Design and validation of a 63K genome-wide SNP-genotyping platform for caribou/reindeer (Rangifer tarandus).

BACKGROUND: Development of large single nucleotide polymorphism (SNP) arrays can make genomic data promptly available for conservation problematic. Medium and high-density panels can be designed with sufficient coverage to offer a genome-wide perspective and the generated genotypes can be used to assess different genetic metrics related to population structure, relatedness, or inbreeding. SNP genotyping could also permit sexing samples with unknown associated metadata as it is often the case when using non-invasive sampling methods favored for endangered species. Genome sequencing of wild species provides the necessary information to design such SNP arrays. We report here the development of a SNP-array for endangered Rangifer tarandus using a multi-platform sequencing approach from animals found in diverse populations representing the entire circumpolar distribution of the species. RESULTS: From a very large comprehensive catalog of SNPs detected over the entire sample set (N = 894), a total of 63,336 SNPs were selected. SNP selection accounted for SNPs evenly distributed across the entire genome (~ every 50Kb) with known minor alleles across populations world-wide. In addition, a subset of SNPs was selected to represent rare and local alleles found in Eastern Canada which could be used for ecotype and population assignments - information urgently needed for conservation planning. In addition, heterozygosity from SNPs located in the X-chromosome and genotyping call-rate of SNPs located into the SRY gene of the Y-chromosome yielded an accurate and robust sexing assessment. All SNPs were validated using a high-throughput SNP-genotyping chip. CONCLUSION: This design is now integrated into the first genome-wide commercially available genotyping platform for Rangifer tarandus. This platform would pave the way to future genomic investigation of populations for this endangered species, including estimation of genetic diversity parameters, population assignments, as well as animal sexing from genetic SNP data for non-invasive samples.

Identification and characterization of miRNAs during early pregnancy in domestic sheep.

MicroRNA resources in sheep are limited compared with those in other domesticated mammalian species. By sequencing small RNAs of sheep corpus luteum and endometrium, we have generated the largest amount of miRNA-seq data and compiled the most comprehensive list thus far of miRNAs (n = 599) in sheep. Additionally, we observed a highly conserved maternally imprinted cluster of miRNAs on chromosome 18 homologous to that found on chromosome 14 in human and several other eutherian mammals.

Comparative mRNA and miRNA expression in European mouflon (Ovis musimon) and sheep (Ovis aries) provides novel insights into the genetic mechanisms for female reproductive success.

Prolific breeds of domestic sheep (Ovis aries) are important genetic resources due to their reproductive performance, which is characterized by multiple lambs per birth and out-of-season breeding. However, the lack of a comprehensive understanding of the genetic mechanisms underlying the important reproductive traits, particularly from the evolutionary genomics perspective, has impeded the efficient advancement of sheep breeding. Here, for the first time, by performing RNA-sequencing we built a de novo transcriptome assembly of ovarian and endometrial tissues in European mouflon (Ovis musimon) and performed an mRNA-miRNA integrated expression profiling analysis of the wild species and a highly prolific domestic sheep breed, the Finnsheep. We identified several novel genes with differentially expressed mRNAs (e.g., EREG, INHBA, SPP1, AMH, TDRD5, and ZP2) between the wild and domestic sheep, which are functionally involved in oocyte and follicle development and fertilization, and are significantly (adjusted P-value < 0.05) enriched in the Gene Ontology (GO) terms of various reproductive process, including the regulation of fertilization, oogenesis, ovarian follicle development, and sperm-egg recognition. Additionally, we characterized 58 differentially expressed miRNAs and 210 associated target genes that are essential for the regulation of female reproduction cycles through specific regulatory networks [e.g., (miR-136, miR-374a, miR-9-5p)-(EREG, INHBA)]. Furthermore, our integrated mRNA and miRNA expression profiling analysis elucidated novel direct and indirect miRNA/mRNA causal regulatory relationships related to the reproductive traits of the Ovis species. This study provides in-depth insights into the genomic evolution underlying the reproductive traits of the Ovis species and valuable resources for ovine genomics.

Integrated ovarian mRNA and miRNA transcriptome profiling characterizes the genetic basis of prolificacy traits in sheep (Ovis aries).

