A chromosome-level genome assembly of Cape hare (Lepus capensis).

The Cape hare (Lepus capensis) is among the most widely distributed hare species globally, inhabiting extensive regions across Africa, the Middle East, and Central Asia. However, evolutionary and genetic research on L. capensis was seriously impeded by the absence of a reference genome. Here, we assembled and constructed a chromosome-level genome of L. capensis (with scaffolds anchored to 25 chromosomes and a total assembled length of 2.9 Gb, achieving a contig N50 length of 124.44 Mb) using PacBio HiFi sequencing and Hi-C assembly technology. Evaluation using BUSCO indicated the genome assembly to be 98.2% complete. The de novo prediction revealed that repetitive sequences constitute 46.13% of the entire genome, and long interspersed nuclear elements (LINEs) constituted the largest portion. We annotated a total of 13, 868 protein-coding genes using transcriptomes from two tissues (muscle and skin). This high-quality reference genome serves as a valuable genomic resource for advancing genetic studies in this species.

Spatial metabolomics, LC-MS and RNA-Seq reveal the effect of red and white muscle on rabbit meat flavor.

Meat quality is a key factor influencing consumer purchasing decisions. Muscle composition consists of various types of myofibers (type I and type IIa, IIb, IIx myofibers), and the relative composition of fiber types has a significant impact on the overall biochemical properties and flavor of fresh meat. However, the relationship between biochemical changes in myofibers and their impact on meat quality remains underexplored. In this study, we compared the differences in meat quality by examining different muscles in rabbits, each containing different muscle fiber types. We focused on the adductor (ADD) and semitendinosus (ST) as our research subjects and investigated skeletal muscle metabolism at the individual myofibers level using Spatial metabolomics. Additionally, we utilized LC-MS and RNA-Seq to explore the molecular mechanisms underlying the metabolic differences between red and white muscle fibers. Our findings demonstrated that variations in myofiber composition significantly influenced meat color, pH, water content, and drip loss. Spatial metabolomics analysis identified 22 unique red and white muscle fingerprint metabolites, while LC-MS analysis revealed 123 differential metabolites, and these differential metabolites were mainly enriched in the pathways of ABC transporters, Biosynthesis of amino acids, glutathione metabolism, and arginine biosynthesis. To further elucidate the molecular mechanism of differential metabolism in ADD and ST, we identified 2248 differentially expressed genes (DEGs) by RNA-Seq and then combined DEGs with DMs for joint analysis. We found that red muscle exhibited higher levels of metabolites such as L-glutamic acid, glutathione, ascorbate, ornithine, oxidized glutathione, gamma-L-glutamyl-L-cysteine, cysteinylglycine, fumaric acid, gamma-aminobutyric acid. Additionally, related metabolic genes such as MGST1, ODC1, MGST3 and PRDX6 were highly expressed in ST muscle. These metabolites and genes were enriched in the glutathione and nicotinamide pathways, and had significant effects on meat color and drip loss. Moreover, red muscle contained more flavor compounds and nutrients, including adenosine monophosphate (AMP), ornithine, citrulline, taurine, acetyl phosphate, L-glutamic acid metabolites, as well as taurine and hypotaurine metabolites. Our results demonstrate that fresh meat with a higher proportion of red muscle fibers exhibited superior meat quality, enhanced flavor, and higher nutrient content. Furthermore, red muscle contains more antioxidant metabolites that can effectively prevent meat oxidation during the production process.

Unraveling the genetic and epigenetic landscape governing intramuscular fat deposition in rabbits: Insights and implications.

