What is a B chromosome? Early definitions revisited.

Since the discovery of B chromosomes, multiple different definitions of these selfish genetic elements have been put forth. We reconsidered early definitions in light of recently published studies. While there are many characteristics that vary among different B chromosomes, such as their evolutionary origins, size, segregation behaviors, gene content, and function, there is one defining trait of all B chromosomes: they are nonessential for the organism. The points raised here may be useful for framing future B chromosome studies and help guide the categorization of new chromosomal elements that are uncovered in genomic studies.

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.

Chromosomal Evolution of the Talpinae.

In recent years, the number of mole species with species status confirmed by genetic methods has been continuously increasing. Unfortunately, cytogenetic data are not yet available for all species. Here, for the first time, a GTG-banded karyotype of the small-toothed mole from Vietnam, Euroscaptor parvidens, a representative of the Eastern clade of the genus Euroscaptor, has been described. Through comparative analysis of available Euroscaptor (Euroscaptor parvidens, Euroscaptor klossi, and Euroscaptor malayana) and Oreoscaptor (Oreoscaptor mizura) karyotypes, we found cytogenetic signatures for each of the studied species. Zoo-FISH with sorted chromosomes of the Siberian mole (Talpa altaica) on chromosome sets of the small-toothed mole (E. parvidens), the small Japanese mole (Mogera imaizumii) from the closely related genus, and the Japanese shrew mole (Urotrichus talpoides) from the tribe Urotrichini made it possible to identify syntenic regions between these species. We propose a possible ancestral karyotype of the tribe and, based on it, traced the features of chromosomal rearrangements accompanying the divergence of moles. The low rates of chromosomal evolution within the species of the genus Talpa-T. altaica and T. europaea-and the high rates of karyotypic reshuffling within the Asian genera of the tribe were confirmed. The karyotype of the Japanese mountain mole O. mizura seems to be the most conserved among the Asian moles. The most frequently occurring types of chromosomal rearrangements in moles are the pericentric inversions and amplification of heterochromatin. The pericentric inversions on four pairs of autosomes are shared between the closely related genera Euroscaptor, Oreoscaptor, and Mogera, while many more apomorphic rearrangements have occurred in each lineage additionally. The highest rate of chromosomal changes, with five rearrangements occurring over approximately 7 million years, was recorded in the lineage of the small-toothed mole.

The Precise Breakpoint Mapping in Paracentric Inversion 10q22.2q23.3 by Comprehensive Cytogenomic Analysis, Multicolor Banding, and Single-Copy Chromosome Sequencing.

Detection and precise genomic mapping of balanced chromosomal abnormalities in patients with impaired fertility or a clinical phenotype represent a challenge for current cytogenomics owing to difficulties with precise breakpoint localization in the regions enriched for DNA repeats and high genomic variation in such regions. Here, we present a comprehensive cytogenomic approach to breakpoint mapping in a rare paracentric inversion on 10q (in a patient with oligoasthenoteratozoospermia and necrozoospermia) that does not affect other phenotype traits. Multicolor banding, chromosomal microarray analysis, chromosome microdissection with reverse painting, and single-copy sequencing of the rearranged chromosome were performed to determine the length and position of the inverted region as well as to rule out a genetic imbalance at the breakpoints. As a result, a paracentric 19.251 Mbp inversion at 10q22.2q23.3 was described. The most probable location of the breakpoints was predicted using the hg38 assembly. The problems of genetic counseling associated with enrichment for repeats and high DNA variability of usual breakpoint regions were discussed. Possible approaches for cytogenomic assessment of couples with balanced chromosome rearrangements and problems like reproductive failures were considered and suggested as useful part of effective genetic counseling.

Identification of Iguania Ancestral Syntenic Blocks and Putative Sex Chromosomes in the Veiled Chameleon (Chamaeleo calyptratus, Chamaeleonidae, Iguania).

