Evolutionary affinities of the enigmatic saola (Pseudoryx nghetinhensis) in the context of the molecular phylogeny of Bovidae.

To elucidate the systematic status of the enigmatic saola (Pseudoryx nghetinhensis), a new bovid genus recently discovered in Vietnam, and to investigate phylogenetic relationships within the family Bovidae, four distinct DNA markers were sequenced. Complete mitochondrial cytochrome b (1143 bp) and 12S rRNA (956 bp) genes and non-coding regions from the nuclear genes for aromatase cytochrome P-450 (199 bp) and lactoferrin (338 bp) have been compared for 25 bovid species and three Cervidae and Antilocapridae outgroups. Independent and/or combined analyses of the four nucleotide matrices through maximum parsimony and maximum-likelihood methods indicated that Bovidae consists of two major lineages, i.e. Bovinac which contains the tribes Bovini, Boselaphini and Tragelaphini, and Antilopinae which encompasses all other bovids. Within Bovinae, the tribe Bovini is divided into buffalo Bovini (Bubalus and Syncerus) and cattle Bovini (Bos and Bison) and Tragelaphini are possibly related to Boselaphini. Pseudoryx is shown to be (i) robustly nested within Bovinae; (ii) strongly associated with Bovini; and (iii) tentatively sharing a sister-group relationship with cattle Bovini. Within Antilopinae, three robust clades are in evidence: (i) Hippotragus and Damaliscus are linked to Ovis; (ii) Aepyceros joins Neotragus; and (iii) Cephalophus clusters with Oreotragus.     

藏绵羊GHR基因5′侧翼区序列特征分析

对欧拉型藏绵羊生长激素受体(GHR)基因5′侧翼区(包括P1启动子和外显子1A)进行了T-A克隆和序列测定(GenBank accession No. EF116490), 在分析其序列结构特征的基础上与GenBank中摩弗伦羊、山羊、普通牛、欧洲野牛进行了比较基因组学和系统进化研究, 结果表明: (1)欧拉型藏绵羊GHR基因启动子P1区存在C/EBP、C/EBPb、SP1、Cap、USF、HFH-2、HNF-3b、Oct-1等多个潜在的转录因子结合位点, 可能与GHR基因的转录调控和起始以及特异表达有关。在该非编码序列中, 重复序列所占比率为2.55%, 不存在SINEs、LINEs、LTR类反转录元件和DNA转座子元件, 而发现存在一(TG)11微卫星位点; (2)在启动子P1区, 藏绵羊与摩弗伦羊、山羊、普通牛、欧洲野牛各物种间同源性大小分别为99.7%、94.2%、85.9%、86.5%; 而在外显子1A区段, 藏绵羊与摩弗伦羊、山羊、普通牛、欧洲野牛各物种间同源性大小分别为 99.0%、97.0%、92.7%、94.6%。物种间欧拉型藏绵羊与摩弗伦羊同源性最高, 而欧拉型藏绵羊与普通牛最低。(3)邻接法(即NJ法)构建的分子系统进化树聚类结果表明, 欧拉型藏绵羊与摩弗伦羊先聚为一类, 再与山羊聚类形成一个大分支, 而普通牛和欧洲野牛先聚类形成另一大分支, 两大分支最后再聚为一起, 其聚类结果与线粒体DNA和动物学分类的研究结果一致。     

The tribal radiation of the family Bovidae (Artiodactyla) and the evolution of the mitochondrial cytochrome b gene

The nucleotide sequence of the complete mitochondrial cytochrome b gene has been determined and compared for 51 species of the family Bovidae and 10 potential pecoran and tragulid outgroups. A detailed saturation analysis at each codon position relative to the maximum parsimony procedure indicates that all transitions on third codon positions do not accumulate in a similar fashion: C-T are more saturated than A-G substitutions. The same trend is observed for second positions but not for first positions where A-G and C-T transitions exhibit roughly the same levels of saturation. Maximum parsimony reconstructions were weighted according to these observations. Maximum parsimony, maximum likelihood, and distance phylogenetic reconstructions all depict a major split within Bovidae. The subfamily Bovinae includes four multifurcating tribes and subtribes: Boselaphini, Tragelaphini, cattle-Bovini (Bos and Bison), and buffalo-Bovini (Bubalus and Syncerus). Its sister group is the subfamily Antilopinae, i.e., all non-Bovinae taxa, represented by seven lineages: Antilopini (including Saiga), Caprini sensu lato (i. e., Caprinae including Pantholops), Hippotragini, Alcelaphini, Reduncini (including Pelea), Aepyceros possibly linked to Neotragus, and Cephalophini possibly linked to Oreotragus (the suni and the klipspringer being members of a polyphyletic Neotragini). These various tribes and major lineages were produced by two noteworthy explosive radiations, which occurred simultaneously between 12.0 and 15.3 MY (Middle Miocene) in the subfamilies Bovinae and Antilopinae.     

