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With the increasing accessibility of cannabis ( Cannabis sativa L., also known as marijuana and hemp), its products are being developed as extracts for both recreational and therapeutic use. This has led to increased scrutiny by regulatory bodies, who aim to understand and regulate the complex chemistry of these products to ensure their safety and efficacy. Regulators use targeted analyses to track the concentration of key bioactive metabolites and potentially harmful contaminants, such as metals and other impurities. However, the metabolic complexity of cannabis metabolic pathways requires a more comprehensive approach. A non-targeted metabolomic analysis of cannabis products is necessary to generate data that can be used to determine their authenticity and efficacy. An authentomics approach, which involves combining the non-targeted analysis of new samples with big data comparisons to authenticated historic datasets, provides a robust method for verifying the quality of cannabis products. To meet International Organization for Standardization (ISO) standards, it is necessary to implement the authentomics platform technology and build an integrated database of cannabis analytical results. This study is the first to review the topic of the authentomics of cannabis and its potential to meet ISO standards.

Objective: Study on the application of high pressure processing (HPP) for dark-firm-dry (DFD) beef was conducted to observe whether HPP has any impact on physical properties and to evaluate oxidative deterioration during refrigerated storage under vacuum., Methods: The longissimus lumborum muscles obtained from Friesian Holstein steers (33±0.5 months old) with 24-h postmortem pH higher than 6.0 were vacuum-packed and subjected to pressurization at 200, 400, and 600 MPa for 180 s at 15°C±2°C; the samples were then stored for 9 days at 4°C±1°C and compared with control (0.1 MPa)., Results: HPP increased meat pH by 0.1 to 0.2 units and the tenderness of cooked DFD beef significantly with no significant effects on meat texture profile. The stability of meat pH was well maintained during refrigerated storage under vacuum. No clear effects were found on the activity of catalase and superoxide dismutase, however, glutathione peroxidase activity was significantly reduced by high pressure. HPP and storage time resulted in aroma changes and the increasing amount of malondialdehyde and metmyoglobin relative composition., Conclusion: Although the increasing amount of malondialdehyde content, metmyoglobin formation and aroma changes in HPP-treated samples could not be avoided, HPP at 200 MPa increased L* and a* values with less discoloration and oxidative deterioration during storage.

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.

Haematological traits play important roles in disease resistance and defence functions. The objective of this study was to locate quantitative trait loci (QTL) and the associated positional candidate genes influencing haematological traits in an F(2) intercross between Landrace and Korean native pigs. Eight blood-related traits (six erythrocyte traits, one leucocyte trait and one platelet trait) were measured in 816 F(2) progeny. All experimental animals were genotyped with 173 informative microsatellite markers located throughout the pig genome. We report that nine chromosomes harboured QTL for the baseline blood parameters: genomic regions on SSC 1, 4, 5, 6, 8, 9, 11, 13 and 17. Eight of twenty identified QTL reached genome-wide significance. In addition, we evaluated the KIT locus, an obvious candidate gene locus affecting variation in blood-related traits. Using dense single nucleotide polymorphism marker data on SSC 8 and the marker-assisted association test, the strong association of the KIT locus with blood phenotypes was confirmed. In conclusion, our study identified both previously reported and novel QTL affecting baseline haematological parameters in pigs. Additionally, the positional candidate genes identified here could play an important role in elucidating the genetic architecture of haematological phenotype variation in swine and in humans., (© 2011 The Authors, Animal Genetics © 2011 Stichting International Foundation for Animal Genetics.)

The genetic consequences of population differentiation and isolation have been the subject of conservation biology. In this study, we analysed the genetic diversity and structure of Mongolian sheep in China. These animals belong to a traditional local breed with high production, extensive adaption, early maturity and roughage resistance. For this purpose, 26 microsatellites were genotyped for five Mongolian sheep populations. The Bayesian clustering indicated five clusters as the most probable genetic structure of the populations investigated. In addition, a clear genetic structure was revealed in three populations distributed at large geographical scales, while the other cluster encompassed UQ and HLBR sheep that displayed no clear differentiation, probably due to their close and small geographical distributions. Overall, our results are helpful in understanding the interplay of population dynamics in these close genetic lineages of Mongolian sheep., (© 2011 The Authors, Animal Genetics © 2011 Stichting International Foundation for Animal Genetics.)