The veiled chameleon (Chamaeleo calyptratus) is a typical member of the family Chamaeleonidae and a promising object for comparative cytogenetics and genomics. The karyotype of C. calyptratus differs from the putative ancestral chameleon karyotype (2n = 36) due to a smaller chromosome number (2n = 24) resulting from multiple chromosome fusions. The homomorphic sex chromosomes of an XX/XY system were described recently using male-specific RADseq markers. However, the chromosomal pair carrying these markers was not identified. Here we obtained chromosome-specific DNA libraries of C. calyptratus by chromosome flow sorting that were assigned by FISH and sequenced. Sequence comparison with three squamate reptiles reference genomes revealed the ancestral syntenic regions in the C. calyptratus chromosomes. We demonstrated that reducing the chromosome number in the C. calyptratus karyotype occurred through two fusions between microchromosomes and four fusions between micro-and macrochromosomes. PCR-assisted mapping of a previously described Y-specific marker indicates that chromosome 5 may be the sex chromosome pair. One of the chromosome 5 conserved synteny blocks shares homology with the ancestral pleurodont X chromosome, assuming parallelism in the evolution of sex chromosomes from two basal Iguania clades (pleurodonts and acrodonts). The comparative chromosome map produced here can serve as the foundation for future genome assembly of chameleons and vertebrate-wide comparative genomic studies.

The Cytogenetic Map of the Nile Crocodile (Crocodylus niloticus, Crocodylidae, Reptilia) with Fluorescence In Situ Localization of Major Repetitive DNAs.

Tandemly arranged and dispersed repetitive DNA sequences are important structural and functional elements that make up a significant portion of vertebrate genomes. Using high throughput, low coverage whole genome sequencing followed by bioinformatics analysis, we have identified seven major tandem repetitive DNAs and two fragments of LTR retrotransposons in the genome of the Nile crocodile (Crocodylus niloticus, 2n = 32). The repeats showed great variability in structure, genomic organization, and chromosomal distribution as revealed by fluorescence in situ hybridization (FISH). We found that centromeric and pericentromeric heterochromatin of C. niloticus is composed of previously described in Crocodylus siamensis CSI-HindIII and CSI-DraI repetitive sequence families, a satellite revealed in Crocodylus porosus, and additionally contains at least three previously unannotated tandem repeats. Both LTR sequences identified here belong to the ERV1 family of endogenous retroviruses. Each pericentromeric region was characterized by a diverse set of repeats, with the exception of chromosome pair 4, in which we found only one type of satellite. Only a few repeats showed non-centromeric signals in addition to their centromeric localization. Mapping of 18S-28S ribosomal RNA genes and telomeric sequences (TTAGGG)n did not demonstrate any co-localization of these sequences with revealed centromeric and pericentromeric heterochromatic blocks.

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.

The Distribution of Puumala orthohantavirus Genome Variants Correlates with the Regional Landscapes in the Trans-Kama Area of the Republic of Tatarstan.

In the European part of Russia, the highest number of hemorrhagic fever with renal syndrome (HFRS) cases are registered in the Volga Federal District (VFD), which includes the Republic of Tatarstan (RT). Puumala orthohantavirus (PUUV) is the main causative agent of HFRS identified in the RT. The goal of the current study is to analyze the genetic variations of the PUUV strains and possible presence of chimeric and reassortant variants among the PUUV strains circulating in bank vole populations in the Trans-Kama area of the RT. Complete S segment CDS as well as partial M and L segment coding nucleotide sequences were obtained from 40 PUUV-positive bank voles and used for the analysis. We found that all PUUV strains belonged to RUS genetic lineage and clustered in two subclades corresponding to the Western and Eastern Trans-Kama geographic areas. PUUV strains from Western Trans-Kama were related to the previously identified strain from Teteevo in the Pre-Kama area. It can be suggested that the PUUV strains were introduced to the Teteevo area as a result of the bank voles' migration from Western Trans-Kama. It also appears that physical obstacles, including rivers, could be overcome by migrating rodents under favorable circumstances. Based on results of the comparative and phylogenetic analyses, we propose that bank vole distribution in the Trans-Kama area occurred upstream along the river valleys, and that watersheds could act as barriers for migrations. As a result, the diverged PUUV strains could be formed in closely located populations. In times of extensive bank vole population growth, happening every 3-4 years, some regions of watersheds may become open for contact between individual rodents from neighboring populations, leading to an exchange of the genetic material between divergent PUUV strains.

