Functional roles for AGPAT6 in milk fat synthesis of buffalo mammary epithelial cells.

AGPAT6 plays a crucial role in the triglyceride (TG) synthesis pathway in mammals. However, its roles in buffalo lactation remain unknown. Therefore, we investigated the functional roles of AGPAT6 in milk fat synthesis by transfecting overexpression and lentivirus interference vectors in buffalo mammary epithelial cells (BuMECs) in vitro. AGPAT6 overexpression in BuMECs significantly enhanced the mRNA expression of FABP4, SLC27A6, ACSL1, DGAT1, DGAT2, LPIN1, INSIG1, CEBPA and SREBF1 genes, and significantly reduced that of XDH, CPT1A, LIPE, INSIG2 and PPARGC1A, but has no significant influence to the mRNA abundance of FABP3, GPAM, PPARG and SREBF2. However, the interference with AGPAT6, the mRNA expression of FABP4, SLC27A6, ACSL1, DGAT1, DGAT2, INSIG1, CEBPA, SREBF1, XDH, CPT1A, LIPE, INSIG2 and PPARGC1A genes in BuMECs changed contrary to the overexpression experiment, and that of GPAM, PPARG and SREBF2 also did not change significantly, but the expression of FABP3 was significantly decreased. In addition, the overexpression/interference of AGPAT6 gene significantly increased/decreased TG content in BuMECs. The results here indicate that AGPAT6 gene is involved in TG synthesis in BuMECs, and affects the expression of major genes associated with FA transport and activation, TG synthesis and transcription regulation, FA oxidation and TG degradation during the lipogenesis of milk.

Elongase of very long chain fatty acids 6 (ELOVL6) promotes lipid synthesis in buffalo mammary epithelial cells.

Recent studies have shown elongase of very-long-chain fatty acids 6 (ELOVL6) is a vital protein for endogenous synthesis of saturated and monounsaturated long-chain fatty acids in some mammals. Nevertheless, its role in lipid synthesis in buffalo mammary gland is still unclear. In this work, the full-length coding sequence (CDS) of ELOVL6 was cloned and identified from buffalo mammary gland. As a result, the CDS of this gene is 795 bp, which encodes a polypeptide of 264 amino acid residues. The buffalo ELOVL6 contains an ELO domain which belongs to the ELO superfamily. Among the 10 tissues of buffalo in peak lactation detected by RT-qPCR, the expression level of ELOVL6 was the highest in the brain, followed by the spleen, and then decreased in the mammary gland, muscle, kidney, heart, liver, rumen, intestine and lung. However, only the expression in the brain and spleen was statistically different from that in other tissues (p < 0.05). Compared with that of the dry-off period, the mRNA abundance of ELOVL6 in the mammary gland was significantly increased in peak lactation. The experiments based on lentivirus transfection in buffalo mammary epithelial cells (BuMECs) displayed that the overexpression of ELOVL6 markedly promoted the expression of INSIG1, INSIG2, SREBP, PPARG, FASN, GPAM, DGAT2 and APGAT6 genes, and the knockdown of ELOVL6 significantly decreased the mRNA abundance of INSIG2, SREBP, FASN, SCD, GPAM, APGAT6 and TIP47 genes. In addition, the increase or decrease of ELOVL6 expression level also caused the corresponding change of total triglyceride content in the BuMECs. The results here suggest that the ELOVL6 can catalyse the synthesis of long-chain fatty acids in the BuMECs, and it can indirectly affect the expression of genes related to milk fat synthesis through its catalytic products to promote the lipid biosynthesis of BuMECs.

Liver X receptor α promotes milk fat synthesis in buffalo mammary epithelial cells by regulating the expression of FASN.