The KIT locus has been suggested to be a strong candidate region linked with whole-body roan in the F(2) population produced by intercrosses between Landrace and Korean Native pigs. In this manuscript, we report the finding of a novel alternative splicing event in the porcine KIT gene that results in the skipping of exon 5 in the I(Rn) allele. KIT mRNAs that lack exon 5 were identified in the large intestine and skin, suggesting that the mechanism responsible for the skipping of exon 5 may be tissue specific. A U(26) repeat in intron 5 showed complete linkage (LOD = 11.8) with the roan phenotype and absolute association with the black phenotype of the Korean Native pig (KNP) population samples, inferring that the repeat pattern may alter the complementary base-pairing-mediated looping-out of introns 4 and 5, which may mediate the exon 5-skipping event. Although the sample size in our study was relatively small, we speculate that the R3 allele containing the U(26) repeat is a causative element for the roan phenotype via alternative control of the exon skipping in our roan pedigree., (© 2011 The Authors, Animal Genetics © 2011 Stichting International Foundation for Animal Genetics.)

Obesity represents a major global public health problem that increases the risk for cardiovascular or metabolic disease. The pigs represent an exceptional biomedical model related to energy metabolism and obesity in humans. To pinpoint causal genetic factors for a common form of obesity, we conducted local genomic de novo sequencing, 18.2 Mb, of a porcine QTL region affecting fatness traits, and carried out SNP association studies for backfat thickness and intramuscular fat content in pigs. In order to relate the association studies in pigs to human obesity, we performed a targeted genome wide association study for subcutaneous fat thickness in a cohort population of 8,842 Korean individuals. These combined association studies in human and pig revealed a significant SNP located in a gene family with sequence similarity 73, member A (FAM73A) associated with subscapular skin-fold thickness in humans (rs4121165, GC-corrected p-value  = 0.0000175) and with backfat thickness in pigs (ASGA0029495, p-value  = 0.000031). Our combined association studies also suggest that eight neuronal genes are responsible for subcutaneous fat thickness: NEGR1, SLC44A5, PDE4B, LPHN2, ELTD1, ST6GALNAC3, ST6GALNAC5, and TTLL7. These results provide strong support for a major involvement of the CNS in the genetic predisposition to a common form of obesity.

The highly polymorphic porcine major histocompatibility complex (MHC), or the swine leukocyte antigens (SLA), has been repeatedly associated with variations in swine immune response to pathogens and vaccines as well as with production traits. The SLA antigens are also important targets for immunological recognition of foreign tissue grafts. We recently established a resource population of Korean native pigs as models for human transplantation and xenotransplantation research. In this study, 115 animals derived from three generations of the Korean native pigs were genotyped for three SLA class I (SLA-2, SLA-3 and SLA-1) and three SLA class II loci (DRB1, DQB1, DQA) using PCR with sequence-specific primers (PCR-SSP) at the allele group resolution. A total of seven SLA haplotypes (Lr-5.34, Lr-7.23, Lr-31.13, Lr-56.23, Lr-56.30, Lr-59.1, Lr-65.34), comprising six unique class I and five unique class II haplotypes, were characterized in the founding animals. Class I haplotype Lr-65.0 and class II haplotype Lr-0.34 were novel; and together with Lr-56.0 these haplotypes appeared to be breed-specific. In the progeny population, Lr-7.23 and Lr-56.30 appeared to be the most prevalent haplotypes with frequencies of 34.7% and 31.6%, respectively; the overall homozygosity was 27.4%. This resource population of SLA-defined Korean native pigs will be useful as large animal models for various transplantation and xenotransplantation experiments, as well as for dissecting the roles of SLA proteins in swine disease resistance and production traits.

Somatic cells from a first-generation red fluorescence protein transgenic cat (first RFP TG cat) were used to produce a recloned RFP transgenic cat (Re-RFP TG cat) (Felis catus) that systemically expressed RFP. A total of 281 RFP cloned embryos were transferred into 13 surrogate mothers (mean=21+/-7.7 embryos/recipient). One surrogate cat was diagnosed pregnant (7.7%) and delivered one live kitten. The presence of the RFP gene in the mRNA and genomic DNA of the Re-RFP TG cat was confirmed by polymerase chain reaction analyses, and red fluorescence was detected in its internal organs and placental tissue samples. Analysis of nine feline-specific microsatellite loci confirmed that the Re-RFP TG cat was genetically identical to the donor cat. To test whether results such as normality of offspring and a low cloning success were due to epigenetic modifications, global methylation of placenta from the two first cloned RFP TG cats (77.08% and 82.29%) and the Re-RFP TG cat (76.38%) were compared by bisulfite mutagenesis sequencing analysis. In conclusion, although cloning efficiency was low, we demonstrated the successful use of a cloned first RFP TG cat as a donor cat to produce a Re-RFP TG cat. These results may facilitate future developments in biomedical models for human therapeutic applications., (Copyright 2010 Elsevier Inc. All rights reserved.)

