Genome sequencing of 2000 canids by the Dog10K consortium advances the understanding of demography, genome function and architecture.

BACKGROUND: The international Dog10K project aims to sequence and analyze several thousand canine genomes. Incorporating 20 × data from 1987 individuals, including 1611 dogs (321 breeds), 309 village dogs, 63 wolves, and four coyotes, we identify genomic variation across the canid family, setting the stage for detailed studies of domestication, behavior, morphology, disease susceptibility, and genome architecture and function. RESULTS: We report the analysis of > 48 M single-nucleotide, indel, and structural variants spanning the autosomes, X chromosome, and mitochondria. We discover more than 75% of variation for 239 sampled breeds. Allele sharing analysis indicates that 94.9% of breeds form monophyletic clusters and 25 major clades. German Shepherd Dogs and related breeds show the highest allele sharing with independent breeds from multiple clades. On average, each breed dog differs from the UU_Cfam_GSD_1.0 reference at 26,960 deletions and 14,034 insertions greater than 50 bp, with wolves having 14% more variants. Discovered variants include retrogene insertions from 926 parent genes. To aid functional prioritization, single-nucleotide variants were annotated with SnpEff and Zoonomia phyloP constraint scores. Constrained positions were negatively correlated with allele frequency. Finally, the utility of the Dog10K data as an imputation reference panel is assessed, generating high-confidence calls across varied genotyping platform densities including for breeds not included in the Dog10K collection. CONCLUSIONS: We have developed a dense dataset of 1987 sequenced canids that reveals patterns of allele sharing, identifies likely functional variants, informs breed structure, and enables accurate imputation. Dog10K data are publicly available.

Iron age genomic data from Althiburos - Tunisia renew the debate on the origins of African taurine cattle.

The Maghreb is a key region for understanding the dynamics of cattle dispersal and admixture with local aurochs following their earliest domestication in the Fertile Crescent more than 10,000 years ago. Here, we present data on autosomal genomes and mitogenomes obtained for four archaeological specimens of Iron Age (∼2,800 cal BP-2,000 cal BP) domestic cattle from the Eastern Maghreb, i.e. Althiburos (El Kef, Tunisia). D-loop sequences were obtained for an additional eight cattle specimens from this site. Maternal lineages were assigned to the elusive R and ubiquitous African-T1 haplogroups found in two and ten Althiburos specimens, respectively. Our results can be explained by post-domestication hybridization of Althiburos cattle with local aurochs. However, we cannot rule out an independent domestication in North Africa considering the shared ancestry of Althiburos cattle with the pre-domestic Moroccan aurochs and present-day African taurine cattle.

Genome-wide assessment of the population structure and genetic diversity of four Portuguese native sheep breeds.

As the effects of global warming become increasingly complex and difficult to manage, the conservation and sustainable use of locally adapted sheep breeds are gaining ground. Portuguese native sheep breeds are important reservoirs of genetic diversity, highly adapted to harsh environments and reared in low input production systems. Genomic data that would describe the breeds in detail and accelerate the selection of more resilient animals to be able to cope with climatic challenges are still lacking. Here, we sequenced the genomes of 37 animals from four Portuguese native sheep breeds (Campaniça, Bordaleira Serra da Estrela, Merino Branco and Merino Preto) and 19 crossbred sheep to make inferences on their genomic diversity and population structure. Mean genomic diversities were very similar across these breeds (.30 ≤ Ho ≤ .34; .30 ≤ He ≤ .35; 1.7 × 10-3 ≤ π ≤ 3.1 × 10-3) and the levels of inbreeding were negligible (.005 ≤ FIS ≤ .038). The Principal Components, Bayesian clustering and Treemix analyses split the Portuguese breeds in two main groups which are consistent with historical records: one comprising Campaniça and Serra da Estrela together with other European and transboundary dairy breeds; and another of the well-differentiated multi-purpose Merino and Merino-related breeds. Runs of homozygosity analyses yielded 1,690 ROH segments covering an average of 2.27 Gb across the genome in all individuals. The overall genome covered by ROH segments varied from 27,75 Mb in Serra da Estrela to 61,29 Mb in Campaniça. The phylogenetic analysis of sheep mitogenomes grouped the Portuguese native breeds within sub-haplogroup B1a along with two animals of the Akkaraman breed from Turkey. This result provides additional support to a direct influence of Southwest Asian sheep in local breeds from the Iberian Peninsula. Our study is a first step pertaining to the genomic characterization of Portuguese sheep breeds and the results emphasize the potential of genomic data as a valid tool to guide conservation efforts in locally adapted sheep breeds. In addition, the genomic data we generated can be used to identify markers for breed assignment and traceability of certified breed-products.

