雷琼牛GH基因第五外显子遗传多样性及其分化

对16头雷琼牛GH基因第5外显子序列进行分析,发现了1个变异位点,定义了2种单倍型。引用巴州牦牛2个个体GH基因同源区序列并结合GenBank中牛属普通牛、其它瘤牛和牦牛3个种群与水牛1个远缘种GH基因同源区序列,分别采用邻接（NJ）法和最大简约（MP）法构建分子系统发育树,得到基本一致的拓扑结构,结果显示GH基因的分化早于雷琼牛（瘤牛）、其它瘤牛、普通牛、牦牛和水牛的分化,瘤牛物种内存在多型,同时证实了GH基因第5外显子区有着较高的突变率。     

Satellite-tagged transcribing sequences in Bubalus bubalis genome undergo programmed modulation in meiocytes: possible implications for transcriptional inactivation.

We cloned and sequenced a 1378 bp BamHI satellite DNA fraction from the water buffalo Bubalus bubalis and have studied its expression in different tissues. The GC-rich sequences of the resultant contig pDS5 crosshybridize only with bovid DNA and are not conserved evolutionarily. Typing of buffalo genomic DNA using pDS5 with several restriction enzymes revealed multilocus monomorphic bands. Similar typing of cattle, buffalo, goat, sheep, and gaur genomic DNA revealed variations in copy number and allele length giving rise to species-specific band patterns. Expression study of pDS5 in bubaline samples by RNA slot-blot, Northern blot, and RT-PCR showed various levels of signal in all the somatic tissues and germline cells except heart. A GenBank database search revealed homology of pDS5 sequences in the 5' region from nt 1-1261 with collagen gene. An AluI typing analysis of DNA from bubaline semen samples showed consistent loss of two bands. The presence of corresponding bands in somatic tissues suggests a sequence modulation within the pDS5 array in meiocytes during spermatogenesis, which is restored in the somatic cells after fertilization. Modulation of the satellite-tagged transcribing sequence in the meiocytes may be a mechanism of its inactivation.     

以Cytb基因部分序列分析大额牛系统地位及其种群现状

对12头大额牛Cytb基因部分序列447bp进行分析,共发现33个变异位点,定义了4种单倍型;结合GenBank中牛属普通牛、瘤牛、牦牛和印度褐牛4个近缘种的Cytb基因同源区序列进行分析,以水牛作为外群,分别采用邻接(NJ)法和最大简约(MP)法构建分子系统发育树,得到基本一致的拓扑结构,结果表明大额牛是独立于普通牛、瘤牛和印度褐牛之外的Bos属的一个独立的种。同时还指出目前我国有相当比例的大额牛受到外来物种的入侵,我国的大额牛保护工作正面临非常严峻的考验。     

Physical organization of the 1.709 satellite IV DNA family in Bovini and Tragelaphini tribes of the Bovidae: sequence and chromosomal evolution

Repetitive DNA in the mammalian genome is a valuable record and marker for evolution, providing information about the order and driving forces related to evolutionary events. The evolutionarily young 1.709 satellite IV DNA family is present near the centromeres of many chromosomes in the Bovidae. Here, we isolated 1.709 satellite DNA sequences from five Bovidae species belonging to Bovini: Bos taurus (BTA, cattle), Bos indicus (BIN, zebu), Bubalus bubalis (BBU, water buffalo) and Tragelaphini tribes: Taurotragus oryx (TOR, eland) and Tragelaphus euryceros (TEU, bongo). Its presence in both tribes shows the sequence predates the evolutionary separation of the two tribes (more than 10 million years ago), and primary sequence shows increasing divergence with evolutionary distance. Genome organization (Southern hybridization) and physical distribution (in situ hybridization) revealed differences in the molecular organization of these satellite DNA sequences. The data suggest that the sequences on the sex chromosomes and the autosomes evolve as relatively independent groups, with the repetitive sequences suggesting that Bovini autosomes and the Tragelaphini sex chromosomes represent the more primitive chromosome forms.     

