Buffalo Genomes Research Articles

Genome-wide copy number variant analysis reveals candidate genes associated with milk production traits in water buffalo (Bubalus bubalis)

Buffaloes are vital contributors to the global dairy industry. Understanding the genetic basis of milk production traits in buffalo populations is essential for breeding programs and improving productivity. In this study, we conducted whole-genome resequencing on 387 buffalo genomes from 29 diverse Asian breeds, including 132 river buffaloes, 129 swamp buffaloes, and 126 crossbred buffaloes. We identified 36,548 copy number variants (CNV) spanning 133.29 Mb of the buffalo genome, resulting in 2,100 CNV regions (CNVR), with 1,993 shared CNVR being found within the studied buffalo types. Analyzing CNVR highlighted distinct genetic differentiation between river and swamp buffalo subspecies, verified by evolutionary tree and principal component analyses. Admixture analysis grouped buffaloes into river and swamp categories, with crossbred buffaloes displaying mixed ancestry. To identify candidate genes associated with milk production traits, we employed 3 approaches. First, we used Vst-based population differentiation, revealing 11 genes within CNVR that exhibited significant divergence between different buffalo breeds, including genes linked to milk production traits. Second, expression quantitative loci analysis revealed differentially expressed CNVR-derived genes (DECG) associated with milk production traits. Notably, known milk production-related genes were among these DECG, validating their relevance. Last, a GWAS identified 3 CNVR significantly linked to peak milk yield. Our study provides comprehensive genomic insights into buffalo populations and identifies candidate genes associated with milk production traits. These findings facilitate genetic breeding programs aimed at increasing milk yield and improving quality in this economically important livestock species.

Disentangling river and swamp buffalo genetic diversity: initial insights from the 1000 Buffalo Genomes Project.

More people in the world depend on water buffalo for their livelihoods than on any other domesticated animals, but its genetics is still not extensively explored. The 1000 Buffalo Genomes Project (1000BGP) provides genetic resources for global buffalo population study and tools to breed more sustainable and productive buffaloes. Here we report the most contiguous swamp buffalo genome assembly (PCC_UOA_SB_1v2) with substantial resolution of telomeric and centromeric repeats, ∼4-fold more contiguous than the existing reference river buffalo assembly and exceeding a recently published male swamp buffalo genome. This assembly was used along with the current reference to align 140 water buffalo short-read sequences and produce a public genetic resource with an average of ∼41 million single nucleotide polymorphisms per swamp and river buffalo genome. Comparison of the swamp and river buffalo sequences showed ∼1.5% genetic differences, and estimated divergence time occurred 3.1 million years ago (95% CI, 2.6-4.9). The open science model employed in the 1000BGP provides a key genomic resource and tools for a species with global economic relevance.

Analysis of genetic diversity and selection characteristics using the whole genome sequencing data of five buffaloes, including Xilin buffalo, in Guangxi, China.

Buffalo is an economically important livestock that renders useful services to manhood in terms of meat, milk, leather, and draught. The Xilin buffalo is among the native buffalo breeds of China. In the present study, the genetic architecture and selection signature signals of Xilin buffalo have been explored. Correlation analysis of the population structure of Xilin buffalo was conducted by constructing NJ tree, PCA, ADMIXTURE and other methods. A total of twenty-five (n = 25) Xilin buffalo whole genome data and data of forty-six (n = 46) buffaloes published data were used. The population structure analysis showed that the Xilin buffalo belong to the Middle-Lower Yangtze. The genome diversity of Xilin buffalo was relatively high. The CLR, π ratio, F ST, and XP-EHH were used to detect the candidate genes characteristics of positive selection in Xilin buffalo. Among the identified genes, most of the enriched signal pathways were related to the nervous system and metabolism. The mainly reported genes were related to the nervous system (GRM5, GRIK2, GRIA4), reproductive genes (CSNK1G2, KCNIP4), and lactation (TP63). The results of this study are of great significance for understanding the molecular basis of phenotypic variation of related traits of Xilin buffalo. We provide a comprehensive overview of sequence variations in Xilin buffalo genomes. Selection signatures were detected in genomic regions that are possibly related to economically important traits in Xilin buffalo and help in future breeding and conservation programs of this important livestock genetic resource.