秦岭羚牛高度重复序列DNA片段的分子克隆和序列分析

羚牛(Budorcas taxicolor)属偶蹄目(Artiodactyla)、牛科(Bovidae),为我国一类大型珍贵保护动物。我们从其基因组中克隆得到若干约800bp的BamHI高度重复序列并对部分克隆进行了序列测定,发现它们显示了很高的同源性。利用其中一个单元为探针,对限制酶消化后的羚牛基因组DNA作杂交分析,发现其杂交谱带不具有个体及亚种间特异性,说明该重复序列在羚牛基因组中具有保守的分布和排列。在牛科动物中,羚牛BamHI片段与绵羊属和山羊属的相关序列具有高度同源性,而与水牛和家牛序列差异较大。这些结果为羚牛与羊亚科物种亲源关系较近的分类学观点提供了分子生物学证据。有证据表明,这些片段可能代表羚牛染色体着丝点的卫星DNA单体。     

秦岭羚牛高度重复序列DNA片段的分子克隆和序列分析

羚牛（Budorcas taxicolor)属偶蹄目（Artiodactyla)、牛科（Bovidae),为我国一类大型珍贵保护动物。我们从其基因组中克隆得到若干约800bp的BamHI高度重复序列并对部分克隆进行了序列测定,发现它们显示了很高的同源性。利用其中一个单元为探针,对限制酶消化后的羚牛基因组DNA作杂交分析,发现其杂交谱带不具有个体及亚种间特异性,说明该重复序列在羚牛基因组中具有保守的分布和排列。在牛科动物中,羚牛BamHI片段与绵羊属和山羊属的相关序列具有高度同源性,而与水牛和家牛序列差异较大。这些结果为羚牛与羊亚科物种亲源关系较近的分类学观点提供了分子生物学证据。有证据表明,这些片段可能代表羚牛染色体着丝点的卫星DNA单体。     

Molecular anatomy of the cytoplasmic domain of bovine growth hormone receptor, a quantitative trait locus

Quantitative trait loci (QTL) studies have indicated growth hormone receptor (GHR) as a candidate gene affecting cattle milk yield and composition. In order to characterize genetic variation at GHR in cattle, we studied European and East African breeds with different histories of selection, and Bos grunniens, Ovis aries, Sus scrofa, Bison bison and Rangifer tarandus as references. We sequenced most of the cytoplasmic domain (900 bp of exon 10), 89 bp of exon 8, including the putative causative mutation for the QTL effect, and 390 bp of intron 8 for comparison. In the cytoplasmic domain, seven synonymous and seven non-synonymous single nucleotide polymorphisms (SNP) were identified in cattle. Three non-synonymous SNPs were found in sheep and one synonymous SNP in yak, while other studied species were monomorphic. Three major haplotypes were observed, one unique to African breeds, one unique to European breeds and one shared. Bison and yak haplotypes are derivatives of the European haplotype lineage. Most of the exon 10 non-synonymous cattle SNPs appear at phylogenetically highly conserved sites. The polymorphisms in exon 10 cluster around a ruminant-specific tyrosine residue, suggesting that this site may act as an additional signalling domain of GHR in ruminants. Alternative explanations for the persistent polymorphism include balancing selection, hitch-hiking, pleiotropic or sexually antagonistic fitness effects or relaxed functional constraints.     