Molecular characterization of cDNA encoding B. taurus cathelicidin-7 like antibiotic peptide from bone marrow cells of Bubalus bubalis.

Cathelicidins represent a diverse family of endogenous cationic antibiotic peptide present in all mammalian species. In the present study, a novel cathelicidin cDNA was identified and characterized from bone marrow cells of buffalo (Bubalus bubalis) using RT-PCR based approach. The cDNA encodes a propeptide of 1.18 kDa with net positive charge at neutral pH. The precursor peptide possesses a signal peptide of 29 amino acids and a biologically active peptide of 34 residues. Comparison of sequences indicates only 66.1 and 64.1% identity at nucleotides and amino acids level respectively, with the already reported cathelicidin congener from the same species. However, high degree of similarity (92.8% nucleotides and 81.9% amino acids) was noticed with cathelicidin 7 sequence of Bos taurus suggesting interspecies conservation of cathelicidin peptides rather than intra-species within bovidae family. Phylogenetic trees analyses also support these data. Our findings, further justify the cloned cDNA as a unique cathelicidin member of B. bubalis, and may reasonably considered to be another example of structural diversity exhibited by cathelicidin-derived peptides as reported from other mammals.     

Analysis of conserved microsatellite sequences suggests closer relationship between water buffalo Bubalus bubalis and sheep Ovis aries.

The distribution and evolutionary pattern of the conserved microsatellite repeat sequences (CA)n, (TGG)6, and (GGAT)4 were studied to determine the divergence time and phylogenetic position of the water buffalo, Bubalus bubalis. The mean allelic frequencies of these repeat loci showed a high level of heterozygosity among the euartiodactyls (buffalo, cattle, sheep, and goat). Genetic distances calculated from the allelic frequencies of these microsatellites were used to position Bubalus bubalis in the phylogenetic tree. The tree topology revealed a closer proximity of the Bubalus bubalis to the Ovis aries (sheep) genome than to other domestic species. The estimated time of divergence of the water buffalo genome relative to cattle, goat, sheep, pig, rabbit, and horse was found to be 21, 0.5, 0.7, 94, 20.3, and 408 million years (Myr), respectively. Although water buffaloes share morphological and biochemical similarities with cattle, our study using the microsatellite sequences places the bubaline species in an entirely new phylogenetic position. Our results also suggest that with respect to these repeat loci, the water buffalo genome shares a common ancestry with sheep and goat after the divergence of subfamily Bovinae (Bos taurus) from the family Bovidae.     

从细胞色素b基因全序列探讨大额牛的分子系统发生

大额牛是一种半野生半家养的珍稀牛种, 有关其起源和系统地位一直存在争议。通过PCR扩增、测序等步骤共获得了11头大额牛细胞色素b(Cyt b)基因全序列(1 140 bp)。应用分析软件, 对大额牛11条Cyt b序列进行了分析, 并结合GenBank中牛属动物6个近缘种的同源序列, 以亚洲水牛(Bubalus bubalis)为外群, 分别采用邻接法(NJ)和最大简约法(MP)构建了牛属动物分子系统发育树。序列分析结果表明, 11条大额牛Cyt b序列1 140位点中, 共发现95个变异位点(占分析位点总数的8.33 %), 定义了6种单倍型, 表明大额牛群体的Cyt b基因遗传多态性比较丰富。构建的NJ和MP分子系统树均显示, 大额牛研究群体明显分为3支, 第1支与普通牛(Bos taurus)相聚, 第2支与瘤牛(Bos indicus)相聚, 第3支与印度野牛(Bos gaurus)相聚。系统发育分析表明, 大额牛很可能是印度野牛的家养型或驯化种, 我国大额牛群体可能曾受到其他牛种血缘的入侵。     

Characterization of PRLR and PPARGC1A genes in buffalo (Bubalus bubalis)

