Assessment of genetic diversity and phylogenetic relationships of Korean native chicken breeds using microsatellite markers.

OBJECTIVE: This study was conducted to investigate the basic information on genetic structure and characteristics of Korean Native chickens (NC) and foreign breeds through the analysis of the pure chicken populations and commercial chicken lines of the Hanhyup Company which are popular in the NC market, using the 20 microsatellite markers. METHODS: In this study, the genetic diversity and phylogenetic relationships of 445 NC from five different breeds (NC, Leghorn [LH], Cornish [CS], Rhode Island Red [RIR], and Hanhyup [HH] commercial line) were investigated by performing genotyping using 20 microsatellite markers. RESULTS: The highest genetic distance was observed between RIR and LH (18.9%), whereas the lowest genetic distance was observed between HH and NC (2.7%). In the principal coordinates analysis (PCoA) illustrated by the first component, LH was clearly separated from the other groups. The correspondence analysis showed close relationship among individuals belonging to the NC, CS, and HH lines. From the STRUCTURE program, the presence of 5 clusters was detected and it was found that the proportion of membership in the different clusters was almost comparable among the breeds with the exception of one breed (HH), although it was highest in LH (0.987) and lowest in CS (0.578). For the cluster 1 it was high in HH (0.582) and in CS (0.368), while for the cluster 4 it was relatively higher in HH (0.392) than other breeds. CONCLUSION: Our study showed useful genetic diversity and phylogenetic relationship data that can be utilized for NC breeding and development by the commercial chicken industry to meet consumer demands.

Microsatellite Analysis of the Genetic Diversity and Population Structure in Dairy Goats in Thailand.

The genetic relationships between different populations and breeds of exotic dairy goats in Thailand were studied using 12 microsatellite markers. Blood samples were obtained from 211 goats from Department of Livestock Development breeding and research farms: 29 Anglonubian (AN), 21 Alpine (AP), 23 Jamunapari (JAM), 50 Saanen (SN), and 88 Toggenburg (TG). Five of the 12 microsatellite markers were found to be polymorphic. A mean of 7.40 alleles per locus was found, with a range from 5 (SPS115 and ETH225) to 11 (TGLA122). We found 24, 27, 19, 32, and 24 alleles in the AN, AP, JAM, SN, and TG breeds, respectively; 37 alleles were present in all breeds. The mean number of alleles in each population ranged from 3.2 (ETH225 locus) to 7.6 (TGLA122 locus). Genetic variability within the breeds was moderate as evidenced by the mean expected heterozygosity of 0.539. The average observed heterozygosity across the 5 markers in all breeds was 0.529 with the maximum observed at the BM1818 locus (0.772) and the minimum at the ETH225 locus (0.248). The observed and expected heterozygosity for all breeds for the 5 microsatellite markers ranged from 0.419 to 0.772 and 0.227 to 0.792, respectively. On the basis of their means, the TGLA122 and BM1818 loci were the most suitable markers for distinguishing genetic diversity among the goats. The estimated average F is value for the breeds ranged from -0.044 (ETH225) to 0.180 (SPS115), while the estimated average F st value ranged from 0.021 (SPS115) to 0.104 (ETH10). These results indicated that TGLA122 and BM1818 markers are suitable to be used for aiding conservation and breeding improvement strategies of dairy.

Antimicrobial peptides in the centipede Scolopendra subspinipes mutilans.

The centipede Scolopendra subspinipes mutilans is an environmentally beneficial and medically important arthropod species. Although this species is increasingly applied as a reliable source of new antimicrobial peptides, the transcriptome of this species is a prerequisite for more rational selection of antimicrobial peptides. In this report, we isolated total RNA from the whole body of adult centipedes, S. subspinipes mutilans, that were nonimmunized and immunized against Escherichia coli, and we generated a total of 77,063 pooled contigs and singletons using high-throughput sequencing. To screen putative antimicrobial peptides, in silico analyses of the S. subspinipes mutilans transcriptome were performed based on the physicochemical evidence of length, charge, isoelectric point, and in vitro and in vivo aggregation scores together with the existence of continuous antimicrobial peptide stretches. Moreover, we excluded some transcripts that showed similarity with both previously known antimicrobial peptides and the human proteome, had a proteolytic cleavage site, and had downregulated expression compared with the nonimmunized sample. As a result, we selected 17 transcripts and tested their antimicrobial activity with a radial diffusion assay. Among them, ten synthetic peptides experimentally showed antimicrobial activity against microbes and no toxicity to mouse erythrocytes. Our results provide not only a useful set of antimicrobial peptide candidates and an efficient strategy for novel antimicrobial peptide development but also the transcriptome data of a big centipede as a valuable resource.