Chromosome-level genome and recombination map of the male buffalo.

The swamp buffalo (Bubalus bubalis carabanesis) is an economically important livestock supplying milk, meat, leather, and draft power. Several female buffalo genomes have been available, but the lack of high-quality male genomes hinders studies on chromosome evolution, especially Y, as well as meiotic recombination. Here, a chromosome-level genome with a contig N50 of 72.2 Mb and a fine-scale recombination map of male buffalo were reported. We found that transposable elements (TEs) and structural variants (SVs) may contribute to buffalo evolution by influencing adjacent gene expression. We further found that the pseudoautosomal region (PAR) of the Y chromosome is subject to stronger purification selection. The meiotic recombination map showed that there were 2 obvious recombination hotspots on chromosome 8, and the genes around them were mainly related to tooth development, which may have helped to enhance the adaption of buffalo to inferior feed. Among several genomic features, TE density has the strongest correlation with recombination rates. Moreover, the TE subfamily, SINE/tRNA, is likely to play a role in driving recombination into SVs. The male genome and sperm sequencing will facilitate the understanding of the buffalo genomic evolution and functional research.

Draft genome of the lowland anoa (Bubalus depressicornis) and comparison with buffalo genome assemblies (Bovidae, Bubalina).

Genomic data for wild species of the genus Bubalus (Asian buffaloes) are still lacking while several whole genomes are currently available for domestic water buffaloes. To address this, we sequenced the genome of a wild endangered dwarf buffalo, the lowland anoa (Bubalus depressicornis), produced a draft genome assembly and made comparison to published buffalo genomes. The lowland anoa genome assembly was 2.56 Gbp long and contained 103,135 contigs, the longest contig being 337.39 kbp long. N50 and L50 values were 38.73 and 19.83 kbp, respectively, mean coverage was 44× and GC content was 41.74%. Two strategies were adopted to evaluate genome completeness: (1) determination of genomic features with de novo and homology-based predictions using annotations of chromosome-level genome assembly of the river buffalo and (2) employment of benchmarking against universal single-copy orthologs (BUSCO). Homology-based predictions identified 94.51% complete and 3.65% partial genomic features. De novo gene predictions identified 32,393 genes, representing 97.14% of the reference's annotated genes, whilst BUSCO search against the mammalian orthologs database identified 71.1% complete, 11.7% fragmented, and 17.2% missing orthologs, indicating a good level of completeness for downstream analyses. Repeat analyses indicated that the lowland anoa genome contains 42.12% of repetitive regions. The genome assembly of the lowland anoa is expected to contribute to comparative genome analyses among bovid species.

High diversity, inbreeding and a dynamic Pleistocene demographic history revealed by African buffalo genomes

Genomes retain records of demographic changes and evolutionary forces that shape species and populations. Remnant populations of African buffalo (Syncerus caffer) in South Africa, with varied histories, provide an opportunity to investigate signatures left in their genomes by past events, both recent and ancient. Here, we produce 40 low coverage (7.14×) genome sequences of Cape buffalo (S. c. caffer) from four protected areas in South Africa. Genome-wide heterozygosity was the highest for any mammal for which these data are available, while differences in individual inbreeding coefficients reflected the severity of historical bottlenecks and current census sizes in each population. PSMC analysis revealed multiple changes in Ne between approximately one million and 20 thousand years ago, corresponding to paleoclimatic changes and Cape buffalo colonisation of southern Africa. The results of this study have implications for buffalo management and conservation, particularly in the context of the predicted increase in aridity and temperature in southern Africa over the next century as a result of climate change.

Whole-Genome Resequencing Reveals Adaptation Prior to the Divergence of Buffalo Subspecies.