More than 40 million households in India depend at least partially on livestock production. Buffaloes are one of the major milk producers in India. The prolactin receptor (PRLR) gene and peroxisome proliferators activated receptor-γ coactivator 1-alpha (PPARGC1A) gene are reportedly associated with milk protein and milk fat yields in Bos taurus. In this study, we sequenced the PRLR and PPARGC1A genes in the water buffalo Bubalus bubalis. The PRLR and PPARGC1A genes coded for 581 and 819 amino acids, respectively. The B. bubalis PRLR gene differed from the corresponding Bos taurus at 21 positions and four differences with an additional arginine at position 620 in the PPARGC1A gene were found in the amino acid sequence. All of the changes were confirmed by cDNA sequencing. Twelve buffalo-specific single nucleotide polymorphisms (SNPs) were identified in both genes, with five of them being non-synonymous.     

Characterization and chromosomal distribution of satellite DNA sequences of the water buffalo (Bubalus bubalis).

Satellite DNA sequences were isolated from the water buffalo (Bubalus bubalis) after digestion with two restriction endonucleases, BamHI and StuI. These satellite DNAs of the water buffalo were classified into two types by sequence analysis: one had an approximately 1,400 bp tandem repeat unit with 79% similarity to the bovine satellite I DNA; the other had an approximately 700 bp tandem repeat unit with 81% similarity to the bovine satellite II DNA. The chromosomal distribution of the satellite DNAs were examined in the river-type and the swamp-type buffaloes with direct R-banding fluorescence in situ hybridization. Both the buffalo satellite DNAs were localized to the centromeric regions of all chromosomes in the two types of buffaloes. The hybridization signals with the buffalo satellite I DNA on the acrocentric autosomes and X chromosome were much stronger than that on the biarmed autosomes and Y chromosome, which corresponded to the distribution of C-band-positive centromeric heterochromatin. This centromere-specific satellite DNA also existed in the interstitial region of the long arm of chromosome 1 of the swamp-type buffalo, which was the junction of the telomere-centromere tandem fusion that divided the karyotype in the two types of buffaloes. The intensity of the hybridization signals with buffalo satellite II DNA was almost the same over all the chromosomes, including the Y chromosome, and no additional hybridization signal was found in noncentromeric sites.     

黄牛、水牛体内人肉孢子虫18S rRNA基因研究及虫种鉴定

本文对自然感染的水牛源的人肉孢子虫以及黄牛源人孢子虫ＤＮＡ的１８Ｓ ｒＲＮＡ基因的ＰＣＲ扩增产物进行了测序。对年获的８６３ｂｐ的１８Ｓ ｒＲＮＡ基因分析比较表明,二者有较高的同源性,因此认为二者可能同是一种肉孢子虫－－人肉孢子虫（Ｓａｒｃｏｃｙｓｔｉｓ ｈｏｍｉｎｉｓ Ｒａｉｌｌｉｅｔ ａｎｄ Ｌｕｃｅｔ,１８９１）。由此推断,不仅黄牛可作为人肉孢子虫的中间宿主,水牛也可作为人肉孢子虫的中间宿主。     

Bubaline versus bovine reproduction

Fertility in water buffalo (Bubalus bubalis) is considerably lower than that in cattle (Bos taurus and Bos indicus). Poor breeding efficiency is attributed to late onset of puberty, seasonality, poor estrus expression, and long calving intervals. Accurate estrus detection is a prerequisite for efficient reproductive management. Established reproductive management techniques in cattle can be successfully applied to water buffalo because of the similarities in the anatomy, physiology, and endocrinology of reproduction between the two genera.     

First radiation hybrid map of the river buffalo X chromosome (BBUX) and comparison with BTAX

