc x Erhualian resource population. RESULTS: A sex-average linkage map with a total length of 2350.3 cM and an average marker interval of 12.84 cM was constructed. Eleven genome-wide significant QTL for TTN were detected on 8 autosomes including pig chromosomes (SSC) 1, 3, 4, 5, 6, 7, 8 and 12. Six suggestive QTL for this trait were detected on SSC6, 9, 13, 14 and 16. Eight chromosomal regions each on SSC1, 3, 4, 5, 6, 7, 8 and 12 showed significant associations with LTN. These regions were also evidenced as significant QTL for RTN except for those on SSC6 and SSC8. The most significant QTL for the 3 traits were all located on SSC7. Erhualian alleles at most of the identified QTL had positive additive effects except for three QTL on SSC1 and SSC7, at which White Duroc alleles increased teat numbers. On SSC1, 6, 9, 13 and 16, significant dominance effects were observed on TTN, and predominant imprinting effect on TTN was only detected on SSC12. CONCLUSION: The results not only confirmed the QTL regions from previous experiments, but also identified five new QTL for the total teat number in swine. Minor differences between the QTL regions responsible for LTN and RTN were validated. Further fine mapping should be focused on consistently identified regions with small confidence intervals, such as those on SSC1, SSC7 and SSC12.

Characterization of the porcine multicopy ribosomal protein SA/37-kDa laminin receptor gene family.

Prions represent a new class of infectious agents. The pathogenic prion protein (PrPSc) is known as the trigger of bovine transmissible spongiform encephalopathy (TSE). By contrast, an oral transmission of PrPSc and an ensuing infection seems to be blocked in non-ruminants such as pigs. Several investigations postulate that the ribosomal protein SA (RPSA) previously named 37-kDa laminin receptor precursor (LRP)/67-kDa laminin receptor (LR) is the candidate for binding and internalization of externally added cellular prion protein in the gut. We isolated a porcine ribosomal protein SA cDNA that consists of 1064 bp with an open reading frame of 885 bp encoding a 295 aa protein. The alignment of vertebrate ribosomal protein SA sequences displayed interspecies differences between cattle and pigs at positions 241 and 272 in the putative indirect PrP interaction site (aa 180-285) on RPSA. A PAC library screen revealed the existence of two processed ribosomal protein SA pseudogenes (RPSAP1 and RPSAP3) and of one non-processed pseudogene (RPSAP2). The pseudogenes have been assigned to SSC6 and SSC1 by hybrid panel analyses and FISH. Compared with the porcine cDNA 3, 7, and 13 insdels, 36, 25, and 57 single nucleotide exchanges and 6, 10, and 8 premature stop codons have been deciphered for RPSAP1, RPSAP2, and RPSAP3. In the 5', 3', and intron like regions, 2 (RPSAP1), 10 (RPSAP2), and 4 (RPSAP3) repeats have been detected. Basically, the repeats belong to one of the class/family LINE/L1, SINE/tRNA-Glu and DNA/MER1_type. We conclude that the pig genome contains multiple copies of the RPSA sequence probably as a consequence to maintain the multifunctionality of the mature protein.

Characterization of the PGK2 associated microsatellite S0719 on SSC7 suitable for parentage and QTL diagnosis.

We isolated and characterized the highly polymorphic tetra-nucleotide microsatellite S0719 on SSC7q14-q15 adjacent to the porcine testis-specific phosphoglycerate kinase 2 (PGK2) gene and assigned it to the USDA-MARC linkage map on SSC7 position 77.5 cM closely linked to markers SW859 (76.3 cM) and SWR2036 (79.0 cM). In a panel of 344 individuals representing 11 pig breeds (European, Chinese, and North American), a total of 32 alleles were observed, and the overall breeds' calculated PIC (polymorphism information content), HE (heterozygosity), and NE (effective allele number) were 0.94, 0.94, and 16.41. Breed-specific PIC and HE ranged from 0.66 to 0.87, whereas NE was as low as 2.95 and as high as 7.96. Considering the high allelic variation of S0719 within and among pig breeds (79% of the genotyped animals were heterozygous), the marker is useful for individual animal identification and parentage determination. Finally, S0719 is also a valuable STS marker for fine-mapping QTL on SSC7 as position 77.5 cM is located in 25 QTL intervals.