We investigated the sperm characteristics of four cloned male cats (Felis catus) to assess their reproductive potential. Fresh and frozen-thawed sperm were assessed for motility, viability, and morphology, and their functional competence was evaluated by in vitro fertilization (IVF) of domestic cat oocytes. All fresh semen characteristics varied among cats and collection times. Sperm concentration (x 10(6)/mL) of Cat A (512+/-140, range 368 to 685) was significantly higher, whereas that of Cat C (335+/-92, range 274 to 469) was significantly lower than that of Cloned B (459+/-159, range 336 to 510) and control cats (680+/-452, range 360 to 479). After thawing, motility and progressive motility of sperm from Cat B were significantly lower than that of the other cloned and control cats. The curvilinear, straight line, and average path velocities of sperm from Cat B were significantly higher, whereas the straightness was lower, than that of the other cloned and control cats. Frozen sperm from Cats A, B, and C successfully fertilized oocytes (cleavage=74.4%, 71.4%, and 86.2%, respectively) and produced embryos that developed to the blastocyst stage after IVF/In vitro culture (IVC) (34.4%, 26.7%, and 48.0%) at frequencies similar to the cleavage rate (82.0%) and blastocyst rate (43.9%) obtained with sperm from the control male. In conclusion, seminal characteristics of cloned male cats did not differ markedly from those of our noncloned, control male cats., (Copyright 2010 Elsevier Inc. All rights reserved.)

Xenotransplantation from pigs provides a possible solution to the shortage of human organs for allotransplantation. Porcine endogenous retroviruses (PERVs) are a possible obstacle to using porcine organs in addition to the immunological barriers. Three main types of PERVs (A, B and C) have been previously investigated in diverse pig breeds. To examine the copy numbers of PERVs and their genomic locations in the Korean native pig genome, we screened a BAC (Bacterial Artificial Chromosome) library with PERV-specific protease primers for initial recognition of PERV-positive clones and three sets of envelope-specific primers for the identification of PERV types. A total of 45 PERV-positive clones, nine PERV-A and 36 PERV-B, have been identified from the library screening and the BAC contigs were constructed using the primers designed from BAC end sequences (BESs). These primers were also used for SCH (Somatic Cell Hybrid) and RH (Radiation Hybrid) mapping of the PERV-positive clones. The results indicate that 45 PERV-positive BAC clones belong to nine contigs and a singleton. SCH and IMpRH (INRA-Minnesota Porcine Radiation Hybrid) mapping results indicated that there are at least eight separate PERV genomic locations, consisting of three PERV-A and five PERV-B. One contig could not be mapped, and two contigs are closely located on SSC7. Southern blotting indicates there may be up to 15 additional sites. Further investigation of these clones will contribute to a general strategy to generate PERV-free lines of pigs suitable for xenotransplantation.

In order to elucidate the precise phylogenetic relationships of Korean wild boar (Sus scrofa coreanus), a partial mtDNA D-loop region (1,274 bp, NC_000845 nucleotide positions 16576-1236) was sequenced among 56 Korean wild boars. In total, 25 haplotypes were identified and classified into four distinct subgroups (K1 to K4) based on Bayesian phylogenetic analysis using Markov chain Monte Carlo methods. An extended analysis, adding 139 wild boars sampled worldwide, confirmed that Korean wild boars clearly belong to the Asian wild boar cluster. Unexpectedly, the Myanmarese/Thai wild boar population was detected on the same branch as Korean wild boar subgroups K3 and K4. A parsimonious median-joining network analysis including all Asian wild boar haplotypes again revealed four maternal lineages of Korean wild boars, which corresponded to the four Korean wild boar subgroups identified previously. In an additional analysis, we supplemented the Asian wild boar network with 34 Korean and Chinese domestic pig haplotypes. We found only one haplotype, C31, that was shared by Chinese wild, Chinese domestic and Korean domestic pigs. In contrast to our expectation that Korean wild boars contributed to the gene pool of Korean native pigs, these data clearly suggest that Korean native pigs would be introduced from China after domestication from Chinese wild boars.

