Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample.

Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (†Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth's death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.

Integration of fluorescence in situ hybridization and chromosome-length genome assemblies revealed synteny map for guinea pig, naked mole-rat, and human.

Descriptions of karyotypes of many animal species are currently available. In addition, there has been a significant increase in the number of sequenced genomes and an ever-improving quality of genome assembly. To close the gap between genomic and cytogenetic data we applied fluorescent in situ hybridization (FISH) and Hi-C technology to make the first full chromosome-level genome comparison of the guinea pig (Cavia porcellus), naked mole-rat (Heterocephalus glaber), and human. Comparative chromosome maps obtained by FISH with chromosome-specific probes link genomic scaffolds to individual chromosomes and orient them relative to centromeres and heterochromatic blocks. Hi-C assembly made it possible to close all gaps on the comparative maps and to reveal additional rearrangements that distinguish the karyotypes of the three species. As a result, we integrated the bioinformatic and cytogenetic data and adjusted the previous comparative maps and genome assemblies of the guinea pig, naked mole-rat, and human. Syntenic associations in the two hystricomorphs indicate features of their putative ancestral karyotype. We postulate that the two approaches applied in this study complement one another and provide complete information about the organization of these genomes at the chromosome level.

Genetic History of the Altai Breed Horses: From Ancient Times to Modernity.

This study focuses on expanding knowledge about the genetic diversity of the Altai horse native to Siberia. While studying modern horses from two Altai regions, where horses were subjected to less crossbreeding, we tested the hypothesis, formulated on the basis of morphological data, that the Altai horse is represented by two populations (Eastern and Southern) and that the Mongolian horse has a greater genetic proximity to Eastern Altai horses. Bone samples of ancient horses from different cultures of Altai were investigated to clarify the genetic history of this horse breed. As a genetic marker, we chose hypervariable region I of mitochondrial DNA. The results of the performed phylogenetic and population genetic analyses of our and previously published data confirmed the hypothesis stated above. As we found out, almost all the haplotypes of the ancient domesticated horses of Altai are widespread among modern Altai horses. The differences between the mitochondrial gene pools of the ancient horses of Altai and Mongolia are more significant than between those of modern horses of the respective regions, which is most likely due to an increase in migration processes between these regions after the Early Iron Age.

Comparative studies of X chromosomes in Cervidae family.

The family Cervidae is the second most diverse in the infraorder Pecora and is characterized by variability in the diploid chromosome numbers among species. X chromosomes in Cervidae evolved through complex chromosomal rearrangements of conserved segments within the chromosome, changes in centromere position, heterochromatic variation, and X-autosomal translocations. The family Cervidae consists of two subfamilies: Cervinae and Capreolinae. Here we build a detailed X chromosome map with 29 cattle bacterial artificial chromosomes of representatives of both subfamilies: reindeer (Rangifer tarandus), gray brocket deer (Mazama gouazoubira), Chinese water deer (Hydropotes inermis) (Capreolinae); black muntjac (Muntiacus crinifrons), tufted deer (Elaphodus cephalophus), sika deer (Cervus nippon) and red deer (Cervus elaphus) (Cervinae). To track chromosomal rearrangements during Cervidae evolution, we summarized new data, and compared them with available X chromosomal maps and chromosome level assemblies of other species. We demonstrate the types of rearrangements that may have underlined the variability of Cervidae X chromosomes. We detected two types of cervine X chromosome-acrocentric and submetacentric. The acrocentric type is found in three independent deer lineages (subfamily Cervinae and in two Capreolinae tribes-Odocoileini and Capreolini). We show that chromosomal rearrangements on the X-chromosome in Cervidae occur at a higher frequency than in the entire Ruminantia lineage: the rate of rearrangements is 2 per 10 million years.

Chromosome-length genome assembly and karyotype of the endangered black-footed ferret (Mustela nigripes).