Molecular characterization and exclusion of porcine GUSB as a candidate gene for congenital hernia inguinalis/scrotalis.

BACKGROUND: Inguinal hernias are usually caused by a congenital defect, which occurs as a weakness of the inguinal canal. Porcine beta-glucuronidase gene (GUSB) was chosen as functional candidate gene because of its involvement in degradation of hyaluronan within gubernacular tissue during descent of testes. Since a genome-wide linkage analysis approach has shown evidence that two regions on porcine chromosome 3 (SSC 3) are involved in the inheritance of hernia inguinalis/scrotalis in German pig breeds, GUSB also attained status as a positional candidate gene by its localization within a hernia-associated chromosomal region. RESULTS: A contig spanning 17,157 bp, which contains the entire GUSB, was assembled. Comparative sequence analyses were conducted for the GUSB gene locus. Single nucleotide polymorphisms (SNPs) located within the coding region of GUSB were genotyped in 512 animals. Results of transmission disequilibrium test (TDT) for two out of a total of five detected SNPs gave no significant association with the outcome of hernia in pigs. CONCLUSION: On the basis of our studies we are able to exclude the two analyzed SNPs within the porcine GUSB gene as causative for the transmission of inguinal hernia.

Targeted oligonucleotide-mediated microsatellite identification (TOMMI) from large-insert library clones.

BACKGROUND: In the last few years, microsatellites have become the most popular molecular marker system and have intensively been applied in genome mapping, biodiversity and phylogeny studies of livestock. Compared to single nucleotide polymorphism (SNP) as another popular marker system, microsatellites reveal obvious advantages. They are multi-allelic, possibly more polymorphic and cheaper to genotype. Calculations showed that a multi-allelic marker system always has more power to detect Linkage Disequilibrium (LD) than does a di-allelic marker system. Traditional isolation methods using partial genomic libraries are time-consuming and cost-intensive. In order to directly generate microsatellites from large-insert libraries a sequencing approach with repeat-containing oligonucleotides is introduced. RESULTS: Seventeen porcine microsatellite markers were isolated from eleven PAC clones by targeted oligonucleotide-mediated microsatellite identification (TOMMI), an improved efficient and rapid flanking sequence-based approach for the isolation of STS-markers. With the application of TOMMI, an average of 1.55 (CA/GT) microsatellites per PAC clone was identified. The number of alleles, allele size distribution, polymorphism information content (PIC), average heterozygosity (HT), and effective allele number (NE) for the STS-markers were calculated using a sampling of 336 unrelated animals representing fifteen pig breeds (nine European and six Chinese breeds). Sixteen of the microsatellite markers proved to be polymorphic (2 to 22 alleles) in this heterogeneous sampling. Most of the publicly available (porcine) microsatellite amplicons range from approximately 80 bp to 200 bp. Here, we attempted to utilize as much sequence information as possible to develop STS-markers with larger amplicons. Indeed, fourteen of the seventeen STS-marker amplicons have minimal allele sizes of at least 200 bp. Thus, most of the generated STS-markers can easily be integrated into multilocus assays covering a broader separation spectrum. Linkage mapping results of the markers indicate their potential immediate use in QTL studies to further dissect trait associated chromosomal regions. CONCLUSION: The sequencing strategy described in this study provides a targeted, inexpensive and fast method to develop microsatellites from large-insert libraries. It is well suited to generate polymorphic markers for selected chromosomal regions, contigs of overlapping clones and yields sufficient high quality sequence data to develop amplicons greater than 250 bases.

Molecular characterization of the porcine testis-specific phosphoglycerate kinase 2 (PGK2) gene and its association with male fertility.

