Characterization of microRNAs from sheep (<Emphasis Type="Italic">Ovis aries</Emphasis>) using computational and experimental analyses

Ovis aries is one of the most important agricultural livestock for meat production, and also is an ideal model organism for biological and comparative genomics studies. Many miRNAs have been reported for their important roles in developmental processes in various animals, but there is limited information about O. aries miRNAs. In this study, combining a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library, we identified 31 miRNAs belong to 24 families in sheep, 2 of which were novel miRNAs which had never been previously identified in any species. Especially, we cloned 12 miRNAs from the sheep skeletal muscle, which were good candidate miRNAs to be studied about the miRNA-dependant regulated process of muscle development, and we identified four pairs of miRNA/miRNA* and one pair of miRNA-3p/miRNA-5p from sheep EST sequences. Expression analysis indicated that some miRNAs were expressed in a specific tissue, and the pair of miRNA-3p/miRNA-5p and one pair of miRNA/miRNA* had a similar relative expression pattern in some tissues, respectively. Further, we predicted 120 potential target genes of 31 oar-miRNAs on the 3′UTR of O. aries genes. Gene ontology analysis showed that most of these genes took part in the cellular process and metabolic process. Our results enriched the O. aries miRNA database and provided useful information for investigating biological functions of miRNAs and miRNA* in sheep.     

MicroRNAome of Porcine Pre- and Postnatal Development

The domestic pig is of enormous agricultural significance and valuable models for many human diseases. Information concerning the pig microRNAome (miRNAome) has been long overdue and elucidation of this information will permit an atlas of microRNA (miRNA) regulation functions and networks to be constructed. Here we performed a comprehensive search for porcine miRNAs on ten small RNA sequencing libraries prepared from a mixture of tissues obtained during the entire pig lifetime, from the fetal period through adulthood. The sequencing results were analyzed using mammalian miRNAs, the precursor hairpins (pre-miRNAs) and the first release of the high-coverage porcine genome assembly (Sscrofa9, April 2009) and the available expressed sequence tag (EST) sequences. Our results extend the repertoire of pig miRNAome to 867 pre-miRNAs (623 with genomic coordinates) encoding for 1,004 miRNAs, of which 777 are unique. We preformed real-time quantitative PCR (q-PCR) experiments for selected 30 miRNAs in 47 tissue-specific samples and found agreement between the sequencing and q-PCR data. This broad survey provides detailed information about multiple variants of mature sequences, precursors, chromosomal organization, development-specific expression, and conservation patterns. Our data mining produced a broad view of the pig miRNAome, consisting of miRNAs and isomiRs and a wealth of information of pig miRNA characteristics. These results are prelude to the advancement in pig biology as well the use of pigs as model organism for human biological and biomedical studies.     

Electrophoretic differentiation of myofibrillar proteins in the pig

1. Starch-gel electrophoretograms of myosin and tropomyosin preparations in 8m-urea, from longissimus dorsi and psoas muscles of the pig, were characterized by laser densitometry. 2. The typical pattern for freshly prepared myosin from both muscles was similar, there being five electrophoretically distinct components. 3. The number of electrophoretically distinct components in both muscles increased after freeze-drying, but the effect of freeze-drying was more marked in longissimus dorsi. 4. Extraction with 8m-urea containing 2% β-mercaptoethanol decreased the number of major electrophoretically distinct components of the fresh myosin of both muscles to four. 5. Although there was also some simplification of the patterns after freeze-drying the greater susceptibility of the myosin from longissimus dorsi was still evident. 6. The typical pattern for freshly prepared tropomyosin in 8m-urea differed in the two muscles: in each case it was more complex than that of the corresponding myosins. 7. The pattern of tropomyosin from neither longissimus dorsi nor psoas was altered significantly after freeze-drying. 8. Electrophoretograms of pig longissimus dorsi tropomyosin in 8m-urea differed from those of longissimus dorsi tropomyosin from sheep, ox and rabbit. 9. Extraction of the tropomyosins in 8m-urea and 2% β-mercaptoethanol simplified the electrophoretic pattern to two major components with samples from pig, sheep and ox, and to one major component with samples from rabbit. 10. It was concluded that classification of skeletal muscles as `red' or `white' is insufficient to account for the degree of functional specialization which the electrophoretograms suggest.     