Intramuscular fat (IMF) content is a predominant factor recognized to affect rabbit meat quality, directly impacting flavor, juiciness, and consumer preference. Despite its significance, the major interplay of genetic and epigenetic factors regulating IMF in rabbits remains largely unexplored. This review sheds light on this critical knowledge gap, offering valuable insights and future directions. We delve into the potential role of established candidate genes from other livestock (e.g. PPARγ, FABP4, and SCD) in rabbits, while exploring the identified novel genes of IMF in rabbits. Furthermore, we explored the quantitative trait loci studies in rabbit IMF and genomic selection approaches for improving IMF content in rabbits. Beyond genetics, this review unveils the exciting realm of epigenetic mechanisms modulating IMF deposition. We explored the potential of DNA methylation patterns, histone modifications, and non-coding RNA-mediation as fingerprints for selecting rabbits with desirable IMF levels. Additionally, we explored the possibility of manipulating the epigenetic landscape through nutraceuticals interventions to promote favorable IMF depositions. By comprehensively deciphering the genomic and epigenetic terrain of rabbit intramuscular fat regulation, this study aims to assess the existing knowledge regarding the genetic and epigenetic factors that control the deposition of intramuscular fat in rabbits. By doing so, we identified gaps in the current research, and suggested potential areas for further investigation that would enhance the quality of rabbit meat. This can enable breeders to develop targeted breeding strategies, optimize nutrition, and create innovative interventions to enhance the quality of rabbit meat, meet consumer demands and increase market competitiveness.

Single-nucleus multiomics unravels the genetic mechanisms underlying musk secretion in Chinese forest musk deer (Moschus berezovskii).

Musk secreted by the musk glands in male forest musk deer (FMD; Moschus berezovskii) is highly valued for its pharmaceutical and perfumery applications. However, the regulatory mechanisms underlying musk secretion are not well understood. This study aimed to investigate the genes and transcription factors involved in musk secretion across different periods and ages. We analyzed the musk glands of adult male FMD during the non-secretory and secretory periods, as well as juvenile and adult male FMD during the secretory period, using single-cell multiome ATAC+gene expression technique. Our analysis identified 13 cell types, including acinar cells of Types 1 and 2. Chromatin accessibility analysis and gene expression data confirmed that the genes Map3k2, Hsd17b12, and Jun are critical for musk secretion. Additionally, EHF, NR4A2, and FOXO1 proteins play crucial regulatory roles. Weighted gene co-expression network analysis (WGCNA) highlighted the importance of GnRH signaling pathway in musk secretion. Gene set enrichment analysis (GSEA) showed that the steroid hormone biosynthesis pathway is notably enriched in acinar cells. Furthermore, intercellular communication appears to influence both the initiation and maintenance of musk secretion. These findings provide valuable insights into the molecular pathways of musk secretion in FMD, offering potential avenues for increasing musk production and developing treatment for inflammation and tumors.

Epigenetic mechanism of Gtl2-miRNAs causes the primitive sheep characteristics found in purebred Merino sheep.

BACKGROUND: It is not uncommon for some individuals to retain certain primitive characteristics even after domestication or long-term intensive selection. Wild ancestors or original varieties of animals typically possess strong adaptability to environmental preservation, a trait that is often lacking in highly artificially selected populations. In the case of the Merino population, a world-renowned fine wool sheep breed, a phenotype with primitive coarse wool characteristic has re-emerged. It is currently unclear whether this characteristic is detrimental to the production of fine wool or whether it is linked to the adaptability of sheep. The underlying genetic/epigenetic mechanisms behind this trait are also poorly understood. RESULTS: This study identified lambs with an ancestral-like coarse (ALC) wool type that emerged during the purebred breeding of Merino fine wool sheep. The presence of this primitive sheep characteristic resulted in better environmental adaptability in lambs, as well as improved fine wool yield in adulthood. Reciprocal cross experiments revealed that the ALC phenotype exhibited maternal genetic characteristics. Transcriptomic SNP analysis indicated that the ALC phenotype was localized to the imprinted Gtl2-miRNAs locus, and a significant correlation was found between the ALC wool type and a newly identified short Interstitial Telomeric Sequences (s-ITSs) at this locus. We further confirmed that a novel 38-nt small RNA transcribed from these s-ITSs, in combination with the previously reported 22-nt small RNAs cluster from the Gtl2-miRNAs locus, synergistically inhibited PI3K/AKT/Metabolic/Oxidative stress and subsequent apoptotic pathways in wool follicle stem cells, resulting in the ALC wool type. The necessity of Gtl2-miRNAs in controlling primary hair follicle morphogenesis, as well as the wool follicle type for ALC wool lambs, was verified using intergenic differentially methylated region-knockout mice. CONCLUSION: The ALC wool type of Merino sheep, which does not reduce wool quality but increases yield and adaptability, is regulated by epigenetic mechanisms in the imprinted Gtl2-miRNAs region on sheep chromosome 18, with the maternally expressed imprinted gene responsible for the ALC phenotype. This study highlights the significance of epigenetic regulation during embryonic and juvenile stages and emphasizes the advantages of early adaptation breeding for maternal parents in enhancing the overall performance of their offspring.

