Genome-wide sequence divergence of satellite DNA could underlie meiotic failure in male hybrids of bighead catfish and North African catfish (Clarias, Clariidae).

Hybrid sterility, a hallmark of postzygotic isolation, arises from parental genome divergence disrupting meiosis. While chromosomal incompatibility is often implicated, the underlying mechanisms remain unclear. This study investigated meiotic behavior and genome-wide divergence in bighead catfish (C. macrocephalus), North African catfish (C. gariepinus), and their sterile male hybrids (important in aquaculture). Repetitive DNA analysis using bioinformatics and cytogenetics revealed significant divergence in satellite DNA (satDNA) families between parental species. Notably, one hybrid exhibited successful meiosis and spermatozoa production, suggesting potential variation in sterility expression. Our findings suggest that genome-wide satDNA divergence, rather than chromosome number differences, likely contributes to meiotic failure and male sterility in these catfish hybrids.

Marsupial genome analysis suggests that satellite DNA formation from walb endogenous retrovirus is an event specific to the red-necked wallaby.

We recently identified walbRep, a satellite DNA residing in the genome of the red-necked wallaby Notamacropus rufogriseus. It originates from the walb endogenous retrovirus and is organized in a manner in which the provirus structure is retained. The walbRep repeat units feature an average pairwise nucleotide identity as high as 99.5%, raising the possibility of a recent origin. The tammar wallaby N. eugenii is a species estimated to have diverged from the red-necked wallaby 2-3 million years ago. In PCR analyses of these two and other related species, walbRep-specific fragment amplification was observed only in the red-necked wallaby. Sequence database searches for the tammar wallaby resulted in sequence alignment lists that were sufficiently powerful to exclude the possibility of walbRep existence. These results suggested that the walbRep formation occurred in the red-necked wallaby lineage after its divergence from the tammar wallaby lineage, thus in a time span of maximum 3 million years.

Hi-C sequencing unravels dynamic three-dimensional chromatin interactions in muntjac lineage: insights from chromosome fusions in Fea's muntjac genome.

Eukaryotes have varying numbers and structures of characteristic chromosomes across lineages or species. The evolutionary trajectory of species may have been affected by spontaneous genome rearrangements. Chromosome fusion drastically alters karyotypes. However, the mechanisms and consequences of chromosome fusions, particularly in muntjac species, are poorly understood. Recent research-based advancements in three-dimensional (3D) genomics, particularly high-throughput chromatin conformation capture (Hi-C) sequencing, have allowed for the identification of chromosome fusions and provided mechanistic insights into three muntjac species: Muntiacus muntjak, M. reevesi, and M. crinifrons. This study aimed to uncover potential genome rearrangement patterns in the threatened species Fea's muntjac (Muntiacus feae), which have not been previously examined for such characteristics. Deep Hi-C sequencing (31.42 × coverage) was performed to reveal the 3D chromatin architecture of the Fea's muntjac genome. Patterns of repeated chromosome fusions that were potentially mediated by high-abundance transposable elements were identified. Comparative Hi-C maps demonstrated linkage homology between the sex chromosomes in Fea's muntjac and autosomes in M. reevesi, indicating that fusions may have played a crucial role in the evolution of the sex chromosomes of the lineage. The species-level dynamics of topologically associated domains (TADs) suggest that TAD organization could be altered by differential chromosome interactions owing to repeated chromosome fusions. However, research on the effect of TADs on muntjac genome evolution is insufficient. This study generated Hi-C data for the Fea's muntjac, providing a genomic resource for future investigations of the evolutionary patterns of chromatin conformation at the chromosomal level.

MicrosatNavigator: exploring nonrandom distribution and lineage-specificity of microsatellite repeat motifs on vertebrate sex chromosomes across 186 whole genomes.