Liver X receptor α (LXRα; NR1H3) is an important transcription factor that can facilitate milk fat synthesis by regulating the transcription of FASN in mice and goats. Nevertheless, the lipid synthesis related to LXRα and its regulation on FASN in the buffalo mammary gland remain elusive. Here, we demonstrated that the mRNA and protein expression of LXRα in buffalo mammary tissue increased in lactation compared with that in the dry-off period. Overexpression of NR1H3 enhanced the lipid droplet formation and triacylglycerol concentration in buffalo mammary epithelial cells (BuMEC), whereas the knockdown of NR1H3 resulted in a decrease in the number of lipid droplets. At the same time, NR1H3 also affected the expression of regulatory factors (INSIG1, INSIG2, SREBF1, and PPARG) related to milk fat synthesis and that of genes involved in de novo synthesis (FASN, ACACA, and SCD), and uptake and transport (LPL, CD36, and FABP3) of fatty acids as well as triacylglycerol synthesis (GPAM, APGAT6, and DGAT1). Luciferase reporter assays indicated that overexpression of NR1H3 resulted in an increase in the activity of FASN promoter, whereas the knockdown of NR1H3 had an opposite effect. When NR1H3 was overexpressed, mutations in LXRE or SRE could decrease the promoter activity of FASN. Furthermore, mutagenesis of both LXRE and SRE within the FASN promoter completely eliminated the induced activity of LXRα. Our results reveal that buffalo LXRα promotes milk fat synthesis through regulating the expression of FASN by directly interacting with FASN promoter and affecting the SREBF1 expression. This study underscores a crucial role of LXRα in regulating lipid synthesis of the buffalo mammary gland.

Negative effect of insulin-induced gene 2 on milk fat synthesis in buffalo mammary epithelial cells.

Insulin-induced gene 2 (INSIG2) is a recently identified gene that is implicated in the regulation of cholesterol metabolism and lipogenesis in mammals. Although the data in goats emphasizes a role for INSIG2 in milk fat synthesis, the regulatory mechanism in buffalo is not clear. In this study, we analyzed the protein abundance of INSIG2 at peak lactation and dry-off period in buffalo mammary tissue. The results indicated that, relative to the peak lactation, the protein abundance of INSIG2 in the dry-off period was higher. To determine the function of INSIG2 in milk fat synthesis, INSIG2 was overexpressed and knocked down by lentiviral transfection in buffalo mammary epithelial cells (BuMECs). The response to overexpressing INSIG2 included down-regulation of SREBP, PPARG, FASN, ELOVL6, SCD, APGAT6 and TIP47 coupled with a decrease in content of triacylglycerol (TAG). However, in response to knockdown of INSIG2, the significant increase in content of TAG along with marked up-regulation of SREBP, PPARG, FASN, ELOVL6, SCD, APGAT6 and TIP47 suggests that INSIG2 negatively affects milk fat synthesis in BuMECs. No significant difference in mRNA abundance of GPAM and DGAT2 in response to overexpression or interference of INSIG2 indicates that they might also be influenced by other regulatory factors. Taken together, our results provide strong support for the negative effect of INSIG2 on milk fat synthesis in BuMECs.

Effect of INSIG1 on the milk fat synthesis of buffalo mammary epithelial cells.

We hypothesized that insulin-induced gene 1 (INSIG1) affects milk fat synthesis in buffalo. For this reason, the protein abundance of INSIG1 in the mammary tissue of buffalo during the peak period of lactation and dry-off period was evaluated. The results showed that the expression of INSIG1 at the peak of lactation was lower than that in the dry-off period. To explore the role of INSIG1 in milk fat synthesis, the buffalo mammary epithelial cells (BMECs) were isolated and purified from buffalo mammary tissue, and INSIG1 gene were overexpressed and knocked down by constructing the recombinant lentivirus vector of INSIG1 gene and transfecting into BMECs. Results revealed that INSIG1 overexpression decreased the expression of INSIG2, SREBP, PPARG, SCD, GPAM, DGAT2 and AGPAT6, which led to reduction of triglycerides (TAG) content in the cell. In contrast, knockdown of INSIG1 had a positive effect on mRNA expression of the above genes. Overall, the data provide strong support for a key role of INSIG1 in the regulation of milk fat synthesis in BMECs.

Genomic Analyses Reveal Potential Independent Adaptation to High Altitude in Tibetan Chickens.

Much like other indigenous domesticated animals, Tibetan chickens living at high altitudes (2,200-4,100 m) show specific physiological adaptations to the extreme environmental conditions of the Tibetan Plateau, but the genetic bases of these adaptations are not well characterized. Here, we assembled a de novo genome of a Tibetan chicken and resequenced whole genomes of 32 additional chickens, including Tibetan chickens, village chickens, game fowl, and Red Junglefowl, and found that the Tibetan chickens could broadly be placed into two groups. Further analyses revealed that several candidate genes in the calcium-signaling pathway are possibly involved in adaptation to the hypoxia experienced by these chickens, as these genes appear to have experienced directional selection in the two Tibetan chicken populations, suggesting a potential genetic mechanism underlying high altitude adaptation in Tibetan chickens. The candidate selected genes identified in this study, and their variants, may be useful targets for clarifying our understanding of the domestication of chickens in Tibet, and might be useful in current breeding efforts to develop improved breeds for the highlands.