Quercetin promotes the secretion of musk by regulating the hormone level and microbial structure of forest musk deer.

Musk is a scarce and precious medical resource secreted by male forest musk deer (FMD). Current research to promote musk secretion in FMD has used almost exclusively hormone injections, but this approach can be detrimental to the health of FMD. In order to conserve this endangered species as much as possible while increasing the production of musk, this study first used bioinformatics methods to predict the function of quercetin, a flavonoid that promotes testosterone (T) production and prevents late-onset male hypogonadism. On the basis of good prediction effect, different concentrations of quercetin were added to the diet of FMD. The results showed that quercetin could change the levels of T, luteinizing hormone releasing hormone, luteinizing hormone, and estradiol, and regulate the structure of intestinal microorganisms and musk microorganisms of FMD. Moreover, there is a correlation among musk components, hormones, intestinal microorganisms, and musk microorganisms, which indicates that the production of musk may be regulated by these three at the same time, and the addition of quercetin with 800 mg per kg diet could significantly increase the yield of muscone (P < 0.05), the most effective ingredient in musk. In addition, quercetin decreased the high level of cortisol during musk secretion, which may relieve the stress on FMD in this process. This may help to protect the health of FMD. Combined with the results of software prediction, we finally proposed a possible mechanism for the complex process of musk secretion in FMD with a view to providing ideas for further studies.

Association of variants and expression levels of MYOD1 gene with carcass and muscle characteristic traits in domestic pigeons.

This study was to investigate the correlations of myogenic differentiation 1 (MYOD1) gene polymorphisms with carcass traits and its expression with breast muscle development in pigeons. Four SNPs were found in the pigeon MYOD1 gene. Correlation analysis showed that individuals with AA genotype at both SNPs g.2967A > G (p < .01) and g.3044G > A (p < .05) have significantly higher live weight (LW), carcass weight (CW), semi-eviscerated weight (SEW), eviscerated weight (EW) and breast muscle weight (BMW). Moreover, the two SNPs also had the same significant effects on MYOD1 mRNA expression levels in breast muscle of pigeons, ie, the AA genotype showed higher MYOD1 mRNA expression levels. The diameter and cross-section area of muscle fibers continuously increased from 0w to 4w (p < .05), accompanied with the increasing expression of MYOD1 gene, while the density decreased (p < .05) dramatically from 0w to 1w and continuously fell over in the next few weeks (p > .05). What's more, the expression level of MYOD1 gene was positively correlated with a diameter (r = 0.937, p < .05) and cross-sectional area (r = 0.956, p < .01) of myofiber, and negatively correlated with density (r = -0.769, p < .01). The results showed that individuals with AA genotype at both SNPs g.2967A > G and g.3044G > A have showed higher carcass traits (LW, CW, SEW, EW, and BMW) and higher MYOD1 mRNA expression level in breast muscle than AB and BB genotypes. Moreover, the expression level of MYOD1 gene was closely correlated with muscle characteristic traits, indicating variants of MYOD1 gene was closely related to muscle development and could be a potential candidate gene in marker-assisted selection of pigeons.

Exploring the growing forest musk deer (Moschus berezovskii) dietary protein requirement based on gut microbiome.

