Adaptive selection signatures in river buffalo with emphasis on immune and major histocompatibility complex genes

River buffalo is an agriculturally important species with many traits, such as disease tolerance, which promote its use worldwide. Highly contiguous genome assemblies of the river buffalo, goat, pig, human and two cattle subspecies were aligned to study gene gains and losses and signs of positive selection. The gene families that have changed significantly in river buffalo since divergence from cattle play important roles in protein degradation, the olfactory receptor system, detoxification and the immune system. We used the branch site model in PAML to analyse single-copy orthologs to identify positively selected genes that may be involved in skin differentiation, mammary development and bone formation in the river buffalo branch. The high contiguity of the genomes enabled evaluation of differences among species in the major histocompatibility complex. We identified a Babesia-like L1 LINE insertion in the DRB1-like gene in the river buffalo and discuss the implication of this finding.     

Preliminary radiation hybrid map for river buffalo chromosome 6 and comparison to bovine chromosome 3

We present the first radiation hybrid (RH) map of river buffalo (Bubalus bubalis) chromosome 6 (BBU6) developed with a recently constructed river buffalo whole-genome RH panel (BBURH(5000)). The preliminary map contains 33 cattle-derived markers, including 12 microsatellites, 19 coding genes and two ESTs, distributed across two linkage groups. Retention frequencies for markers ranged from 14.4% to 40.0%. Most of the marker orders within the linkage groups on BBU6 were consistent with the cattle genome sequence and RH maps. This preliminary RH map is the starting point for comparing gene order between river buffalo and cattle, presenting an opportunity for the examination of micro-rearrangements of these chromosomes. Also, resources for positional candidate cloning in river buffalo are enhanced.     

A high‐resolution radiation hybrid map of the river buffalo major histocompatibility complex and comparison with BoLA

The major histocompatibility complex (MHC) in mammals codes for antigen‐presenting proteins. For this reason, the MHC is of great importance for immune function and animal health. Previous studies revealed this gene‐dense and polymorphic region in river buffalo to be on the short arm of chromosome 2, which is homologous to cattle chromosome 23. Using cattle‐derived STS markers and a river buffalo radiation hybrid (RH) panel (BBURH₅₀₀₀), we generated a high‐resolution RH map of the river buffalo MHC region. The buffalo MHC RH map (cR₅₀₀₀) was aligned with the cattle MHC RH map (cR₁₂₀₀₀) to compare gene order. The buffalo MHC had similar organization to the cattle MHC, with class II genes distributed in two segments, class IIa and class IIb. Class IIa was closely associated with the class I and class III regions, and class IIb was a separate cluster. A total of 53 markers were distributed into two linkage groups based on a two‐point LOD score threshold of ≥8. The first linkage group included 32 markers from class IIa, class I and class III. The second linkage group included 21 markers from class IIb. Bacterial artificial chromosome clones for seven loci were mapped by fluorescence in situ hybridization on metaphase chromosomes using single‐ and double‐color hybridizations. The order of cytogenetically mapped markers in the region corroborated the physical order of markers obtained from the RH map and served as anchor points to align and orient the linkage groups.     

Comparative Analysis of the Shadoo Gene between Cattle and Buffalo Reveals Significant Differences

Background

While prions play a central role in the pathogenesis of transmissible spongiform encephalopathies, the biology of these proteins and the pathophysiology of these diseases remain largely unknown. Since no case of bovine spongiform encephalopathy (BSE) has ever been reported in buffalo despite their phylogenetic proximity to cattle, genetic differences may be driving the different susceptibilities of these two species to BSE. We thus hypothesized that differences in expression of the most recently identified member of the prion family or Shadoo (SPRN) gene may relate to these species-specific differences.

Principal Findings

We first analyzed and compared the polymorphisms of the SPRN gene (∼4.4 kb), including the putative promoter, coding and 3′ regions, and further verified the entire ORF and putative promoter. This yielded a total of 117 fixed differences, remarkably: 1) a 12-bp insertion/deletion polymorphism in the hydrophobic domain of the cattle but not buffalo gene, introducing a four amino acid expansion/contraction in a series of 5 tandem Ala/Gly-containing repeats; 2) two fixed missense mutations (102Ser→Gly and 119Thr→Ala), and three missense mutations (92Pro>Thr/Met, 122Thr>Ile and 139Arg>Trp) in the coding region presenting different (P<0.05) genotypic and allelic frequency distributions between cattle and buffalo; and, 3) functional luciferase-reporter experiments for the predicted promoter region, consistent with a significantly higher activity in buffalo than cattle. Supporting these findings, immunoblotting revealed higher relative expression levels of Sho protein in cerebrum from buffalo than from cattle. In addition, for cattle, highest Sho expression was detected in obex, as compared to cerebrum or cerebellum.