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.

Whole-chromosome fusions in the karyotype evolution of Sceloporus (Iguania, Reptilia) are more frequent in sex chromosomes than autosomes.

Whole-chromosome fusions play a major role in the karyotypic evolution of reptiles. It has been suggested that certain chromosomes tend to fuse with sex chromosomes more frequently than others. However, the comparative genomic synteny data are too scarce to draw strong conclusions. We obtained and sequenced chromosome-specific DNA pools of Sceloporus malachiticus, an iguanian species which has experienced many chromosome fusions. We found that four of seven lineage-specific fusions involved sex chromosomes, and that certain syntenic blocks which constitute the sex chromosomes, such as the homologues of the Anolis carolinensis chromosomes 11 and 16, are repeatedly involved in sex chromosome formation in different squamate species. To test the hypothesis that the karyotypic shift could be associated with changes in recombination patterns, we performed a synaptonemal complex analysis in this species and in Sceloporus variabilis (2n = 34). It revealed that the sex chromosomes in S. malachiticus had two distal pseudoautosomal regions and a medial differentiated region. We found that multiple fusions little affected the recombination rate in S. malachiticus. Our data confirm more frequent involvement of certain chromosomes in sex chromosome formation, but do not reveal a connection between the gonosome-autosome fusions and the evolution of recombination rate. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)'.

Geographical and Tick-Dependent Distribution of Flavi-Like Alongshan and Yanggou Tick Viruses in Russia.

The genus Flavivirus includes related, unclassified segmented flavi-like viruses, two segments of which have homology with flavivirus RNA-dependent RNA polymerase NS5 and RNA helicase-protease NS3. This group includes such viruses as Jingmen tick virus, Alongshan virus, Yanggou tick virus and others. We detected the Yanggou tick virus in Dermacentor nuttalli and Dermacentor marginatus ticks in two neighbouring regions of Russia. The virus prevalence ranged from 0.5% to 8.0%. We detected RNA of the Alongshan virus in 44 individuals or pools of various tick species in eight regions of Russia. The virus prevalence ranged from 0.6% to 7.8%. We demonstrated the successful replication of the Yanggou tick virus and Alongshan virus in IRE/CTVM19 and HAE/CTVM8 tick cell lines without a cytopathic effect. According to the phylogenetic analysis, we divided the Alongshan virus into two groups: an Ixodes persulcatus group and an Ixodes ricinus group. In addition, the I. persulcatus group can be divided into European and Asian subgroups. We found amino acid signatures specific to the I. ricinus and I. persulcatus groups and also distinguished between the European and Asian subgroups of the I. persulcatus group.

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.

Bridging the Gap between Vertebrate Cytogenetics and Genomics with Single-Chromosome Sequencing (ChromSeq).

The study of vertebrate genome evolution is currently facing a revolution, brought about by next generation sequencing technologies that allow researchers to produce nearly complete and error-free genome assemblies. Novel approaches however do not always provide a direct link with information on vertebrate genome evolution gained from cytogenetic approaches. It is useful to preserve and link cytogenetic data with novel genomic discoveries. Sequencing of DNA from single isolated chromosomes (ChromSeq) is an elegant approach to determine the chromosome content and assign genome assemblies to chromosomes, thus bridging the gap between cytogenetics and genomics. The aim of this paper is to describe how ChromSeq can support the study of vertebrate genome evolution and how it can help link cytogenetic and genomic data. We show key examples of ChromSeq application in the refinement of vertebrate genome assemblies and in the study of vertebrate chromosome and karyotype evolution. We also provide a general overview of the approach and a concrete example of genome refinement using this method in the species Anolis carolinensis.

An 8.22 Mb Assembly and Annotation of the Alpaca (Vicugna pacos) Y Chromosome.