Fematrin-1 Is Involved in Fetomaternal Cell-to-Cell Fusion in Bovinae Placenta and Has Contributed to Diversity of Ruminant Placentation

During placentation, mammals employ different strategies for nourishing and supporting fetuses. Members of the Bovidae family, consisting of cloven-hoofed ruminants, utilize multiple maternal attachment points on the placenta, known as cotyledons, and hybrid cells, named trinucleate cells or syncytial plaques, made up of a fusion of fetal trophoblasts and maternal endometrial cells to provide essential hormones and maintain long gestation periods. These hybrid cells are unique to the Bovidae, as fetomaternal borders are clearly separated by syncytiotrophoblasts or epithelial cells in the placenta of other mammals. Recently, it was reported that Syncytin-Rum1 was inserted into ruminant genomes, including cattle and sheep, and was possibly involved in fetomaternal cell-to-cell fusion in both species. However, Syncytin-Rum1 alone is insufficient to explain the morphological diversity of the fetomaternal hybrids between Bovinae and Caprinae (i.e., trinucleate cells in Bovinae and syncytial plaques in Caprinae). Here we report that the bovine endogenous retrovirus K1 (BERV-K1) envelope, which we term Fematrin-1, was specifically expressed in binucleated trophoblasts throughout gestation in cattle and induced fusion with bovine endometrial cells in vitro at a significantly higher level than Syncytin-Rum1 under physiological conditions. Fematrin-1 was found to be integrated into intron 18 of FAT tumor suppressor homolog 2 (FAT2) about 18.3 to 25.4 million years ago and has been subject to purifying selection through the evolution of Bovinae. Phylogenetically, Fematrin-1 is distinct from Syncytin genes found in other mammalian species that form syncytiotrophoblasts. Our results suggest that the newly acquired endogenous retroelement has contributed to generating placentation diversity through ruminant evolution.     

Phylogenetics of the Caprinae Based on Cytochrome b Sequence

Relationships within the subfamily Caprinae have never been fully resolved. Phylogenies have been proposed based on morphological, behavioral, ecological, and some molecular comparisons. Because of the relatively poor fossil record of the Caprinae, paleontological evidence has not been extensively used in phylogenetic reconstruction for this group. Traditionally, four tribes: Saigini, Ovibovini, Rupicaprini, and Caprini, have been recognized. We investigated relationships within the Caprinae by comparing sequences of the cytochrome b gene of mitochondrial DNA from 11 species of Caprinae and 1 Bovinae species. Our analyses suggest that revisions to previous phylogenies, including the dissolution of the Ovibovini, are warranted.     

Cytogenetic aspects of phylogeny in the Bovidae. I. G-banding

An extensive G-banding study of karyotypes of 12 species of Bovidae has been undertaken in an attempt to trace homologies and patterns of evolution of karyotype phenotypes throughout the family. G-banding profiles revealed a considerable degree of chromosome-arm homology throughout the group, which also extended into the related superfamilies, the Giraffoidea and Cervoidea. The conservation of banding patterns in chromosome arms strongly indicates that Robertsonian translocation type rearrangements have provided the major source of interspecies karyotype differences, with inversions and reciprocal and tandem translocations providing relatively minor contributions. Examples of individuals carrying newly arisen Robertsonian translocations are not infrequent, and in one instance there was evidence that two similar rearrangements had arisen independently in two species. Despite the extensive changes in karyotype organization, subfamilies within the Bovidae were characterized by the presence of common rearrangements, and those involving autosomal pairs 11 and 12 of the ox, as well as the X chromosome, separate the Bovinae from the Caprinae and Hippotraginae.     

Sequence of specific mitochondrial 16S rRNA gene fragment from Egyptian buffalo is used as a pattern for discrimination between river buffaloes,cattle, sheep and goats