Isolation, sequence characterization, and tissue transcription profiles of two novel buffalo genes: INSIG1 and INSIG2.

Insulin-induced genes (INSIGs) are recently discovered genes that are involved in the metabolism of cholesterol and lipogenesis in animal tissues. In this study, two INSIG genes (INSIG1 and INSIG2) were isolated and characterized in 11 buffalo. The full-length coding sequence (CDS) of the buffalo INSIG1 consists of 831 bp which encodes a 276 amino acid protein with molecular mass 29.55 kD. And the INSIG2 CDS is 678 bp in length which encodes a 225 amino acid protein with molecular mass 24.87 kD. No polymorphisms were found in the CDSs of the buffalo INSIGs, but seven and two nucleotide differences were found in the CDSs between buffalo and other bovine species. Phylogenetic analyses based on the INSIG amino acid sequences showed that buffalo was grouped with other members in the Bovidae family. Four types of putative modification sites were detected in buffalo INSIG proteins. And two predicted microRNA target sites were found respectively in the CDSs of buffalo INSIG1 and INSIG2. The tissue expression analyses by quantitative PCR (qPCR) revealed that the buffalo INSIG1 was expressed in ten tissues tested. Among these tissues, the liver and mammary gland showed high expression levels. And the INSIG2 was only expressed in the brain, mammary glands, pituitary, abomasum, heart, and liver. Among these tissues, the mammary gland, brain, and pituitary demonstrated a high expression levels. These data provide the primary foundation for further insights into the buffalo INSIG genes.

LEP Gene Promotes Milk Fat Synthesis via the JAK2-STAT3 and mTOR Signaling Pathways in Buffalo Mammary Epithelial Cells.

Leptin (LEP), a protein hormone well-known for its role in metabolic regulation, has recently been linked to lipid metabolism in cattle. However, its function in buffalo mammary glands remains unclear. To address this issue, we isolated and identified the LEP gene and conducted experiments to investigate its function in buffalo mammary epithelial cells (BuMECs). In this study, two transcript variants of LEP, designated as LEP_X1 and LEP_X2, were identified. The coding sequences (CDS) of LEP_X1 and LEP_X2 are 504 bp and 579 bp in length, encoding 167 and 192 amino acid residues, respectively. Bioinformatics analysis revealed that LEP_X2 is a hydrophobic protein with an isoelectric point below 7 and contains a signal peptide, while LEP_X1 is hydrophilic and lacks a signal peptide. Our study found that LEP gene expression in lactating BuMECs was significantly higher than in non-lactating cells, with LEP_X2 expression remarkably higher than LEP_X1 in lactating BuMECs. Overexpression of both LEP_X1 and LEP_X2 significantly promoted the expression of genes related to milk fat synthesis in lactating BuMECs, including STAT3, PI3K, mTOR, SCD, and SREBF1, accompanied by an increase in cellular triglycerides (TG). Interestingly, LEP_X2 overexpression significantly suppressed LEP_X1 expression while increasing intracellular TG concentration by 12.10-fold compared to LEP_X1 overexpression, suggesting an antagonistic relationship between the two variants and supposing LEP_X2 plays a dominant role in milk fat synthesis in lactating BuMECs. Additionally, four nucleotide substitutions were identified in the buffalo LEP CDS, including a nonsynonymous substitution c.148C>T (p.Arg50Cys), which was predicted to decrease the stability of the LEP protein without affecting its function. These results collectively underscore the significant role of LEP in milk fat synthesis and can provide a basis for molecular breeding strategies of buffalo.

Exceptionally high cumulative percentage of NUMTs originating from linear mitochondrial DNA molecules in the Hydra magnipapillata genome.