Significance

Our findings support Sho as a non-PrP specific marker for prion infections, with obex as the best tissue source for the detection of Sho in TSE rapid tests. Moreover, these discoveries may prove advantageous for further understanding the biology of prion diseases.     

MHC-DRB exon 2 allele polymorphism in Indian river buffalo (Bubalus bubalis)

The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.     

The complete coding region sequence of river buffalo (Bubalus bubalis) SRY gene.

The Y-linked SRY gene is responsible for testis determination in mammals. Mutations in this gene can lead to XY Gonadal Dysgenesis, an abnormal sexual phenotype described in humans, cattle, horses and river buffalo. We report here the complete river buffalo SRY sequence in order to enable the genetic diagnosis of this disease. The SRY sequence was also used to confirm the evolutionary divergence time between cattle and river buffalo 10 million years ago.     

Draft genome of the river water 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.     

Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel and preliminary RH mapping of chromosomes 3 and 10

The buffalo (Bubalus bubalis) is a source of milk and meat, and also serves as a draft animal. In this study, a 5000-rad whole-genome radiation hybrid (RH) panel for river buffalo was constructed and used to build preliminary RH maps for BBU3 and BBU10 chromosomes. The preliminary maps contain 66 markers, including coding genes, cattle expressed sequence tags (ESTs) and microsatellite loci. The RH maps presented here are the starting point for mapping additional loci that will allow detailed comparative maps between buffalo, cattle and other species whose genomes may be mapped in the future. A large quantity of DNA has been prepared from the cell lines forming the river buffalo RH panel and will be made publicly available to the international community both for the study of chromosome evolution and for the improvement of traits important to the role of buffalo in animal agriculture.     

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 1 (BBU1)

The largest chromosome in the river buffalo karyotype, BBU1, is a submetacentric chromosome with reported homology between BBU1q and bovine chromosome 1 and between BBU1p and BTA27. We present the first radiation hybrid map of this chromosome containing 69 cattle derived markers including 48 coding genes, 17 microsatellites and four ESTs distributed in two linkage groups spanning a total length of 1330.1 cR(5000). The RH map was constructed based on analysis of a recently developed river buffalo-hamster whole genome radiation hybrid (BBURH(5000)) panel. The retention frequency of individual markers across the panel ranged from 17.8 to 52.2%. With few exceptions, the order of markers within linkage groups is identical to the order established for corresponding cattle RH maps. The BBU1 map provides a starting point for comparison of gene order rearrangements between river buffalo chromosome 1 and its bovine homologs.     

Lactate dehydrogenase β processed pseudogene in river buffalo and other mammalian species

In the present study a primer pair originally designed to amplify a DNA segment of the lactate dehydrogenase β (LDHβ) parent gene was tested in river buffalo. The primer pair amplified a 318 bp DNA segment. The DNA sequence of this segment was determined and compared with the mammalian whole genome sequences in Genbank database for human, cattle and mouse. Blast data analysis showed that the sequence of the buffalo amplified DNA segment aligns with LDHβ parent genes of cattle, mouse and human at four scattered sites representing the last 23 bases of exon 2, exon 3, exon 4 and the first three bases of exon 5. Results also revealed that the sequence of buffalo DNA segment is 98%, 88% and 85% similar to a DNA segment of LDHβ processed pseudogene (LDHβP) of cattle, mouse and humans, respectively. These findings indicate that the amplified DNA segment does not belong to LDHβ parent gene and that as in human, cattle and mouse, the river buffalo has an LDHβ pseudogene of the processed type.     

Polymorphism and haplotype structure in River Buffalo (Bubalus bubalis) toll-like receptor 5 (TLR5) coding sequence

Most of the 160 million river buffalo in the world are in Asia where they are used extensively, both as a food source and for draught power. Only recently have investigations begun exploring the buffalo genome for variation that might influence health and productivity of these economically important animals. This paper describes the sequence variability of the toll-like receptor 5 (TLR5) gene, which recognizes bacterial flagellin and is a key player in the immune system. TLR5 is comprised of a single exon that is 2577?bp and codes 858?amino acids. We examined single-nucleotide polymorphisms (SNPs) located within the coding region. Overall, 17 SNPs were discovered, seven of which are non-synonymous. Our study population yielded four different haplotypes. We examined predicted protein domain structure and found that river buffalo, swamp buffalo, and African Forest buffalo shared the same protein domain structure and are more similar to each other than they are to cattle and American bison, which are similar to each other. PolyPhen 2 analysis revealed one amino acid substitution in the river buffalo population with potential functional significance.     