The application of high-throughput genotyping or sequencing data helps us to understand the genomic response to natural and artificial selection. In this study, we scanned the genomes of five indigenous buffalo populations belong to three recognized breeds, adapted to different geographical and agro-ecological zones in Iran, to unravel the extent of genomic diversity and to localize genomic regions and genes underwent past selection. A total of 46 river buffalo whole genomes, from West and East Azerbaijan, Gilan, Mazandaran, and Khuzestan provinces, were resequenced. Our sequencing data reached to a coverage above 99% of the river buffalo reference genome and an average read depth around 9.2× per sample. We identified 20.55 million SNPs, including 63,097 missense, 707 stop-gain, and 159 stop-loss mutations that might have functional consequences. Genomic diversity analyses showed modest structuring among Iranian buffalo populations following frequent gene flow or admixture in the recent past. Evidence of positive selection was investigated using both differentiation (Fst) and fixation (Pi) metrics. Analysis of fixation revealed three genomic regions in all three breeds with aberrant polymorphism contents on BBU2, 20, and 21. Fixation signal on BBU2 overlapped with the OCA2-HERC2 genes, suggestive of adaptation to UV exposure through pigmentation mechanism. Further validation using resequencing data from other five bovine species as well as the Axiom Buffalo Genotyping Array 90K data of river and swamp buffaloes indicated that these fixation signals persisted across river and swamp buffaloes and extended to taurine cattle, implying an ancient evolutionary event occurred before the speciation of buffalo and taurine cattle. These results contributed to our understanding of major genetic switches that took place during the evolution of modern buffaloes.

Selection signatures of Fuzhong Buffalo based on whole-genome sequences

BackgroundFuzhong buffalo, a native breed of Guangxi Zhuang Autonomous Region, is traditionally used as a draft animal to provide farm power in the rice cultivation. In addition, the Fuzhong buffalo also prepared for the bullfighting festival organized by the locals. The detection of the selective signatures in its genome can help in elucidating the selection mechanisms in its stamina and muscle development of a draft animal.ResultsIn this study, we analyzed 27 whole genomes of buffalo (including 15 Fuzhong buffalo genomes and 12 published buffalo genomes from Upper Yangtze region). The ZHp, ZFst, π-Ratio, and XP-EHH statistics were used to identify the candidate signatures of positive selection in Fuzhong buffalo. Our results detected a set of candidate genes involving in the pathways and GO terms associated with the response to exercise (e.g., ALDOA, STAT3, AKT2, EIF4E2, CACNA2D2, TCF4, CDH2), immunity (e.g., PTPN22, NKX2-3, PIK3R1, ITK, TMEM173), nervous system (e.g., PTPN21, ROBO1, HOMER1, MAGI2, SLC1A3, NRG3, SNAP47, CTNNA2, ADGRL3). In addition, we also identified several genes related to production and growth traits (e.g., PHLPP1, PRKN, MACF1, UCN3, RALGAPA1, PHKB, PKD1L). Our results depicted several pathways, GO terms, and candidate genes to be associated with response to exercise, immunity, nervous system, and growth traits.ConclusionsThe selective sweep analysis of the Fuzhong buffalo demonstrated positive selection pressure on potential target genes involved in behavior, immunity, and growth traits, etc. Our findings provided a valuable resource for future research on buffalo breeding and an insight into the mechanisms of artificial selection.

Genomic Identification, Evolution, and Expression Analysis of Collagen Genes Family in Water Buffalo during Lactation