Characterization of molecular markers and the development of better assays for precise and rapid detection of domestic species are always in demand. This is particularly due to recent food scares and the crisis of biodiversity resulting from the huge ongoing illegal traffic of endangered species. The aim of this study was to develop a new and easy method for domestic species identification (river buffalo, cattle, sheep and goat) based on the analysis of a specific mitochondrial nucleotide sequence. For this reason, a specific fragment of Egyptian buffalo mitochondrial 16S rRNA gene (422 bp) was amplified by PCR using two universal primers. The sequence of this specific fragment is completely conserved between all tested Egyptian buffaloes and other river buffaloes in different places in the world. Also, the lengths of the homologous fragments were less by one nucleotide (421 bp) in case of goats and two nucleotides (420 bp) in case of both cattle and sheep. The detection of specific variable sites between investigated species within this fragment was sufficient to identify the biological origin of the samples. This was achieved by alignment between the unknown homologous sequence and the reference sequences deposited in GenBank database (accession numbers, FJ748599–FJ748607). Considering multiple alignment results between 16S rRNA homologous sequences obtained from GenBank database with the reference sequence, it was shown that definite nucleotides are specific for each of the four studied species of the family Bovidae. In addition, other nucleotides are detected which can allow discrimination between two groups of animals belonging to two subfamilies of family Bovidae, Group one (closely related species like cattle and buffalo, Subfamily Bovinae) and Group two (closely related species like sheep and goat, Subfamily Caprinae). This 16S DNA barcode character-based approach could be used to complement cytochrome c oxidase I (COI) in DNA barcoding. Also, it is a good tool for identification of unknown sample belonging to one of the four domestic animal species of family Bovidae quickly and easily.     

A Cladistic Analysis of Mitochondrial Ribosomal DNA from the Bovidae

There is a huge data base of genetic information for the domestic artiodactyl speciesBos taurus(cow),Ovis aries(sheep), andCapra hircus(goat). However, the phylogenetic relationships of these economically critical taxa and their close relatives, family Bovidae, remain for the most part unresolved. In this report, we aligned new mitochondrial (mt) 12S and 16S ribosomal (r) DNA sequences from 26 bovid taxa with published sequences. Phylogenetic analyses of the more than 64 kilobases of mt rDNA from 57 taxa support a basal division in the Bovidae that separatesBosand its close relatives fromCapra, Ovis,and their kin. As suggested by previous molecular and morphological studies, “antelopes” are a paraphyletic assemblage. Caprinae (sheep, goats, goat antelopes, and musk oxen) groups consistently with hippotragine and alcelaphine antelopes, while Bovini (cattle and buffaloes) clusters with tragelaphine and boselaphine antelopes. The traditional tribal subdivisions of Bovidae are supported in most cases, but there are exceptions within Caprinae and Antilopinae (gazelles and close relatives). The rDNA data consistently place the enigmatic generaPelea, Pantholops,andSaiga,but the origin ofAepyceros,the impala, remains obscure. Combined phylogenetic analyses of the rDNA data with the skeletal characters of Gentry (1992) were used to assess the stability of the molecular results.     

Molecular insights into the evolution of the family Bovidae: a nuclear DNA perspective.

The evolutionary history of the family Bovidae remains controversial despite past comprehensive morphological and genetic investigations. In an effort to resolve some of the systematic uncertainties within the group, a combined molecular phylogeny was constructed based on four independent nuclear DNA markers (2,573 characters) and three mitochondrial DNA genes (1,690 characters) for 34 bovid taxa representing all seven of the currently recognized bovid subfamilies. The nuclear DNA fragments were analyzed separately and in combination after partition homogeneity tests were performed. There was no significant rate heterogeneity among lineages, and retention index values indicated the general absence of homoplasy in the nuclear DNA data. The conservative nuclear DNA data were remarkably effective in resolving associations among bovid subfamilies, which had a rapid radiation dating back to approximately 23 MYA. All analyses supported the monophyly of the Bovinae (cow, nilgai, and kudu clade) as a sister lineage to the remaining bovid subfamilies, and the data convincingly suggest that the subfamilies Alcelaphinae (hartebeest, tsessebe, and wildebeest group) and Hippotraginae (roan, sable, and gemsbok clade) share a close evolutionary relationship and together form a sister clade to the more primitive Caprinae (represented by sheep, goat, and muskox). The problematic Reduncinae (waterbuck, reedbuck) seem to be the earliest-diverging group of the Caprinae/Alcelaphinae/Hippotraginae clade, whereas the Antilopinae (gazelle and dwarf antelope clade) were always polyphyletic. The sequence data suggest that the initial diversification of the Bovidae took place in Eurasia and that lineages such as the Cephalophinae and other enigmatic taxa (impala, suni, and klipspringer) most likely originated, more or less contemporaneously, in Africa.     