Genetic traceability of black pig meats using microsatellite markers.

Pork from Jeju black pig (population J) and Berkshire (population B) has a unique market share in Korea because of their high meat quality. Due to the high demand of this pork, traceability of the pork to its origin is becoming an important part of the consumer demand. To examine the feasibility of such a system, we aim to provide basic genetic information of the two black pig populations and assess the possibility of genetically distinguishing between the two breeds. Muscle samples were collected from slaughter houses in Jeju Island and Namwon, Chonbuk province, Korea, for populations J and B, respectively. In total 800 Jeju black pigs and 351 Berkshires were genotyped at thirteen microsatellite (MS) markers. Analyses on the genetic diversity of the two populations were carried out in the programs MS toolkit and FSTAT. The population structure of the two breeds was determined by a Bayesian clustering method implemented in structure and by a phylogenetic analysis in Phylip. Population J exhibited higher mean number of alleles, expected heterozygosity and observed heterozygosity value, and polymorphism information content, compared to population B. The FIS values of population J and population B were 0.03 and -0.005, respectively, indicating that little or no inbreeding has occurred. In addition, genetic structure analysis revealed the possibility of gene flow from population B to population J. The expected probability of identify value of the 13 MS markers was 9.87×10(-14) in population J, 3.17×10(-9) in population B, and 1.03×10(-12) in the two populations. The results of this study are useful in distinguishing between the two black pig breeds and can be used as a foundation for further development of DNA markers.

Whole transcriptome analyses of six thoroughbred horses before and after exercise using RNA-Seq.

BACKGROUND: Thoroughbred horses are the most expensive domestic animals, and their running ability and knowledge about their muscle-related diseases are important in animal genetics. While the horse reference genome is available, there has been no large-scale functional annotation of the genome using expressed genes derived from transcriptomes. RESULTS: We present a large-scale analysis of whole transcriptome data. We sequenced the whole mRNA from the blood and muscle tissues of six thoroughbred horses before and after exercise. By comparing current genome annotations, we identified 32,361 unigene clusters spanning 51.83 Mb that contained 11,933 (36.87%) annotated genes. More than 60% (20,428) of the unigene clusters did not match any current equine gene model. We also identified 189,973 single nucleotide variations (SNVs) from the sequences aligned against the horse reference genome. Most SNVs (171,558 SNVs; 90.31%) were novel when compared with over 1.1 million equine SNPs from two SNP databases. Using differential expression analysis, we further identified a number of exercise-regulated genes: 62 up-regulated and 80 down-regulated genes in the blood, and 878 up-regulated and 285 down-regulated genes in the muscle. Six of 28 previously-known exercise-related genes were over-expressed in the muscle after exercise. Among the differentially expressed genes, there were 91 transcription factor-encoding genes, which included 56 functionally unknown transcription factor candidates that are probably associated with an early regulatory exercise mechanism. In addition, we found interesting RNA expression patterns where different alternative splicing forms of the same gene showed reversed expressions before and after exercising. CONCLUSION: The first sequencing-based horse transcriptome data, extensive analyses results, deferentially expressed genes before and after exercise, and candidate genes that are related to the exercise are provided in this study.

Identification and analysis of MC4R polymorphisms and their association with economic traits of Korean cattle (Hanwoo).

