Progress on genetic mapping and genetic mechanism of cattle and sheep horns.

Horns are cranial appendages, which are unique in ruminants. Cattle (Bos taurus) and sheep (Ovis aries) cranial appendages exhibit various forms of morphology, including wild-type two-horn phenotype, polled phenotype and scur phenotype. These animals provide an ideal model for studies on the underlying relationship between quality and quantitative traits of cattle and sheep horn and the molecular mechanisms of horn phenotype as a polygenic regulation for quality traits. In recent years, some research progresses of cattle and sheep horns are successively reported, which helps us better understand the evolutionary origin of new organ, the effects of natural selection, sex selection and artificial selection on horn phenotypes. In this review, we introduce in details the recent advances on the research of horn traits in cattle and sheep, and summarize the genetic mapping of multi-horned phenotypes, the genetic mapping of polled locus, and studies on scur phenotype. Moreover, we discuss potential problems in such research, thereby providing a reference for investigation on the genetic mechanisms of horn traits in ruminants.

Expression profiling and functional characterization of miR-192 throughout sheep skeletal muscle development.

MicroRNAs (miRNAs) are evolutionarily conserved, small, non-coding RNAs that have emerged as key regulators of myogenesis. Here, we examined the miRNA expression profiles of developing sheep skeletal muscle using a deep sequencing approach. We detected 2,396 miRNAs in the sheep skeletal muscle tissues. Of these, miR-192 was found to be up-regulated in prenatal skeletal muscle, but was down-regulated postnatally. MiR-192 expression also decreased during the myogenic differentiation of sheep satellite cells (SCs). MiR-192 overexpression significantly attenuated SCs myogenic differentiation but promoted SCs proliferation, whereas miR-192 inhibition enhanced SCs differentiation but suppressed SCs proliferation. We found that miR-192 targeted retinoblastoma 1 (RB1), a known regulator of myogenesis. Furthermore, knockdown of RB1 in cultured cells significantly inhibited SCs myogenic differentiation but accelerated SCs proliferation, confirming the role of RB1 in myogenesis. Taken together, our findings enrich the ovine miRNA database, and outline the miRNA transcriptome of sheep during skeletal muscle development. Moreover, we show that miR-192 affects SCs proliferation and myogenic differentiation via down-regulation of RB1.

Mitogenomic Meta-Analysis Identifies Two Phases of Migration in the History of Eastern Eurasian Sheep.

Despite much attention, history of sheep (Ovis aries) evolution, including its dating, demographic trajectory and geographic spread, remains controversial. To address these questions, we generated 45 complete and 875 partial mitogenomic sequences, and performed a meta-analysis of these and published ovine mitochondrial DNA sequences (n = 3,229) across Eurasia. We inferred that O. orientalis and O. musimon share the most recent female ancestor with O. aries at approximately 0.790 Ma (95% CI: 0.637-0.934 Ma) during the Middle Pleistocene, substantially predating the domestication event (∼8-11 ka). By reconstructing historical variations in effective population size, we found evidence of a rapid population increase approximately 20-60 ka, immediately before the Last Glacial Maximum. Analyses of lineage expansions showed two sheep migratory waves at approximately 4.5-6.8 ka (lineages A and B: ∼6.4-6.8 ka; C: ∼4.5 ka) across eastern Eurasia, which could have been influenced by prehistoric West-East commercial trade and deliberate mating of domestic and wild sheep, respectively. A continent-scale examination of lineage diversity and approximate Bayesian computation analyses indicated that the Mongolian Plateau region was a secondary center of dispersal, acting as a "transportation hub" in eastern Eurasia: Sheep from the Middle Eastern domestication center were inferred to have migrated through the Caucasus and Central Asia, and arrived in North and Southwest China (lineages A, B, and C) and the Indian subcontinent (lineages B and C) through this region. Our results provide new insights into sheep domestication, particularly with respect to origins and migrations to and from eastern Eurasia.

Genetic phylogeography and maternal lineages of 18 Chinese black goat breeds.