Collagens, as extracellular matrix proteins, support cells for structural integrity and contribute to support mammary basic structure and development. This study aims to perform the genomic identification, evolution, and expression analyses of the collagen gene family in water buffalo (Bubalus bubalis) during lactation. A total of 128 buffalo collagen protein sequences were deduced from the 45 collagen genes identified in silico from buffalo genome, which classified into six groups based on their phylogenetic relationships, conserved motifs, and gene structure analyses. The identified collagen sequences were unequally distributed on 16 chromosomes. The tandem duplicated genes were found within three chromosomes, while only one segmental event occurred between Chr3 and Chr8. Collinearity analysis revealed that a total of 36 collagen gene pairs were orthologous between buffalo and cattle genomes despite having different chromosome numbers. Comparative transcription analyses revealed that a total of 23 orthologous collagen genes were detected in the milk samples at different lactation periods between the two species. Notably, the duplicated gene pair of COL4A1-COL4A2 during lactation had a higher mRNA expression level than that of cattle, while a higher expression level of COL6A1-COL6A2 pair was found in cattle compared with that of buffalo. The present study provides useful information for investigating the potential functions of the collagen family in buffalo during lactation and helps in the functional characterization of collagen genes in additional research.

Genomewide identification and annotation of SNPs in Bubalus bubalis

The present study was carried out to identify and annotate the genome wide SNPs in Murrah buffalo genome. A total of 21.2 million raw reads from 4 pooled female Murrah buffalo samples were obtained using restriction enzyme digestion followed by sequencing with Illumina Hiseq 2000. After quality filtration, the reads were aligned to Murrah buffalo genome (ICAR-NBAGR) and Water buffalo genome (UMD_CASPUR_WB_2.0) which resulted in 99.37% and 99.67% of the reads aligning, respectively. A total of 130,688 high quality SNPs along with 35,110 indels were identified versus the Murrah bufffalo genome. Similarly 219,856 high quality SNPs along with 15,201 indels were identified versus the Water buffalo genome. We report 483 SNPs in 66 genes affecting Milk Production, 436 SNPs in 38 genes affecting fertility and 559 SNPs in 72 genes affecting other major traits. The average genome coverage was 13.4% and 14.8% versus the Murrah and Water buffalo genomes, respectively.

Design and validation of a 90K SNP genotyping assay for the water buffalo (Bubalus bubalis).

BackgroundThe availability of the bovine genome sequence and SNP panels has improved various genomic analyses, from exploring genetic diversity to aiding genetic selection. However, few of the SNP on the bovine chips are polymorphic in buffalo, therefore a panel of single nucleotide DNA markers exclusive for buffalo was necessary for molecular genetic analyses and to develop genomic selection approaches for water buffalo. The creation of a 90K SNP panel for river buffalo and testing in a genome wide association study for milk production is described here.MethodsThe genomes of 73 buffaloes of 4 different breeds were sequenced and aligned against the bovine genome, which facilitated the identification of 22 million of sequence variants among the buffalo genomes. Based on frequencies of variants within and among buffalo breeds, and their distribution across the genome, inferred from the bovine genome sequence, 90,000 putative single nucleotide polymorphisms were selected to create an Axiom® Buffalo Genotyping Array 90K.ResultsThis 90K “SNP-Chip” was tested in several river buffalo populations and found to have ∼70% high quality and polymorphic SNPs. Of the 90K SNPs about 24K were also found to be polymorphic in swamp buffalo. The SNP chip was used to investigate the structure of buffalo populations, and could distinguish buffalo from different farms. A Genome Wide Association Study identified genomic regions on 5 chromosomes putatively involved in milk production.ConclusionThe 90K buffalo SNP chip described here is suitable for the analysis of the genomes of river buffalo breeds, and could be used for genetic diversity studies and potentially as a starting point for genome-assisted selection programmes. This SNP Chip could also be used to analyse swamp buffalo, but many loci are not informative and creation of a revised SNP set specific for swamp buffalo would be advised.

Comparison of Copy Number of HSF Genes in Two Buffalo Genomes

ABSTRACTThe copy number variation (CNV) is the number of copies of a particular gene in the genotype of an individual. Recent evidences show that the CNVs can vary in frequency and occurrence between breeds. These variations reportedly allowed different breeds to adapt to different environments. As copy number variations follow Mendelian pattern of inheritance, identification and distribution of these variants between populations can be used to infer the evolutionary history of the species. In this study, we have examined the absolute copy number of four Heat shock factor genes viz. HSF-1, 2, 4, and 5 in two different breeds of buffalo species using real-time PCR. Here, we report that the absolute copy number of HSF2 varies between the two breeds. In contrast no significant difference was observed in the copy number for HSF-1, 4, and 5 between the two breeds. Our results provide evidence for the presence of breed specific differences in HSF2 genomic copy number. This seems to be the first step in delineating the genetic factors underlying environmental adaptation between the two breeds. Nevertheless, a more detailed study is needed to characterize the functional consequence of this variation.