Signaling by the melanocortin-4 receptor (MC4R) is important for mediation the effect of leptin on food intake and energy homeostasis, and is associated with obesity, energy homeostasis and control of feeding behavior. Presently, the bovine MC4R gene was characterized to detect genetic variation at this locus and to relate it to economic traits in Korean cattle (Hanwoo). Five single nucleotide polymorphisms (SNPs) were identified in the coding region (G709A, C927T, C1069G, C1343A, and C1786T). G709A changed amino acid 166 of the MC4R protein from valine to methionine and C1069G changed amino acid 286 of the MC4R protein from leucine to valine. A SNP at C927T significantly influenced the Marbling score, SNP markers C1069G and C1343A significantly affected the Backfat thickness, and the SNP marker C1786T significantly influenced backfat and Marbling score. The MC4R gene may thus be a candidate gene for carcass traits with MC4R SNPs being potentially valuable as genetic markers for economic traits in Hanwoo.

Analysis of genetic diversity and structure of Mongolian horse using microsatellite markers.

Mongolian horses are one of the oldest horse breeds, and are very important livestock in Mongolia as they are used in various fields such as transportation, food (milk, meat), and horse racing. In addition, research and preservation on pure Mongolian breeds are being promoted under the implementation of the new Genetics of Livestock Resources' act in Mongolia. However, despite the implementation of this act, genetic research on Mongolian horses using microsatellites (MS) has not progressed enough. Therefore, this study was conducted to analyze the genetic polymorphism of five breeds (Gobi shankh, Tes, Gal shar, Darkhad, and Undurshil) using 14 MS markers recommended by International Society for Animal Genetics (ISAG). The mean number of alleles (MNA) was 8.29, expected heterozygosity frequency (HExp) was 0.767, observed heterozygosity frequency (HObs) was 0.752, and polymorphism information content (PIC) was 0.729. The Nei's genetic distance analysis showed that the genetic distance between Gobi shankh and Darkhad horses was the farthest, and the other three breeds, Tes, Gal shar, and Undurshil were found to be close to each other. Similarly, the principal coordinate analysis (PCoA) and factorial correspondence analysis (FCA) showed that the Gobi shankh and Darkhad horses were genetically distinct from other breeds. On the other hand, it appears that Tes, Gal shar, and Undurshil horses, which are genetically similar, most likely interbred with each other. Therefore, it is expected that these results will help the conservation of genetic resources in Mongolia and the establishment of policies related to Mongolian horses.

Genetic diversity and population structure of Mongolian regional horses with 14 microsatellite markers.

OBJECTIVE: This study aimed to identify the genetic diversity and population structure of Mongolian horse populations according to the province of residence (Khentii, KTP; Uvs, USP; Omnogovi and Dundgovi, GOP; Khovsgol, KGP) using 14 microsatellite (MS) markers. METHODS: A total of 269 whole blood samples were obtained from the four populations (KTP, USP, GOP, KGP) geographically distinct provinces. Multiplex polymerase chain reaction (PCR) was conducted using 14 MS markers (AHT4, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, and VHL20), as recommended by the International Society for Animal Genetics. Capillary electrophoresis was conducted using the amplified PCR products, alleles were determined. Alleles were used for statistical analysis of genetic variability, Nei's DA genetic distance, principal coordinate analysis (PCoA), factorial corresponding analysis (FCA), and population structure. RESULTS: On average, the number of alleles, expected heterozygosity (HExp), observed heterozygosity (HObs), and polymorphic information content among all populations were 11.43, 0.772, 0.757, and 0.737, respectively. In the PCoA and FCA, GOP, and KGP were genetically distinct from other populations, and the KTP and USP showed a close relationship. The two clusters identified using Nei's DA genetic distance analysis and population structure highlighted the presence of structurally clear genetic separation. CONCLUSION: Overall, the results of this study suggest that genetic diversity between KTP and USP was low, and that between GOP and KGP was high. It is thought that these results will help in the effective preservation and improvement of Mongolian horses through genetic diversity analysis and phylogenetic relationships.

Effects of L-glutamine supplementation on degradation rate and rumen fermentation characteristics in vitro.