BACKGROUND: In contrast to most animal genomes, mitochondrial genomes in species belonging to the phylum Cnidaria show distinct variations in genome structure, including the mtDNA structure (linear or circular) and the presence or absence of introns in protein-coding genes. Therefore, the analysis of nuclear insertions of mitochondrial sequences (NUMTs) in cnidarians allows us to compare the NUMT content in animals with different mitochondrial genome structures. RESULTS: NUMT identification in the Hydra magnipapillata, Nematostella vectensis and Acropora digitifera genomes showed that the NUMT density in the H. magnipapillata genome clearly exceeds that in other two cnidarians with circular mitochondrial genomes. We found that H. magnipapillata is an exceptional ancestral metazoan with a high NUMT cumulative percentage but a large genome, and its mitochondrial genome linearisation might be responsible for the NUMT enrichment. We also detected the co-transposition of exonic and intronic fragments within NUMTs in N. vectensis and provided direct evidence that mitochondrial sequences can be transposed into the nuclear genome through DNA-mediated fragment transfer. In addition, NUMT expression analyses showed that NUMTs are co-expressed with adjacent protein-coding genes, suggesting the relevance of their biological function. CONCLUSIONS: Taken together, our results provide valuable information for understanding the impact of mitochondrial genome structure on the interaction of mitochondrial molecules and nuclear genomes.

Molecular Characteristics and Polymorphisms of Buffalo (Bubalus bubalis) ABCG2 Gene and Its Role in Milk Fat Synthesis.

The ATP-binding cassette subfamily G member 2 (ABCG2) serves crucial roles in secreting riboflavin and biotin vitamins into the milk of cattle, mice, and humans, as well as in the transportation of xenotoxic and cytostatic drugs across the plasma membrane. However, the specific role of the ABCG2 gene in water buffaloes (Bubalus bubalis), especially its effect on milk fat synthesis in buffalo mammary epithelial cells (BuMECs), remains inadequately understood. In this study, the full-length CDS of the buffalo ABCG2 gene was isolated and identified from the mammary gland in buffaloes. A bioinformatics analysis showed a high degree of similarity in the transcriptional region, motifs, and conservative domains of the buffalo ABCG2 with those observed in other Bovidae species. The functional role of buffalo ABCG2 was associated with the transportation of solutes across lipid bilayers within cell membranes. Among the 11 buffalo tissues detected, the expression levels of ABCG2 were the highest in the liver and brain, followed by the mammary gland, adipose tissue, heart, and kidney. Notably, its expression in the mammary gland was significantly higher during peak lactation than during non-lactation. The ABCG2 gene was identified with five SNPs in river buffaloes, while it was monomorphic in swamp buffaloes. Functional experiments revealed that ABCG2 increased the triglyceride (TAG) content by affecting the expression of liposynthesis-related genes in BuMECs. The results of this study underscore the pivotal role of the ABCG2 gene in influencing the milk fat synthesis in BuMECs.

CEBPA-Regulated Expression of SOCS1 Suppresses Milk Protein Synthesis through mTOR and JAK2-STAT5 Signaling Pathways in Buffalo Mammary Epithelial Cells.

Milk protein content is a key quality indicator of milk, and therefore elucidating its synthesis mechanism has been the focus of research in recent years. Suppressor of cytokine signaling 1 (SOCS1) is an important inhibitor of cytokine signaling pathways that can inhibit milk protein synthesis in mice. However, it remains elusive whether SOCS1 plays roles in the milk protein synthesis in the buffalo mammary gland. In this study, we found that the mRNA and protein expression levels of SOCS1 in buffalo mammary tissue during the dry-off period was significantly lower than those during lactation. Overexpression and knockdown experiments of SOCS1 showed that it influenced the expression and phosphorylation of multiple key factors in the mTOR and JAK2-STAT5 signaling pathways in buffalo mammary epithelial cells (BuMECs). Consistently, intracellular milk protein content was significantly decreased in cells with SOCS1 overexpression, while it increased significantly in the cells with SOCS1 knockdown. The CCAAT/enhancer binding protein α (CEBPA) could enhance the mRNA and protein expression of SOCS1 and its promoter activity in BuMECs, but this effect was eliminated when CEBPA and NF-κB binding sites were deleted. Therefore, CEBPA was determined to promote SOCS1 transcription via the CEBPA and NF-κB binding sites located in the SOCS1 promoter. Our data indicate that buffalo SOCS1 plays a significant role in affecting milk protein synthesis through the mTOR and JAK2-STAT5 signaling pathways, and its expression is directly regulated by CEBPA. These results improve our understanding of the regulation mechanism of buffalo milk protein synthesis.

LPIN1 promotes triglycerides synthesis and is transcriptionally regulated by PPARG in buffalo mammary epithelial cells.