To understand the origin and genetic phylogeography of Chinese black goats, variations of mitochondrial DNA were characterised with 394 goats from 18 breeds, including 91 new individuals from regions poorly studied until now. Comparison of a 481-bp segment revealed a total of 192 haplotypes with 141 variable sites. The haplotype and nucleotide diversities ranged from 0.782 ± 0.079 to 1.000 ± 0.020 and from 0.009 ± 0.001 to 0.045 ± 0.006, respectively, indicating a relatively high genetic diversity in Chinese black goats. Phylogenetic analyses identified five haplogroups (A, B1, B2, C and D). The dominant haplogroups A, B1 and B2 were distributed in most of breeds, while the haplogroups C and D were only presented in the breeds located in north or northwest of China. Analysis of molecular variance and multidimensional scaling plot of F ST analyses indicated no obvious geographic structure among breeds. Furthermore, the migration rates revealed that a wide range of gene flow or gene exchange occurred among breeds, which may result in the weak geographic structure of Chinese black goats. Population expansion analysis based on mismatch distribution indicated that two expansion events in Chinese black goats occurred at 10 and 28 mutational time units. Finally, our findings indicate the multiple maternal origins of Chinese black goats and more gene flow (female-mediated) which occurred during their domestic and breeding histories.

Dynamic analysis of Ca²+ level during bovine oocytes maturation and early embryonic development.

Mammalian oocyte maturation and early embryo development processes are Ca(2+)-dependent. In this study, we used confocal microscopy to investigate the distribution pattern of Ca(2+) and its dynamic changes in the processes of bovine oocytes maturation, in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryo development. During the germinal vesicle (GV) and GV breakdown stage, Ca(2+) was distributed in the cortical ooplasm and throughout the oocytes from the MI to MII stage. In IVF embryos, Ca(2+) was distributed in the cortical ooplasm before the formation of the pronucleus. In 4-8 cell embryos and morulas, Ca(2+) was present throughout the blastomere. In PA embryos, Ca(2+) was distributed throughout the blastomere at 48 h, similar to in the 4-cell and 8-cell phase and the morula. At 6 h after activation, there was almost no distribution of Ca(2+) in the SCNT embryos. However, Ca(2+) was distributed in the donor nucleus at 10 h and it was distributed throughout the blastomere in the 2-8 cell embryos. In this study, Ca(2+) showed significant fluctuations with regularity of IVF and SCNT groups, but PA did not. Systematic investigation of the Ca(2+) location and distribution changes during oocyte maturation and early embryo development processes should facilitate a better understanding of the mechanisms involved in oocyte maturation, reconstructed embryo activation and development, ultimately improving the reconstructed embryo development rate.

Establishment and biological characteristics of Ujumqin sheep fibroblast line.

A Ujumqin sheep ear marginal tissue (USEM) fibroblast line, frozen in 147 cryovials with 4 x 10(6) cell each, was successfully established from 33 Ujumqin sheep ear marginal tissues using explant culture and cryopreservation techniques. The cells were morphologically consistent with fibroblasts. The growth curve was typical S-shape and the cell population passed through a lag phase, a logarithmic phase and a plateau phase. The population doubling time (PDT) was approximately 72 h. Tests for bacteria, fungi, viruses and mycoplasma were all negative. Isoenzyme polymorphism indicated that the genetic characteristics of the cell line were stable in vitro. Karyotyping analysis indicated that the chromosome number of a normal cell was of 2n = 54 and 95.4% of the entire population was diploid. The transfection efficiencies of six fluorescent proteins (pEGFP-N3, pEGFP-C1, pDsRed-N1, pEYFP-N1, pECFP-N1 and pECFP-mito) optimal at 48 h were from 18.5% to 30.1%. The cell line met all criteria from the American Type Culture Collection (ATCC). Not only has the germline of this important sheep breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somatic cloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.

[Genetic relationships between Tuva population and the neighboring populations in the Altai Region of Xinjiang Uygur Autonomous Region].

In the Hanasi scenic spot of the Altai Region, Xinjiang Uygur Autonomous Region, China, there is a special population known as Xinjiang Tuvinians for short. These Tuvinians were classified as Mongolians in the early 1950s by the National Ethnic Affairs Commission of China, but they claimed that they have an independent origin. To resolve this dispute and their genetic relationships with the people in the neighboring regions, we randomly selected 150 male Tuvinians in the Altai Region. Fourteen Y chromosomal markers were genotyped and eleven haplogroups were constructed. The frequencies of the haplogroups K-M9 and Q-M242 were higher in Xinjiang Tuvinians or Tuvinians in the Tuva Republic than those in the other populations (e.g., Mongolians and Kazakh). Principal component analysis , multi-dimensional scaling analysis and further phylogenetic tree analysis revealed that the Xinjiang Tuvinians were far separated from Mongolians and Kazakh. Based on these results, we proposed that Xinjiang Tuvinians are genetically distinct from Mongolians and Kazakh.