Single nucleotide polymorphisms in exon 10 of the chinchilla growth hormone receptor (GHR) gene

The aim of this study was to detect SNPs in exon 10 of the chinchilla growth hormone receptor gene (GHR) by comparative sequencing. Sixty females of the same breed (Standard) were analysed. Four new SNPs were identified, which cause 3 amino acid substitutions in the intracellular domain of the receptor: G/C at position 135 bp (in relation to the total sequence of exon 10) (gln/his), CAG/AAA at 352 bp and 354 bp (gln/lys), and C/A at 641 bp (thr/asn).     

Phylogenetic analyses and improved resolution of the family Bovidae based on complete mitochondrial genomes

Efforts have been made to investigate the phylogeny of the family Bovidae; however, the relationships within this group still remain controversial. To further our understanding of the relationships, we sequenced the mitochondrial genome of the Himalayan goral, Naemorhedus goral, an IUCN Redlist near threatened conservation dependent species. Then we conducted molecular phylogenetic relationships of the Bovidae based on Bayesian and Maximum Likelihood methods. The results indicate that the basal divergence within the Bovidae is between the Bovinae and a strongly supported clade of the remaining Bovidae species. The two Neotragus species (the suni and pygmy antelope) clustered with the impala, Aepyceros melampus (Aepycerotinae), and together they formed the most basal of the non-Bovinae. All the genera of the Antilopinae clustered together except Neotragus, which suggested that the Antilopinae was a paraphyletic subfamily. The present study confirmed a close relationship between the genera Capricornis and Naemorhedus while supporting their designation as separate genera and suggested that the Capricornis-Naemorhedus-Ovibos clade (serows, gorals, and the muskox) should be placed in the Caprinae. Bison, Bos, and Tragelaphus (bison & cattle and kudus and nyalas) were paraphyletic. The very close relationship between Bison and Bos suggested that Bos and Bison should be integrated into a single Bos genus. Saiga and Pantholops (the Chiru or Tibetan Antelope), unique genera which have sometimes been lumped together, were placed in different groups: Saiga within the Antilopinae and Pantholops at the base of the Caprinae. Our results also supported a new taxonomy which places the three species of Hemitragus into three monospecific genera: the genus Hemitragus is restricted to the Himalayan tahr, and two new genera are created: Arabitragus for the Arabian tahr and Nilgiritragus for the Nilgiri tahr.     

Mutation rates of TGFBR2 and ACVR2 coding microsatellites in human cells with defective DNA mismatch repair

Microsatellite instability promotes colonic tumorigenesis through generating frameshift mutations at coding microsatellites of tumor suppressor genes, such as TGFBR2 and ACVR2. As a consequence, signaling through these TGFbeta family receptors is abrogated in DNA Mismatch repair (MMR)-deficient tumors. How these mutations occur in real time and mutational rates of these human coding sequences have not previously been studied. We utilized cell lines with different MMR deficiencies (hMLH1-/-, hMSH6-/-, hMSH3-/-, and MMR-proficient) to determine mutation rates. Plasmids were constructed in which exon 3 of TGFBR2 and exon 10 of ACVR2 were cloned +1 bp out of frame, immediately after the translation initiation codon of an enhanced GFP (EGFP) gene, allowing a -1 bp frameshift mutation to drive EGFP expression. Mutation-resistant plasmids were constructed by interrupting the coding microsatellite sequences, preventing frameshift mutation. Stable cell lines were established containing portions of TGFBR2 and ACVR2, and nonfluorescent cells were sorted, cultured for 7-35 days, and harvested for flow cytometric mutation detection and DNA sequencing at specific time points. DNA sequencing revealed a -1 bp frameshift mutation (A9 in TGFBR2 and A7 in ACVR2) in the fluorescent cells. Two distinct fluorescent populations, M1 (dim, representing heteroduplexes) and M2 (bright, representing full mutants) were identified, with the M2 fraction accumulating over time. hMLH1 deficiency revealed 11 (5.91 x 10(-4)) and 15 (2.18 x 10(-4)) times higher mutation rates for the TGFBR2 and ACVR2 microsatellites compared to hMSH6 deficiency, respectively. The mutation rate of the TGFBR2 microsatellite was approximately 3 times higher in both hMLH1 and hMSH6 deficiencies than the ACVR2 microsatellite. The -1 bp frameshift mutation rates of TGFBR2 and ACVR2 microsatellite sequences are dependent upon the human MMR background.