We report the first radiation hybrid map of the river buffalo X chromosome generated from a recently constructed river buffalo ( Bubalus bubalis ) whole-genome radiation hybrid panel (BBURH₅₀₀₀). This map contains a total of 33 cattle-derived markers, including 10 genes, four ESTs and 19 microsatellites. The markers are distributed in two linkage groups: LG1 contains eight markers spanning 125.6 cR, and LG2 contains 25 markers spanning 366.3 cR. LG1 contains six markers in common with bovine sequence assembly build 3.1. With the exception of BMS2152 , the order of these markers on our BBUX map is shuffled when compared to the cow X chromosome ( Bos taurus ; BTAX). From LG2, two markers ( AMELX and BL22 ) map to a more distal portion of BTAX compared to BBUX. In addition, two pairs of LG2 markers exhibit inversions compared to BTAX ( ILSTS017 and ATRX ; XBM38 and PPEF1 ). Alternatively, when compared to the most recent bovine RH map (Bov-Gen 3000rads), BL1098 and BMS2227 from LG1 as well as PLS3 and BMS1820 from LG2 showed inverted positions on the BBUX map. These discrepancies in buffalo and cattle maps may reflect evolutionary divergence of the chromosomes or mapping errors in one of the two species. Although the set of mapped markers does not cover the entire X chromosome, this map is a starting point for the construction of a high-resolution map, which is necessary for characterization of small rearrangements that might have occurred between the Bubalus bubalis and Bos taurus X chromosomes.     

Partial purification and isoelectric focusing patterns of the buffalo (Bubalus bubalis) and the Kedah-Kelantan cattle (Bos indicus) glutathione transferases.

1. Glutathione transferases from the liver, lung and kidney tissues of the buffalo (Bubalus bubalis) and the Kedah-Kelantan cattle (Bos indicus) were partially purified by ammonium sulphate precipitation and Sephadex G-75 gel filtration. 2. Liver tissue contains the highest enzyme activity when compared to the lung and kidney tissues. 3. The activity in cattle is higher than that in the buffalo. 4. Isoelectric focusing separates the activities into the acidic, near neutral and basic fractions. 5. The focused patterns are different for each of the tissues and in each of the species investigated.     

Dominance of Methanomicrobium phylotype in methanogen population present in Murrah buffaloes (Bubalus bubalis)

Aims:  To study the diversity of rumen methanogens in Murrah buffaloes ( Bubalus bubalis ) from North India by using 16S rRNA gene libraries obtained from the pooled rumen content from four animals and using suitable software analysis.
Methods and Results:  Genomic DNA was isolated and PCR was set up by using specific primers. Amplified product was cloned into a suitable vector and the positive clones were selected on the basis of blue–white screening and sequenced. The resulting nucleotide sequences were arranged in the phylogenetic tree. A total of 108 clones were examined, revealing 17 different 16S rRNA gene sequences or phylotypes. Of the 17 phylotypes, 15 (102 of 108 clones) belonged to the genus Methanomicrobium , indicating that the genus Methanomicrobium is the most dominant component of methanogen populations in Murrah buffaloes ( Bubalus bubalis ) from North India. The largest group of clones (102 clones) was more than 98% similar to Methanomicrobium mobile . BLAST analysis of the rumen contents from individual animals also revealed 17 different phylotypes with a range of 3–10 phylotypes per animal.
Conclusion:  Methanomicrobium phylotype is the most dominant phylotype of methanogens present in Murrah buffaloes ( Bubalus bubalis ).
Significance and Impact of the Study:  Effective strategies can be made to inhibit the growth of Methanomicrobium phylotype to reduce the methane emission from rumen contents and thus help in preventing global warming.     

Mitomycin C-induced sister chromatid exchange in X chromosomes of Bovidae

Sister chromatid exchanges (SCEs) in early- and late-replicating X chromosomes of seven female cattle (Bos taurus L.) and five female river buffalo (Bubalus bubalis L.) were studied in untreated lymphocytes and lymphocytes treated with mitomycin C (MMC). In the experiment, 577 SCEs on X chromosomes of MMC-untreated cells and 825 SCEs on X chromosomes of MMC-treated cells from both species were observed. No significant differences between the number of SCEs in early- and late-replicating X chromosomes were found even when singular species and subjects were considered.     