Consequences of breed formation on patterns of genomic diversity and differentiation: the case of highly diverse peripheral Iberian cattle.

BACKGROUND: Iberian primitive breeds exhibit a remarkable phenotypic diversity over a very limited geographical space. While genomic data are accumulating for most commercial cattle, it is still lacking for these primitive breeds. Whole genome data is key to understand the consequences of historic breed formation and the putative role of earlier admixture events in the observed diversity patterns. RESULTS: We sequenced 48 genomes belonging to eight Iberian native breeds and found that the individual breeds are genetically very distinct with FST values ranging from 4 to 16% and have levels of nucleotide diversity similar or larger than those of their European counterparts, namely Jersey and Holstein. All eight breeds display significant gene flow or admixture from African taurine cattle and include mtDNA and Y-chromosome haplotypes from multiple origins. Furthermore, we detected a very low differentiation of chromosome X relative to autosomes within all analyzed taurine breeds, potentially reflecting male-biased gene flow. CONCLUSIONS: Our results show that an overall complex history of admixture resulted in unexpectedly high levels of genomic diversity for breeds with seemingly limited geographic ranges that are distantly located from the main domestication center for taurine cattle in the Near East. This is likely to result from a combination of trading traditions and breeding practices in Mediterranean countries. We also found that the levels of differentiation of autosomes vs sex chromosomes across all studied taurine and indicine breeds are likely to have been affected by widespread breeding practices associated with male-biased gene flow.

New insights into the genetic composition and phylogenetic relationship of wolves and dogs in the Iberian Peninsula.

This study investigates the gene pool of Portuguese autochthonous dog breeds and their wild counterpart, the Iberian wolf subspecies (Canis lupus signatus), using standard molecular markers. A combination of paternal and maternal molecular markers was used to investigate the genetic composition, genetic differentiation and genetic relationship of native Portuguese dogs and the Iberian wolf. A total of 196 unrelated dogs, including breed and village dogs from Portugal, and other dogs from Spain and North Africa, and 56 Iberian wolves (wild and captive) were analyzed for nuclear markers, namely Y chromosome SNPs, Y chromosome STR loci, autosomal STR loci, and a mitochondrial fragment of the control region I. Our data reveal new variants for the molecular markers and confirm significant genetic differentiation between Iberian wolf and native domestic dogs from Portugal. Based on our sampling, no signs of recent introgression between the two subspecies were detected. Y chromosome data do not reveal genetic differentiation among the analyzed dog breeds, suggesting they share the same patrilineal origin. Moreover, the genetic distinctiveness of the Iberian wolf from other wolf populations is further confirmed with the description of new mtDNA variants for this endemism. Our research also discloses new molecular markers for wolf and dog subspecies assignment, which might become particularly relevant in the case of forensic or noninvasive genetic studies. The Iberian wolf represents a relic of the once widespread wolf population in Europe and our study reveals that it is a reservoir of unique genetic diversity of the grey wolf, Canis lupus. These results stress the need for conservation plans that will guarantee the sustainability of this threatened top predator in Iberia.

Treatment with low doses of polyclonal immunoglobulin improves B cell function during immune reconstitution in a murine model.