POU5F1 and NANOG play important roles in the maintenance of embryonic stem cell pluripotency. Recently, we isolated cat embryonic stem (ES)-like cells from cat blastocysts generated in vivo. In an effort to identify genetic markers for the characterization of cat ES-like cells, we have determined the coding sequences (CDSs) of cat POU5F1 (cPOU5F1) and NANOG (cNANOG). The sequence identities of cPOU5F1 with orthologous genes of the human and mouse were 92 and 82%, respectively, at the nucleotide level and 94 and 83%, respectively, at the amino acid level. We identified POU-specific and POU homeodomain sequences in the CDS of cPOU5F1. The sequence identities of cNANOG with its human and mouse orthologs were 69 and 68%, respectively, at the nucleotide level and 69 and 58%, respectively, at the amino acid level. We identified a homeodomain, SMAD4 domain and tryptophan repeat domain (W/QXXXX) in the CDS of cNANOG. We examined the expression of cPOU5F1 and cNANOG mRNA in ES-like cells and fibroblast feeder cells by RT-PCR. Transcripts of cPOU5F1 and cNANOG were detected at a high level in ES-like cells. However, these two genes were undetectable in cat fibroblast feeder cells and 6 adult tissues. We also examined ES-like cells by immunocytochemistry and demonstrated that cPOU5F1 and cNANOG are present at high levels in cat ES-like cells and are undetectable in cat fibroblast feeder cells. These results confirmed that cat ES-like cells can be successfully isolated from in vivo-produced blastocysts and that the expression of cPOU5F1 and cNANOG can be used as a biomarker for characterization of cat ES-like cells.

Mesenchymal stem cells (MSCs) are pluripotent adult stem cells. It has been shown that MSCs secrete neurotrophic factors involving nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Also, these neurotrophic factors can upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells and neural stem cells. Here, we investigated the effect of co-culturing rat E13.5 ventral mesencephalic cells (VMCs) with MSCs from rat bone marrow on TH expression and dopamine (DA) content. The study consisted of 3 groups: MSC, VMC and a combined MSC+VMC group. All groups were cultured in serum-free neuro-basal medium for 3 days. Thereafter, each group was analyzed by RT-PCR, western blotting, and HPLC. The co-culture group showed a higher expression at TH and DA than the VMC group. However, TH and DA were not present in the MSC group. These observations suggest that MSCs could be an alternative source for treating neurodegenerative diseases such as Parkinson's disease (PD).

Eight swine leucocyte antigen (SLA) gene (SLA-1, SLA-2, SLA-3, SLA-6, DRA, DRB1, DQA, DQB1) alleles were identified using sequence-based typing method in three Korean native pigs used for breeding at the National Institute of Animal Science in Korea. Six new alleles in class I genes and three new alleles in class II genes have been identified in this breed and can give valuable information for xenotransplantation and disease resistance.

Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells, which can differentiation into cells of connective tissue and neural lineages. This study investigated the potential for neuronal differentiation of red fluorescent protein (RFP)-transgenic cat UCB-derived MSCs. The cells were cultured in pre-induction medium for 24 hr and in neuronal-induction medium for 72 hr. Immunofluorescent staining showed that 6.85% of the total cells were beta III-tubulin-positive, 3.37% were neurofilament light (NF-L)-positive and 7.04% were neurofilament medium (NF-M)-positive. A beta III-tubulin band was detected by western blot analysis. Our results demonstrate that RFP-transgenic UCB-derived MSCs can be differentiated into neuronal cells in vitro. Thus, RFP-transgenic MSCs could provide alternative tracing material for stem cell transplantation.

Three isoforms of pig PDE4B were cloned and classified as two forms: PDE4B1 and PDE4B3, which contain UCR1 and UCR2; and PDE4B2, which contains only UCR2. The amino acid sequences of each isoform showed good conservation in human and rat. PDE4B2 is expressed in a wide range of tissues, but PDE4B1 and PDE4B3 are not. Using an informative SNP for the Iberian x Landrace intercross detected from intron 12, a linkage map was constructed. The location of PDE4B was estimated at 123.6 cM outside of the QTL-CI (124-128 cM) for IMF. However, the QTL-CI for IMF was reconfirmed with high significance, and its position was narrowed down to an interval of 4 cM (the region defined by markers PDE4B and SW1881). Using radiation hybrid mapping, LEPR, LEPROT, DNAJC6, AK3L1 and AK3L2 were selected as positional and/or functional candidates related to the QTL.

Copy number variations (CNVs) have effects on phenotypes by altering transcription levels of genes and may have major impacts on protein sequence, structure and function. Therefore, CNV screening and analysis focused on the identification of CNV-genetic disease relations are actively progressing. CNVs can be detected and analyzed by various methodologies at the genome-wide and locus-specific levels. The genome-wide analysis of CNVs has been enhanced by bioinformatic tools for long-range sequence analysis, and comparative genome hybridization using microarrays containing either single nucleotide polymorphisms or bacterial artificial chromosome clones that represent the whole genome. RFLP followed by Southern blot analysis, quantitative real-time PCR, pyrosequencing, ligation detection reaction and the invader assay have become the main tools for locus-specific analysis so far. In this review, we present a brief principle, application history, and strengths and weaknesses of the methods used to detect CNVs at the genome-wide and locus-specific levels., (Copyright 2009 S. Karger AG, Basel.)

