Limited evidence for extensive genetic differentiation between X and Y chromosomes in Hybognathus amarus (Cypriniformes: Leuciscidae).

Sex determination systems and genetic sex differentiation across fishes are highly diverse but are unknown for most Cypriniformes, including Rio Grande silvery minnow (Hybognathus amarus). In this study, we aimed to detect and validate sex-linked markers to infer sex determination system and to demonstrate the utility of combining several methods for sex-linked marker detection in nonmodel organisms. To identify potential sex-linked markers, Nextera-tagmented reductively amplified DNA (nextRAD) libraries were generated from 66 females, 64 males, and 60 larvae of unknown sex. These data were combined with female and male de novo genomes from Nanopore long-read sequences. We identified five potential unique male nextRAD-tags and one potential unique male contig, suggesting an XY sex determination system. We also identified two single-nucleotide polymorphisms (SNPs) in the same contig with values of FST, allele frequencies, and heterozygosity conforming with expectations of an XY system. Through PCR we validated the marker containing the sex-linked SNPs and a single nextRAD-tag sex-associated marker but it was not male specific. Instead, more copies of this locus in the male genome were suggested by enhanced amplification in males. Results are consistent with an XY system with low differentiation between sex-determining regions. Further research is needed to confirm the level of differentiation between the sex chromosomes. Nonetheless, this study highlighted the power of combining reduced representation and whole-genome sequencing for identifying sex-linked markers, especially when reduced representation sequencing does not include extensive variation between sexes, either because such variation is not present or not captured.

Evolutionary origin and establishment of a dioecious diploid-tetraploid complex.

Polyploids recurrently emerge in angiosperms, but most polyploids are likely to go extinct before establishment due to minority cytotype exclusion, which may be specifically a constraint for dioecious plants. Here we test the hypothesis that a stable sex-determination system and spatial/ecological isolation facilitate the establishment of dioecious polyploids. We determined the ploidy levels of 351 individuals from 28 populations of the dioecious species Salix polyclona, and resequenced 190 individuals of S. polyclona and related taxa for genomic diversity analyses. The ploidy survey revealed a frequency 52% of tetraploids in S. polyclona, and genomic k-mer spectra analyses suggested an autopolyploid origin for them. Comparisons of diploid male and female genomes identified a female heterogametic sex-determining factor on chromosome 15, which probably also acts in the dioecious tetraploids. Phylogenetic analyses revealed two diploid clades and a separate clade/grade of tetraploids with a distinct geographic distribution confined to western and central China, where complex mountain systems create higher levels of environmental heterogeneity. Fossil-calibrated phylogenies showed that the polyploids emerged during 7.6-2.3 million years ago, and population demographic histories largely matched the geological and climatic history of the region. Our results suggest that inheritance of the sex-determining system from the diploid progenitor as intrinsic factor and spatial isolation as extrinsic factor may have facilitated the preservation and establishment of polyploid dioecious populations.

Exploration of an XX/XY Sex Determination System and Development of PCR-Based Sex-specific Markers in Procambarus clarkii Based on Next-Generation Sequencing Data.

Many economic crustacean species have sex dimorphisms during their growth. Exploring the sex determination system and developing sex-specific molecular marker(s) are very helpful for carrying out sex control breeding, and next-generation sequencing has been used as an efficient way to explore them in recent years. In this study, first, the genetic sex determination system of P. clarkii was explored as an XX/XY system by analyzing the 2b-RAD sequencing data. Furthermore, DNA samples of male and female individuals from a P. clarkii family were pooled separately for whole-genome resequencing. Based on the data of whole-genome resequencing, the 9,163 male- and female-specific bias sites with higher feasibility were obtained based on the assumption of the XX/XY sex determination system, and four sites were selected to design the sex-specific marker primers. One efficient sex-specific marker was identified with a sex discrimination rate of 99.49% (195/196) when applied to five different geographical groups with 196 individuals. The results of this study would provide a foundation for the realization of P. clarkii sex control and could provide some reference for investigating the sex determination system and sex molecular marker(s) of other crustacean species based on next-generation sequencing data.

Sex-Specific Genomic Region Identification and Molecular Sex Marker Development of Rock Bream (Oplegnathus fasciatus).

Rock bream (Oplegnathus fasciatus) is a valuable commercial marine teleost species, which exhibits sexual dimorphism in growth performance. However, the absence of a rapid and cost-effective sex identification method based on sex-specific genetic marker has impeded study on sex determination mechanisms and breeding applications. In the present study, we firstly developed the PCR method for identifying potential sex-specific sequences in Oplegnathus fasciatus with the next-generation sequencing. Sex-specific genomic regions/loci for sex determination were discovered on Chr2 and Chr6 by genome-wide association analysis, sequencing depth, and heterozygosity comparison between females and males. Candidate sex-determining genes (CCDC63, ITR, WNT4) were furtherly detected in transcriptome data of testes and ovaries. Taken together, a male-specific 34-bp deletion on the Chr2 was identified and developed into molecular marker of sex for O. fasciatus. After validation in individuals with known phenotypic sexes, the accuracy was 100%. This study gives an insight into the mechanism of sex determination in O. fasciatus, and the gender marker is crucial both for future genomic research and for development of efficient and sustainable aquaculture practice.

Genome-Wide SNP Analysis of Hybrid Clariid Fish Reflects the Existence of Polygenic Sex-Determination in the Lineage.