It is necessary to assess the appropriate dietary protein level of the forest musk deer (FMD), as nutritional needs are unclear. The microbiome in gastrointestinal tracts plays an important role in regulating nutrient utilization, absorption and host growth or development. Thus, we aimed to evaluate growth performance, nutrient digestibility and fecal microbiome of growing FMD supplied with different protein levels of diets. Eighteen 6-month-old male FMD with an initial weight 5.0 ± 0.2 kg were used in a 62-day trial. The animals were randomly distributed to three groups, the dietary crude protein (CP) level was 11.51% (L), 13.37% (M), and 15.48% (H). The results showed that the CP digestibility decreased as dietary CP level increased (p < 0.01). Compared with group L and H, FMD in M group has higher average daily gain, feed efficiency and neutral detergent fiber digestibility. For the fecal bacterial community, the percentage of Firmicutes was increased, Bacteroidetes was decreased and the diversity of microbiota significantly reduced (p < 0.05) with the increasing of dietary protein. The proportion of Ruminococcaceae_005, Ruminococcaceae_UCG-014 and uncultured_bacterium_f_Lachnospiraceae were significantly increased wtih rising CP, the proportions of Bacteroides and Rikenellaceae_RC9_gut_group were significantly decrease nevertheless at the genus level. The higher abundance of f_Prevotellaceae and g_Prevotellaceae_UCG_004 were found at M group by LEfSe analysis. The relative abundance of uncultured_bacterium_f_Ruminococcaceae was positively correlated with the average daily gain and feed conversion ratio (p < 0.05), whereas Family_XIII_AD3011_group was negatively correlated with feed conversion ratio (p < 0.05). The UPGMA tree showed L and M groups were closer in clustering relationship, while H group was clustered separately into a branch, which indicated that the bacterial structure had changed greatly with protein level increased from 13.37 to 15.48%. Overall, our results indicated that the optimum dietary CP for the growing FMD was 13.37%.

Association of blood APMAP content and meat quality trait in Rex rabbits.

APMAP is single transmembrane arylesterase which plays a cardinal role in adipogenesis. In this experiment, three tissue and blood samples of Rex rabbits at 3 growing periods were selected. The expression levels of APMAP gene in different tissues were detected by real-time fluorescence quantitative PCR and the content of APMAP in the blood was detected by Elisa. The results showed that fat deposition, the expression of APMAP in muscle and the content of APMAP in the blood increased rapidly during the growth of Rex rabbits. The correlation analysis showed that the correlation coefficient between APMAP content in the blood and the expression level of APMAP gene in longissimus lumborum muscle was 0.75(p < 0.05); the correlation coefficients between APMAP content in the blood and intramuscular fat and 24-hour pH were 0.90 (p < 0.01) and 0.75 (p < 0.05), respectively. According to the analysis results, we inferred APMAP content in the blood in Rex rabbits may influence meat quality and the meat quality of high APMAP content in the blood in Rex rabbits is better. These results revealed APMAP content in the blood may be one of the important signs for meat quality traits of molecular markers.

Detection the eDNA of Batrachuperus taibaiensis from the Zhouzhi Heihe River Using a Nested PCR Method and DNA Barcoding.

The Taibai stream salamander (Batrachuperus taibaiensis) is a recently described species of the genus Batrachuperus that occurs in the Zhouzhi Heihe River and is endangered in its native range. Here, we have established a method for water environmental DNA (eDNA) analysis of Batrachuperus using a series of optimizations. We have designed a specific set of primers for the genus Batrachuperus to amplify a 160 bp fragment of Cytb. The sequences were obtained from nested PCR on eDNA extracted from water samples, after which DNA barcoding was performed according to sequence analysis to determine the presence of the target species in the water. The method was validated using water from the Zhouzhi Heihe River with known B. taibaiensis populations and found that B. taibaiensis eDNA can move at least 150 m downstream from its point of origin. This study is the first to establish an optimal method for obtaining the eDNA of Batrachuperus from water samples, which provides a theoretical basis for resource investigation and the protection of B. taibaiensis in future research. It is also an example of the eDNA extraction of other species that live in similar waters and are less genetically diverse between species.

Exploring the genomic resources and analysing the genetic diversity and population structure of Chinese indigenous rabbit breeds by RAD-seq.