牛科动物HSL基因序列分析及其分子进化研究

在对牛科中4种动物即牦牛、瘤牛、普通牛和水牛HSL基因外显子Ⅰ部分核苷酸序列进行测定的基础上,与Gen-Bank中其他物种相应基因核苷酸序列、氨基酸序列进行了比对分析,并构建了牦牛与其他物种间分子系统进化树。结果表明:牦牛与普通牛、瘤牛、水牛、猪、人、小鼠、大鼠7个物种HSL基因外显子Ⅰ部分核苷酸序列间保守性较高,同源性大小依次为99.8%、99.6%、97.4%、90.6%、88.4%、83.5%、82.3%。相应氨基酸序列间保守性更高,同源性分别为100%、100%、98.2%、94.0%、92.2%、89.8%、89.8%。牦牛与各物种该基因部分核苷酸序列间碱基变异类型主要表现为碱基转换和颠换,无碱基插入和缺失发生,碱基转换的频率高于颠换的频率;在核苷酸水平上的多数碱基替换都是同义替换;序列间单碱基变异位点大多出现在同一位点,多发生在密码子第3位,其次是第1位,最少发生在第2位,符合分子进化的中性学说。HSL基因外显子Ⅰ部分核苷酸序列进行多序列对位排列构建的各物种间分子系统进化树结果表明,普通牛和瘤牛首先聚为一类,再分别与牦牛、水牛、猪、人聚类,最后与大鼠、小鼠聚为一类。该聚类结果与动物学上的分类结果一致,表明HSL基因外显子Ⅰ部分核苷酸序列适合于构建物种间分子系统进化树。研究表明,牦牛、普通牛和瘤牛3个物种间的遗传距离大小相近,牦牛和水牛间的遗传距离与普通牛、瘤牛和水牛间的遗传距离大小相当。牦牛、普通牛和瘤牛3个物种间的遗传距离远小于它们各自与水牛这一物种的遗传距离,它们三者之间的亲缘关系也相对于它们各自与水牛间的亲缘关系都较近,故将牦牛、普通牛和瘤牛划分在同一个属——牛属(Bos)更为合理。     

Polymorphism and gene organization of water buffalo MHC-DQB genes show homology to the BoLA DQB region

In cattle (Bos taurus), there is evidence of more than 50 alleles of BoLA-DQB (bovine lymphocyte antigen DQB) that are distributed across at least five DQB loci, making this region one of the most complex in the BoLA gene family. In this study, DQB alleles were analysed for the water buffalo (Bubalus bubalis), another economically important bovine species. Twelve alleles for Bubu-DQB (Bubalis bubalis DQB) were determined by nucleotide sequence analysis. A phylogenetic analysis revealed numerous trans-species polymorphisms, with alleles from water buffalo assigned to at least three different loci (BoLA-DQB1, BoLA-DQB3 and BoLA-DQB4) that are also found in cattle. These presumptive loci were analysed for patterns of synonymous (d(S)) and non-synonymous (d(N)) substitution. Like BoLA-DQB1, Bubu-DQB1 was observed to be under strong positive selection for polymorphism. We conclude that water buffalo and cattle share the current arrangement of their DQB region because of their common ancestry.     

Evolution of ruminant hemoglobins. Thermodynamic divergence of ox and buffalo hemoglobins.

The ligand-binding properties of hemoglobins from two homozygote phenotypes (AA and BB) of water buffalo (Bubalus bubalis) have been characterized by equilibrium and kinetic techniques. In the case of the BB phenotype, the two constituent hemoglobins have been purified and separately analysed. Buffalo hemoglobins display the reduced sensitivity to organic phosphates characteristic of ruminant hemoglobins, their physiological effector probably being the chloride ion. In contrast to the other known hemoglobins from ruminants, all the hemoglobins from the water buffalo display a significant temperature sensitivity, the delta H for oxygen binding in the presence of physiological effectors approaching that of human hemoglobin (delta H = -30.5 kJ/mol O2). This discrepancy with the other ruminant hemoglobins (e.g. ox, delta H = -10.4 kJ/mol O2), whose primary structure is very similar to that of buffalo, hemoglobins might be correlated to the different habitat and phylogenetic history of the two subfamilies (Bos and Bubalus) of Bovidae.     

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.