BACKGROUND: The highly prolific breeds of domestic sheep (Ovis aries) are globally valuable genetic resources for sheep industry. Genetic, nutritional and other environmental factors affect prolificacy traits in sheep. To improve our knowledge of the sheep prolificacy traits, we conducted mRNA-miRNA integrated profiling of ovarian tissues from two pure breeds with large (Finnsheep) vs. small (Texel) litter sizes and their F1 crosses, half of which were fed a flushing diet. RESULTS: Among the samples, 16,402 genes (60.6% known ovine genes) were expressed, 79 novel miRNAs were found, and a cluster of miRNAs on chromosome 18 was detected. The majority of the differentially expressed genes between breeds were upregulated in the Texel with low prolificacy, owing to the flushing diet effect, whereas a similar pattern was not detected in the Finnsheep. F1 ewes responded similarly to Finnsheep rather than displaying a performance intermediate between the two pure breeds. CONCLUSIONS: The identification and characterization of differentially expressed genes and miRNAs in the ovaries of sheep provided insights into genetic and environmental factors affecting prolificacy traits. The three genes (CST6, MEPE and HBB) that were differentially expressed between the group of Finnsheep and Texel ewes kept in normal diet appeared to be candidate genes of prolificacy traits and will require further validation.

Identification and characterization of miRNAs in the ovaries of a highly prolific sheep breed.

Until recently, there have been few studies concerning miRNAs or miRNA-mediated biological processes in sheep (Ovis aries). In the present study, we used a deep-sequencing approach to examine ovarian miRNAs and the mRNA transcriptomes in two ewes of a highly prolific breed, Finnsheep. We identified 113 known sheep miRNAs, 131 miRNAs conserved in other mammals and 60 novel miRNAs, the expression levels of which accounted for 78.22%, 21.73% and 0.05% of the total respectively. Furthermore, the 10 most abundantly expressed miRNAs in the two libraries were characterized in detail, and the putative target genes of these miRNAs were annotated using GO annotation and KEGG pathway enrichment analyses. Among the target genes, intracellular transducers (SMAD1, SMAD4, SMAD5 and SMAD9) and bone morphogenetic protein (BMP) receptors (BMPR1B and BMPR2) were involved in the transforming growth factor ß (TGFß) signaling pathway in the reproductive axis, and the most significant GO terms were intracellular part (GO:0044424), binding (GO:0005488) and biological_process (GO:0008150) for cellular component, molecular function and biological process respectively. Thus, these results expanded the sheep miRNA database and provided additional information on the prolificacy trait regulated through specific miRNAs in sheep and other mammals.

Inferences about the population history of Rangifer tarandus from Y chromosome and mtDNA phylogenies.

Reindeer, called caribou in North America, has a circumpolar distribution and all extant populations belong to the same species (Rangifer tarandus). It has survived the Holocene thanks to its immense adaptability and successful coexistence with humans in different forms of hunting and herding cultures. Here, we examine the paternal and maternal history of Rangifer based on robust Y-chromosomal and mitochondrial DNA (mtDNA) trees representing Eurasian tundra reindeer, Finnish forest reindeer, Svalbard reindeer, Alaska tundra caribou, and woodland caribou. We first assembled Y-chromosomal contigs, representing 1.3 Mb of single-copy Y regions. Based on 545 Y-chromosomal and 458 mtDNA SNPs defined in 55 males, maximum parsimony trees were created. We observed two well separated clades in both phylogenies: the "EuroBeringian clade" formed by animals from Arctic Islands, Eurasia, and a few from North America and the "North American clade" formed only by caribou from North America. The time calibrated Y tree revealed an expansion and dispersal of lineages across continents after the Last Glacial Maximum. We show for the first time unique paternal lineages in Svalbard reindeer and Finnish forest reindeer and reveal a circumscribed Y haplogroup in Fennoscandian tundra reindeer. The Y chromosome in domesticated reindeer is markedly diverse indicating that several male lineages have undergone domestication and less intensive selection on males. This study places R. tarandus onto the list of species with resolved Y and mtDNA phylogenies and builds the basis for studies of the distribution and origin of paternal and maternal lineages in the future.

Whole-genome sequencing provides novel insights into the evolutionary history and genetic adaptation of reindeer populations in northern Eurasia.

Domestic reindeer (Rangifer tarandus) play a vital role in the culture and livelihoods of indigenous people across northern Eurasia. These animals are well adapted to harsh environmental conditions, such as extreme cold, limited feed availability and long migration distances. Therefore, understanding the genomics of reindeer is crucial for improving their management, conservation and utilisation. In this study, we have generated a new genome assembly for the Fennoscandian domestic reindeer with high contiguity, making it the most complete reference genome for reindeer to date. The new genome assembly was utilised to explore genetic diversity, population structure and selective sweeps in Eurasian Rangifer tarandus populations which was based on the largest population genomic dataset for reindeer, encompassing 58 individuals from diverse populations. Phylogenetic analyses revealed distinct genetic clusters, with the Finnish wild forest reindeer (Rangifer tarandus fennicus) standing out as a unique subspecies. Divergence time estimates suggested a separation of ~ 52 thousand years ago (Kya) between the northern European Rangifer tarandus fennicus and Rangifer tarandus tarandus. Our study identified four main genetic clusters: Fennoscandian, the eastern/northern Russian and Alaskan group, the Finnish forest reindeer, and the Svalbard reindeer. Furthermore, two independent reindeer domestication processes were inferred, suggesting separate origins for the domestic Fennoscandian and eastern/northern Russian reindeer. Notably, shared genes under selection, including retroviral genes, point towards molecular domestication processes that aided adaptation of this species to diverse environments.

Differences in Adipose Gene Expression Profiles between Male and Female Even Reindeer (Rangifer tarandus) in Sakha (Yakutia).

Reindeer are native to harsh northern Eurasian environments which are characterized by long and cold winters, short summers, and limited pasture vegetation. Adipose tissues play a significant role in these animals by modulating energy metabolism, immunity, and reproduction. Here, we have investigated the transcriptome profiles of metacarpal, perirenal, and prescapular adipose tissues in Even reindeer and searched for genes that were differentially expressed in male and female individuals. A total of 15,551 genes were expressed, where the transcriptome profile of metacarpal adipose tissue was found to be distinct from that of perirenal and prescapular adipose tissues. Interestingly, 10 genes, including PRDM9, which is known to have an important role in adaptation and speciation in reindeer, were always upregulated in all three tissues of male reindeer.

Adipose gene expression profiles reveal insights into the adaptation of northern Eurasian semi-domestic reindeer (Rangifer tarandus).

Reindeer (Rangifer tarandus) are semi-domesticated animals adapted to the challenging conditions of northern Eurasia. Adipose tissues play a crucial role in northern animals by altering gene expression in their tissues to regulate energy homoeostasis and thermogenic activity. Here, we perform transcriptome profiling by RNA sequencing of adipose tissues from three different anatomical depots: metacarpal (bone marrow), perirenal, and prescapular fat in Finnish and Even reindeer (in Sakha) during spring and winter. A total of 16,212 genes are expressed in our data. Gene expression profiles in metacarpal tissue are distinct from perirenal and prescapular adipose tissues. Notably, metacarpal adipose tissue appears to have a significant role in the regulation of the energy metabolism of reindeer in spring when their nutritional condition is poor after winter. During spring, genes associated with the immune system are upregulated in the perirenal and prescapular adipose tissue. Blood and tissue parameters reflecting general physiological and metabolic status show less seasonal variation in Even reindeer than in Finnish reindeer. This study identifies candidate genes potentially involved in immune response, fat deposition, and energy metabolism and provides new information on the mechanisms by which reindeer adapt to harsh arctic conditions.

Gene Expression Profiling of Corpus luteum Reveals Important Insights about Early Pregnancy in Domestic Sheep.

The majority of pregnancy loss in ruminants occurs during the preimplantation stage, which is thus the most critical period determining reproductive success. Here, we performed a comparative transcriptome study by sequencing total mRNA from corpus luteum (CL) collected during the preimplantation stage of pregnancy in Finnsheep, Texel and F1 crosses. A total of 21,287 genes were expressed in our data. Highly expressed autosomal genes in the CL were associated with biological processes such as progesterone formation (STAR, CYP11A1, and HSD3B1) and embryo implantation (e.g., TIMP1, TIMP2 and TCTP). Among the list of differentially expressed genes, sialic acid-binding immunoglobulin (Ig)-like lectins (SIGLEC3, SIGLEC14, SIGLEC8), ribosomal proteins (RPL17, RPL34, RPS3A, MRPS33) and chemokines (CCL5, CCL24, CXCL13, CXCL9) were upregulated in Finnsheep, while four multidrug resistance-associated proteins (MRPs) were upregulated in Texel ewes. A total of 17 known genes and two uncharacterized non-coding RNAs (ncRNAs) were differentially expressed in breed-wise comparisons owing to the flushing diet effect. The significantly upregulated TXNL1 gene indicated potential for embryonic diapause in Finnsheep and F1. Moreover, we report, for the first time in any species, several genes that are active in the CL during early pregnancy (including TXNL1, SIGLEC14, SIGLEC8, MRP4, and CA5A).

Genome sequence and comparative analysis of reindeer (Rangifer tarandus) in northern Eurasia.

Reindeer are semi-domesticated ruminants that have adapted to the challenging northern Eurasian environment characterized by long winters and marked annual fluctuations in daylight. We explored the genetic makeup behind their unique characteristics by de novo sequencing the genome of a male reindeer and conducted gene family analyses with nine other mammalian species. We performed a population genomics study of 23 additional reindeer representing both domestic and wild populations and several ecotypes from various geographic locations. We assembled 2.66 Gb (N50 scaffold of 5 Mb) of the estimated 2.92 Gb reindeer genome, comprising 27,332 genes. The results from the demographic history analysis suggested marked changes in the effective population size of reindeer during the Pleistocene period. We detected 160 reindeer-specific and expanded genes, of which zinc finger proteins (n = 42) and olfactory receptors (n = 13) were the most abundant. Comparative genome analyses revealed several genes that may have promoted the adaptation of reindeer, such as those involved in recombination and speciation (PRDM9), vitamin D metabolism (TRPV5, TRPV6), retinal development (PRDM1, OPN4B), circadian rhythm (GRIA1), immunity (CXCR1, CXCR2, CXCR4, IFNW1), tolerance to cold-triggered pain (SCN11A) and antler development (SILT2). The majority of these characteristic reindeer genes have been reported for the first time here. Moreover, our population genomics analysis suggested at least two independent reindeer domestication events with genetic lineages originating from different refugial regions after the Last Glacial Maximum. Taken together, our study has provided new insights into the domestication, evolution and adaptation of reindeer and has promoted novel genomic research of reindeer.

Whole-Genome Sequencing of Three Native Cattle Breeds Originating From the Northernmost Cattle Farming Regions.

Northern Fennoscandia and the Sakha Republic in the Russian Federation represent the northernmost regions on Earth where cattle farming has been traditionally practiced. In this study, we performed whole-genome sequencing to genetically characterize three rare native breeds Eastern Finncattle, Western Finncattle and Yakutian cattle adapted to these northern Eurasian regions. We examined the demographic history, genetic diversity and unfolded loci under natural or artificial selection. On average, we achieved 13.01-fold genome coverage after mapping the sequencing reads on the bovine reference genome (UMD 3.1) and detected a total of 17.45 million single nucleotide polymorphisms (SNPs) and 1.95 million insertions-deletions (indels). We observed that the ancestral species (Bos primigenius) of Eurasian taurine cattle experienced two notable prehistorical declines in effective population size associated with dramatic climate changes. The modern Yakutian cattle exhibited a higher level of within-population variation in terms of number of SNPs and nucleotide diversity than the contemporary European taurine breeds. This result is in contrast to the results of marker-based cattle breed diversity studies, indicating assortment bias in previous analyses. Our results suggest that the effective population size of the ancestral Asiatic taurine cattle may have been higher than that of the European cattle. Alternatively, our findings could indicate the hybrid origins of the Yakutian cattle ancestries and possibly the lack of intensive artificial selection. We identified a number of genomic regions under selection that may have contributed to the adaptation to the northern and subarctic environments, including genes involved in disease resistance, sensory perception, cold adaptation and growth. By characterizing the native breeds, we were able to obtain new information on cattle genomes and on the value of the adapted breeds for the conservation of cattle genetic resources.

The structure and catalytic cycle of a sodium-pumping pyrophosphatase.

Membrane-integral pyrophosphatases (M-PPases) are crucial for the survival of plants, bacteria, and protozoan parasites. They couple pyrophosphate hydrolysis or synthesis to Na(+) or H(+) pumping. The 2.6-angstrom structure of Thermotoga maritima M-PPase in the resting state reveals a previously unknown solution for ion pumping. The hydrolytic center, 20 angstroms above the membrane, is coupled to the gate formed by the conserved Asp(243), Glu(246), and Lys(707) by an unusual "coupling funnel" of six α helices. Comparison with our 4.0-angstrom resolution structure of the product complex suggests that helix 12 slides down upon substrate binding to open the gate by a simple binding-change mechanism. Below the gate, four helices form the exit channel. Superimposing helices 3 to 6, 9 to 12, and 13 to 16 suggests that M-PPases arose through gene triplication.