BACKGROUND: Chinese indigenous rabbits have distinct characteristics, such as roughage resistance, stress resistance and environmental adaptability, which are of great significance to the sustainable development of the rabbit industry in China. Therefore, it is necessary to study the genetic diversity and population structure of this species and develop genomic resources. RESULTS: In this study, we used restriction site-associated DNA sequencing (RAD-seq) to obtain 1,006,496 SNP markers from six Chinese indigenous rabbit breeds and two imported rabbit breeds. Jiuyishan and Fujian Yellow rabbits showed the highest nucleotide diversity (π) and decay of linkage disequilibrium (LD), as well as higher observed heterozygosity (Ho) and expected heterozygosity (He), indicating higher genetic diversity than other rabbits. The inbreeding coefficient (FIS) of New Zealand rabbits and Belgian rabbits was higher than that of other rabbits. The neighbour-joining (NJ) tree, principal component analysis (PCA), and population structure analysis of autosomes and Y chromosomes showed that Belgian, New Zealand, Wanzai, Sichuan White, and Minxinan Black rabbits clustered separately, and Fujian Yellow, Yunnan Colourful, and Jiuyishan rabbits clustered together. Wanzai rabbits were clearly separated from other populations (K = 3), which was consistent with the population differentiation index (FST) analysis. The selection signature analysis was performed in two populations with contrasting coat colours. With Sichuan White and New Zealand rabbits as the reference populations and Minxinan Black and Wanzai rabbits as the target populations, 408, 454, 418, and 518 genes with a selection signature, respectively, were obtained. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on the genes with a selection signature. The results showed that the genes with a selection signature were enriched in the melanogenesis pathway in all four sets of selection signature analyses. CONCLUSIONS: Our study provides the first insights into the genetics and genomics of Chinese indigenous rabbit breeds and serves as a valuable resource for the further effective utilization of the species.

An EAV-HP insertion in the promoter region of SLCO1B3 has pleiotropic effects on chicken liver metabolism based on the transcriptome and proteome analysis.

Solute carrier organic anion transporter 1B3 (SLCO1B3) is an important liver primarily highly expressed gene, its encoded protein (OATP1B3) involved in the transport of multi-specific endogenous and exogenous substances. We previously reported that an EAV-HP inserted mutation (IM+) in the 5' flanking region of SLCO1B3 was the causative mutation of chicken blue eggs, and a further research showed that IM+ significantly reduced the expression of SLCO1B3 in liver. Herein, we confirmed a cholate response element (IR-1) played an important role in activating SLCO1B3 and in vitro experiments showed that the activation of IR-1 can be significantly reduced by the EAV-HP IM+ . We performed transcriptome and proteomic analysis using the same set of IM+ and IM- liver tissues from Yimeng hens (a Chinese indigenous breed) to study the effect of SLCO1B3 and OATP1B3 expression reduction on chicken liver function. The results showed that common differential expression pathways were screened out from both transcriptome and proteome, in which fatty acid metabolism and drug metabolism-cytochrome P450 were significantly enriched in the KEGG analysis. The lipid-related metabolism was weakened in IM+ group, which was validated by serum biochemical assay. We unexpectedly found that EAV-HP fragment was highly expressed in the liver of the IM+ chickens. We cloned the EAV-HP full-length transcript and obtained the complete open reading frame. It is worth noting that there was some immune related differential expressed genes, such as NFKBIZ, NFKBIA, and IL1RL1, which were higher expressed in the IM+ group, which may due to the high expression of EAV-HP. Our study showed that EAV-HP IM+ reduced the expression of SLCO1B3 in liver, resulting in the decrease of fatty metabolism and exogenous substance transport capacity. The mutation itself also expressed in the liver and may be involved in the immune process. The mechanism needs further study.

An EAV-HP insertion in the 5' flanking region of SLCO1B3 is associated with its tissue-expression profile in blue-eggshell Yimeng chickens (Gallus gallus).