Microsatellites are short tandem DNA repeats, ubiquitous in genomes. They are believed to be under selection pressure, considering their high distribution and abundance beyond chance or random accumulation. However, limited analysis of microsatellites in single taxonomic groups makes it challenging to understand their evolutionary significance across taxonomic boundaries. Despite abundant genomic information, microsatellites have been studied in limited contexts and within a few species, warranting an unbiased examination of their genome-wide distribution in distinct versus closely related-clades. Large-scale comparisons have revealed relevant trends, especially in vertebrates. Here, "MicrosatNavigator", a new tool that allows quick and reliable investigation of perfect microsatellites in DNA sequences, was developed. This tool can identify microsatellites across the entire genome sequences. Using this tool, microsatellite repeat motifs were identified in the genome sequences of 186 vertebrates. A significant positive correlation was noted between the abundance, density, length, and GC bias of microsatellites and specific lineages. The (AC)n motif is the most prevalent in vertebrate genomes, showing distinct patterns in closely related species. Longer microsatellites were observed on sex chromosomes in birds and mammals but not on autosomes. Microsatellites on sex chromosomes of non-fish vertebrates have the lowest GC content, whereas high-GC microsatellites (≥ 50 M% GC) are preferred in bony and cartilaginous fishes. Thus, similar selective forces and mutational processes may constrain GC-rich microsatellites to different clades. These findings should facilitate investigations into the roles of microsatellites in sex chromosome differentiation and provide candidate microsatellites for functional analysis across the vertebrate evolutionary spectrum.

Small but Mighty: Genetic Diversity of the Thai Ridgeback Dog Population.

Originating in Thailand, the Thai Ridgeback dog is known for its unique fur ridge that grows in the opposite direction along its back. Selective breeding and a limited populations in Thailand have led to significant close inbreeding among related individuals. The current Thai Ridgeback population is assumed to have experienced a loss of genetic diversity and bottleneck events. Furthermore, studies on the genetic diversity and structure of Thai Ridgeback dogs are limited. Therefore, the aim of this study was to assess the genetic diversity in Thai Ridgeback dogs. Microsatellite genotyping and mitochondrial DNA D-loop sequences were used to assess genetic diversity in 105 Thai Ridgeback dogs from various farms throughout Thailand. Significant genetic diversity and minimal inbreeding were observed in the current Thai Ridgeback population. Signs of bottlenecks were not observed because the exchange of genetic material among Thai Ridgeback owners effectively preserved the genetic diversity. Moreover, the genetic parameters in this study supported owner-to-owner exchanges animals for mating programs. To sustain the genetic diversity of Thai Ridgeback dogs, the use of genetic parameters to manage genetic closeness while preserving breed characteristics is essential. These data are crucial for ensuring demographic stability, which is pivotal for long-term conservation and effective population management.

Replacement of owl monkey centromere satellite by a newly evolved variant was a recent and rapid process.

Alpha satellite DNA is a major DNA component of primate centromeres. We previously reported that Azara's owl monkey has two types of alpha satellite DNA, OwlAlp1 and OwlAlp2. OwlAlp2 (344 bp) exhibits a sequence similarity throughout its entire length with alpha satellite DNA of closely related species. OwlAlp1 (185 bp) corresponds to the part of OwlAlp2. Based on the observation that the CENP-A protein binds to OwlAlp1, we proposed that OwlAlp1 is a relatively new repetitive DNA that replaced OwlAlp2 as the centromeric satellite DNA. However, a detailed picture of the evolutionary process of this centromere DNA replacement remains largely unknown. Here, we performed a phylogenetic analysis of OwlAlp1 and OwlAlp2 sequences, and also compared our results to alpha satellite DNA sequences of other primate species. We found that: (i) OwlAlp1 exhibits a higher similarity to OwlAlp2 than to alpha satellite DNA of other species, (ii) OwlAlp1 has a single origin, and (iii) sequence variation is lower in OwlAlp1 than in OwlAlp2. We conclude that OwlAlp1 underwent a recent and rapid expansion in the owl monkey lineage. This centromere DNA replacement could have been facilitated by the heterochromatin reorganization that is associated with the adaptation of owl monkeys to a nocturnal lifestyle.

Marsupial satellite DNA as faithful reflections of long-terminal repeat retroelement structure.