Polymorphism and Haplotype Structure in River Buffalo (Bubalus bubalis) Toll-Like Receptor 5 (TLR5) Coding Sequence

Most of the 160 million river buffalo in the world are in Asia where they are used extensively, both as a food source and for draught power. Only recently have investigations begun exploring the buffalo genome for variation that might influence health and productivity of these economically important animals. This paper describes the sequence variability of the toll-like receptor 5 (TLR5) gene, which recognizes bacterial flagellin and is a key player in the immune system. TLR5 is comprised of a single exon that is 2577 bp and codes 858 amino acids. We examined single-nucleotide polymorphisms (SNPs) located within the coding region. Overall, 17 SNPs were discovered, seven of which are non-synonymous. Our study population yielded four different haplotypes. We examined predicted protein domain structure and found that river buffalo, swamp buffalo, and African Forest buffalo shared the same protein domain structure and are more similar to each other than they are to cattle and American bison, which are similar to each other. PolyPhen 2 analysis revealed one amino acid substitution in the river buffalo population with potential functional significance.     

The river buffalo (Bubalus bubalis, 2n = 50) cytogenetic map: assignment of 64 loci by fluorescence in situ hybridization and R-banding

Sixty-four genomic BAC-clones mapping five type I (ADCYAP1, HRH1, IL3, RBP3B and SRY) and 59 type II loci, previously FISH-mapped to goat (63 loci) and cattle (SRY) chromosomes, were fluorescence in situ mapped to river buffalo R-banded chromosomes, noticeably extending the physical map of this species. All mapped loci from 26 bovine syntenic groups were located on homeologous chromosomes and chromosome regions of river buffalo and goat (cattle) chromosomes, confirming the high degree of chromosome homeologies among bovids. Furthermore, an improved cytogenetic map of the river buffalo with 293 loci from all 31 bovine syntenic groups is reported.     

Interferon-alpha genes from Bos and Bubalus bubalus

Interferon-a genes were cloned from six breeds of three species of two genera (three Chinese native cattle breeds of yellow cattle, wild yak and HuanHu domestic yak, one European breed of Holstein cow, and two water buffalo breeds of FuAn water buffalo and FuZhong water buffalo) by direct PCR. The PCR products were directly inserted into the expression vector to be sequenced and expressed. Sequence analysis showed that IFN-a genes of six clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids. Compared with the published BoIFN-a subtypes, the IFN-a gene of Holstein cow had only one point mutation with the BoIFN-aA subtype. The IFN-a gene of yellow cattle was similar to the BoIFN-aD subtype with amino acid identity of 97.0% and may be considered as a new subtype, namely, BoIFN-aD1. The other four IFN-a genes, cloned from wild yak and HuanHu domestic yak, FuAn water buffalo, and FuZhong water buffalo, represented four new subtypes, namely, BoIFN-aI, BoIFN-aJ, BuIFN-a1, and BuIFN-a2, respectively. Each of the six clones was expressed in E. coli with molecular weight of approximately 20 kDa by SDS-PAGE and Western blot analyses. Antiviral activity assays showed that the six recombinant IFN-a (rIFN-a) all exhibited 1,000 times higher antiviral activity in the MDBK/VSV cell line than in the CEF/VSV one. Moreover, the rIFN-as could inhibit infectious bovine rhinotracheitis virus replication in the MDBK cell line using CPE inhibition method. The results suggested that rIFN-as a potential agent for clinical application against virus diseases in cattle industry.     