Genome-wide cross-amplification of domestic sheep microsatellites in bighorn sheep and mountain goats

We tested for cross‐species amplification of microsatellite loci located throughout the domestic sheep (Ovis aries) genome in two north American mountain ungulates (bighorn sheep, Ovis canadensis, and mountain goats, Oreamnos americanus). We identified 247 new polymorphic markers in bighorn sheep (≥ 3 alleles in one of two study populations) and 149 in mountain goats (≥ 2 alleles in a single study population) using 648 and 576 primer pairs, respectively. Our efforts increased the number of available polymorphic microsatellite markers to 327 for bighorn sheep and 180 for mountain goats. The average distance between successive polymorphic bighorn sheep and mountain goat markers inferred from the Australian domestic sheep genome linkage map (mean ± 1 SD) was 11.9 ± 9.2 and 15.8 ± 13.8 centimorgans, respectively. The development of genomic resources in these wildlife species enables future studies of the genetic architecture of trait variation.     

Identification of microRNAs in Macaca fascicularis (Cynomolgus Monkey) by Homology Search and Experimental Validation by Small RNA-Seq and RT-qPCR Using Kidney Cortex Tissues

MicroRNAs (miRNAs) present in tissues and biofluids are emerging as sensitive and specific safety biomarkers. MiRNAs have not been thoroughly described in M. fascicularis, an animal model used in pharmaceutical industry especially in drug safety evaluation. Here we investigated the miRNAs in M. fascicularis. For Macaca mulatta, a closely related species of M. fascicularis, 619 stem-loop precursor miRNAs (pre-miRNAs) and 914 mature miRNAs are available in miRBase version 21. Using M. mulatta miRNAs as a reference list and homology search tools, we identified 604 pre-miRNAs and 913 mature miRNAs in the genome of M. fascicularis. In order to validate the miRNAs identified by homology search we attempted to sequence miRNAs expressed in kidney cortex from M. fascicularis. MiRNAs expressed in kidney cortex may indeed be released in urine upon kidney cortex damage and be potentially used to monitor drug induced kidney injury. Hence small RNA sequencing libraries were prepared using kidney cortex tissues obtained from three naive M. fascicularis and sequenced. Analysis of sequencing data indicated that 432 out of 913 mature miRNAs were expressed in kidney cortex tissues. Assigning these 432 miRNAs to pre-miRNAs revealed that 273 were expressed from both the -5p and -3p arms of 150 pre-miRNAs and 159 miRNAs expressed from either the -5p or -3p arm of 176 pre-miRNAs. Mapping sequencing reads to pre-miRNAs also facilitated the detection of twenty-two new miRNAs. To substantiate miRNAs identified by small RNA sequencing, 313 miRNAs were examined by RT-qPCR. Expression of 262 miRNAs in kidney cortex tissues ware confirmed by TaqMan microRNA RT-qPCR assays. Analysis of kidney cortex miRNA targeted genes suggested that they play important role in kidney development and function. Data presented in this study may serve as a valuable resource to assess the renal safety biomarker potential of miRNAs in Cynomolgus monkeys.     