Long terminal repeat (LTR) retroelements, including endogenous retroviruses, are one of the origins of satellite DNAs. However, the vast majority of satellite DNAs originating from LTR retroelements consists of parts of the element. In addition, they frequently contain sequences unrelated to that element. Here we report a novel marsupial satellite DNA (named walbRep) that contains, and consists solely of, the entire sequence of an LTR retroelement (the walb element). As is common with LTR retroelements, walb copies exhibit length variation. We focused on the abundance of copies of a specific length (2.7 kb) in the genome of the red-necked wallaby. Cloning and analyses of long genomic DNA fragments revealed a satellite DNA in which the LTR sequence (0.4 kb) and the sequence of the internal region of a nonautonomous walb copy (2.3 kb) were repeated alternately. The junctions between these two components exhibited the same end-to-end arrangements as those in the walb element. This satellite organization could be accounted for by a simple formation model that includes slippage during chromosome pairing followed by homologous recombination but does not invoke any other types of rearrangements. We discuss the possible reasons why satellite DNAs having such structures are rarely found in mammals.

An endogenous retrovirus presumed to have been endogenized or relocated recently in a marsupial, the red-necked wallaby.

An albino infant wallaby was born to a mother with wild-type body color. PCR and sequencing analyses of TYR (encoding tyrosinase, which is essential for melanin biosynthesis) of this albino wallaby revealed a 7.1-kb-long DNA fragment inserted in the first exon. Since the fragment carried long terminal repeats, we assumed it to be a copy of an endogenous retrovirus, which we named walb. We cloned other walb copies residing in the genomes of this species and of another wallaby species. The copies exhibited length variation, and the longest copy (>8.0 kb) contained open reading frames whose deduced amino acid sequences were well aligned with those of gag, pol, and env of retroviruses. It is unknown through which of the following likely processes the walb copy was inserted into TYR: endogenization (infection of a germline cell by an exogenous virus), reinfection (infection by a virus produced from a previously endogenized provirus), or retrotransposition (intracellular relocation of a provirus). In any case, the insertion into TYR is considered to have been a recent event on an evolutionary timescale because albino mutant alleles generally do not persist for long because of their deleterious effects in wild circumstances.

The medaka fish Tol2 transposable element is in an early stage of decay: identification of a nonautonomous copy.

The majority of DNA-based transposable elements comprise autonomous and nonautonomous copies, or only nonautonomous copies, where the autonomous copy contains an intact gene for a transposase protein and the nonautonomous copy does not. Even if autonomous copies coexist, they are generally less frequent. The Tol2 element of medaka fish is one of the few elements for which a nonautonomous copy has not yet been found. Here, we report the presence of a nonautonomous Tol2 copy that was identified by surveying the medaka genome sequence database. This copy contained three local sequence alterations that affected the deduced amino acid sequence of the transposase: a deletion of 15 nucleotides resulting in a deletion of 5 amino acids, a base substitution causing a single amino acid change, and another base substitution giving rise to a stop codon. Transposition assays using cultured human cells revealed that transposase activity was reduced by the 15-nucleotide deletion and abolished by the nonsense mutation. This is the first example of a nonautonomous Tol2 copy. Thus, Tol2 is in an early stage of decay in the medaka genome, and is therefore a unique element to observe an almost complete decay process that progresses in natural populations.

Alpha satellite DNA-repeat OwlAlp1 forms centromeres in Azara's owl monkey.

Centromeres play crucial roles in faithful chromosome segregation and genome integrity. In simian primates, centromeres possess tandem array of alpha satellite DNA (also referred to as alphoid DNA). Average sizes of alpha satellite repeat units vary between species, for example, 171 bp in human and 343-344 bp in many platyrrhini species (New World monkeys). Interestingly, Azara's owl monkey (Aotus azarae), a platyrrhini species, possesses alpha satellite DNA of two distinct unit sizes, OwlAlp1 (185 bp) and OwlAlp2 (344 bp), both of which present as megasatellite DNAs in the genome. It is, however, unknown which repeat sequence is responsible for functional centromere formation. To investigate the localization of centromeres in vivo, we carried out chromatin immunoprecipitation (ChIP) assay using Azara's owl monkey cells. We found that CENP-A, a histone H3 variant essential for centromere formation, was enriched at OwlAlp1, but not at OwlAlp2. Moreover, CENP-A was detected only at constricted regions of chromosomes by immunofluorescent microscopy. In contrast, trimethylation of histone H3-K9 (H3K9me3), a marker of heterochromatin, was enriched at both OwlAlp1 and OwlAlp2. Our results show that the shorter alpha satellite repeat, OwlAlp1, is selectively used for centromere formation in this monkey.