We previously reported that blue eggshell color in chickens is associated with a partial endogenous retroviral (EAV-HP) insertion in the promoter region of the solute carrier organic anion transporter family member 1B3 (SLCO1B3) gene. The EAV-HP sequence includes numerous regulatory elements, which may modulate the expression of adjacent genes. To determine whether this insertion influences the expression of neighboring genes, we screened the expression of solute carrier organic anion transporter family members 1C1, 1B1 (SLCO1C1, SLCO1B1), and SLCO1B3 in 13 and 10 tissues from female and male Yimeng chickens, respectively. We observed that the insertion only significantly modulated the expression of SLCO1B3 and did not majorly affect that of SLCO1C1 and SLCO1B1. High expression of SLCO1B3 was detected in the shell gland, magnum, isthmus, and vagina of the oviduct in female blue-eggshell chickens. We also observed ectopic expression of SLCO1B3 in the testes of male chickens. SLCO1B3 is typically highly expressed in the liver; however, the EAV-HP insertion significantly reduces SLCO1B3 expression. As a liver-specific transporter, a reduction in the expression of SLCO1B3 may affect liver metabolism, particularly that of bile acids. We also detected higher ectopic expression of SLCO1B3 in the lungs of birds heterozygous for the EAV-HP insertion than in homozygous genotypes. In conclusion, we confirmed that the EAV-HP insertion modifies SLCO1B3 expression, and showed, for the first time, similar expression profile of this gene in all parts of the oviduct in females and testis in males. We also observed different levels of SLCO1B3 expression in the liver, which were associated with the EAV-HP insertion, and significantly higher expression in the lungs of birds with heterozygous genotype. The effects of these changes in the SLCO1B3 expression pattern on the function of the tissues warrant further investigation.

Genomic Analysis Reveals Pleiotropic Alleles at EDN3 and BMP7 Involved in Chicken Comb Color and Egg Production.

Artificial selection is often associated with numerous changes in seemingly unrelated phenotypic traits. The genetic mechanisms of correlated phenotypes probably involve pleiotropy or linkage of genes related to such phenotypes. Dongxiang blue-shelled chicken, an indigenous chicken breed of China, has segregated significantly for the dermal hyperpigmentation phenotype. Two lines of the chicken have been divergently selected with respect to comb color for over 20 generations. The red comb line chicken produces significantly higher number of eggs than the dark comb line chicken. The objective of this study was to explore potential mechanisms involved in the relationship between comb color and egg production among chickens. Based on the genome-wide association study results, we identified a genomic region on chromosome 20 involving EDN3 and BMP7, which is associated with hyperpigmentation of chicken comb. Further analyses by selection signatures in the two divergent lines revealed that several candidate genes, including EDN3, BMP7, BPIFB3, and PCK1, closely located on chromosome 20 are involved in the development of neural crest cell and reproductive system. The two genes EDN3 and BMP7 have known roles in regulating both ovarian function and melanogenesis, indicating the pleiotropic effect on hyperpigmentation and egg production in blue-shelled chickens. Association analysis for egg production confirmed the pleiotropic effect of selected loci identified by selection signatures. The study provides insights into phenotypic evolution due to genetic variation across the genome. The information might be useful in the current breeding efforts to develop improved breeds for egg production.

Genome-wide profiling of RNA editing sites in sheep.

BACKGROUND: The widely observed RNA-DNA differences (RDDs) have been found to be due to nucleotide alteration by RNA editing. Canonical RNA editing (i.e., A-to-I and C-to-U editing) mediated by the adenosine deaminases acting on RNA (ADAR) family and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) family during the transcriptional process is considered common and essential for the development of an individual. To date, an increasing number of RNA editing sites have been reported in human, rodents, and some farm animals; however, genome-wide detection of RNA editing events in sheep has not been reported. The aim of this study was to identify RNA editing events in sheep by comparing the RNA-seq and DNA-seq data from three biological replicates of the kidney and spleen tissues. RESULTS: A total of 607 and 994 common edited sites within the three biological replicates were identified in the ovine kidney and spleen, respectively. Many of the RDDs were specific to an individual. The RNA editing-related genes identified in the present study might be evolved for specific biological functions in sheep, such as structural constituent of the cytoskeleton and microtubule-based processes. Furthermore, the edited sites found in the ovine BLCAP and NEIL1 genes are in line with those in previous reports on the porcine and human homologs, suggesting the existence of evolutionarily conserved RNA editing sites and they may play an important role in the structure and function of genes. CONCLUSIONS: Our study is the first to investigate RNA editing events in sheep. We screened out 607 and 994 RNA editing sites in three biological replicates of the ovine kidney and spleen and annotated 164 and 247 genes in the kidney and spleen, respectively. The gene function and conservation analysis of these RNA editing-related genes suggest that RNA editing is associated with important gene function in sheep. The putative functionally important RNA editing sites reported in the present study will help future studies on the relationship between these edited sites and the genetic traits in sheep.

The Origin of a Coastal Indigenous Horse Breed in China Revealed by Genome-Wide SNP Data.