OBJECTIVE: Two follow-up studies (exp. 1 and 2) were conducted to determine the effects of L-glutamine (L-Gln) supplementation on degradation and rumen fermentation characteristics in vitro. METHODS: First, rumen liquor from three cannulated cows was used to test L-Gln (50 mM) degradation rate and ammonia-N production at 6, 12, 24, 36, and 48 h after incubation (exp. 1). Second, rumen liquor from two cannulated steers was used to assess the effects of five levels of L-Gln including 0% (control), 0.5%, 1%, 2%, and 3% at 0, 3, 6, 12, 24, 36, and 48 h after incubation on fermentation characteristics, gas production, and degradability of nutrients (exp. 2). RESULTS: In exp. 1, L-Gln degradation rate and ammonia-N concentrations increased over time (p<0.001). In exp. 2, pH was reduced significantly as incubation time elapsed (p<0.001). Total gas production tended to increase in all groups as incubation time increased. Acetate and propionate tended to increase by increasing glutamine (Gln) levels, whereas levels of total volatile fatty acids (VFAs) were the highest in 0.5% and 3% Gln groups (p<0.001). The branched-chain VFA showed both linear and quadratic effects showing the lowest values in the 1% Gln group particularly after 6 h incubation (p<0.001). L-Gln increased crude protein degradability (p<0.001), showing the highest degradability in the 0.5% Gln group regardless of incubation time (p<0.05). Degradability of acid detergent fiber and neutral detergent fiber showed a similar pattern showing the highest values in 0.5% Gln group (p<0.10). CONCLUSION: Although L-Gln showed no toxicity when it was supplemented at high dosages (2% to 3% of DM), 0.5% L-Gln demonstrated the positive effects on main factors including VFAs production in-vitro. The results of this study need to be verified in further in-vivo study.

Estimation of Genetic Parameters and Correlation between Yearling Ultrasound Measurements and Carcass Traits in Hanwoo Cattle.

Genetic parameters have a significant role in designing a breeding program and are required to evaluate economically important traits. The objective of this study was to estimate heritability and genetic correlation between yearling ultrasound measurements, such as backfat thickness (UBFT), eye muscle area (UEMA), intramuscular fat content (UIMF), and carcass traits, such as backfat thickness (BFT), carcass weight (CW), eye muscle area (EMA), marbling score (MS) at approximately 24 months of age, as well as yearling weight (YW) in Hanwoo bulls (15,796) and steers (5682). The (co) variance components were estimated using a multi-trait animal model. Moderate to high heritability estimates were obtained and were 0.42, 0.50, 0.56, and 0.59 for CW, EMA, BFT, and MS, respectively. Heritability estimates for yearling measurements of YW, UEMA, UBFT, and UIMF were 0.31, 0.32, 0.30, and 0.19, respectively. Favorable and strong genetic correlations were observed between UIMF and MS (0.78), UBFT and BFT (0.63), and UEMA and EMA (0.65). Moreover, the estimated genetic correlation between YW and CW was high (0.84) and relatively moderate between YW and EMA (0.43). These results suggest that genetic improvement can be achieved for carcass traits when using yearling ultrasound measurements as selection criteria in ongoing Hanwoo breeding programs.

Genetic diversity of Halla horses using microsatellite markers.

BACKGROUND: Currently about 26,000 horses are breeding in Korea and 57.2% (14,776 horses) of them are breeding in Jeju island. According to the statistics published in 2010, the horses breeding in Jeju island are subdivided into Jeju horse (6.1%), Thoroughbred (18.8%) and Halla horse (75.1%). Halla horses are defined as a crossbreed between Jeju and Thoroughbred horses and are used for horse racing, horse riding and horse meat production. However, little research has been conducted on Halla horses because of the perception of crossbreed and people's weighted interest toward Jeju horses. METHOD: Using 17 Microsatellite (MS) Markers recommended by International Society for Animal Genetics (ISAG), genomic DNAs were extracted from the hair roots of 3,880 Halla horses breeding in Korea and genetic diversity was identified by genotyping after PCR was performed. RESULTS AND CONCLUSION: In average, 10.41 alleles (from 6 alleles in HTG7 to 17 alleles in ASB17) were identified after the analysis using 17 MS Markers. The mean value of Hobs was 0.749 with a range from 0.612(HMS1) to 0.857(ASB2). Also, it was found that Hexp and PIC values were lowest in HMS1 (0.607 and 0.548, respectively), and highest in LEX3(0.859 and 0.843, respectively), and the mean value of Hexp was 0.760 and that of PIC was 0.728. 17 MS markers used in this studies were considered as appropriate markers for the polymorphism analysis of Halla horses. The frequency for the appearance of identical individuals was 5.90 × 10-20 when assumed as random mating population and when assumed as half-sib and full-sib population, frequencies were 4.08 × 10-15 and 3.56 × 10-8, respectively. Based on these results, the 17 MS markers can be used adequately for the Individual Identification and Parentage Verification of Halla horses. Remarkably, allele M and Q of ASB23 marker, G of HMS2 marker, H and L of HTG6 marker, L of HTG7 marker, E of LEX3 marker were the specific alleles unique to Halla horses.

