The swamp type of the Asian water buffalo is assumed to have been domesticated by about 4000 years BP, following the introduction of rice cultivation. Previous localizations of the domestication site were based on mitochondrial DNA (mtDNA) variation within China, accounting only for the maternal lineage. We carried out a comprehensive sampling of China, Taiwan, Vietnam, Laos, Thailand, Nepal and Bangladesh and sequenced the mtDNA Cytochrome b gene and control region and the Y-chromosomal ZFY, SRY and DBY sequences. Swamp buffalo has a higher diversity of both maternal and paternal lineages than river buffalo, with also a remarkable contrast between a weak phylogeographic structure of river buffalo and a strong geographic differentiation of swamp buffalo. The highest diversity of the swamp buffalo maternal lineages was found in south China and north Indochina on both banks of the Mekong River, while the highest diversity in paternal lineages was in the China/Indochina border region. We propose that domestication in this region was later followed by introgressive capture of wild cows west of the Mekong. Migration to the north followed the Yangtze valley as well as a more eastern route, but also involved translocations of both cows and bulls over large distances with a minor influence of river buffaloes in recent decades. Bayesian analyses of various migration models also supported domestication in the China/Indochina border region. Coalescence analysis yielded consistent estimates for the expansion of the major swamp buffalo haplogroups with a credibility interval of 900 to 3900 years BP. The spatial differentiation of mtDNA and Y-chromosomal haplotype distributions indicates a lack of gene flow between established populations that is unprecedented in livestock.

The mammary gland is the production organ in mammals that is of great importance for milk production and quality. However, characterization of the buffalo mammary gland transcriptome and identification of the valuable candidate genes that affect milk production is limited. Here, we performed the differential expressed genes (DEGs) analysis of mammary gland tissue on day 7, 50, 140, and 280 after calving and conducted gene-based genome-wide association studies (GWAS) of milk yield in 935 Mediterranean buffaloes. We then employed weighted gene co-expression network analysis (WGCNA) to identify specific modules and hub genes related to milk yield based on gene expression profiles and GWAS data. The results of the DEGs analysis showed that a total of 1,420 DEGs were detected across different lactation points. In the gene-based analysis, 976 genes were found to have genome-wide association (P ≤ 0.05) that could be defined as the nominally significant GWAS geneset (NSGG), 9 of which were suggestively associated with milk yield (P < 10−4). Using the WGCNA analysis, 544 and 225 genes associated with milk yield in the turquoise module were identified from DEGs and NSGG datasets, respectively. Several genes (including BNIPL, TUBA1C, C2CD4B, DCP1B, MAP3K5, PDCD11, SRGAP1, GDPD5, BARX2, SCARA3, CTU2, and RPL27A) were identified and considered as the hub genes because they were involved in multiple pathways related to milk production. Our findings provide an insight into the dynamic characterization of the buffalo mammary gland transcriptome, and these potential candidate genes may be valuable for future functional characterization of the buffalo mammary gland.

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Linkage disequilibrium (LD) is a useful parameter for guiding the accuracy and power of both genome-wide association studies (GWAS) and genomic selection (GS) among different livestock species. The present study evaluated the extent of LD, persistence of phase and effective population size (Ne) for the purebred (Mediterranean buffalo; n = 411) and crossbred [Mediterranean × Jianghan × Nili-Ravi buffalo, n = 9; Murrah × Nili-Ravi × local (Xilin or Fuzhong) buffalo, n = 36] buffalo populations using the 90K Buffalo SNP genotyping array. The results showed that the average square of correlation coefficient (r2) between adjacent SNP was 0.13 ± 0.19 across all autosomes for purebred and 0.09 ± 0.13 for crossbred, and the most rapid decline in LD was observed over the first 200 kb. Estimated r2 ≥ 0.2 extended up to ~50 kb in crossbred and 170 kb in purebred populations, while average r2 values ≥0.3 were respectively observed in the ~10 and 60 kb in the crossbred and purebred populations. The largest phase correlation (RP, C = 0.47) was observed at the distance of 100 kb, suggesting that this phase was not actively preserved between the two populations. Estimated Ne for the purebred and crossbred population at the current generation was 387 and 113 individuals, respectively. These findings may provide useful information to guide the GS and GWAS in buffaloes.

The Chinese swamp buffalo (Bubalis bubalis) is vital to the lives of small farmers and has tremendous economic importance. However, a lack of genomic information has hampered research on augmenting marker assisted breeding programs in this species. Thus, a high-throughput transcriptomic sequencing of B. bubalis was conducted to generate transcriptomic sequence dataset for gene discovery and molecular marker development. Illumina paired-end sequencing generated a total of 54,109,173 raw reads. After trimming, de novo assembly was performed, which yielded 86,017 unigenes, with an average length of 972.41 bp, an N50 of 1,505 bp, and an average GC content of 49.92%. A total of 62,337 unigenes were successfully annotated. Among the annotated unigenes, 27,025 (43.35%) and 23,232 (37.27%) unigenes showed significant similarity to known proteins in NCBI non-redundant protein and Swiss-Prot databases (E-value < 1.0E-5), respectively. Of these annotated unigenes, 14,439 and 15,813 unigenes were assigned to the Gene Ontology (GO) categories and EuKaryotic Ortholog Group (KOG) cluster, respectively. In addition, a total of 14,167 unigenes were assigned to 331 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Furthermore, 17,401 simple sequence repeats (SSRs) were identified as potential molecular markers. One hundred and fifteen primer pairs were randomly selected for amplification to detect polymorphisms. The results revealed that 110 primer pairs (95.65%) yielded PCR amplicons and 69 primer pairs (60.00%) presented polymorphisms in 35 individual buffaloes. A phylogenetic analysis showed that the five swamp buffalo populations were clustered together, whereas two river buffalo breeds clustered separately. In the present study, the Illumina RNA-seq technology was utilized to perform transcriptome analysis and SSR marker discovery in the swamp buffalo without using a reference genome. Our findings will enrich the current SSR markers resources and help spearhead molecular genetic research studies on the swamp buffalo.

Water buffalo (Bubalus bubalis), a large‐sized member of the Bovidae family, is considered as an important livestock species throughout Southeast Asia. In order to better understand the molecular basis of buffalo improvement and breeding, we sequenced and assembled the genome (2n=50) of a river buffalo species Bubalus bubalis from Bangladesh. Its genome size is 2.77 Gb, with a contig N50 of 25 kb and the scaffold N50 of 6.9 Mbp. Based on the assembled genome, we annotated 24,613 genes for future functional genomics studies. Phylogenetic tree analysis of cattle and water buffalo lineages showed that they diverged about 5.8–9.8 million years ago. Our findings provide an insight into the water buffalo genome which will contribute in further research on buffalo such as molecular breeding, understanding complex traits, conservation, and biodiversity.

Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, <4 mm; and large, >8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment.

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