Baboon bearing resemblance in pigmentation pattern to Siamese cat carries a missense mutation in the tyrosinase gene.

An infant hamadryas baboon exhibiting an albino phenotype-white body hair and red eyes-was born to parents with wild-type body color. Pigmentation on some parts of its body surfaced during childhood and progressed with age. This baboon in adulthood has gray hair on parts of its body, such as the tail, distal portion of the legs, and face, with the remainder being white. This pigmentation pattern resembles that of the Siamese cat and the Himalayan variants of the mouse and the mink. The distinguishing phenotypes in these animals are known to be caused by a temperature-sensitive activity of tyrosinase, an enzyme essential for biosynthesis of melanin. We sequenced all the five exons of the tyrosinase (TYR) gene of this albino baboon, which were amplified by PCR, and found a base substitution leading to alteration of the 365th amino acid from Ala to Thr. Tyrosinase requires copper as a cofactor for its enzyme function. It has two copper-binding sites, the second of which contains His residues in positions 363 and 367 that are critical to its function. Thus, p.(Ala365Thr) due to a mutation in the TYR gene is a likely candidate for the cause of the albino phenotype in this baboon.

Complex intragene deletion leads to oculocutaneous albinism in tanuki (Japanese raccoon dog).

Tanuki (Nyctereutes procyonoides viverrinus), or Japanese raccoon dog, is a canine native to Japan. Tanuki with complete oculocutaneous albinism are relatively frequent in mountainous areas of mainland Japan. Tyrosinase, which is encoded by the TYR gene, is an enzyme essential for the biosynthesis of melanin pigment. We examined the structure and nucleotide sequence of TYR in an albino tanuki and found that the third exon was removed due to a deletion of approximately 11 kb. In addition, two nonsynonymous nucleotide substitutions were found in the fifth exon. These mutations are possible causes of the albino phenotype; however, the order of occurrence is unclear. Even if the 11-kb deletion was not the first of these mutations, it is considered to cause a total loss of the tyrosinase function because the third exon carries codons for one of the two copper-binding sites of tyrosinase and these sites are essential for the enzyme function. Intriguingly, the deletion was not a simple removal of an 11-kb segment: an internal portion was retained as a segment in the reverse orientation. We propose possible formation processes for this mutation that involve multiple DNA scission events, or an inversion followed by a deletion.

Structural variations of subterminal satellite blocks and their source mechanisms as inferred from the meiotic configurations of chimpanzee chromosome termini.

African great apes have large constitutive heterochromatin (C-band) blocks in subtelomeric regions of the majority of their chromosomes, but humans lack these. Additionally, the chimpanzee meiotic cell division process demonstrates unique partial terminal associations in the first meiotic prophase (pachytene). These are likely formed as a result of interaction among subtelomeric C-band blocks. We thus conducted an extensive study to define the features in the subtelomeric heterochromatic regions of chimpanzee chromosomes undergoing mitotic metaphase and meiotic cell division. Molecular cytogenetic analyses with probes of both subterminal satellite DNA (a main component of C-band) and rDNA demonstrated principles of interaction among DNA arrays. The results suggest that homologous and ectopic recombination through persistent subtelomeric associations (post-bouquet association observed in 32% of spermatocytes in the pachytene stage) appears to create variability in heterochromatin patterns and simultaneously restrain subtelomeric genome polymorphisms. That is, the meeting of non-homologous chromosome termini sets the stage for ectopic pairing which, in turn, is the mechanism for generating variability and genomic dispersion of subtelomeric C-band blocks through a system of concerted evolution. Comparison between the present study and previous reports indicated that the chromosomal distribution rate of sutelomeric regions seems to have antagonistic correlation with arm numbers holding subterminal satellite blocks in humans, chimpanzees, and gorillas. That is, the increase of subterminal satellite blocks probably reduces genomic diversity in the subtelomeric regions. The acquisition vs. loss of the subtelomeric C-band blocks is postulated as the underlying engine of this chromosomal differentiation yielded by meiotic chromosomal interaction.

Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry.

The cerebellum is involved in some forms of motor coordination and motor learning. Here we isolated transgenic (Tg) zebrafish lines that express a modified version of Gal4-VP16 (GFF) in the cerebellar neural circuits: granule, Purkinje, or eurydendroid cells, Bergmann glia, or the neurons in the inferior olive nuclei (IO) which send climbing fibers to Purkinje cells, with the transposon Tol2 system. By combining GFF lines with Tg lines carrying a reporter gene located downstream of Gal4 binding sequences (upstream activating sequence: UAS), we investigated the anatomy and developmental processes of the cerebellar neural circuitry. Combining an IO-specific Gal4 line with a UAS reporter line expressing the photoconvertible fluorescent protein Kaede demonstrated the contralateral projections of climbing fibers. Combining a granule cell-specific Gal4 line with a UAS reporter line expressing wheat germ agglutinin (WGA) confirmed direct and/or indirect connections of granule cells with Purkinje cells, eurydendroid cells, and IO neurons in zebrafish. Time-lapse analysis of a granule cell-specific Gal4 line revealed initial random movements and ventral migration of granule cell nuclei. Transgenesis of a reporter gene with another transposon Tol1 system visualized neuronal structure at a single cell resolution. Our findings indicate the usefulness of these zebrafish Gal4 Tg lines for studying the development and function of cerebellar neural circuits.

CENP-B box, a nucleotide motif involved in centromere formation, occurs in a New World monkey.

Centromere protein B (CENP-B) is one of the major proteins involved in centromere formation, binding to centromeric repetitive DNA by recognizing a 17 bp motif called the CENP-B box. Hominids (humans and great apes) carry large numbers of CENP-B boxes in alpha satellite DNA (AS, the major centromeric repetitive DNA of simian primates). Only negative results have been reported regarding the presence of the CENP-B box in other primate taxa. Consequently, it is widely believed that the CENP-B box is confined, within primates, to the hominids. We report here that the common marmoset, a New World monkey, contains an abundance of CENP-B boxes in its AS. First, in a long contig sequence we constructed and analysed, we identified the motif in 17 of the 38 alpha satellite repeat units. We then sequenced terminal regions of additional clones and found the motif in many of them. Immunostaining of marmoset cells demonstrated that CENP-B binds to DNA in the centromeric regions of chromosomes. Therefore, functional CENP-B boxes are not confined to hominids. Our results indicate that the efficiency of identification of the CENP-B box may depend largely on the sequencing methods used, and that the CENP-B box in centromeric repetitive DNA may be more common than researchers previously thought.

Reduction in the structural instability of cloned eukaryotic tandem-repeat DNA by low-temperature culturing of host bacteria.

Summary For accurate analyses of eukaryotic tandem-repeat DNA, it is often required to clone a genomic DNA fragment into a bacterial plasmid. It is, however, a serious problem that tandem-repeat DNA is frequently subjected to structural changes during maintenance or amplification in the host bacteria. Here, we show an example of a clear difference in the instability of tandem-repeat DNA between different culturing temperatures. A fragment of monkey centromeric DNA carried by pUC19 was considerably degraded by culturing bacteria at 37 °C, but the damage was reduced at 25 °C. Thus, culturing temperature is a significant factor for avoiding degradation, in addition to the genotype of the host bacteria.

Spontaneous germline excision of Tol1, a DNA-based transposable element naturally occurring in the medaka fish genome.