: The Jinjiang horse is a unique Chinese indigenous horse breed distributed in the southern coastal areas, but the ancestry of Jinjiang horses is not well understood. Here, we used Equine SNP70 Bead Array technology to genotype 301 horses representing 10 Chinese indigenous horse breeds, and we integrated the published genotyped data of 352 individuals from 14 foreign horse breeds to study the relationships between Jinjiang horses and horse breeds from around the world. Principal component analysis (PCA), linkage disequilibrium (LD), runs of homozygosity (ROH) analysis, and ancestry estimating methods were conducted to study the population relationships and the ancestral sources and genetic structure of Jinjiang horses. The results showed that there is no close relationship between foreign horse breeds and Jinjiang horses, and Jinjiang horses shared a similar genetic background with Baise horses. TreeMix analysis revealed that there was gene flow from Chakouyi horses to Jinjiang horses. The ancestry analysis showed that Baise horses and Chakouyi horses are the most closely related ancestors of Jinjiang horses. In conclusion, our results showed that Jinjiang horses have a native origin and that Baise horses and Chakouyi horses were key ancestral sources of Jinjiang horses. The study also suggested that ancient trade activities and the migration of human beings had important effects on indigenous horse breeds in China.

Genome-wide association study and a post replication analysis revealed a promising genomic region and candidate genes for chicken eggshell blueness.

The eggshell blueness is an interesting object for chicken genetic studies and blue-shelled chicken industry, especially after the discovery of the causative mutation of chicken blue eggshell. In the present study, genome wide association study (GWAS) was conducted in Chinese Dongxiang blue-shelled chicken underlying four traits of blue eggshell pigments: quantity of biliverdin (QB), quantity of protoporphyrin (QP), quantity of total pigment (QT), and color density trait (CD). A total of 139 individuals were randomly collected for GWAS. We detected two SNPs in genome-wise significance and 35 in suggestive significance, 24 out of the 37 SNP were located either within intron/exon or near 15 genes in a range of ~1.17 Mb on GGA21. For further confirmation of the identified SNP loci by GWAS, the follow-up replication studies were performed in two populations. A total of 146 individuals of the second generation derived from the former GWAS population, as well as 280 individuals from an alternative independent population were employed for genotyping by MALDI-TOF MS in a genotype-phenotype association study. Eighteen SNPs evenly distributed on the GGA21 significant region were successfully genotyped in the two populations, of which 4 and 6 SNP loci were shown significantly associated with QB, QT and QP in the two repeat populations, respectively. Further, the SNPs were narrowed down to a region of ~ 653.819 Kb on GGA21 that harbors five candidate genes: AJAP1, TNFRSF9, C1ORF174, CAMTA1, and CEP104. Shell gland of chickens laying dark and light blue eggshell was chosen for detection of mRNA expression of the five candidate genes. The results showed differential expression levels of these genes in the two groups. The specific function of these genes has not yet been defined clearly in chickens and further in-depth studies are needed to explore the new functional role in chicken eggshell blueness.

Molecular cloning and characterization of the endothelin 3 gene in black bone sheep.

BACKGROUND: Black bone sheep was first discovered in Yunnan province of China in 1970, with unique black pigmentation on the body and internal organs. Endothelin 3 (EDN3) has been known as a key gene causing hyperpigmentation in black bone chicken, the Silky fowl. METHODS: In this study, EDN3 was employed as a candidate gene for regulating black color pigmentation. First, EDN3 was cloned from sheep to obtain the full-length cDNA by using the rapid amplification of cDNA ends (RACE). Genomic EDN3 was screened and a total of thirty predicted single nucleotide polymorphisms (SNPs) were genotyped for allele and genotype frequency analysis in a case-control study involving two black bone sheep populations. Genomic copy number analysis of EDN3 in sheep was conducted to measure the variation in copy number. EDN3 expression levels were observed among the groups in adult liver, lymph node, and kidney tissues, as well as embryo kidney samples. Also, among the tissues of black bone and non-black bone sheep. RESULTS: The size of the full-length cDNA was 1,578 bp, which included 426 bp of 5'-untranslated region (5'-UTR), an open reading frame (ORF) of 639 bp encoding a protein of 212 amino acids, and a 3'-UTR of 513 bp. Genotype and allele frequencies of all the discovered SNPs were found insignificantly different in black bone and non-black bone sheep (P > 0.05). Genomic copy number analysis of EDN3 in sheep revealed no significant difference between the two sheep groups. No significant variations were found in the adult liver and kidney embryo samples. However, the expression in lymph node and kidney tissue was significantly higher in black bone sheep than that in non-black bone sheep (P < 0.05). Significant variations in the EDN3 expression levels were observed among the tissues of non-black bone sheep. CONCLUSIONS: The findings of the present study indicate that unlike in Silky chickens, EDN3 is not responsible for hyperpigmentation but may play a key functional role in immune and excretory systems of black bone sheep.