Background: Aside from single nucleotide polymorphisms, copy number variations (CNVs) are the most important factors in susceptibility to genetic disorders because they affect expression levels of genes. In previous studies, pyrosequencing, mini-sequencing, real-time PCR, invader assays and other techniques have been used to detect CNVs. However, the higher the copy number in a genome, the more difficult it is to resolve the copies, so a more accurate method for measuring CNVs and assigning genotype is needed., Results: PCR followed by a quantitative oligonucleotide ligation assay (qOLA) was developed for quantifying CNVs. The accuracy and precision of the assay were evaluated for porcine KIT, which was selected as a model locus. Overall, the root mean squares of bias and standard deviation of qOLA were 2.09 and 0.45, respectively. These values are less than half of those in the published pyrosequencing assay for analyzing CNV in porcine KIT. Using a combined method of qOLA and another pyrosequencing for quantitative analysis of KIT copies with spliced forms, we confirmed the segregation of KIT alleles in 145 F1 animals with pedigree information and verified the correct assignment of genotypes. In a diagnostic test on 100 randomly sampled commercial pigs, there was perfect agreement between the genotypes obtained by grouping observations on a scatter plot and by clustering using the nearest centroid sorting method implemented in PROC FASTCLUS of the SAS package. In a test on 159 Large White pigs, there were only two discrepancies between genotypes assigned by the two clustering methods (98.7% agreement), confirming that the quantitative ligation assay established here makes genotyping possible through the accurate measurement of high KIT copy numbers (>4 per diploid genome). Moreover, the assay is sensitive enough for use on DNA from hair follicles, indicating that DNA from various sources could be used., Conclusion: We have established a high resolution quantification method using an oligonucleotide ligation assay to measure CNVs, and verified the reliability of genotype assignment for random animal samples using the nearest centroid sorting method. This new method will make it more practical to determine KIT CNV and to genotype the complicated Dominant White/KIT locus in pigs. This procedure could have wide applications for studying gene or segment CNVs in other species.

A method of reliably producing developmentally competent cat embryos in vitro is a prerequisite for study of the physiology of early development and application of assisted reproductive techniques. Oocytes were collected and then cultured in TCM-199 + 10% FBS for 4 h. The matured oocytes were activated with a 20 microsec electric pulse at 1.2 kV/mm. The activated oocytes were incubated in 2 mM of 6-dimethylaminopurine (6-DMAP) for 4 h and were then divided randomly among the treatment groups. In experiment 1, we compared the effects of three culture systems (TCM-199, CR1-aa and Tyrode's) on the in vitro development of parthenogenetically activated cat oocytes. In experiment 2, we investigated the effect of addition of Iloprost (a stable prostaglandin I(2) analogue) to Tyrode's medium on in vitro development of parthenogenetically activated oocytes. As a control, we recovered in vivo produced blastocysts and determined their average cell number. In experiment 1, the cleavage frequency of the oocytes cultured in TCM199, CR1-aa and Tyrode's media were similar (74, 72 and 83%, respectively). However, the incidence of in vitro development to the blastocyst stage was significantly higher in Tyrode's medium (20.4%) than in TCM-199 (2.4%) or CR1-aa (11.1%). Likewise, the average cell number of in vitro activated blastocysts was higher in Tyrode's than in CR1-aa or TCM-199 (106.5 +/- 45.2 vs. 68.3 +/- 25.4 and 35.0 +/- 7.7, respectively; P<0.05). In experiment 2, the percentage of parthenogenetically activated oocytes that underwent in vitro blastocyst development was significantly improved by addition of Iloprost to the culture medium (33.6 vs. 19.1%; P<0.05). The average cell number of in vivo blastocysts (909.0 +/- 226.4) was significantly higher than those of in vitro blastocysts cultured in Tyrode's medium supplemented with or without Iloprost (103.2 +/- 31.3 and 112.2 +/- 39.3, respectively; P<0.05). This result indicated that the current culture method for cat pathogenetically activated oocytes requires further improvement.