Studies on 3T3-L1 cells and HepG2 hepatocytes have shown that phosphatidic acid phosphohydrolase1 (LPIN1) plays a key role in adipogenesis, acting as a co-activator of peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1a) to regulate fatty acid metabolism. However, the functional role and regulatory mechanism of LPIN1 gene in milk fat synthesis of buffalo are still unknown. In this study, overexpression of buffalo LPIN1 gene transfected with recombinant fusion expression vector significantly increased the expression of AGPAT6, DGAT1, DGAT2, GPAM and BTN1A1 genes involved in triglyceride (TAG) synthesis and secretion, as well as PPARG and SREBF1 genes regulating fatty acid metabolism in the buffalo mammary epithelial cells (BMECs), while the lentivirus-mediated knockdown of buffalo LPIN1 dramatically decreased the relative mRNA abundance of these genes. Correspondingly, total cellular TAG content in the BMECs increased significantly after LPIN1 overexpression, but decreased significantly after LPIN1 knockdown. In addition, the overexpression or knockdown of PPARG also enhanced or reduced the expression of LPIN1 and the transcriptional activity of its promoter. The core region of buffalo LPIN1 promoter spans from - 666 bp to + 42 bp, and two PPAR response elements (PPREs: PPRE1 and PPRE2) were identified in this region. Site mutagenesis analysis showed that PPARG directly regulated the transcription of buffalo LPIN1 by binding to the PPRE1 and PPRE2 on its core promoter. The results here reveal that the LPIN1 gene is involved in the milk fat synthesis of BMECs, and one of the important pathways is to participate in this process through direct transcriptional regulation of PPARG, which in turn significantly affects the content of TAG in BMECs.

Identification and characterization of alternative splicing variants of buffalo LXRα expressed in mammary gland.

Liver X receptor α (LXRα) is a ligand-dependent transcription factor and plays an important role in the regulation of cholesterol homeostasis, fatty acid biosynthesis and glucose metabolism. In this study, transcripts of LXRα gene were cloned and characterized from buffalo mammary gland, and three alternative splicing transcripts of buffalo LXRα gene were identified, named LXRα1, LXRα2 and LXRα3. The structure of the LXRα transcripts of buffalo and cattle was highly similar. Bioinformatics analysis showed that LXRα1 contains two complete functional domains of LXRα, one is the DNA-binding domain (NR_DBD_LXR) and the other is the ligand-binding domain (NR_LBD_LXR). The reading frame of LXRα2 is altered due to the skipping of exon 9, which truncates its encoding protein prematurely at the 400th amino acid residue, making it contain a complete DNA-binding domain and part of a ligand-binding domain. Due to the deletion of exon 4, the protein encoded by LXRα3 lacks 89 amino acid residues and contains only a complete ligand-binding domain, which makes it lose its transcriptional regulation function. In addition, motifs and conserved domains of three LXRα variants of buffalo were highly consistent with those of corresponding transcripts from other mammal species. Subcellular localization analysis showed that LXRα1 plays a functional role in the nucleus of buffalo mammary epithelial cells, while LXRα2 and LXRα3 are distributed in the nucleus and cytoplasm. Compared with non-lactating period, the mRNA abundance of the three LXRα transcripts in the mammary gland tissue of buffalo increased during lactating period, revealing that they play a key role in the synthesis of buffalo milk fat. Among the three LXRα transcripts, LXRα1 has the highest expression in the mammary gland, indicating that it is the major transcript in the mammary gland and has important regulatory functions, while LXRα2 and LXRα3 may have regulatory effects on the function of LXRα1. This study highlights the key role of LXRα alternative splicing in the post-transcriptional regulation of buffalo lactation.

Genome-wide identification of long noncoding RNA genes and their potential association with mammary gland development in water buffalo.

OBJECTIVE: Water buffalo, an important domestic animal in tropical and subtropical regions, play an important role in agricultural economy. It is an important source for milk, meat, horns, skin, and draft power, especially its rich milk that is the great source of cream, butter, yogurt, and many cheeses. In recent years, long noncoding RNAs (lncRNAs) have been reported to play pivotal roles in many biological processes. Previous studies for the mammary gland development of water buffalo mainly focus on protein coding genes. However, lncRNAs of water buffalo remain poorly understood, and the regulation relationship between mammary gland development/milk production traits and lncRNA expression is also unclear. METHODS: Here, we sequenced 22 samples of the milk somatic cells from three lactation stages and integrated the current annotation and identified 7,962 lncRNA genes. RESULTS: By comparing the lncRNA genes of the water buffalo in the early, peak, and late different lactation stages, we found that lncRNA gene lnc-bbug14207 displayed significantly different expression between early and late lactation stages. And lnc-bbug14207 may regulate neighboring milk fat globule-EGF factor 8 (MFG-E8) and hyaluronan and proteoglycan link protein 3 (HAPLN3) protein coding genes, which are vital for mammary gland development. CONCLUSION: This study provides the first genome-wide identification of water buffalo lncRNAs and unveils the potential lncRNAs that impact mammary gland development.

