Genome-wide analyses of the Jeju,Thoroughbred, and Jeju crossbred horse populations using the high density SNP array

The Jeju horse is an indigenous Korean horse breed that is currently registered with the Food and Agriculture Organization of the United Nations. However, there is severe lack of genomic studies on Jeju horse. This study was conducted to investigate genetic characteristics of horses including Jeju horse, Thoroughbred and Jeju crossbred (Jeju?×?Thoroughbred) populations. We compared the genomes of three horse populations using the Equine SNP70 Beadchip array. Short-range Linkage disequilibrium was the highest in Thoroughbred, whereas r² values were lowest in Jeju horse. Expected heterozygosity was the highest in Jeju crossbred (0.351), followed by the Thoroughbred (0.337) and Jeju horse (0.311). The level of inbreeding was slightly higher in Thoroughbred (??0.009) than in Jeju crossbred (??0.035) and Jeju horse (??0.038). F_ST value was the highest between Jeju horse and Thoroughbred (0.113), whereas Jeju crossbred and Thoroughbred showed the lowest value (0.031). The genetic relationship was further assessed by principal component analysis, suggesting that Jeju crossbred is more genetically similar to Thoroughbred than Jeju horse population. Additionally, we detected potential selection signatures, for example, in loci located on LCORL/NCAPG and PROP1 genes that are known to influence body. Genome-wide analyses of the three horse populations showed that all the breeds had somewhat a low level of inbreeding within each population. In the population structure analysis, we found that Jeju crossbred was genetically closer to Thoroughbred than Jeju horse. Furthermore, we identified several signatures of selection which might be associated with traits of interest. To our current knowledge, this study is the first genomic research, analyzing genetic relationships of Jeju horse, Thoroughbred and Jeju crossbred.     

Genetic characterization of horse bone excavated from the Kwakji archaeological site,Jeju, Korea

We determined the nucleotide sequences of the hypervariable D-loop region of mitochondrial DNA (mtDNA) from horse bone (humerus, A.D. 700 to A.D. 800) that was excavated from the Kwakji archaeological site, Jeju, Korea. We compared them with ones from extant horses. We designed three pairs of oligonucleotide primers from the tRNA-Thr and tRNA-Phe gene regions of mtDNA that are highly conserved among many other animal species. We cloned 232, 336, and 644 bp from the horse bone in order to determine the mtDNA D-loop sequence. The sequence was 1,124 bp long; the middle contained 19 tandem repeats of an 8-bp sequence (TGTGCACC) that is specific to equines. The mtDNA D-loop region contained each base (total number, percentage of total) as follows: A (317, 28.20%), C (336, 29.89%), G (169, 15.04%), and T (302, 26.87%). This sequence, like those of other horse populations, was AT rich. Sequence divergence was the lowest (1.71%) between the ancient horse bone and that of the Thoroughbred horse 1. The neighbor-joining and strict consensus tree of three of the most parsimonious trees also suggested that the ancient bone was considerably unrelated to native Jeju horses. The molecular phylogenetic characteristics of the horse bone that was excavated from the Kwakji archaeological site (Jeju, Korea) showed that some horse breeds may have existed on Jeju Island, Korea before Mongolian horses were introduced. The horse bone that was excavated from the Kwakji archaeological site may aid future research on the origin and ancestry of native Jeju horses.     

Complete mitochondrial genome sequences of Korean native horse from Jeju Island: uncovering the spatio-temporal dynamics

The Korean native horse (Jeju horse) is one of the most important animals in Korean historical, cultural, and economical viewpoints. In the early 1980s, the Jeju horse was close to extinction. The aim of this study is to explore the phylogenomics of Korean native horse focusing on spatio-temporal dynamics. We determined complete mitochondrial genome sequences for the first Korean native (n?=?6) and additional Mongolian (n?=?2) horses. Those sequences were analyzed together with 143 published ones using Bayesian coalescent approach as well as three different phylogenetic analysis methods, Bayesian inference, maximum likelihood, and neighbor-joining methods. The phylogenomic trees revealed that the Korean native horses had multiple origins and clustered together with some horses from four European and one Middle Eastern breeds. Our phylogenomic analyses also supported that there was no apparent association between breed or geographic location and the evolution of global horses. Time of the most recent common ancestor of the Korean native horse was approximately 13,200–63,200 years, which was much younger than 0.696 My of modern horses. Additionally, our results showed that all global horse lineages including Korean native horse existed prior to their domestication events occurred in about 6000–10,000 years ago. This is the first study on phylogenomics of the Korean native horse focusing on spatio-temporal dynamics. Our findings increase our understanding of the domestication history of the Korean native horses, and could provide useful information for horse conservation projects as well as for horse genomics, emergence, and the geographical distribution.     

Genome-wide detection and characterization of positive selection in Korean Native Black Pig from Jeju Island

Background

In the 1980s, Korean native black pigs from Jeju Island (Jeju black pigs) served as representative sample of Korean native black pigs, and efforts were made to help the species rebound from the brink of extinction, which occurred as a result of the introduction of Western pig breeds. Geographical separation of Jeju Island from the Korean peninsula has allowed Jeju black pigs not only to acquire unique characteristics but also to retain merits of rare Korean native black pigs.