Global profiling of miRNAs and the hairpin precursors: insights into miRNA processing and novel miRNA discovery

MicroRNAs (miRNAs) constitute an important class of small regulatory RNAs that are derived from distinct hairpin precursors (pre-miRNAs). In contrast to mature miRNAs, which have been characterized in numerous genome-wide studies of different organisms, research on global profiling of pre-miRNAs is limited. Here, using massive parallel sequencing, we have performed global characterization of both mouse mature and precursor miRNAs. In total, 87 369 704 and 252 003 sequencing reads derived from 887 mature and 281 precursor miRNAs were obtained, respectively. Our analysis revealed new aspects of miRNA/pre-miRNA processing and modification, including eight Ago2-cleaved pre-miRNAs, eight new instances of miRNA editing and exclusively 5′ tailed mirtrons. Furthermore, based on the sequences of both mature and precursor miRNAs, we developed a miRNA discovery pipeline, miRGrep, which does not rely on the availability of genome reference sequences. In addition to 239 known mouse pre-miRNAs, miRGrep predicted 41 novel ones with high confidence. Similar as known ones, the mature miRNAs derived from most of these novel loci showed both reduced abundance following Dicer knockdown and the binding with Argonaute2. Evaluation on data sets obtained from Caenorhabditis elegans and Caenorhabditis sp.11 demonstrated that miRGrep could be widely used for miRNA discovery in metazoans, especially in those without genome reference sequences.     

Sequence and evolution of related bovine and caprine satellite DNAs: Identification of a short DNA sequence potentially involved in satellite DNA amplification

The satellite II DNAs of the domestic ox Bos taurus and sheep Ovis aries have been sequenced, and that of the domestic goat Capra hircus partially sequenced. All three are related, and consist of repeat units of about 700 base-pairs. There is no evidence of internal repetition within these repeat units. When matched for maximum homology, the goat and sheep sequences show 83% homology, whereas the ox and sheep sequences share only 70% homology. Factors contributing to the uncertainty of the exact homology between these sequences are discussed, but the results are nevertheless consistent with their progenitor sequence being present in the common ancestor of cattle and sheep. Goat satellite II DNA is shown to contain another, unrelated, tandemly repeated sequence, which is composed of 22 base-pair repeat units. Both this sequence and a region of ox satellite II share good homology with the 11 base-pair progenitor sequence of ox 1.706 g/cm³ satellite DNA. It is suggested that this shared sequence could play a role in bovine satellite DNA amplification.     

11个绵羊品种MSTN基因非翻译区的变异

利用PCR-RFLP技术对特克塞尔羊、夏洛莱羊、小尾寒羊、蒙古羊、乌珠穆沁羊、阿勒泰羊、呼伦贝尔羊、塔什库尔干羊、多浪羊、湖羊和岗巴羊11个品种的345个个体的肌肉生长抑制素(Myostatin, MSTN)基因非翻译区(UTR)的变异进行了多态性分析。结果表明大小为271 bp和1 003 bp的扩增片段经限制性内切酶MboⅡ和BsaⅠ酶切后表现多态, 经卡方检验所有品种在该基因座位均处于平衡状态(P>0.05), 3种基因型在11个绵羊品种中的分布差异极显著(P<0.01)。通过限制性内切酶HpyCH4Ⅳ 酶切实验, 证明我国9个地方绵羊品种不存在特克塞尔绵羊中发现的导致肌肉发达的SNP位点, 并在3′UTR区发现了个别碱基突变位点能够形成miRNA作用的靶基序, 测序表明3′UTR区的突变频率较高。     

Beta‐globin gene evolution in the ruminants: evidence for an ancient origin of sheep haplotype B

Domestic sheep (Ovis aries) can be divided into two groups with significantly different responses to hypoxic environments, determined by two allelic beta‐globin haplotypes. Haplotype A is very similar to the goat beta‐globin locus, whereas haplotype B has a deletion spanning four globin genes, including beta‐C globin, which encodes a globin with high oxygen affinity. We surveyed the beta‐globin locus using resequencing data from 70 domestic sheep from 42 worldwide breeds and three Ovis canadensis and two Ovis dalli individuals. Haplotype B has an allele frequency of 71.4% in O. aries and was homozygous (BB) in all five wild sheep. This shared ancestry indicates haplotype B is at least 2–3 million years old. Approximately 40 kb of the sequence flanking the ~37‐kb haplotype B deletion had unexpectedly low identity between haplotypes A and B. Phylogenetic analysis showed that the divergent region of sheep haplotype B is remarkably distinct from the beta‐globin loci in goat and cattle but still groups with the Ruminantia. We hypothesize that this divergent ~40‐kb region in haplotype B may be from an unknown ancestral ruminant and was maintained in the lineage to O. aries, but not other Bovidae, evolving independently of haplotype A. Alternatively, the ~40‐kb sequence in haplotype B was more recently acquired by an ancestor of sheep from an unknown non‐Bovidae ruminant, replacing part of haplotype A. Haplotype B has a lower nucleotide diversity than does haplotype A, suggesting a recent bottleneck, whereas the higher frequency of haplotype B suggests a subsequent spread through the global population of O. aries.     

The effect of maternal nutrition level during the periconception period on fetal muscle development and plasma hormone concentrations in sheep

The effect of maternal nutrition level during the periconception period on the muscle development of fetus and maternal–fetal plasma hormone concentrations in sheep were examined. Estrus was synchronized in 55 Karayaka ewes and were either fed ad libitum (well-fed, WF, n=23) or 0.5×maintenance (under-fed, UF, n=32) 6 days before and 7 days after mating. Non-pregnant ewes (WF, n=13; UF, n=24) and ewes carrying twins (WF, n=1) and female (WF, n=1; UF, n=3) fetuses were removed from the experiment. The singleton male fetuses from well-fed (n=8) and under-fed (n=5) ewes were collected on day 90 of gestation and placental characteristics, fetal BWs and dimensions, fetal organs and muscles weights were recorded. Maternal (on day 7 after mating) and fetal (on day 90 of pregnancy) blood samples were collected to analyze plasma hormone concentrations. Placental characteristics, BW and dimensions, organs and muscles weights of fetuses were not affected by maternal feed intake during the periconception period. Maternal nutrition level did not affect fiber numbers and the muscle cross-sectional area of the fetal longissimus dorsi (LD), semitendinosus (ST) muscles, but the cross-sectional area of the secondary fibers in the fetal LD and ST muscles from the UF ewes were higher than those from the WF ewes (P<0.05). Also, the ratio of secondary to primary fibers in the ST muscle were tended to be lower in the fetuses from the UF ewes (P=0.07). Maternal nutrition level during the periconception period did not cause any significant changes in fetal plasma insulin and maternal and fetal plasma IGF-I, cortisol, progesterone, free T3 and T4 concentrations. However, maternal cortisol concentrations were lower while insulin concentrations were higher in the WF ewes than those in the UF ewes (P<0.05). These results indicate that the reduced maternal feed intake during the periconception period may alter muscle fiber diameter without affecting fiber types, fetal weights and organ developments and plasma hormone concentrations in the fetus.     

Harnessing cross-species alignment to discover SNPs and generate a draft genome sequence of a bighorn sheep (Ovis canadensis)

Background

Whole genome sequences (WGS) have proliferated as sequencing technology continues to improve and costs decline. While many WGS of model or domestic organisms have been produced, a growing number of non-model species are also being sequenced. In the absence of a reference, construction of a genome sequence necessitates de novo assembly which may be beyond the ability of many labs due to the large volumes of raw sequence data and extensive bioinformatics required. In contrast, the presence of a reference WGS allows for alignment which is more tractable than assembly. Recent work has highlighted that the reference need not come from the same species, potentially enabling a wide array of species WGS to be constructed using cross-species alignment. Here we report on the creation a draft WGS from a single bighorn sheep (Ovis canadensis) using alignment to the closely related domestic sheep (Ovis aries).

Results

Two sequencing libraries on SOLiD platforms yielded over 865 million reads, and combined alignment to the domestic sheep reference resulted in a nearly complete sequence (95% coverage of the reference) at an average of 12x read depth (104 SD). From this we discovered over 15 million variants and annotated them relative to the domestic sheep reference. We then conducted an enrichment analysis of those SNPs showing fixed differences between the reference and sequenced individual and found significant differences in a number of gene ontology (GO) terms, including those associated with reproduction, muscle properties, and bone deposition.

Conclusion

Our results demonstrate that cross-species alignment enables the creation of novel WGS for non-model organisms. The bighorn sheep WGS will provide a resource for future resequencing studies or comparative genomics.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1618-x) contains supplementary material, which is available to authorized users.     

Hair diameter distribution in sheep interspecies hybrids

Hair diameter distribution was studied in wild sheep species (argali—Ovis ammon, mouflon—O. Musimon; domestic sheep—O. aries) and their hybrids. In wild species and their hybrids, rather distinctly separated subpopulations of thin (underwool) hairs (10–22 μm diameter) and thick (kemp) hairs (85–302 μm) were detected, and intermediate diameters were rare. In domestic sheep, the kemp subpopulation was very poor, not distinctly separated from the underwool, while maximal hair diameter was <150 μm. In wild × domestic crosses, the kemp subpopulation was clearly expressed, shifted to lower diameter range, and maximal diameters was <200 μm. The same restriction was found in two-month-old wild species lambs compared with adults.     

The microRNA expression profile in porcine skeletal muscle is changed by constant heat stress

Heat stress has profound effects on animal performance and muscle function, and microRNAs (miRNAs) play a critical role in muscle development and stress responses. To characterize the changes in miRNAs in skeletal muscle responding to heat stress, the miRNA expression profiles of longissimus dorsi muscles of pigs raised under constant heat stress (30 °C; n = 8) or control temperature (22 °C; n = 8) for 21 days were analyzed by Illumina deep sequencing. A total of 58 differentially expressed miRNAs were identified with 30 down‐regulated and 28 up‐regulated, and 63 differentially expressed target genes were predicted by miRNA–mRNA joint analysis. GO and KEGG analyses showed that the genes regulated by differentially expressed miRNAs were enriched in glucose metabolism, cytoskeletal structure and function and stress response. Real‐time PCR showed that the mRNA levels of PDK4, HSP90 and DES were significantly increased, whereas those of SCD and LDHA significantly decreased by heat exposure. The protein levels of CALM1, DES and HIF1α were also significantly increased by constant heat. These results demonstrated that the change in miRNA expression in porcine longissimus dorsi muscle underlies the changes in muscle structure and metabolism in porcine skeletal muscle affected by constant heat stress.     

Genome-wide scan reveals important additive and non-additive genetic effects associated with resistance to Haemonchus contortus in Florida Native sheep

Florida Native sheep is among the sheep breeds best adapted to humid and hot climatic conditions such as those of Florida, USA, and have shown a superior ability to regulate nematode burdens. This is one of the oldest sheep breeds in North America and is an endangered species. To ensure genetic diversity and long-term survival of the breed, protection of the current genetic stock is critical and conservation efforts are required to promote its breeding and production. The objective of the present study was to investigate the importance of additive and non-additive genetic effects on resistance to natural Haemonchus contortus infections in Florida Native sheep using a whole genome scan. A total of 200 sheep were evaluated in the present study. Phenotypic records included faecal egg count (FEC, eggs/gram), FAMACHA® score, packed cell volume (PCV, %), body condition score and average daily gain (ADG, kg). Sheep were genotyped using the GGP Ovine 50K SNP chip and 45.2 k single nucleotide polymorphism (SNP) markers spanning the entire genome were available for quality control procedures. Mixed models were used to analyse the response variables and included the identity by state matrix to control for population structure. Bonferroni correction was used to control for multiple testing and a second arbitrary threshold (0.1 × 10^?3) was used. Fifteen SNPs with additive and non-additive genetic effects and located in Ovis aries chromosome OAR1, 2, 3, 6, 8, 10, 11, 12, 13 and 21 were associated with FEC, FAMACHA® score, PCV and ADG. These SNPs could be potential genetic markers for resistance to natural H. contortus exposure in Florida Native sheep.