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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

Molecular characterization, tissue expression and polymorphisms of buffalo PPARGC1A gene.

PPARGC1A exerts important functions in activating many nuclear receptors and transcription factors that are related to energy balance. Previous studies have shown that PPARGC1A gene is associated with lactation traits of dairy cattle. However, the functional role of the buffalo PPARGC1A gene is still unknown. In this work, the complete coding sequence (CDS) of buffalo PPARGC1A was isolated and characterized for swamp and river buffalo. The CDS length of PPARGC1A for both types of buffalo was the same, which was composed of 2394 nucleotides and encoded a peptide composed of 797 amino acid residues. This protein belonged to a hydrophilic protein and contained one RRM_PPARGC1A domain (AA 674-764) without a signal peptide or a transmembrane domain. The differential expressions of this gene in 10 buffalo tissues in lactation and non-lactation displayed that the PPARGC1A was highly expressed in the muscle, heart, liver, brain and kidney of both non-lactating and lactating periods, but its expression was significantly different in the muscle, heart, liver, small intestine, mammary gland, rumen, spleen and lung between the two periods. Eight single nucleotide polymorphisms (SNPs) were found in buffalo, in which the c.778C > T, c.1257G > A and c.1311G > A were shared by two types of buffalo with similar allele frequencies, while the c.419C > T, c.759A > G, c.920C > A, c.926G > A and c.1509A > T were only observed in river buffalo. The SNP419, SNP920 and SNP926 were non-synonymous, which led to the amino acid changes of p.Ser140Phe, p.Pro307His and p.Arg309Lys. Seven nucleotide differential sites were identified in the PPARGC1A gene between buffalo and other Bovidae species. Phylogenetic analysis indicated that buffaloes were independently clustered into one branch, but they were closely related to the species of the Bos genus. The results indicate that buffalo PPARGC1A is an inducible transcriptional coactivator involved in regulating carbohydrate and fat metabolism. It can exert a functional role in a variety of buffalo tissues and may participate in milk fat synthesis and development in the mammary gland.

Isolation, identification, expression and subcellular localization of PPARG gene in buffalo mammary gland.

Peroxisome proliferator-activated receptor gamma (PPARG), as a member of the nuclear receptor superfamily, plays an important role in adipocyte differentiation and regulation of lipid and glucose metabolism. In this study, the transcripts of PPARG gene were isolated and identified in buffalo mammary gland. The results showed that two types of transcripts (PPARG1 and PPARG2) of PPARG gene produced by alternative 5' end use were expressed in buffalo mammary gland, and each of them had four different alternative splicing variants. The PPARG1 includes PPARG1a, PPARG1b, PPARG1c and PPARG1d, while the PPARG2 contains PPARG2a, PPARG2b, PPARG2c and PPARG2d. Among them, only PPARG1a, PPARG2a and PPARG2d can encode complete functional proteins with three complete functional domains, and the rest encode truncated proteins with incomplete functional domains. All the eight variants of PPARG protein do not contain transmembrane regions and signal peptides, but their conserved domain, secondary and tertiary structure and subcellular localization were different. Subcellular localization confirmed that the main transcripts PPARG1a and PPARG2a played a functional role in the nucleus, which was consistent with the results by in silico prediction. RT-qPCR analysis of buffalo mammary tissue showed that the mRNA expression levels of PPARG1 and PPARG2 in lactation were higher than those in non-lactation, and the expression levels of transcripts PPARG2d and PPARG1b + PPARG2b in lactating stage were also higher than those in non-lactating stage, but the mRNA abundance of transcripts PPARG1c, PPARG1d and PPARG2c in non-lactating period was higher than that in lactating period. The results of this study suggest that PPARG1 and PPARG2 may play important role in buffalo milk fat synthesis, and the eight alternative splicing variants found here are likely to be related to the post-transcriptional regulation of lactation.

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.

Polymorphism and molecular characteristics of the CSN1S2 gene in river and swamp buffalo.

The α S 2 -casein ( α S 2 -CN) is a member of the casein family associated with milk traits in ruminants, but so far the buffalo CSN1S2 gene has not been well understood. In this work, the polymorphisms of CSN1S2 in river and swamp buffalo were detected using direct sequencing of polymerase chain reaction (PCR) products. As a result, 13 single nucleotide polymorphisms (SNPs) were identified in the coding sequence (CDS) of CSN1S2 in two types of buffalo, of which eight SNPs were non-synonymous. The amino acid changes caused by c.580T > C and c.642C > G may affect the function of buffalo α S 2 -CN. A total of 11 CSN1S2 CDS haplotypes were defined, and accordingly 11 variants of buffalo α S 2 -CN were inferred and named. The CSN1S2 CDSs of both types of buffalo were 669 nucleotides, which encoded a precursor of 222 amino acids (AAs), and the first 15 AAs constitute a signal peptide. The composition and physicochemical characteristics of two types of buffalo α S 2 -CNs were similar but slightly different from those of cattle α S 2 -CN. The α S 2 -CN mature peptides of buffalo and the species of Bos genus contained a casein domain, and their secondary structures were highly consistent, indicating that they are functionally similar. The results here provide initial insights into the variation, characteristics and biological function of buffalo CSN1S2.

Understanding divergent domestication traits from the whole-genome sequencing of swamp- and river-buffalo populations.

Domesticated buffaloes have been integral to rice-paddy agro-ecosystems for millennia, yet relatively little is known about the buffalo genomics. Here, we sequenced and assembled reference genomes for both swamp and river buffaloes and we re-sequenced 230 individuals (132 swamp buffaloes and 98 river buffaloes) sampled from across Asia and Europe. Beyond the many actionable insights that our study revealed about the domestication, basic physiology and breeding of buffalo, we made the striking discovery that the divergent domestication traits between swamp and river buffaloes can be explained with recent selections of genes on social behavior, digestion metabolism, strengths and milk production.