PURPOSE: [corrected] After autologous stem cell transplantation (ASCT) the immunological B cell compartment recovers slowly. Delays on the recovery of B cell function after autologous stem cell transplantation are due to the low lymphocytes count and to their intrinsic dysfunction. METHODS: We studied the in vivo B cell reconstitution after ASCT examining the independent effect of polyclonal IgG (PolyIg), Fab or Fc fragments infusions in a murine animal model during a period of 12 weeks. These molecules were used in low doses, mimicking the recommended use of IVIg in the case of hypogammaglobulinemia in humans. Flow cytometry analysis and ELISA tests were conducted to monitor the reconstitution of B cells and serum immunoglobulin production. Panama blot and PCA factor 1 analysis were used to study the kinetics of immunoglobulin repertoires reconstitution. Mechanistic studies were also performed using in vitro cell culture. RESULTS: During follow-up after ASCT, peripheral B cells expand independently of treatment, correcting the immediate increase in sBAFF (soluble B cell activating factor) induced by previous intense myeloablation. Treatments with Fab and Fc fragments infusions promote significant IgM and IgG production comparing to control. Although the complete recovery of antibody repertoire is only achieved at the end of follow-up after ASCT, there is an earlier and significantly stronger recovery in the treated mice, which is evident at 9 weeks after ASCT. At 30 weeks after ASCT, normal values of antibody repertoire were detected in all individuals. Mechanistic studies show that Fab and Fc fragments promote IgG1 production by indirect pathways. CONCLUSIONS: The results presented here demonstrate that polyclonal immunoglobulin indirectly improves the function of the reconstituted B cells and their IgG production by means of Fc-mediated effects on bystander cells. These results further stimulate the discussion about the advantages of IVIg therapy during immune reconstitution after human ASCT.

Treatment with polyclonal immunoglobulin during T-cell reconstitution promotes naive T-cell proliferation.

Natural antibodies are unique self molecules endowed with both suppressive and activating functions on various cells of the immune system and are recognized as a fundamental link between the adaptive and innate immune system. Here, we examine the role of natural antibodies, using polyclonal immunoglobulins (Ig), as a promoter of T-cell reconstitution in a context of lymphopenia. We have established a mouse model to mimic immunologic recovery in adult patients with severe hypothymic function subjected to autologous hematopoietic precursor cell transplantation. Thymectomized mice were transplanted and treated with low doses of Ig or its Fab or Fc fragments. The animals displayed, during early phases of Ig treatment, a significant increase of T-cell reconstitution displaying a naive CD4(+) phenotype. In addition, the Ig-treated animals exhibited an increase dilution of single-joint T-cell receptor excision circles (sjTRECs) in peripheral blood, suggesting an early increase in proliferation of T cells stimulated by the natural antibodies. These results unveil a novel and considerable effect of intravenous Ig treatment in situations of severe lymphopenia as a stimulator of proliferation of peripheral naive T cells, possibly protecting diverse immune repertoires.

Mitochondrial DNA sequence variation in Portuguese native dog breeds: diversity and phylogenetic affinities.

In an extensive survey of the genetic diversity in Portuguese dogs, we have examined an 887-bp fragment of the mitochondrial DNA (mtDNA) from 8 Portuguese, 1 Spanish, and 2 North African native dog breeds, including village dogs from Portugal and Tunisia. Forty-nine haplotypes were found in the 164 individuals analyzed, with private haplotypes being found in several breeds. For example, the Castro Laboreiro Watchdog, a rare breed from a small and isolated region in Portugal, was monomorphic for mtDNA and possessed a new haplotype, which may be provisionally considered a breed-specific marker. Phylogenetic analyses recapitulated 4 major clades identified in other studies, but new haplotypes, grouping within a clade that was previously thought as geographically restricted, were detected in Portugal and Morocco. Portuguese village dogs showed no genetic differentiation from nonnative dogs or from local breeds of the areas in which the village dogs were sampled. Although Iberian and North African dog breeds possessed breed-specific mtDNA haplotypes, no significant geographic structure could be detected among them. There is no evidence for introgression of North African haplotypes in Iberian dogs, contrary to previous results for other domestic animals.