The black-footed ferret (Mustela nigripes) narrowly avoided extinction to become an oft-cited example of the benefits of intensive management, research, and collaboration to save a species through ex situ conservation breeding and reintroduction into its former range. However, the species remains at risk due to possible inbreeding, disease susceptibility, and multiple fertility challenges. Here, we report the de novo genome assembly of a male black-footed ferret generated through a combination of linked-read sequencing, optical mapping, and Hi-C proximity ligation. In addition, we report the karyotype for this species, which was used to anchor and assign chromosome numbers to the chromosome-length scaffolds. The draft assembly was ~2.5 Gb in length, with 95.6% of it anchored to 19 chromosome-length scaffolds, corresponding to the 2n = 38 chromosomes revealed by the karyotype. The assembly has contig and scaffold N50 values of 148.8 kbp and 145.4 Mbp, respectively, and is up to 96% complete based on BUSCO analyses. Annotation of the assembly, including evidence from RNA-seq data, identified 21,406 protein-coding genes and a repeat content of 37.35%. Phylogenomic analyses indicated that the black-footed ferret diverged from the European polecat/domestic ferret lineage 1.6 million yr ago. This assembly will enable research on the conservation genomics of black-footed ferrets and thereby aid in the further restoration of this endangered species.

Chromosome-length genome assemblies and cytogenomic analyses of pangolins reveal remarkable chromosome counts and plasticity.

We report the first chromosome-length genome assemblies for three species in the mammalian order Pholidota: the white-bellied, Chinese, and Sunda pangolins. Surprisingly, we observe extraordinary karyotypic plasticity within this order and, in female white-bellied pangolins, the largest number of chromosomes reported in a Laurasiatherian mammal: 2n = 114. We perform the first karyotype analysis of an African pangolin and report a Y-autosome fusion in white-bellied pangolins, resulting in 2n = 113 for males. We employ a novel strategy to confirm the fusion and identify the autosome involved by finding the pseudoautosomal region (PAR) in the female genome assembly and analyzing the 3D contact frequency between PAR sequences and the rest of the genome in male and female white-bellied pangolins. Analyses of genetic variability show that white-bellied pangolins have intermediate levels of genome-wide heterozygosity relative to Chinese and Sunda pangolins, consistent with two moderate declines of historical effective population size. Our results reveal a remarkable feature of pangolin genome biology and highlight the need for further studies of these unique and endangered mammals.

Chromosome-Length Assembly of the Baikal Seal (Pusa sibirica) Genome Reveals a Historically Large Population Prior to Isolation in Lake Baikal.

Pusa sibirica, the Baikal seal, is the only extant, exclusively freshwater, pinniped species. The pending issue is, how and when they reached their current habitat-the rift lake Baikal, more than three thousand kilometers away from the Arctic Ocean. To explore the demographic history and genetic diversity of this species, we generated a de novo chromosome-length assembly, and compared it with three closely related marine pinniped species. Multiple whole genome alignment of the four species compared with their karyotypes showed high conservation of chromosomal features, except for three large inversions on chromosome VI. We found the mean heterozygosity of the studied Baikal seal individuals was relatively low (0.61 SNPs/kbp), but comparable to other analyzed pinniped samples. Demographic reconstruction of seals revealed differing trajectories, yet remarkable variations in Ne occurred during approximately the same time periods. The Baikal seal showed a significantly more severe decline relative to other species. This could be due to the difference in environmental conditions encountered by the earlier populations of Baikal seals, as ice sheets changed during glacial-interglacial cycles. We connect this period to the time of migration to Lake Baikal, which occurred ~3-0.3 Mya, after which the population stabilized, indicating balanced habitat conditions.

Maps of Constitutive-Heterochromatin Distribution for Four Martes Species (Mustelidae, Carnivora, Mammalia) Show the Formative Role of Macrosatellite Repeats in Interspecific Variation of Chromosome Structure.

Constitutive-heterochromatin placement in the genome affects chromosome structure by occupying centromeric areas and forming large blocks. To investigate the basis for heterochromatin variation in the genome, we chose a group of species with a conserved euchromatin part: the genus Martes [stone marten (M. foina, 2n = 38), sable (M. zibellina, 2n = 38), pine marten (M. martes, 2n = 38), and yellow-throated marten (M. flavigula, 2n = 40)]. We mined the stone marten genome for the most abundant tandem repeats and selected the top 11 macrosatellite repetitive sequences. Fluorescent in situ hybridization revealed distributions of the tandemly repeated sequences (macrosatellites, telomeric repeats, and ribosomal DNA). We next characterized the AT/GC content of constitutive heterochromatin by CDAG (Chromomycin A3-DAPI-after G-banding). The euchromatin conservatism was shown by comparative chromosome painting with stone marten probes in newly built maps of the sable and pine marten. Thus, for the four Martes species, we mapped three different types of tandemly repeated sequences critical for chromosome structure. Most macrosatellites are shared by the four species with individual patterns of amplification. Some macrosatellites are specific to a species, autosomes, or the X chromosome. The variation of core macrosatellites and their prevalence in a genome are responsible for the species-specific variation of the heterochromatic blocks.

Comparative Studies of Karyotypes in the Cervidae Family.

The family Cervidae is the second most diverse family in the infraorder Pecora and is characterized by a striking variability in the diploid chromosome numbers among species, ranging from 6 to 70. Chromosomal rearrangements in Cervidae have been studied in detail by chromosome painting. There are many comparative cytogenetic data for both subfamilies (Cervinae and Capreolinae) based on homologies with chromosomes of cattle and Chinese muntjac. Previously it was found that interchromosomal rearrangements are the major type of rearrangements occurring in the Cervidae family. Here, we build a detailed chromosome map of a female reindeer (Rangifer tarandus, 2n = 70, Capreolinae) and a female black muntjac (Muntiacus crinifrons, 2n = 8, Cervinae) with dromedary homologies to find out what other types of rearrangements may have underlined the variability of Cervidae karyotypes. To track chromosomal rearrangements and the distribution of nucleolus organizer regions not only during Cervidae but also Pecora evolution, we summarized new data and compared them with chromosomal maps of other already studied species. We discuss changes in the pecoran ancestral karyotype in the light of new painting data. We show that intrachromosomal rearrangements in autosomes of Cervidae are more frequent than previously thought: at least 13 inversions in evolutionary breakpoint regions were detected.

Phylogeography of ancient and modern brown bears from eastern Eurasia.

The brown bear (Ursus arctos) is an iconic carnivoran species of the Northern Hemisphere. Its population history has been studied extensively using mitochondrial markers, which demonstrated signatures of multiple waves of migration, arguably connected with glaciation periods. Among Eurasian brown bears, Siberian populations remain understudied. We have sequenced complete mitochondrial genomes of four ancient (~4.5-40 kya) bears from South Siberia and 19 modern bears from South Siberia and the Russian Far East. Reconstruction of phylogenetic relationships between haplotypes and evaluation of modern population structure have demonstrated that all the studied samples belong to the most widespread Eurasian clade 3. One of the ancient haplotypes takes a basal position relative to the whole of clade 3; the second is basal to the haplogroup 3a (the most common subclade), and two others belong to clades 3a1 and 3b. Modern Siberian bears retain at least some of this diversity; apart from the most common haplogroup 3a, we demonstrate the presence of clade 3b, which was previously found mainly in mainland Eurasia and Northern Japan. Our findings highlight the importance of South Siberia as a refugium for northern Eurasian brown bears and further corroborate the hypothesis of several waves of migration in the Pleistocene.

Performance and automation of ancient DNA capture with RNA hyRAD probes.

DNA hybridization-capture techniques allow researchers to focus their sequencing efforts on preselected genomic regions. This feature is especially useful when analysing ancient DNA (aDNA) extracts, which are often dominated by exogenous environmental sources. Here, we assessed, for the first time, the performance of hyRAD as an inexpensive and design-free alternative to commercial capture protocols to obtain authentic aDNA data from osseous remains. HyRAD relies on double enzymatic restriction of fresh DNA extracts to produce RNA probes that cover only a fraction of the genome and can serve as baits for capturing homologous fragments from aDNA libraries. We found that this approach could retrieve sequence data from horse remains coming from a range of preservation environments, including beyond radiocarbon range, yielding up to 146.5-fold on-target enrichment for aDNA extracts showing extremely low endogenous content (<1%). Performance was, however, more limited for those samples already characterized by good DNA preservation (>20%-30%), while the fraction of endogenous reads mapping on- and off-target was relatively insensitive to the original endogenous DNA content. Procedures based on two instead of a single round of capture increased on-target coverage up to 3.6-fold. Additionally, we used methylation-sensitive restriction enzymes to produce probes targeting hypomethylated regions, which improved data quality by reducing post-mortem DNA damage and mapping within multicopy regions. Finally, we developed a fully automated hyRAD protocol utilizing inexpensive robotic platforms to facilitate capture processing. Overall, our work establishes hyRAD as a cost-effective strategy to recover a set of shared orthologous variants across multiple ancient samples.

New Data on Comparative Cytogenetics of the Mouse-Like Hamsters (Calomyscus Thomas, 1905) from Iran and Turkmenistan.

The taxonomy of the genus Calomyscus remains controversial. According to the latest systematics the genus includes eight species with great karyotypic variation. Here, we studied karyotypes of 14 Calomyscus individuals from different regions of Iran and Turkmenistan using a new set of chromosome painting probes from a Calomyscus sp. male (2n = 46, XY; Shahr-e-Kord-Soreshjan-Cheshme Maiak Province). We showed the retention of large syntenic blocks in karyotypes of individuals with identical chromosome numbers. The only rearrangement (fusion 2/21) differentiated Calomyscus elburzensis, Calomyscus mystax mystax, and Calomyscus sp. from Isfahan Province with 2n = 44 from karyotypes of C. bailwardi, Calomyscus sp. from Shahr-e-Kord, Chahar Mahal and Bakhtiari-Aloni, and Khuzestan-Izeh Provinces with 2n = 46. The individuals from Shahdad tunnel, Kerman Province with 2n = 51-52 demonstrated non-centric fissions of chromosomes 4, 5, and 6 of the 46-chromosomal form with the formation of separate small acrocentrics. A heteromorphic pair of chromosomes in a specimen with 2n = 51 resulted from a fusion of two autosomes. C-banding and chromomycin A3-DAPI staining after G-banding showed extensive heterochromatin variation between individuals.

Draft de novo Genome Assembly of the Elusive Jaguarundi, Puma yagouaroundi.

The Puma lineage within the family Felidae consists of 3 species that last shared a common ancestor around 4.9 million years ago. Whole-genome sequences of 2 species from the lineage were previously reported: the cheetah (Acinonyx jubatus) and the mountain lion (Puma concolor). The present report describes a whole-genome assembly of the remaining species, the jaguarundi (Puma yagouaroundi). We sequenced the genome of a male jaguarundi with 10X Genomics linked reads and assembled the whole-genome sequence. The assembled genome contains a series of scaffolds that reach the length of chromosome arms and is similar in scaffold contiguity to the genome assemblies of cheetah and puma, with a contig N50 = 100.2 kbp and a scaffold N50 = 49.27 Mbp. We assessed the assembled sequence of the jaguarundi genome using BUSCO, aligned reads of the sequenced individual and another published female jaguarundi to the assembled genome, annotated protein-coding genes, repeats, genomic variants and their effects with respect to the protein-coding genes, and analyzed differences of the 2 jaguarundis from the reference mitochondrial genome. The jaguarundi genome assembly and its annotation were compared in quality, variants, and features to the previously reported genome assemblies of puma and cheetah. Computational analyzes used in the study were implemented in transparent and reproducible way to allow their further reuse and modification.

Karyotypic and molecular evidence supports the endemic Tibetan hamsters as a separate divergent lineage of Cricetinae.

The genus status of Urocricetus was defined recently based on morphological and molecular data. Even though the amount of evidence for a separate phylogenetic position of this genus among Cricetinae continues to increase, there is still no consensus on its relationship to other groups. Here we give the first comprehensive description of the U. kamensis karyotype (2n = 30, NFa = 50) including results of comparative cytogenetic analysis and detailed examination of its phylogenetic position by means of numerous molecular markers. The molecular data strongly indicated that Urocricetus is a distant sister group to Phodopus. Comparative cytogenetic data showed significant reorganization of the U. kamensis karyotype compared to karyotypes of all other hamsters investigated earlier. The totality of findings undoubtedly means that Urocricetus belongs to a separate divergent lineage of Cricetinae.

Traces of Late Bronze and Early Iron Age Mongolian Horse Mitochondrial Lineages in Modern Populations.

The Mongolian horse is one of the most ancient and relatively unmanaged horse breeds. The population history of the Mongolian horse remains poorly understood due to a lack of information on ancient and modern DNA. Here, we report nearly complete mitochondrial genome data obtained from five ancient Mongolian horse samples of the Khereksur and Deer Stone culture (late 2nd to 1st third of the 1st millennium BC) and one ancient horse specimen from the Xiongnu culture (1st century BC to 1st century AD) using target enrichment and high-throughput sequencing methods. Phylogenetic analysis involving ancient, historical, and modern mitogenomes of horses from Mongolia and other regions showed the presence of three mitochondrial haplogroups in the ancient Mongolian horse populations studied here and similar haplotype composition of ancient and modern horse populations of Mongolia. Our results revealed genetic continuity between the Mongolian horse populations of the Khereksur and Deer Stone culture and those of the Xiongnu culture owing to the presence of related mitotypes. Besides, we report close phylogenetic relationships between haplotypes of the Khereksur and Deer Stone horses and the horses of indigenous breeds of the Middle East (Caspian and Iranian), China (Naqu, Yunnan, and Jinjiang), and Italy (Giara) as well as genetic similarity between the Xiongnu Mongolian horses and those of the most ancient breeds of the Middle East (Arabian) and Central Asia (Akhal-Teke). Despite all the migrations of the Mongolian peoples over the past 3000 years, mitochondrial haplogroup composition of Mongolian horse populations remains almost unchanged.

Evolution of Tandemly Arranged Repetitive DNAs in Three Species of Cyprinoidei with Different Ploidy Levels.

Polyploid species represent a challenge for both cytogenetic and genomic studies due to their high chromosome numbers and the morphological similarity between their paralogous chromosomes. This paper describes the use of low-coverage high-throughput sequencing to identify the 14 most abundant tandemly arranged repetitive elements in the paleotetraploid genome of the crucian carp (Carassius carassius, 2n = 100). These repetitive elements were then used for molecular cytogenetic studies of a closely related functionally triploid form of the Prussian carp (Carassius gibelio, 3n = 150 + Bs) and a relatively distant diploid species, the tench (Tinca tinca, 2n = 48). According to their distribution on the chromosomes of the 3 aforementioned species, the repetitive elements here identified can be divided into 5 groups: (1) those specific to a single genomic locus in both Carassius species, despite the recent carp-specific genome duplication; (2) those located in a single genomic locus of T. tinca, but amplified in one or both Carassius species; (3) those massively amplified in the B chromosomes of C. gibelio; (4) those located in a single locus in C. gibelio, but amplified in many blocks in C. carassius; and (5) those located in multiple pericentromeric loci in both Carassius species. Our data indicate that some of the repetitive elements are highly conserved in cyprinoid species and may serve as good cytogenetic and genomic markers for discriminating paralogous chromosomes, while others are evolutionarily recent, and their amplification may be related to the last whole-genome duplication event.

Demographic History, Adaptation, and NRAP Convergent Evolution at Amino Acid Residue 100 in the World Northernmost Cattle from Siberia.