The unique evolutionary dynamics and complex structure make the Y chromosome the most diverse and least understood region in the mammalian genome, despite its undisputable role in sex determination, development, and male fertility. Here we present the first contig-level annotated draft assembly for the alpaca (Vicugna pacos) Y chromosome based on hybrid assembly of short- and long-read sequence data of flow-sorted Y. The latter was also used for cDNA selection providing Y-enriched testis transcriptome for annotation. The final assembly of 8.22 Mb comprised 4.5 Mb of male specific Y (MSY) and 3.7 Mb of the pseudoautosomal region. In MSY, we annotated 15 X-degenerate genes and two novel transcripts, but no transposed sequences. Two MSY genes, HSFY and RBMY, are multicopy. The pseudoautosomal boundary is located between SHROOM2 and HSFY. Comparative analysis shows that the small and cytogenetically distinct alpaca Y shares most of MSY sequences with the larger dromedary and Bactrian camel Y chromosomes. Most of alpaca X-degenerate genes are also shared with other mammalian MSYs, though WWC3Y is Y-specific only in alpaca/camels and the horse. The partial alpaca Y assembly is a starting point for further expansion and will have applications in the study of camelid populations and male biology.

Evolution of MicroRNA Biogenesis Genes in the Sterlet (Acipenser ruthenus) and Other Polyploid Vertebrates.

MicroRNAs play a crucial role in eukaryotic gene regulation. For a long time, only little was known about microRNA-based gene regulatory mechanisms in polyploid animal genomes due to difficulties of polyploid genome assembly. However, in recent years, several polyploid genomes of fish, amphibian, and even invertebrate species have been sequenced and assembled. Here we investigated several key microRNA-associated genes in the recently sequenced sterlet (Acipenser ruthenus) genome, whose lineage has undergone a whole genome duplication around 180 MYA. We show that two paralogs of drosha, dgcr8, xpo1, and xpo5 as well as most ago genes have been retained after the acipenserid-specific whole genome duplication, while ago1 and ago3 genes have lost one paralog. While most diploid vertebrates possess only a single copy of dicer1, we strikingly found four paralogs of this gene in the sterlet genome, derived from a tandem segmental duplication that occurred prior to the last whole genome duplication. ago1,3,4 and exportins1,5 look to be prone to additional segment duplications producing up to four-five paralog copies in ray-finned fishes. We demonstrate for the first time exon microsatellite amplification in the acipenserid drosha2 gene, resulting in a highly variable protein product, which may indicate sub- or neofunctionalization. Paralogous copies of most microRNA metabolism genes exhibit different expression profiles in various tissues and remain functional despite the rediploidization process. Subfunctionalization of microRNA processing gene paralogs may be beneficial for different pathways of microRNA metabolism. Genetic variability of microRNA processing genes may represent a substrate for natural selection, and, by increasing genetic plasticity, could facilitate adaptations to changing environments.

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.

Chromosome Distribution of Highly Conserved Tandemly Arranged Repetitive DNAs in the Siberian Sturgeon (Acipenser baerii).

Polyploid genomes present a challenge for cytogenetic and genomic studies, due to the high number of similar size chromosomes and the simultaneous presence of hardly distinguishable paralogous elements. The karyotype of the Siberian sturgeon (Acipenser baerii) contains around 250 chromosomes and is remarkable for the presence of paralogs from two rounds of whole-genome duplications (WGD). In this study, we applied the sterlet-derived acipenserid satDNA-based whole chromosome-specific probes to analyze the Siberian sturgeon karyotype. We demonstrate that the last genome duplication event in the Siberian sturgeon was accompanied by the simultaneous expansion of several repetitive DNA families. Some of the repetitive probes serve as good cytogenetic markers distinguishing paralogous chromosomes and detecting ancestral syntenic regions, which underwent fusions and fissions. The tendency of minisatellite specificity for chromosome size groups previously observed in the sterlet genome is also visible in the Siberian sturgeon. We provide an initial physical chromosome map of the Siberian sturgeon genome supported by molecular markers. The application of these data will facilitate genomic studies in other recent polyploid sturgeon species.