Marbling of cattle meat is dependent on the coordinated expression of multiple genes. Cattle dramatically increase their intramuscular fat content in the longissimus dorsi muscle between 12 and 27 months of age. We used the annealing control primer (ACP)-differential display RT-PCR method to identify differentially expressed genes (DEGs) that may participate in the development of intramuscular fat between early (12 months old) and late fattening stages (27 months old). Using 20 arbitrary ACP primers, we identified and sequenced 14 DEGs. BLAST searches revealed that expression of the MDH, PI4-K, ferritin, ICER, NID-2, WDNMI, telethonin, filamin, and desmin (DES) genes increased while that of GAPD, COP VII, ACTA1, CamK II, and nebulin decreased during the late fattening stage. The results of functional categorization using the Gene Ontology database for 14 known genes indicated that MDH, GAPD, and COP VII are involved in metabolic pathways such as glycolysis and the TCA cycle, whereas telethonin, filamin, nebulin, desmin, and ACTA1 contribute to the muscle contractile apparatus, and PI4-K, CamK II, and ICER have roles in signal transduction pathways regulated by growth factor or hormones. The final three genes, NID-2, WDNMI, and ferritin, are involved in iron transport and extracellular protein inhibition. The expression patterns were confirmed for seven genes (MDH, PI4-K, ferritin, ICER, nebulin, WDNMI, and telethonin) using real-time PCR. We found that the novel transcription repressor ICER gene was highly expressed in the late fattening stage and during bovine preadipocyte differentiation. This information may be helpful in selecting candidate genes that participate in intramuscular fat development in cattle.

Low fat sausages were prepared with added hydrated oatmeal or tofu as texture-modifying agents at levels of 10%, 15%, and 25% (w/w), respectively. The effects of the type and level of texture-modifying agents on the physical and sensory properties of low fat sausages were investigated. The water-holding capacity in sausage products increased by increasing the hydrated oatmeal level, but no significant differences was observed by the addition of tofu. The higher level of the agents produced a sausage product with less cooking loss and with a softer texture. The moisture absorption measurements suggest that the decrease in hardness of oatmeal-added sausage products may be due to the higher water-retention properties of oatmeal in response to heat treatment, while that of tofu-added sausage products may be associated with a weaker internal structure of tofu than the pork loin. The sensory evaluations indicated that the greatest overall acceptability in a low fat sausage was attained when the hydrated oatmeal or tofu were at their 15% addition level, respectively.

Ancient cattle bones were excavated from archaeological sites in Jeju, Korea. We used molecular genetic techniques to identify the species and establish its relationship to extant cattle breeds. Ancient DNA was extracted from four sources: a humerus (Gonae site, A.D. 700-800), two fragments of radius, and a tooth (Kwakji site, A.D. 0-900). The mitochondrial DNA (mtDNA) D-loop regions were cloned, sequenced, and compared with previously reported sequences of various cattle breeds (9 Asian, 8 European, and 3 African). The results revealed that these bones were of the breed, Bos taurus, and a phylogenetic tree indicated that the four cattle bones formed a monophyletic group with Jeju native black cattle. However, the patterns of sequence variation and reports from archaeological sites suggest that a few wild cattle, with a different maternal lineage, may have existed on Jeju Island. Our results will contribute to further studies of the origin of Jeju native cattle and the possible existence of local wild cattle.

A porcine genomic bacterial artificial chromosome (BAC) library was constructed by cloning partial EcoRI-digested high-molecular-weight DNA from a Korean native boar into the EcoRI site of the pBACe3.6 vector. The library consists of about 165,000 clones with an average insert size of 125 kb, representing about seven genome equivalents of coverage. About 130,000 clones (corresponding to fivefold genome coverage) were arrayed in 14 superpools which were organized as four dimensional pools. The library was further characterized by PCR screening of 38 microsatellite probes. An average of 4.84 positive clones were selected per marker. This indicates that the library is unbiased and will be useful for initiating fine scale physical mapping of major QTL in pigs. The library is being used to isolate specific clones by screening with type I and type II marker clones located in the QTL region affecting intramuscular fat content on SSC6.