Identification, Molecular Characterization, and Tissue Expression Profiles of Three Smad Genes from Water Buffalo (Bubalus bubalis).

Smads are involved in a variety of biological activities by mediating bone morphogenetic protein (BMP) signals. The full-length coding sequences (CDSs) of buffalo Smads 1, 4, and 5 were isolated and identified through RT-PCR in this study. Their lengths are 1398 bp, 1662 bp, and 1398 bp, respectively. In silico analysis showed that their transcriptional region structures, as well as their amino acid sequences, physicochemical characteristics, motifs, conserved domains, and three-dimensional structures of their encoded proteins are highly consistent with their counterparts in the species of Bovidae. The three Smad proteins are all hydrophilic without the signal peptides and transmembrane regions. Each of them has an MH1 domain and an MH2 domain. A nuclear localization sequence was found in the MH1 domain of buffalo Smads 1 and 5. Prediction showed that the function of the three Smads is mainly protein binding, and they can interact with BMPs and their receptors. The three genes were expressed in all 10 buffalo tissues assayed, and their expression in the mammary gland, gonad, and spleen was relatively high. The results here indicate that the three buffalo Smads may be involved in the transcriptional regulation of genes in a variety of tissues.

Peroxisome proliferator-activated receptor gamma regulates genes involved in milk fat synthesis in mammary epithelial cells of water buffalo.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical transcription factor regulating lipid and glucose metabolism. However, the regulatory effect of PPARγ on milk fat synthesis in buffalo mammary gland is not clear. In order to explore the role of buffalo PPARG gene in milk fat synthesis, lentivirus-mediated interference was used to knock it down and then the recombinant fusion expression vector was transfected into buffalo mammary epithelial cell (BMEC) to overexpress it. PPARG gene knockdown significantly decreased the expression of CD36, FABP3, FABP4, ACSS2, ELOVL6, DGAT2, BTN1A1, AGPAT6, LPIN1, ABCG2, PPARGC1A, INSIG1, FASN, and SREBF2 genes and significantly upregulated the expression of INSIG2 gene but had no significant effect on the expression of ACSL1, GPAM, and SREBF1 genes. PPARG overexpression significantly increased the relative mRNA abundance of CD36, FABP3, FABP4, ACSS2, ELOVL6, DGAT2, BTN1A1, AGPAT6, LPIN1, PPARGC1A, INSIG1, and SREBF2 genes and significantly downregulated the expression of INSIG2 gene but had no significant effect on the expression of ACSL1, GPAM, ABCG2, FASN, and SREBF1 genes. In addition, knockdown/overexpression of PPARG gene significantly decreased/increased triacylglycerol (TAG) content in BMECs. This study revealed that buffalo PPARG gene is a key gene regulating buffalo milk fat synthesis.

Mitochondrial DNA diversity and demographic history of Black-boned chickens in China.

Black-boned chickens (Gallus domesticus, herein abbreviated BBCs) are well known for their unique appearance and medicinal properties and have a long breeding history in China. However, the genetic diversity and demographic history of BBCs remain unclear. In this study, we analyzed 844 mitochondrial DNA D-loop sequences, including 346 de novo sequences and 498 previously published sequences from 20 BBC breeds. We detected a generally high level of genetic diversity among the BBCs, with average haplotype and nucleotide diversities of 0.917 ± 0.0049 and 0.01422, respectively. Nucleotide diversity was highest in populations from Southwest China (0.01549 ± 0.00026), particularly in Yunnan Province (0.01624 ± 0.00025). Significant genetic divergence was detected between most breeds, particularly between Yunnan chickens and those from all other provinces. Haplogroups F and G had the highest levels of genetic diversity and were restricted to Southwest China, particularly Yunnan Province. Based on neutrality tests and mismatch distribution analyses, we did not obtain evidence for rapid population expansions and observed similar demographic histories in BBCs and local non-BBCs. Our results suggest that Chinese BBCs have complex breeding histories and may be selected in situ from local domestic chickens. These results improve our understanding of the genetic heritage and breeding histories of these desirable chickens.