Hyperpigmentation Results in Aberrant Immune Development in Silky Fowl (Gallus gallus domesticus Brisson).

The Silky Fowl (SF) is known for its special phenotypes and atypical distribution of melanocytes among internal organs. Although the genes associated with melanocyte migration have been investigated substantially, there is little information on the postnatal distribution of melanocytes in inner organs and the effect of hyperpigmentation on the development of SF. Here, we analyzed melanocyte distribution in 26 tissues or organs on postnatal day 1 and weeks 2, 3, 4, 6, 10, and 23. Except for the liver, pancreas, pituitary gland, and adrenal gland, melanocytes were distributed throughout the body, primarily around blood vessels. Interaction between melanocytes and the tissue cells was observed, and melanin was transported by filopodia delivery through engulfed and internalized membrane-encapsulated melanosomes. SFs less than 10 weeks old have lower indices of spleen, thymus, and bursa of Fabricius than White Leghorns (WLs). The expression levels of interferon-γ and interlukin-4 genes in the spleen, and serum antibody levels against H5N1 and infectious bursal disease virus were lower in SF than in WL. We also found immune organ developmental difference between Black-boned and non-Black- boned chickens from SFs and WLs hybrid F2 population. However, degeneration of the thymus and bursa of Fabricius occurred later in SF than in WL after sexual maturity. Analysis of apoptotic cells and apoptosis-associated Bax and Bcl-2 proteins indicated that apoptosis is involved in degeneration of the thymus and bursa of Fabricius. Therefore, these results suggest that hyperpigmentation in SF may have a close relationship with immune development in SF, which can provide an important animal model to investigate the roles of melanocyte.

Molecular phylogenetic analysis of Chinese indigenous blue-shelled chickens inferred from whole genomic region of the SLCO1B3 gene.

In total, 246 individuals from 8 Chinese indigenous blue- and brown-shelled chicken populations (Yimeng Blue, Wulong Blue, Lindian Blue, Dongxiang Blue, Lushi Blue, Jingmen Blue, Dongxiang Brown, and Lushi Brown) were genotyped for 21 SNP markers from the SLCO1B3 gene to evaluate phylogenetic relationships. As a representative of nonblue-shelled breeds, White Leghorn was included in the study for reference. A high proportion of SNP polymorphism was observed in Chinese chicken populations, ranging from 89% in Jingmen Blue to 100% in most populations, with a mean of 95% across all populations. The White Leghorn breed showed the lowest polymorphism, accounting for 43% of total SNPs. The mean expected heterozygosity varied from 0.11 in Dongxiang Blue to 0.46 in Yimeng Blue. Analysis of molecular variation (AMOVA) for 2 groups of Chinese chickens based on eggshell color type revealed 52% within-group and 43% between-group variations of the total genetic variation. As expected, FST and Reynolds' genetic distance were greatest between White Leghorn and Chinese chicken populations, with average values of 0.40 and 0.55, respectively. The first and second principal coordinates explained approximately 92% of the total variation and supported the clustering of the populations according to their eggshell color type and historical origins. STRUCTURE analysis showed a considerable source of variation among populations for the clustering into blue-shelled and nonblue-shelled chicken populations. The low estimation of genetic differentiation (FST) between Chinese chicken populations is possibly due to a common historical origin and high gene flow. Remarkably similar population classifications were obtained with all methods used in the study. Aligning endogenous avian retroviral (EAV)-HP insertion sequences showed no difference among the blue-shelled chickens.