The PRKAG3 gene encodes a muscle-specific isoform of the regulatory gamma subunit of AMP-activated protein kinase (AMPK). A major part of the coding PRKAG3 sequence was isolated from horse muscle cDNA using reverse-transcriptase (RT)-PCR analysis. Horse-specific primers were used to amplify genomic fragments containing 12 exons. Comparative sequence analysis of horse, pig, mouse, human, Fugu, and zebrafish was performed to establish the exon/intron organization of horse PRKAG3 and to study the homology among different isoforms of AMPK gamma genes in vertebrates. The results showed conclusively that the three different isoforms (gamma1, gamma2, and gamma3) were established already in bony fishes. Seven single nucleotide polymorphisms (SNPs), five causing amino acid substitutions, were identified in a screening across horse breeds with widely different phenotypes as regards muscle development and intended performance. The screening of a major part of the PRKAG3 coding sequence in a small case/control material of horses affected with polysaccharide storage myopathy did not reveal any mutation that was exclusively associated with this muscle storage disease. The breed comparison revealed several potentially interesting SNPs. One of these (Pro258Leu) occurs at a residue that is highly conserved among AMPK gamma genes. In an SNP screening, the variant allele was only found in horse breeds that can be classified as heavy (Belgian) or moderately heavy (North Swedish Trotter, Fjord, and Swedish Warmblood) but not in light horse breeds selected for speed or racing performance (Standardbred, Thoroughbred, and Quarter horse) or in ponies (Icelandic horses and Shetland pony). The results will facilitate future studies of the possible functional significance of PRKAG3 polymorphisms in horses., (Copyright 2003 S. Karger AG, Basel)

The PRKAG3 gene encodes the gamma3 chain of AMP-activated protein kinase (AMPK). A non-conservative missense mutation in the PRKAG3 gene causes a dominant phenotype involving abnormally high glycogen content in pig skeletal muscle. We have determined >126 kb (in 13 contigs) of porcine genomic sequence surrounding the PRKAG3 gene and the corresponding mouse region covering the gene. A comparison of these PRKAG3 sequences and the human sequence was conducted and used to predict evolutionarily conserved regions, including regulatory regions. A comparison of the human genomic sequence and a porcine BAC sequence containing the PRKAG3 gene, revealed a conserved organization and the presence of three additional genes, CYP27A1 (cytochrome P450, family 27, subfamily A, polypeptide 1), STK36 (Serine Threonine Kinase 36), and the homolog of the unidentified human mRNA KIAA0173. Interspersed repetitive elements constituted 51.4 and 38.6% of this genomic region in human and pig, respectively. We were able to reliably align 12.6 kb of orthologous repeats shared between pig and human and these showed an average sequence identity of 72.4%. Our analysis revealed that the human KIAA0173 gene harbors alternative 5' untranslated exons originating from repetitive elements. This provides an obvious example how transposable elements may affect gene evolution., (Copyright 2003 S. Karger AG, Basel)

This article describes the construction of a high-resolution radiation hybrid map of Hsap 2q35 by using the TNG RH panel generated by irradiation with 50,000 rads. We were able to build a framework map of 1300 cR(50,000) including 34 markers ordered with odds higher than 1000:1. The comprehensive map includes 77 loci and describes a region of 3 Mb around the SLC11A1 gene. Because of the very small size of the fragments retained and a reduced retention frequency, it was difficult to build a high-resolution multi-point map of this region by using the TNG RH panel. Nevertheless, this study confirmed the very high potential of this RH panel for constructing a human, high-resolution physical map (2.3 kb/cR(50,000)). Moreover, human ESTs from Hsap 2q35 were hybridized with porcine BAC contigs to establish a porcine transcript map of the homologous region Sscr 15q25 (greater than 2.5 Mb). We identified 17 new genes in this porcine chromosomal region. We were able to compare the location of 26 genes mapped in both species. The gene order was similar except for two possible minor discrepancies in the Desmin sub-region, suggesting the existence of a porcine micro-region between TNP1 and IL8RB with an unknown origin.

The poorly developed transcript maps and the limited resources for genome analysis hamper positional cloning of trait loci in farm animals. This study demonstrates that this will now be easier by the combined use of BAC contigs and the import of the near complete human transcript map. The conclusion was obtained by a comparative analysis of a 2.4-Mb BAC contig of the RN region in pigs. The contig was constructed as part of a successful positional cloning project, which identified PRKAG3 as the causative gene for the RN phenotype. A comparative map including the corresponding regions on human chromosome 2q35 and mouse chromosome 1 (region 36-44 cM) is reported. Sixteen coding sequences were mapped on the BAC contig. The majority of these were identified by BLAST searches of BAC end sequences and BAC shotgun sequences generated during the positional cloning project. Map data for the orthologues in humans were available for 12 of the 16 coding sequences, and all 12 have been assigned to 2q35. Furthermore, no evidence for any rearrangement in gene order was obtained. The extensive linkage conservation indicates that the near complete human transcript map will be an invaluable resource for positional cloning projects in pigs and other domestic animals., (Copyright 2001 Academic Press.)

A high proportion of purebred Hampshire pigs carries the dominant RN- mutation, which causes high glycogen content in skeletal muscle. The mutation has beneficial effects on meat content but detrimental effects on processing yield. Here, it is shown that the mutation is a nonconservative substitution (R200Q) in the PRKAG3 gene, which encodes a muscle-specific isoform of the regulatory gamma subunit of adenosine monophosphate-activated protein kinase (AMPK). Loss-of-function mutations in the homologous gene in yeast (SNF4) cause defects in glucose metabolism, including glycogen storage. Further analysis of the PRKAG3 signaling pathway may provide insights into muscle physiology as well as the pathogenesis of noninsulin-dependent diabetes mellitus in humans, a metabolic disorder associated with impaired glycogen synthesis.

The porcine RN locus affects muscle glycogen content and meat quality. We previously mapped the RN locus to chromosome 15. This study describes the identification of polymorphisms for four class I and four class II markers located in the RN region. Resource families were genotyped with F-SSCP markers (fluorescent single strand conformation polymorphism) and microsatellite markers. Subsequent multipoint linkage analysis revealed the order FN1-IGFBP5-S1000-S1001-IL8RB-VIL1-RN-Sw936-Sw906. The gene order is identical to the previously reported porcine RH map of the same region. The described map will facilitate positional cloning of the RN gene.

The domestic pig originates from the Eurasian wild boar (Sus scrofa). We have sequenced mitochondrial DNA and nuclear genes from wild and domestic pigs from Asia and Europe. Clear evidence was obtained for domestication to have occurred independently from wild boar subspecies in Europe and Asia. The time since divergence of the ancestral forms was estimated at approximately 500,000 years, well before domestication approximately 9,000 years ago. Historical records indicate that Asian pigs were introduced into Europe during the 18th and early 19th centuries. We found molecular evidence for this introgression and the data indicated a hybrid origin of some major "European" pig breeds. The study is an advance in pig genetics and has important implications for the maintenance and utilization of genetic diversity in this livestock species.

We recently constructed a 7000-rad porcine whole-genome radiation hybrid (RH) panel with the primary objective of integrating linkage maps of microsatellites with evolutionary conserved genes into one ordered map. In order to evaluate the resolution of this RH panel, we have now constructed a radiation hybrid map of the Chromosome (Chr) 15q2.3-q2.6 region containing the RN gene. This gene has large effects on glycogen content in muscle and meat quality. Ten microsatellites covering a region of 55 centiMorgans and eight genes (AE3, FN1, IGFBP5, INHA, IRS1, PAX3, TNP1, and VIL1) were placed on the Sscr15 RH map. All the genes, except IRS1, were mapped on the RH map between microsatellites located in 15q2.5. The relative order of AE3 and INHA was inverted on the porcine physical map in comparison with the mouse linkage map. The order of other genes already mapped in the mouse (FN1, IGFBP5, TNP1, VIL1, INHA/AE3, and PAX3) was identical in pigs. We found no clear difference between the gene order on pig Chr 15 and human Chr 2q.

Six YAC clones representing five microsatellite markers from the RN region were mapped by fluorescent in situ hybridization (FISH) on pig metaphase chromosomes and their relative order was determined by pairwise multicolour FISH. Two of the microsatellites viz., Sw120 and Sw936 flank RN as well as the remaining three microsatellites Sw1683, Sw2083 and Sw1309. The results assigned the RN locus to the distal part of the 15q25 band. The linear order of the microsatellites was compared with the available linkage mapping data.

Somatic cell hybrid (SCH) panels and radiation hybrid (RH) panels are powerful resources for comparative gene mapping because gene assignments are made without the detection of genetic polymorphism as needed for linkage mapping. A frequently encountered problem, however, is that the gene specific primers may amplify homologous PCR products of equal length from the donor and recipient species of the panel. Here, we describe a simple solution to this problem in which we utilize the formation of interspecies heteroduplexes that can be easily distinguished from the corresponding homoduplexes by native polyacrylamide gel electrophoresis. We denote these DNA-DNA interspecies hybrids, xenoduplexes (xeno = Gr. Xenos, foreigner). A merit of the method is that the formation of xenoduplexes strongly suggests that the PCR products from the two species represent homologous sequences. The method is thus particularly useful for comparative gene mapping when the PCR primers have been designed by use of sequence information from other species. In this study we have successfully used xenoduplex analysis and a pig-rodent SCH panel to map seven porcine genes (ACADM, AT3, HOXD, IL8RB, LEPR, PAX8, PKLR) for which no previous sequence information was available. The assignment of the leptin receptor gene (LEPR) to pig chromosome 6q32-35 excluded LEPR as a candidate gene for a QTL on pig chromosome 4 with a major effect on fatness.