Results

To further analyze the Jeju black pig genome, we performed whole-genome re-sequencing (average read depth of 14×) of 8 Jeju black pig and 6 Korean pigs (which live on the Korean peninsula) to compare and identify putative signatures of positive selection in Jeju black pig, the true and pure Korean native black pigs. The candidate genes potentially under positive selection in Jeju black pig support previous reports of high marbling score, rare occurrence of pale, soft, exudative (PSE) meat, but low growth rate and carcass weight compared to Western breeds.

Conclusions

Several candidate genes potentially under positive selection were involved in fatty acid transport and may have contributed to the unique characteristics of meat quality in JBP. Jeju black pigs can offer a unique opportunity to investigate the true genetic resource of once endangered Korean native black pigs. Further genome-wide analyses of Jeju black pigs on a larger population scale are required in order to define a conservation strategy and improvement of native pig resources.

Electronic supplementary material

The online version of this article (doi:10.1186/s12863-014-0160-1) contains supplementary material, which is available to authorized users.     

mRNA sequence analysis and quantitative expression of the ADAMTS4 gene in the thoroughbred horse

Cartilage increases flexibility of motion and helps protect the body from physical shock. Strong physical shock or some biological factor could cause joint disease. ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) has been related to degradation of aggrecans in cartilage. It has been associated with joint disease, which could influence the ability of horses to exercise. Here, we performed sequence analysis and expression profiling of the ADAMTS4 gene in thoroughbred horses. Quantitative real-time RT-PCR data indicated that higher expression of the ADAMTS4 gene appeared in the cartilage tissues compared to those of pancreas, stomach, lung and colon. The expression pattern was also higher in the muscle tissues after exercise than before exercise. These data could be of great use for further studies in relation to both horse racing and joint disease.     

An Approach to Identify SNPs in the Gene Encoding Acetyl-CoA Acetyltransferase-2 (ACAT-2) and Their Proposed Role in Metabolic Processes in Pig

The novel liver protein acetyl-CoA acetyltransferase-2 (ACAT2) is involved in the beta-oxidation and lipid metabolism. Its comprehensive relative expression, in silico non-synonymous single nucleotide polymorphism (nsSNP) analysis, as well as its annotation in terms of metabolic process with another protein from the same family, namely, acetyl-CoA acyltransferase-2 (ACAA2) was performed in Sus scrofa. This investigation was conducted to understand the most important nsSNPs of ACAT2 in terms of their effects on metabolic activities and protein conformation. The two most deleterious mutations at residues 122 (I to V) and 281 (R to H) were found in ACAT2. Validation of expression of genes in the laboratory also supported the idea of differential expression of ACAT2 and ACAA2 conceived through the in silico analysis. Analysis of the relative expression of ACAT2 and ACAA2 in the liver tissue of Jeju native pig showed that the former expressed significantly higher (P<0.05). Overall, the computational prediction supported by wet laboratory analysis suggests that ACAT2 might contribute more to metabolic processes than ACAA2 in swine. Further associations of SNPs in ACAT2 with production traits might guide efforts to improve growth performance in Jeju native pigs.     

Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.)

Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions.     

Genomic structure and expression analyses of the PYGM gene in the thoroughbred horse

Muscle glycogen Phosphorylase (PYGM) has been shown to catalyze the degradation of glycogen to glucose-1-phosphate. The PYGM gene can contribute to providing energy to the body by disassembling the glycogen in muscle. Here, we analyzed the genomic structure and expression of the PYGM gene in the thoroughbred horse. The PYGM gene, containing several transposable elements (MIRs, LINEs, and MERs), was highly conserved in mammalian genomes. In order to understand the expression of the horse PYGM gene, we performed quantitative RT-PCR using 11 thoroughbred horse tissue samples. The horse PYGM gene was broadly expressed in all tissues tested. In particular, the highest expression of the horse PYGM gene was observed in skeletal muscle tissue relative to the other tissues. Interestingly, the horse PYGM gene contains fewer mobile elements than its human ortholog, resulting in an increase in the structural stability of the PYGM gene sequence. This study provides insights into the genomic structure of the horse PYGM gene that may be useful in future studies of its association with exercise capability.     

Genome-Wide Analysis of DNA Methylation before-and after Exercise in the Thoroughbred Horse with MeDIP-Seq

Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits.     

Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Drosophila suzukii (Diptera: Drosophilidae)

To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR) data, normalization relative to reliable reference gene(s) is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1β, TBP, NADH, HSP22, GAPDH, Actin, α-Tubulin), were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population), and abiotic (photoperiod, temperature) conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper) and one web-based comprehensive tool (RefFinder) were used to normalize analysis of the ten candidate reference genes identified α-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (α-Tubulin, TBP and AK) and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes α-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii.