Identification and characterization of structural variants related to meat quality in pigs using chromosome-level genome assemblies.

BACKGROUND: Many studies have been performed to identify various genomic loci and genes associated with the meat quality in pigs. However, the full genetic architecture of the trait still remains unclear in part because of the lack of accurate identification of related structural variations (SVs) which resulted from the shortage of target breeds, the limitations of sequencing data, and the incompleteness of genome assemblies. The recent generation of a new pig breed with superior meat quality, called Nanchukmacdon, and its chromosome-level genome assembly (the NCMD assembly) has provided new opportunities. RESULTS: By applying assembly-based SV calling approaches to various genome assemblies of pigs including Nanchukmacdon, the impact of SVs on meat quality was investigated. Especially, by checking the commonality of SVs with other pig breeds, a total of 13,819 Nanchukmacdon-specific SVs (NSVs) were identified, which have a potential effect on the unique meat quality of Nanchukmacdon. The regulatory potentials of NSVs for the expression of nearby genes were further examined using transcriptome- and epigenome-based analyses in different tissues. CONCLUSIONS: Whole-genome comparisons based on chromosome-level genome assemblies have led to the discovery of SVs affecting meat quality in pigs, and their regulatory potentials were analyzed. The identified NSVs will provide new insights regarding genetic architectures underlying the meat quality in pigs. Finally, this study confirms the utility of chromosome-level genome assemblies and multi-omics analysis to enhance the understanding of unique phenotypes.

The transcriptomic blueprint of molt in rooster using various tissues from Ginkkoridak (Korean long-tailed chicken).

BACKGROUND: Annual molt is a critical stage in the life cycle of birds. Although the most extensively documented aspects of molt are the renewing of plumage and the remodeling of the reproductive tract in laying hens, in chicken, molt deeply affects various tissues and physiological functions. However, with exception of the reproductive tract, the effect of molt on gene expression across the tissues known to be affected by molt has to date never been investigated. The present study aimed to decipher the transcriptomic effects of molt in Ginkkoridak, a Korean long-tailed chicken. Messenger RNA data available across 24 types of tissue samples (9 males) and a combination of mRNA and miRNA data on 10 males and 10 females blood were used. RESULTS: The impact of molt on gene expression and gene transcript usage appeared to vary substantially across tissues types in terms of histological entities or physiological functions particularly related to nervous system. Blood was the tissue most affected by molt in terms of differentially expressed genes in both sexes, closely followed by meninges, bone marrow and heart. The effect of molt in blood appeared to differ between males and females, with a more than fivefold difference in the number of down-regulated genes between both sexes. The blueprint of molt in roosters appeared to be specific to tissues or group of tissues, with relatively few genes replicating extensively across tissues, excepted for the spliceosome genes (U1, U4) and the ribosomal proteins (RPL21, RPL23). By integrating miRNA and mRNA data, when chickens molt, potential roles of miRNA were discovered such as regulation of neurogenesis, regulation of immunity and development of various organs. Furthermore, reliable candidate biomarkers of molt were found, which are related to cell dynamics, nervous system or immunity, processes or functions that have been shown to be extensively modulated in response to molt. CONCLUSIONS: Our results provide a comprehensive description at the scale of the whole organism deciphering the effects of molt on the transcriptome in chicken. Also, the conclusion of this study can be used as a valuable resource in transcriptome analyses of chicken in the future and provide new insights related to molt.

Non-Coding Transcriptome Maps across Twenty Tissues of the Korean Black Chicken, Yeonsan Ogye.

Yeonsan Ogye is a rare Korean domestic chicken breed whose entire body, including feathers and skin, has a unique black coloring. Although some protein-coding genes related to this unique feature have been examined, non-coding elements have not been widely investigated. Thus, we evaluated coding and non-coding transcriptome expression and identified long non-coding RNAs functionally linked to protein-coding genes in Ogye. High-throughput RNA sequencing and DNA methylation sequencing were performed to profile the expression of 14,264 Ogye protein-coding and 6900 long non-coding RNA (lncRNA) genes and detect DNA methylation in 20 different tissues of an individual Ogye. Approximately 75% of Ogye lncRNAs and 45% of protein-coding genes showed tissue-specific expression. For some genes, tissue-specific expression levels were inversely correlated with DNA methylation levels in their promoters. Approximately 39% of tissue-specific lncRNAs displayed functional associations with proximal or distal protein-coding genes. Heat shock transcription factor 2-associated lncRNAs appeared to be functionally linked to protein-coding genes specifically expressed in black skin tissues, more syntenically conserved in mammals, and differentially expressed in black relative to in white tissues. Pending experimental validation, our findings increase the understanding of how the non-coding genome regulates unique phenotypes and can be used for future genomic breeding of chickens.

A genome-wide assessment of genetic diversity and population structure of Korean native cattle breeds.

BACKGROUND: The native cattle breeds are an important genetic resource for meat and milk production throughout Asia. In Asia cattle were domesticated around 10,000 years ago and in Korea cattle are being raised since 2000 B.C. There are three native breeds of cattle in Korea viz. Brown Hanwoo, Brindle Hanwoo and Jeju Black. While one of these breeds, Brown Hanwoo, is a part of a Food and Agricultural Organization and national genetic evaluation plans, others get little attention. This study is an effort to understand and provide a detailed insight into the population structure and genetic variability of the Korean cattle breeds along with other Asian breeds using various methods. In this study we report the genetic variation and structure of the Korean cattle breeds and their comparison with five other Asian cattle breeds along with a panel of animals from European taurine, African taurine and indicine cattle breeds. RESULTS: Asian cattle were found to be least differentiated which reflects their recent history. Amongst the Asian breeds Hainan, which is an indicine breed, had the lowest gene diversity while Yanbian had the highest followed by Mongolian and Korean cattle. Amongst the Korean breeds Brown Hanwoo had the highest diversity followed by Brindle Hanwoo and Jeju Black. The genetic diversity in Asian cattle breeds was found comparable to the European taurines and more than the African taurines and Zebu cattle. Korean cattle breed, Brown Hanwoo was consistently found to be closer to Yanbian, a Chinese cattle breed. We found low divergence and moderate levels of genetic diversity among the native Korean breeds. Indicine introgression from Hainan was seen in other Asian breeds. From Europe, Limousin, Holstein and Hereford introgression was found in Asian breeds. CONCLUSIONS: In this study we provide a genome-wide insight into the genetic history of the native cattle breeds of Korea. The outcomes of this study will help in prioritization and designing of the conservation plans.

Genomic structure, expression and association study of the porcine FSD2.

The fibronectin type III and SPRY domain containing 2 (FSD2) on porcine chromosome 7 is considered a candidate gene for pork quality, since its two domains, which were present in fibronectin and ryanodine receptor. The fibronectin type III and SPRY domains were first identified in fibronectin and ryanodine receptor, respectively, which are candidate genes for meat quality. The aim of this study was to elucidate the genomic structure of FSD2 and functions of single nucleotide polymorphisms (SNPs) within FSD2 that are related to meat quality in pigs. Using a bacterial artificial chromosome clone sequence, we revealed that porcine FSD2 consisted of 13 exons encoding 750 amino acids. In addition, FSD2 was expressed in heart, longissimus dorsi muscle, psoas muscle, and tendon among 23 kinds of porcine tissues tested. A total of ten SNPs, including four missense mutations, were identified in the exonic region of FSD2, and two major haplotypes were obtained based on the SNP genotypes of 633 Berkshire pigs. Both haplotypes were associated significantly with intramuscular fat content (IMF, P < 0.020) and moisture percentage (MP, P < 0.002). Moreover, haplotype 2 was associated with meat color, affecting yellowness (P = 0.002). These haplotype effects were further supported by the alteration of putative protein structures with amino acid substitutions. Taken together, our results suggest that FSD2 haplotypes are involved in regulating meat quality including IMF, MP, and meat color in pigs, and may be used as meaningful molecular makers to identify pigs with preferable pork quality.

Gene Expression Patterns Associated with Peroxisome Proliferator-activated Receptor (PPAR) Signaling in the Longissimus dorsi of Hanwoo (Korean Cattle).

Adipose tissue deposited within muscle fibers, known as intramuscular fat (IMF or marbling), is a major determinant of meat quality and thereby affects its economic value. The biological mechanisms that determine IMF content are therefore of interest. In this study, 48 genes involved in the bovine peroxisome proliferator-activated receptor signaling pathway, which is involved in lipid metabolism, were investigated to identify candidate genes associated with IMF in the longissimus dorsi of Hanwoo (Korean cattle). Ten genes, retinoid X receptor alpha, peroxisome proliferator-activated receptor gamma (PPARG), phospholipid transfer protein, stearoyl-CoA desaturase, nuclear receptor subfamily 1 group H member 3, fatty acid binding protein 3 (FABP3), carnitine palmitoyltransferase II, acyl-Coenzyme A dehydrogenase long chain (ACADL), acyl-Coenzyme A oxidase 2 branched chain, and fatty acid binding protein 4, showed significant effects with regard to IMF and were differentially expressed between the low- and high-marbled groups (p<0.05). Analysis of the gene co-expression network based on Pearson's correlation coefficients identified 10 up-regulated genes in the high-marbled group that formed a major cluster. Among these genes, the PPARG-FABP4 gene pair exhibited the strongest correlation in the network. Glycerol kinase was found to play a role in mediating activation of the differentially expressed genes. We categorized the 10 significantly differentially expressed genes into the corresponding downstream pathways and investigated the direct interactive relationships among these genes. We suggest that fatty acid oxidation is the major downstream pathway affecting IMF content. The PPARG/RXRA complex triggers activation of target genes involved in fatty acid oxidation resulting in increased triglyceride formation by ATP production. Our findings highlight candidate genes associated with the IMF content of the loin muscle of Korean cattle and provide insight into the biological mechanisms that determine adipose deposition within muscle.

Homology modeling study of bovine µ-calpain inhibitor-binding domains.

The activated mammalian CAPN-structures, the CAPN/CAST complex in particular, have become an invaluable target model using the structure-based virtual screening of drug candidates from the discovery phase to development for over-activated CAPN linked to several diseases, such as post-ischemic injury and cataract formation. The effect of Ca²âº-binding to the enzyme is thought to include activation, as well as the dissociation, aggregation, and autolysis of small regular subunits. Unfortunately, the Ca²âº-activated enzyme tends to aggregate when provided as a divalent ion at the high-concentration required for the protease crystallization. This is also makes it very difficult to crystallize the whole-length enzyme itself, as well as the enzyme-inhibitor complex. Several parameters that influence CAPN activity have been investigated to determine its roles in Ca²âº-modulation, autoproteolysis, phosphorylation, and intracellular distribution and inhibition by its endogenous inhibitor CAST. CAST binds and inhibits CAPN via its CAPN-inhibitor domains (four repeating domains 1-4; CAST1-4) when CAPN is activated by Ca²âº-binding. An important key to understanding CAPN1 inhibition by CAST is to determine how CAST interacts at the molecular level with CAPN1 to inhibit its protease activity. In this study, a 3D structure model of a CAPN1 bound bovine CAST4 complex was built by comparative modeling based on the only known template structure of a rat CAPN2/CAST4 complex. The complex model suggests certain residues of bovine CAST4, notably, the TIPPKYQ motif sequence, and the structural elements of these residues, which are important for CAPN1 inhibition. In particular, as CAST4 docks near the flexible active site of CAPN1, conformational changes at the interaction site after binding could be directly related to CAST4 inhibitory activity. These functional interfaces can serve as a guide to the site-mutagenesis in research on bovine CAPN1 structure-function relationships for the design of small molecules inhibitors to prevent uncontrolled and unspecific degradation in the proteolysis of key protease substrates.

Understanding the interaction determinants of CAPN1 inhibition by CAST4 from bovines using molecular modeling techniques.

HCV-induced CAPN activation and its effects on virus-infected cells in a host-immune system have been studied recently. It has been shown that the HCV-nonstructural 5A protein acts as both an inducer and a substrate for host CAPN protease; it participates in suppressing the TNF-α-induced apoptosis response and downstream IFN-induced antiviral processes. However, little is known regarding the disturbance of antiviral responses generated by bovine CAPN activation by BVDV, which is a surrogate model of HCV and is one of the most destructive diseases leading to great economic losses in cattle herds worldwide. This is also thought to be associated with the effects of either small CAPN inhibitors or the natural inhibitor CAST. They mainly bind to the binding site of CAPN substrate proteins and competitively inhibit the binding of the enzyme substrates to possibly defend against the two viruses (HCV and BVDV) for anti-viral immunity. To devise a new stratagem to discover lead candidates for an anti-BVDV drug, we first attempted to understand the bovine CAPN-CAST interaction sites and the interaction constraints of local binding architectures, were well reflected in the geometry between the pharmacophore features and its shape constraints identified using our modeled bovine CAPN1/CAST4 complex structures. We propose a computer-aided molecular design of an anti-BVDV drug as a mimetic CAST inhibitor to develop a rule-based screening function for adjusting the puzzle of relationship between bovine CAPN1 and the BVDV nonstructural proteins from all of the data obtained in the study.

Gene Co-expression Analysis to Characterize Genes Related to Marbling Trait in Hanwoo (Korean) Cattle.

Marbling (intramuscular fat) is an important trait that affects meat quality and is a casual factor determining the price of beef in the Korean beef market. It is a complex trait and has many biological pathways related to muscle and fat. There is a need to identify functional modules or genes related to marbling traits and investigate their relationships through a weighted gene co-expression network analysis based on the system level. Therefore, we investigated the co-expression relationships of genes related to the 'marbling score' trait and systemically analyzed the network topology in Hanwoo (Korean cattle). As a result, we determined 3 modules (gene groups) that showed statistically significant results for marbling score. In particular, one module (denoted as red) has a statistically significant result for marbling score (p = 0.008) and intramuscular fat (p = 0.02) and water capacity (p = 0.006). From functional enrichment and relationship analysis of the red module, the pathway hub genes (IL6, CHRNE, RB1, INHBA and NPPA) have a direct interaction relationship and share the biological functions related to fat or muscle, such as adipogenesis or muscle growth. This is the first gene network study with m.logissimus in Hanwoo to observe co-expression patterns in divergent marbling phenotypes. It may provide insights into the functional mechanisms of the marbling trait.

Identification of recently selected mutations driven by artificial selection in hanwoo (korean cattle).

Hanwoo have been subjected over the last seventy years to intensive artificial selection with the aim of improving meat production traits such as marbling and carcass weight. In this study, we performed a signature of selection analysis to identify recent positive selected regions driven by a long-term artificial selection process called a breeding program using whole genome SNP data. In order to investigate homozygous regions across the genome, we estimated iES (integrated Extended Haplotype Homozygosity SNP) for the each SNPs. As a result, we identified two highly homozygous regions that seem to be strong and/or recent positive selection. Five genes (DPH5, OLFM3, S1PR1, LRRN1 and CRBN) were included in this region. To go further in the interpretation of the observed signatures of selection, we subsequently concentrated on the annotation of differentiated genes defined according to the iES value of SNPs localized close or within them. We also described the detection of the adaptive evolution at the molecular level for the genes of interest. As a result, this analysis also led to the identification of OLFM3 as having a strong signal of selection in bovine lineage. The results of this study indicate that artificial selection which might have targeted most of these genes was mainly oriented towards improvement of meat production.

Case report: Investigation of genetic mutations in a case of schistosomus reflexus in a Holstein dairy cattle fetus in Korea.

Schistosomus reflexus (SR) is one of the most common congenital anomalies found in cases of cattle dystocia; this disorder occurs mostly in cattle. Congenital anomalies such as SR are caused by various genetic and environmental factors, but no specific cause has been elucidated for SR. This study reports a case of SR in a Holstein dairy cattle fetus with congenital anomalies in Korea. Grossly, a distinct spine curvature was observed between the thoracic and lumbar vertebrae, accompanied by a consequential malformation from the sacrum to the occipital bone. Furthermore, the thoracic and abdominal organs were exposed. In computed tomography (CT) images, mild and severe kyphoscoliosis was observed in T1~11 and L1~6, respectively. Additionally, vertebral dysplasia was observed in S1~5 and Cd 1~5. To pinpoint the causal genes and mutations, we leveraged a custom 50K Hanwoo SNP-Chip and the Online Mendelian Inheritance in Animals (OMIA) database. As a result, we identified a nonsense mutation in apoptotic protease activating factor 1 (APAF1) within HH1 that was associated with a decrease in conception rate and an increase in abortion in Holstein dairy cattle. The genotype of the SR case was A/A, and most of the 1,142 normal Holstein dairy cattle tested as a control group had the genotype G/G. In addition, the A/A genotype did not exist in the control group. Based on the pathological, genetic, and radiological findings, the congenital abnormalities observed were diagnosed as SR.

Genome-wide association study revealed the genomic regions associated with skin pigmentation in an Ogye x White Leghorn F2 chicken population.

Skin color in chickens is an economically important trait that determines the first impression of a consumer toward a broiler and can ultimately affect consumer choice in the market. Therefore, identification of genomic regions associated with skin color is crucial for increasing the sales value of chickens. Although previous studies have attempted to reveal the genetic markers associated with the skin coloration in chickens, most were limited to investigations of candidate genes, such as melanin-related genes, and focused on case/control studies based on a single or small population. In this study, we performed a genome-wide association study (GWAS) on 770 F2 intercrosses produced by an experimental population of 2 chicken breeds, namely Ogye and White Leghorns, with different skin colors. The GWAS demonstrated that the L* value among the 3 skin color traits is highly heritable, and the genomic regions located on 2 chromosomes (20 and Z) were detected to harbor SNPs significantly associated with the skin color trait, accounting for most of the total genetic variance. Particular genomic regions spanning a ∼2.94 Mb region on GGA Z and a ∼3.58 Mb region on GGA 20 were significantly associated with skin color traits, and in these regions, certain candidate genes, including MTAP, FEM1C, GNAS, and EDN3, were found. Our findings could help elucidate the genetic mechanisms underlying chicken skin pigmentation. Furthermore, the candidate genes can be used to provide a valuable breeding strategy for the selection of specific chicken breeds with ideal skin coloration.

The idiosyncratic genome of Korean long-tailed chicken as a valuable genetic resource.

Today, breeds with ornamental traits such as exceptionally long tail feathers are economically valuable. However, the genetic basis of long-tail feathers is yet to be understood. To provide better understanding of long tail feathers, we sequenced Korean long-tailed chicken (KLC) genomes and compared them with genomes of other chicken breeds. We first analyzed the genome structure of KLC and its genomic relationship with other chickens and observed unique characteristics. Subsequently, we searched for genomic regions under selection. Feather keratin 1-like enriched region and several genes were found to have novel putative functions and effects on the long tail trait in KLC. Our findings support the value of KLC as a unique genetic resource and cast light on the genetic basis of long tail traits in avian species. We expect this novel knowledge to provide new genomic evidence and options for designing and implementing genetic improvements of ornamental chicken productivity through precision crossbreeding aids.

A chromosome-level genome assembly of the Korean crossbred pig Nanchukmacdon (Sus scrofa).

As plentiful high-quality genome assemblies have been accumulated, reference-guided genome assembly can be a good approach to reconstruct a high-quality assembly. Here, we present a chromosome-level genome assembly of the Korean crossbred pig called Nanchukmacdon (the NCMD assembly) using the reference-guided assembly approach with short and long reads. The NCMD assembly contains 20 chromosome-level scaffolds with a total size of 2.38 Gbp (N50: 138.77 Mbp). Its BUSCO score is 93.1%, which is comparable to the pig reference assembly, and a total of 20,588 protein-coding genes, 8,651 non-coding genes, and 996.14 Mbp of repetitive elements are annotated. The NCMD assembly was also used to close many gaps in the pig reference assembly. This NCMD assembly and annotation provide foundational resources for the genomic analyses of pig and related species.

Blood transcriptome comparison between sexes and their function in 4-week Rhode Island red chickens.

Sex is a major biological factor in the development and physiology of a sexual reproductive organism, and its role in the growing process is needed to be investigated in various species. We compare blood transcriptome between 5 males and 5 females in 4-week-old Rhode Island Red chickens and perform functional annotation of differentially expressed genes (DEGs). The results are as follows. 141 and 109 DEGs were located in autosomes and sex chromosomes, respectively. The gene ontology (GO) terms are significantly (p < 0.05) enriched, which were limb development, inner ear development, positive regulation of dendrite development, the KEGG pathway the TGF-beta signaling pathway, and melanogenesis (p < 0.05). These pathways are related to morphological maintenance and growth of the tissues. In addition, the SMAD2W and the BMP5 were involved in the TGF-beta signaling pathway, and both play an important role in maintaining tissue development. The major DEGs related to the development of neurons and synapses include the up-regulated NRN1, GDF10, SLC1A1, BMP5, NBEA, and NRXN1. Also, 7 DEGs were validated using RT-qPCR with high correlation (r 2 = 0.74). In conclusion, the differential expression of blood tissue in the early growing chicken was enriched in TGF-beta signaling and related to the development of neurons and synapses including SMAD2W and BMP5. These results suggest that blood in the early growing stage is differentially affected in tissue development, nervous system, and pigmentation by sex. For future research, experimental characterization of DEGs and a holistic investigation of various tissues and growth stages will be required.

Structure and function of the porcine TAP protein and its inhibition by the viral immune evasion protein ICP47.

The binding mode to TAP (i.e., the peptide transporter associated with antigen processing) from a viral peptide thus far has been unknown in the field of antiviral immunity, but an interfering mode from a virus-encoded TAP inhibitor has been well documented with respect to blocking the TAP function. In the current study, we predicted the structure of the pig TAP transporter and its inhibition complex by the small viral protein ICP47 of the herpes simplex virus (HSV) encoded by the TAP inhibitor to exploit inhibition of the TAP transporter as the host's immune evasion strategy. We found that the hot spots (residues Leu5, Tyr22, and Leu51) on the ICP47 inhibitor interface tended to prevail over the favored Leu and Tyr, which contributed to significant functional binding at the C-termini recognition principle of the TAP. We further characterized the specificity determinants of the peptide transporter from the pig TAP by the ICP47 inhibitor effects and multidrug TmrAB transporter from the Thermus thermophillus and its immunity regarding its structural homolog of the pig TAP. The specialized structure-function relationship from the pig TAP exporter could provide insight into substrate specificity of the unique immunological properties from the host organism. The TAP disarming capacity from all five viral inhibitors (i.e., the five virus-encoded TAP inhibitors of ICP47, UL49.5, U6, BNLF2a, and CPXV012 proteins) was linked to the infiltration of the TAP functional structure in an unstable conformation and the mounting susceptibility caused by the host's TAP polymorphism. It is anticipated that the functional characterization of the pig TAP transporter based on the pig genomic variants will lead to additional insights into the genotype and single nucleotide polymorphism (SNP) in relation to antiviral resistance and disease susceptibility.

Prediction of Hanwoo Cattle Phenotypes from Genotypes Using Machine Learning Methods.

Hanwoo was originally raised for draft purposes, but the increase in local demand for red meat turned that purpose into full-scale meat-type cattle rearing; it is now considered one of the most economically important species and a vital food source for Koreans. The application of genomic selection in Hanwoo breeding programs in recent years was expected to lead to higher genetic progress. However, better statistical methods that can improve the genomic prediction accuracy are required. Hence, this study aimed to compare the predictive performance of three machine learning methods, namely, random forest (RF), extreme gradient boosting method (XGB), and support vector machine (SVM), when predicting the carcass weight (CWT), marbling score (MS), backfat thickness (BFT) and eye muscle area (EMA). Phenotypic and genotypic data (53,866 SNPs) from 7324 commercial Hanwoo cattle that were slaughtered at the age of around 30 months were used. The results showed that the boosting method XGB showed the highest predictive correlation for CWT and MS, followed by GBLUP, SVM, and RF. Meanwhile, the best predictive correlation for BFT and EMA was delivered by GBLUP, followed by SVM, RF, and XGB. Although XGB presented the highest predictive correlations for some traits, we did not find an advantage of XGB or any machine learning methods over GBLUP according to the mean squared error of prediction. Thus, we still recommend the use of GBLUP in the prediction of genomic breeding values for carcass traits in Hanwoo cattle.

Corrigendum: Genomic Prediction Accuracy Using Haplotypes Defined by Size and Hierarchical Clustering Based on Linkage Disequilibrium.

[This corrects the article DOI: 10.3389/fgene.2020.00134.].