[Study on single nucleotide polymorphisms of H-FABP gene in sheep].

PCR-SSCP and DNA sequencing approaches were applied to assess the single nucleotide polymorphisms (SNPs) and analyze the genetic polymorphisms at partial exon 2 and intron 2 of H-FABP(Heart fatty acid-binding protein)gene, and five sheep populations that comprised of Small-Tailed Han sheep (SH, 48), Ningxia Tan sheep (Tan, 121), Tan x SH F1 (23), Poll Dorset (48) and Suffolk (24) sheep were screened in this study. The result showed: (1)four SNPs at 981(G/A), 1014(A/C) 1019(T/C) and 1058 (-/G ) and nine genotypes (AA, BB, CC, AB, AC, BC, AD, CD and BD) were detected using primer 2, the AA was the predominant genotype. A chi-square analysis suggested that the allele frequencies and genotype frequencies of H-FABP were in Hardy-Weinberg equilibrium except the Tan and Suffolk populations. Statistical analysis revealed a low polymorphism information content (PIC) in the Suffolk and Tan x SH F1 populations (PIC amp; 0.25) but an intermediate PIC in the remaining three populations (0.25 amp; PIC amp;0.50). It meant that the fragment of H-FABP had polymorphisms, which could be used as a candidate gene associated gene with phenotypic traits like intramuscular fat content in different sheep populations. (2)Three genotypes (HH, Hh and hh) determined by a SNP at 2407(T-C) were detected using primer 4. The genotype frequencies were in the order of HHHhhh. A chi-square analysis suggested that the allele frequencies and genotype frequencies of H-FABP were in Hardy-Weinberg equilibrium in the Tan and Poll Dorset populations, and the PIC values were low (PIC amp; 0.25). However, there was no polymorphisms in SH, Tan x SH F1 and Suffolk populations.

A fine map for maternal lineage analysis by mitochondrial hypervariable region in 12 Chinese goat breeds.

As the fast pace of genomic research continues to identify mitochondrial lineages in animals, it has become apparent that many independent studies are needed to support a robust phylogenetic inference. The aim of this study was thus to further characterize the maternal lineage, proposed to originate in southwestern region of China, using a wider survey of diverse goat breeds in China. To this end, we sequenced the mitochondrial hypervariable region 1 (HVR1) of the mtDNA control region in 145 goats of 12 Chinese breeds. Phylogenetic analysis revealed that Chinese goats were classified into four distinct lineages (A, B, C and D) as previously reported. A Mantel test and the analysis of Analysis of Molecular Variance (ANOVA) indicated that there was not an obvious geographic structure among Chinese goat breeds. Population expansion analysis based on mismatch distribution and Fu's Fs statistic indicate that two expansion events in Chinese goats occurred respectively at about 11 and 29 mutational time units ago, revealing two star-like subclades in lineage B corresponding to two population expansion events. Moreover, lineage B sequences were presented only in the breeds of southwestern or surrounding regions of China. Multiple lines of evidence from this study and previous studies indicate that for Chinese goats mtDNA lineage B originated from the southwestern region of China.

[Variation of MSTN gene UTR in eleven sheep breeds].

PCR-RFLP was applied to analyze the polymorphism of MSTN gene UTR in 345 sheep that comprised of eleven sheep breeds, namely Texel sheep, Charolais sheep, Small-tailed Han sheep, Monggolian sheep, Ujumqin sheep, Altay Fat-rumped sheep, Hulunbeir sheep, Tashikurgan sheep, Duolang sheep, Hu sheep, and Gangba sheep. A 271 bp and a 1 003 bp long PCR products were digested with Mboand Bsato demonstrate polymorphism in the eleven sheep breeds, which were all at Hardy-Weinberg equilibrium (P>0.05). The distribution of 3 genotypes in 11 sheep breeds was significantly different (P<0.01). Digestion of the PCR products with HpyCH4 proved that 9 domestic local sheep breeds were different from Texel sheep in the SNP site that was associated with muscularity. The individual mutation base could generate the motifs for miRNA in the 3'UTR, and sequencing analysis demonstrated high frequency of mutation in the 3'UTR region.