Transcriptome Analysis of the Tadpole Shrimp (Triops longicaudatus) by Illumina Paired-End Sequencing: Assembly, Annotation, and Marker Discovery.

The tadpole shrimp (Triops longicaudatus) is an aquatic crustacean that helps control pest populations. It inhabits freshwater ponds and pools and has been described as a living fossil. T. longicaudatus was officially declared an endangered species South Korea in 2005; however, through subsequent protection and conservation management, it was removed from the endangered species list in 2012. The limited number of available genetic resources on T. longicaudatus makes it difficult to obtain valuable genetic information for marker-aided selection programs. In this study, whole-transcriptome sequencing of T. longicaudatus generated 39.74 GB of clean data and a total of 269,822 contigs using the Illumina HiSeq 2500 platform. After clustering, a total of 208,813 unigenes with an N50 length of 1089 bp were generated. A total of 95,105 unigenes were successfully annotated against Protostome (PANM), Unigene, Eukaryotic Orthologous Groups (KOG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases using BLASTX with a cut-off of 1E-5. A total of 57,731 unigenes were assigned to GO terms, and 7247 unigenes were mapped to 129 KEGG pathways. Furthermore, 1595 simple sequence repeats (SSRs) were detected from the unigenes with 1387 potential SSR markers. This is the first report of high-throughput transcriptome analysis of T. longicaudatus, and it provides valuable insights for genetic research and molecular-assisted breeding of this important species.

Differences in serum protein 2D gel electrophoresis patterns of Przewalski's (Mongolian wild horse) and thoroughbred horses.

The objective of this study was to assess differences in serum protein expression profiles of Przewalski's (Mongolian wild horse) and thoroughbred horses using proteome analysis. The serum proteins were separated by two-dimensional electrophoresis (2-DE) and five different gene products were identified. Proteins represented by the five spots were identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS)/MS technology. The identities of all proteins were deduced based on their similarity to proteins in the human plasma protein database. Three proteins (a haptoglobin-2 alpha glycoprotein and two haptoglobin-2beta glycoproteins with different accession numbers) were downregulated in Przewalski's horse sera compared to thoroughbred horse sera. Moreover, two proteins (tetraspanin-18 and pM5) were upregulated in Przewalski's horses compared to thoroughbred horses. Haptoglobin-2 alpha and haptoglobin-2beta may serve as candidate molecules in future studies of inflammation, coagulation, immune modulation and pro-oxidant and antioxidant activity with consequential effects on the entire metabolism of the horse.

Peeling back the evolutionary layers of molecular mechanisms responsive to exercise-stress in the skeletal muscle of the racing horse.

The modern horse (Equus caballus) is the product of over 50 million yrs of evolution. The athletic abilities of the horse have been enhanced during the past 6000 yrs under domestication. Therefore, the horse serves as a valuable model to understand the physiology and molecular mechanisms of adaptive responses to exercise. The structure and function of skeletal muscle show remarkable plasticity to the physical and metabolic challenges following exercise. Here, we reveal an evolutionary layer of responsiveness to exercise-stress in the skeletal muscle of the racing horse. We analysed differentially expressed genes and their co-expression networks in a large-scale RNA-sequence dataset comparing expression before and after exercise. By estimating genome-wide dN/dS ratios using six mammalian genomes, and FST and iHS using re-sequencing data derived from 20 horses, we were able to peel back the evolutionary layers of adaptations to exercise-stress in the horse. We found that the oldest and thickest layer (dN/dS) consists of system-wide tissue and organ adaptations. We further find that, during the period of horse domestication, the older layer (FST) is mainly responsible for adaptations to inflammation and energy metabolism, and the most recent layer (iHS) for neurological system process, cell adhesion, and proteolysis.