Native cattle breeds represent an important cultural heritage. They are a reservoir of genetic variation useful for properly responding to agriculture needs in the light of ongoing climate changes. Evolutionary processes that occur in response to extreme environmental conditions could also be better understood using adapted local populations. Herein, different evolutionary histories of the world northernmost native cattle breeds from Russia were investigated. They highlighted Kholmogory as a typical taurine cattle, whereas Yakut cattle separated from European taurines approximately 5,000 years ago and contain numerous ancestral and some novel genetic variants allowing their adaptation to harsh conditions of living above the Polar Circle. Scans for selection signatures pointed to several common gene pathways related to adaptation to harsh climates in both breeds. But genes affected by selection from these pathways were mostly different. A Yakut cattle breed-specific missense mutation in a highly conserved NRAP gene represents a unique example of a young amino acid residue convergent change shared with at least 16 species of hibernating/cold-adapted mammals from six distinct phylogenetic orders. This suggests a convergent evolution event along the mammalian phylogenetic tree and fast fixation in a single isolated cattle population exposed to a harsh climate.

Karyotype Evolution in 10 Pinniped Species: Variability of Heterochromatin versus High Conservatism of Euchromatin as Revealed by Comparative Molecular Cytogenetics.

Pinnipedia karyotype evolution was studied here using human, domestic dog, and stone marten whole-chromosome painting probes to obtain comparative chromosome maps among species of Odobenidae (Odobenus rosmarus), Phocidae (Phoca vitulina, Phoca largha, Phoca hispida, Pusa sibirica, Erignathus barbatus), and Otariidae (Eumetopias jubatus, Callorhinus ursinus, Phocarctos hookeri, and Arctocephalus forsteri). Structural and functional chromosomal features were assessed with telomere repeat and ribosomal-DNA probes and by CBG (C-bands revealed by barium hydroxide treatment followed by Giemsa staining) and CDAG (Chromomycin A3-DAPI after G-banding) methods. We demonstrated diversity of heterochromatin among pinniped karyotypes in terms of localization, size, and nucleotide composition. For the first time, an intrachromosomal rearrangement common for Otariidae and Odobenidae was revealed. We postulate that the order of evolutionarily conserved segments in the analyzed pinnipeds is the same as the order proposed for the ancestral Carnivora karyotype (2n = 38). The evolution of conserved genomes of pinnipeds has been accompanied by few fusion events (less than one rearrangement per 10 million years) and by novel intrachromosomal changes including the emergence of new centromeres and pericentric inversion/centromere repositioning. The observed interspecific diversity of pinniped karyotypes driven by constitutive heterochromatin variation likely has played an important role in karyotype evolution of pinnipeds, thereby contributing to the differences of pinnipeds' chromosome sets.

High genetic diversity of ancient horses from the Ukok Plateau.

A growing number of researchers studying horse domestication come to a conclusion that this process happened in multiple locations and involved multiple wild maternal lines. The most promising approach to address this problem involves mitochondrial haplotype comparison of wild and domestic horses from various locations coupled with studies of possible migration routes of the ancient shepherds. Here, we sequenced complete mitochondrial genomes of six horses from burials of the Ukok plateau (Russia, Altai Mountains) dated from 2.7 to 1.4 thousand years before present and a single late Pleistocene wild horse from the neighboring region (Denisova cave). Sequencing data indicates that the wild horse belongs to an extinct pre-domestication lineage. Integration of the domestic horse data with known Eurasian haplotypes of a similar age revealed two distinct groups: the first one widely distributed in Europe and presumably imported to Altai, and the second one specific for Altai Mountains and surrounding area.

Evolutionary rearrangements of X chromosomes in voles (Arvicolinae, Rodentia).

Euchromatic segments of the X chromosomes of placental mammals are the most conservative elements of the karyotype, only rarely subjected to either inter- or intrachromosomal rearrangements. Here, using microdissection-derived set of region-specific probes of Terricola savii we detailed the evolutionary rearrangements found in X chromosomes in 20 vole species (Arvicolinae, Rodentia). We show that the evolution of X chromosomes in this taxon was accompanied by multiple para- and pericentric inversions and centromere shifts. The contribution of intrachromosomal rearrangements to the karyotype evolution of Arvicolinae species was approximately equivalent in both the separate autosomal conserved segments and the X chromosomes. Intrachromosmal rearrangements and structural reorganization of the X chromosomes was likely accompanied by an accumulation, distribution, and evolution of repeated sequences.