DNA-based transposable elements are ubiquitous constituents of eukaryotic genomes. Vertebrates are, however, exceptional in that most of their DNA-based elements appear to be inactivated. The Tol1 element of the medaka fish, Oryzias latipes, is one of the few elements for which copies containing an undamaged gene have been found. Spontaneous transposition of this element in somatic cells has previously been demonstrated, but there is only indirect evidence for its germline transposition. Here, we show direct evidence of spontaneous excision in the germline. Tyrosinase is the key enzyme in melanin biosynthesis. In an albino laboratory strain of medaka fish, which is homozygous for a mutant tyrosinase gene in which a Tol1 copy is inserted, we identified de novo reversion mutations related to melanin pigmentation. The gamete-based reversion rate was as high as 0.4%. The revertant fish carried the tyrosinase gene from which the Tol1 copy had been excised. We previously reported the germline transposition of Tol2, another DNA-based element that is thought to be a recent invader of the medaka fish genome. Tol1 is an ancient resident of the genome. Our results indicate that even an old element can contribute to genetic variation in the host genome as a natural mutator.

Novel variable number of tandem repeats of gibbon MAOA gene and its evolutionary significance.

Variable number of tandem repeats (VNTRs) are scattered throughout the primate genome, and genetic variation of these VNTRs have been accumulated during primate radiation. Here, we analyzed VNTRs upstream of the monoamine oxidase A (MAOA) gene in 11 different gibbon species. An abundance of truncated VNTR sequences and copy number differences were observed compared to those of human VNTR sequences. To better understand the biological role of these VNTRs, a luciferase activity assay was conducted and results indicated that selected VNTR sequences of the MAOA gene from human and three different gibbon species (Hylobates klossii, Hylobates lar, and Nomascus concolor) showed silencing ability. Together, these data could be useful for understanding the evolutionary history and functional significance of MAOA VNTR sequences in gibbon species.

Genetic insights: mapping sex-specific loci in Siamese cobra (Naja kaouthia) sheds light on the putative sex determining region.

The location of female-specific/linked loci identified in Siamese cobra (Naja kaouthia) previously has been determined through in silico chromosome mapping of the Indian cobra genome (N. naja) as a reference genome. In the present study, we used in silico chromosome mapping to identify sex-specific and linked loci in Siamese cobra. Many sex-specific and sex-linked loci were successfully mapped on the Z sex chromosome, with 227 of the 475 specific loci frequently mapped in a region covering 57 Mb and positioned at 38,992,675-95,561,177 bp of the Indian cobra genome (N. naja). This suggested the existence of a putative sex-determining region (SDR), with one specific locus (PA100000600) homologous to the TOPBP1 gene. The involvement of TOPBP1 gene may lead to abnormal synaptonemal complexes and meiotic chromosomal defects, resulting in male infertility. These findings offer valuable insights into the genetic basis and functional aspects of sex-specific traits in the Siamese cobra, which will contribute to our understanding of snake genetics and evolutionary biology.

Disclosing the hidden nucleotide sequences: a journey into DNA barcoding of raptor species in public repositories.

BACKGROUND: In nucleotide public repositories, studies discovered data errors which resulted in incorrect species identification of several accipitrid raptors considered for conservation. Mislabeling, particularly in cases of cryptic species complexes and closely related species, which were identified based on morphological characteristics, was discovered. Prioritizing accurate species labeling, morphological taxonomy, and voucher documentation is crucial to rectify spurious data. OBJECTIVE: Our study aimed to identify an effective DNA barcoding tool that accurately reflects the efficiency status of barcodes in raptor species (Accipitridae). METHODS: Barcode sequences, including 889 sequences from the mitochondrial cytochrome c oxidase I (COI) gene and 1052 sequences from cytochrome b (Cytb), from 150 raptor species within the Accipitridae family were analyzed. RESULTS: The highest percentage of intraspecific nearest neighbors from the nearest neighbor test was 88.05% for COI and 95.00% for Cytb, suggesting that the Cytb gene is a more suitable marker for accurately identifying raptor species and can serve as a standard region for DNA barcoding. In both datasets, a positive barcoding gap representing the difference between inter-and intra-specific sequence divergences was observed. For COI and Cytb, the cut-off score sequence divergences for species identification were 4.00% and 3.00%, respectively. CONCLUSION: Greater accuracy was demonstrated for the Cytb gene, making it the preferred primary DNA barcoding marker for raptors.