We have isolated and characterized the porcine testis-specific phosphoglycerate kinase 2 (PGK2) gene, and 1665 bp of full-length PGK2 cDNA were also compiled using modified rapid amplification 5'-RACE and 3'-RACE information. The results of genomic and cDNA sequences of the porcine PGK2 gene demonstrated that it is a single-exon intronless gene with a complete open reading frame of 1251 bp encoding a PGK protein of 417 amino acids. Real-time quantitative PCR results showed that PGK2 mRNA was solely expressed in the testis. There was a lower amount of PGK2 expression in the testis of a 10-month-old herniated boar and a very small amount of PGK2 expression in the testis of an 8-week-old cryptorchid piglet compared to an adult boar. Two SNPs in the PGK2 gene (SNP-A: T427C; SNP-B: C914A) resulting in amino acid substitutions (SNP-A: Ser102-Pro102; SNP-B: Thr264-Lys264) were detected and genotyped among six pig breeds. The nucleotide C at SNP-A responsible for the amino acid exchange to proline could lead to the loss of a casein kinase II (CK2) phosphorylation site in the PGK2 peptide. Association analyses between PGK2 genotypes and several traits of sperm quantity and quality were performed. The results showed that SNP-B has a positive significant effect on semen volume in the breed Pietrain (p = 0.08), i.e., boars carrying genotype CC revealed an increased volume of 49 ml compared with boars having the genotype AA.

Isolation and molecular characterization of the porcine stearoyl-CoA desaturase gene.

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme in the biosynthesis of unsaturated fatty acids. So far only a partial porcine SCD sequence is available. Here we described the isolation and molecular characterization of the full-length cDNA and the determination of the genomic DNA sequence of the porcine SCD gene. The 5134-bp cDNA contains a 1080-bp open reading frame (ORF) encoding a protein of 359 amino acids with a calculated molecular mass of 41.3 kDa and a theoretical isoelectric point of 9.4. The porcine SCD protein shares high identity (>80%) with the other mammalian SCD. To further elucidate the genomic structure of the porcine SCD gene, we sequenced 20,985 bp of genomic DNA sequence encompassing the complete pig SCD gene. Similar to the other mammalian orthologs, particularly in term of exon size and exon/intron boundaries, the porcine SCD gene spans a transcription unit of 16,186 bp, consisting of six exons with sizes ranging from 131 to 4048 bp, and five introns varying in size from 518 to 4784 bp. The gene reveals a 176-bp-long 5' UTR and possesses an unusually long 3'UTR of 3848 bp in the last exon. Comparison of different mammalian SCD promoters identified some regulatory domains required for the transcription regulation in the 5' flanking sequence of the porcine SCD gene, such as the conserved polyunsaturated fatty acid response region (PUFA-RE). A total of 21 gene polymorphisms were revealed in the 21-kb DNA sequence, including 19 single nucleotide polymorphisms (SNPs), a 24-bp-long fragment length polymorphism in the fourth intron and a triplet nucleotide insertion in the fifth intron. Reverse transcription (RT)-PCR result indicates that the SCD gene is expressed ubiquitously in pigs.

Structural and expression analysis of the porcine FUS2 gene.

The putative porcine tumor suppressor gene FUS2 or N-acetyltransferase (Nat6) assigned to SSC13q21 spans 864-basepairs (bp) of genomic DNA, consisting of a single exon encoding a protein of 288 amino acids (aa), with 73% identity to the human and 74% to the mouse protein. Similar to man and mouse, the gene possesses an N-acetyltransferase domain, but the cell attachment motif arginine-glycine-aspartate (RGD) is exclusively found in the pig gene. Expression studies of the gene in several organs by RT-amplification and by quantitative polymerase chain reaction (Q-PCR) showed that FUS2 is widely expressed in porcine tissues. A point mutation was detected at position 836 of the coding sequence (G to A) leading to an amino acid substitution from cystein (C) to tyrosine (Y) at position 278 of the protein. Genes of the tumor suppressor gene (TSG) cluster act together to suppress tumor growth through their functional activation of tumor suppressing pathways. Studies in humans have proven that mutations in N-acetyltransferase genes are associated with some kind of cancers. Knowledge of structure and function of the respective porcine genes and proteins is important. Pigs-in particular minipigs-will be the non-rodent biomodels for human oncology and cancer therapy in the future.