水牛、大额牛和牦牛抑制素α亚基编码基因外显子1多态性分析

为了揭示牛科物种INHA基因的遗传特征,该文采用PCR产物直接测序法对水牛、大额牛和牦牛INHA基因外显子1及其侧翼序列进行多态性检测,并结合已发表的包括牛科物种在内的一些哺乳动物数据进行了比较分析。结果表明,在水牛INHA基因外显子1中存在c.73C>A替换,为同义替换,河流型和沼泽型水牛编码产物一致;在大额牛的INHA基因外显子1中发现c.62C>T、c.187G>A替换,分别引起INHA中氨基酸发生p.P21L、p.V63M改变,两者均为相同性质氨基酸的替换;在牦牛中发现c.62C>T、c.129A>G替换,前者也引起编码氨基酸发生p.P21L替换,后者为同义替换。在INHA基因5’侧翼区所测出的序列中,水牛、大额牛和牦牛等物种内均未发现SNP位点,但在种间发现存在c.-6T>G的替换,大额牛、牦牛和普通牛均为c.-6G,而水牛为c.-6T。在INHA基因内含子中,水牛的第31～36位核苷酸处发现有6个碱基的缺失,即c.262+31₂62+36delTCTGAC;该位点在河流型水牛中野生型（+/+）占主体,而在沼泽型水牛中则缺失型（-/-）占主体。在大额牛、牦牛和普通牛等其它牛科物种的内含子中均未发现该缺失,但与水牛相比,大额牛、牦牛和普通牛内含子中发现缺失c.262+78₂62+79delTG。序列比对显示,INHA基因外显子1序列中c.43A和c.67G为水牛中所特有,而c.173A和c.255G为大额牛、牦牛和普通牛所共有,c.24C、c.47G、c.174T和c.206T为山羊所特有。大额牛、牦牛和普通牛间INHA基因外显子1序列差异较小,而山羊和水牛与它们间的差异相对较大。     

Interferon-α Genes from Bos and Bubalus bubalus

Interferon-α genes were cloned from six breeds of three species of two genera (three Chinese native cattle breeds of yellow cattle, wild yak and HuanHu domestic yak, one European breed of Holstein cow, and two water buffalo breeds of FuAn water buffalo and FuZhong water buffalo) by direct PCR. The PCR products were directly inserted into the expression vector to be sequenced and expressed. Sequence analysis showed that IFN-α genes of six clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids. Compared with the published BoIFN-α subtypes, the IFN-α gene of Holstein cow had only one point mutation with the BoIFN-αA subtype. The IFN-α gene of yellow cattle was similar to the BoIFN-αD subtype with amino acid identity of 97.0% and may be considered as a new subtype, namely, BoIFN-αD1. The other four IFN-α genes, cloned from wild yak and HuanHu domestic yak, FuAn water buffalo, and FuZhong water buffalo, represented four new subtypes, namely, BoIFN-αI, BoIFN-αJ, BuIFN-α1, and BuIFN-α2, respectively. Each of the six clones was expressed in E. coli with molecular weight of ～ 20kDa by SDS-PAGE and Western blot analyses. Antiviral activity assays showed that the six recombinant IFN-α (rIFN-α) all exhibited 1000 times higher antiviral activity in the MDBK/VSV cell line than in the CEF/VSV one. Moreover, the rIFN-αs could inhibit infectious bovine rhinotracheitis virus replication in the MDBK cell line using CPE inhibition method. The results suggested that rIFN-αs a potential agent for clinical application against virus diseases in cattle industry.     

Karyotypic evolution of ribosomal sites in buffalo subspecies and their crossbreed

Domestic buffaloes are divided into two group based on cytogenetic characteristics and habitats: the “river buffaloes” with 2n = 50 and the “swamp buffaloes”, 2n = 48. Nevertheless, their hybrids are viable, fertile and identified by a 2n = 49. In order to have a better characterization of these different cytotypes of buffaloes, and considering that NOR-bearing chromosomes are involved in the rearrangements responsible for the karyotypic differences, we applied silver staining (Ag-NOR) and performed fluorescent in situ hybridization (FISH) experiments using 18S rDNA as probe. Metaphases were obtained through blood lymphocyte culture of 21 individuals, including river, swamp and hybrid cytotypes. Ag-NOR staining revealed active NORs on six chromosome pairs (3p, 4p, 6, 21, 23, 24) in the river buffaloes, whereas the swamp buffaloes presented only five NOR-bearing pairs (4p, 6, 20, 22, 23). The F1 cross-breed had 11 chromosomes with active NORs, indicating expression of both parental chromosomes. FISH analysis confirmed the numerical divergence identified with Ag-NOR. This result is explained by the loss of the NOR located on chromosome 4p in the river buffalo, which is involved in the tandem fusion with chromosome 9 in this subspecies. A comparison with the ancestral cattle karyotype suggests that the NOR found on the 3p of the river buffalo may have originated from a duplication of ribosomal genes, resulting in the formation of new NOR sites in this subspecies.