Genomic Analyses of Unveil Helmeted Guinea Fowl (Numida meleagris) Domestication in West Africa.

Domestication of the helmeted guinea fowl (HGF; Numida meleagris) in Africa remains elusive. Here we report a high-quality de novo genome assembly for domestic HGF generated by long- and short-reads sequencing together with optical and chromatin interaction mapping. Using this assembly as the reference, we performed population genomic analyses for newly sequenced whole-genomes for 129 birds from Africa, Asia, and Europe, including domestic animals (n = 89), wild progenitors (n = 34), and their closely related wild species (n = 6). Our results reveal domestication of HGF in West Africa around 1,300-5,500 years ago. Scanning for selective signals characterized the functional genes in behavior and locomotion changes involved in domestication of HGF. The pleiotropy and linkage in genes affecting plumage color and fertility were revealed in the recent breeding of Italian domestic HGF. In addition to presenting a missing piece to the jigsaw puzzle of domestication in poultry, our study provides valuable genetic resources for researchers and breeders to improve production in this species.

The complete mitochondrial genomes of the whistling duck (Dendrocygna javanica) and black swan (Cygnus atratus): dating evolutionary divergence in Galloanserae.

Galloanserae is an ancient and diverse avian group, for which comprehensive molecular evidence relevant to phylogenetic analysis in the context of molecular chronology is lacking. In this study, we present two additional mitochondrial genome sequences of Galloanserae (the whistling duck, Dendrocygna javanica, and the black swan, Cygnus atratus) to broaden the scope of molecular phylogenetic reconstruction. The lengths of the whistling duck's and black swan's mitochondrial genomes are 16,753 and 16,748 bases, respectively. Phylogenetic analyses suggest that Dendrocygna is more likely to be in a basal position of the branch consisting of Anatinae and Anserinae, an affiliation that does not conform to its traditional classification. Bayesian approaches were employed to provide a rough timescale for Galloanserae evolution. In general, a narrow range of 95% confidence intervals gave younger estimates than those based on limited genes and estimated that at least two lineages originated before the Coniacian epoch around 90 MYA, well before the Cretaceous-Tertiary boundary. In addition, these results, which were compatible with estimates from fossil evidence, also imply that the origin of numerous genera in Anseriformes took place in the late Oligocene to early Miocene. Taken together, the results presented here provide a working framework for future research on Galloanserae evolution, and they underline the utility of whole mitochondrial genome sequences for the resolution of deep divergence.

Identification, molecular characteristics, and tissue differential expression of DGAT2 full-CDS cDNA sequence in Binglangjiang buffalo (Bubalus bubalis).

It has been found that diacylglycerol acyltransferase-2 (DGAT2) plays a crucial role in the synthesis of triglycerides (TGs) in some mammals, but its role in buffalo lactation is unclear. In the present study, the DGAT2 full-CDS cDNA sequence of Binglangjiang buffalo was isolated, and the physicochemical characteristics and structure of its encoding protein were characterized. Furthermore, the differential expressions of this gene in 10 tissues of lactating and non-lactating buffalo were analyzed by real-time quantitative PCR (RT-qPCR). The results showed that the coding region (CDS) of this gene was 1086 bp in length, encoding a peptide composed of 361 amino acid residues. The deduced amino acid sequence shared more than 98.6 % identity with that of cattle, zebu, yak, and bison in the Bovidae family. Buffalo DGAT2 protein is a slightly hydrophobic protein with a transmembrane region, which functions in membrane of endoplasmic reticulum. Besides, this protein belongs to the LPLAT_MGAT-like family and contains a conserved domain of DAGAT that has a function in the synthesis of TGs. The multi-tissue differential expression analysis demonstrated that DGAT2 was expressed in the heart, liver, mammary gland, and muscle in both non-lactating and lactating buffalo. And its expression level in the heart, liver, and mammary gland during lactation was significantly higher than that during non-lactation. The results indicate that buffalo DGAT2 may be involved in milk fat synthesis. This study can establish a foundation for further elucidating mechanisms of the buffalo DGAT2 gene in milk fat synthesis.