Evaluation of a combinatorial RNAi lentivirus vector targeting foot-and-mouth disease virus in vitro and in vivo.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals, which leads to serious economical losses. FMDV is not adequately controlled by vaccination or biosecurity measures. To generate genetically modified FMDV-resistant animals, a combinatorial expression cassette producing three short hairpin (sh) RNAs was constructed using the lentivirus (LV) vector, LV-3shRNA. The three shRNAs were expressed under the regulation of DNA polymerase III promoters from a buffalo and a bovine source, with one targeted to the non-structural protein 3B, and the other two targeted to the viral polymerase protein 3D of FMDV, respectively. The role of LV-3shRNA in the inhibition of the replication of FMDV was determined in BHK-21 cells and in suckling mice. The results revealed that LV-3shRNA reduced viral growth 3-fold (24 h post-infection) when the cells were challenged with 107-times the tissue culture infective dose (TCID50)/ml of O serotype FMDV. The suckling mice pretreated with LV-3shRNA were completely protected on administration of 5-times the dose of FMDV otherwise sufficient to kill 50% of the experimental animals (LD50). These results demonstrated that the LV-mediated dual expression of three FMDV-specific shRNAs provided a novel strategy towards combating FMDV, which facilitates the permanent introduction of novel disease-resistance traits into the buffalo and bovine genomes in the future.

RsaI repetitive DNA in Buffalo Bubalus bubalis representing retrotransposons, conserved in bovids, are part of the functional genes

BackgroundRepetitive sequences are the major components of the eukaryotic genomes. Association of these repeats with transcribing sequences and their regulation in buffalo Bubalus bubalis has remained largely unresolved.ResultsWe cloned and sequenced RsaI repeat fragments pDp1, pDp2, pDp3, pDp4 of 1331, 651, 603 and 339 base pairs, respectively from the buffalo, Bubalus bubalis. Upon characterization, these fragments were found to represent retrotransposons and part of some functional genes. The resultant clones showed cross hybridization only with buffalo, cattle, goat and sheep genomic DNA. Real Time PCR, detected ~2 × 104 copies of pDp1, ~ 3000 copies of pDp2 and pDp3 and ~ 1000 of pDp4 in buffalo, cattle, goat and sheep genomes, respectively. RsaI repeats are transcriptionally active in somatic tissues and spermatozoa. Accordingly, pDp1 showed maximum expression in lung, pDp2 and pDp3 both in Kidney, and pDp4 in ovary. Fluorescence in situ hybridization showed repeats to be distributed all across the chromosomes.ConclusionsThe data suggest that RsaI repeats have been incorporated into the exonic regions of various transcribing genes, possibly contributing towards the architecture and evolution of the buffalo and related genomes. Prospects of our present work in the context of comparative and functional genomics are highlighted.

Detection of genome specific monomorphic loci in Bos taurus and Bubalus bubalis with oligodeoxyribonucleotide probe

A synthetic oligodeoxyribonucleotide probe (OAT36) comprising nine repeats of 5'GACA 3' and several enzymes were used to analyse cow, (Bos taurus) and buffalo (Bubalus bubalis) genomes and a number of monomorphic loci were detected in both the species. Different animals from the same species showed an almost 'similar' monomorphic hybridization pattern but animals from two separate species showed a different 'genome specific' pattern. The overall hybridization with any enzyme and probe combination was found to be unique to one species. This forms the basis of genome specific hybridization which is substantiated by our zoo-blot hybridization studies. The evolutionary aspect of these loci in the context of sequence polymorphisms is discussed.