The African catfish (Clarias gariepinus) may exhibit the co-existence of XX/XY and ZZ/ZW sex-determination systems (SDSs). However, the SDS of African catfish might be influenced by a polygenic sex-determination (PSD) system, comprising multiple independently segregating sex "switch" loci to determine sex within a species. Here, we aimed to detect the existence of PSD using hybrid. The hybrid produced by crossing male African catfish with female bighead catfish (C. macrocephalus, XX/XY) is a good animal model to study SDSs. Determining the SDS of hybrid catfish can help in understanding the interactions between these two complex SDS systems. Using the genotyping-by-sequencing "DART-seq" approach, we detected seven moderately male-linked loci and seventeen female-linked loci across all the examined hybrid specimens. Most of these loci were not sex-linked in the parental species, suggesting that the hybrid exhibits a combination of different alleles. Annotation of the identified sex-linked loci revealed the presence of one female-linked locus homologous with the B4GALNT1 gene, which is involved in the spermatogenesis pathway and hatchability. However, this locus was not sex-linked in the parental species, and the African catfish might also exhibit PSD.

High karyotypic variation in Orthemis Hagen, 1861 species, with insights about the neo-XY in Orthemisambinigra Calvert, 1909 (Libellulidae, Odonata).

The American dragonfly genus Orthemis Hagen, 1861 is mainly found in the Neotropical region. Seven of 28 taxonomically described species have been reported from Argentina. Chromosome studies performed on this genus showed a wide variation in chromosome number and a high frequency of the neoXY chromosomal sex-determination system, although the sexual pair was not observed in all cases. This work analyzes the spermatogenesis of Orthemisdiscolor (Burmeister, 1839), O.nodiplaga Karsch, 1891 and O.ambinigra Calvert, 1909 in individuals from the provinces of Misiones and Buenos Aires, Argentina. Orthemisdiscolor has 2n=23, n=11+X and one larger bivalent. Orthemisnodiplaga exhibits the largest chromosome number of the order, 2n=41, n=20+X and small chromosomes. Orthemisambinigra shows a reduced complement, 2n=12, n=5+neo-XY, large-sized chromosomes, and a homomorphic sex bivalent. Fusions and fragmentations are the main evolutionary mechanisms in Odonata, as well as in other organisms with holokinetic chromosomes. Orthemisnodiplaga would have originated by nine autosomal fragmentations from the ancestral karyotype of the genus (2n=22A+X in males). We argue that the diploid number 23 in Orthemis has a secondary origin from the ancestral karyotype of family Libellulidae (2n=25). The complement of O.ambinigra would have arisen from five autosomal fusions and the insertion of the X chromosome into a fused autosome. C-banding and DAPI/CMA3 staining allowed the identification of the sexual bivalent, which revealed the presence of constitutive heterochromatin. We propose that the chromosome with intermediate C-staining intensity and three medial heterochromatic regions corresponds to the neo-Y and that the neo-system of this species has an ancient evolutionary origin. Moreover, we discuss on the mechanisms involved in the karyotypic evolution of this genus, the characteristics of the neo sex-determining systems and the patterns of heterochromatin distribution, quantity and base pair richness.

A new linkage group involved in sex determination in haplotilapiine cichlids.

The high diversity of sex chromosomes and sex determination systems among haplotilapiines suggests that this large cichlid clade is a good model for investigating the evolution of genetics of sex determination. Nonetheless, information about sex determination in this clade remains sparse. The present study reports a microsatellite marker that is closely associated with sex in Xenotilapia rotundiventralis from Lake Tanganyika, Africa. This study is the first to suggest the role of linkage group 17 in sex determination in haplotilapiine cichlids.

Female-specific genomic regions and molecular sex identification of the clearhead icefish (Protosalanx hyalocranius).

BACKGROUND: The clearhead icefish, Protosalanx hyalocranius, is an economically important fishery species in China. Since 1980s, P. hyalocranius was widely introduced into lakes and reservoirs of northern China for aquaculture. However, the lack of a rapid and cost-effective sex identification method based on sex specific genetic markers has hindered study on sex determination mechanisms and breeding applications. RESULTS: Female-specific genomic regions were discovered by comparing whole genome re-sequencing data of both males and females. Two female-specific genomic regions larger than 50 bp were identified, and one (598 bp) contained a putative FOXI gene, which was paralogous to another FOXI gene with sex-associated SNPs. The two FOXI sequences displayed significant length difference with nine deletions of total length of 230 bp. This deletion-type structural variation could be easily and efficiently detected by traditional PCR and agarose gel electrophoresis with one 569 bp band for males and two bands (569 and 339 bp) for females, which were validated in 50 females and 40 males with known phenotypic sexes. CONCLUSIONS: The results provided structural genomic evidence for the ZZ/ZW sex determination system in P. hyalocranius discovered in our previous study with association analysis of SNPs. Moreover, the female-specific markers and rapid and cost-effective PCR-based genetic sex identification method should have applications in further studies of sex determination mechanism for this species.

No evidence for genetic sex determination in Daphnia magna.

Mechanisms of sex determination (SD) differ widely across the tree of life. In genotypic sex determination (GSD), genetic elements determine whether individuals are male or female, while in environmental sex determination (ESD), external cues control the sex of the offspring. In cyclical parthenogens, females produce mostly asexual daughters, but environmental stimuli such as crowding, temperature or photoperiod may cause them to produce sons. In aphids, sons are induced by ESD, even though GSD is present, with females carrying two X chromosomes and males only one (X0 SD system). By contrast, although ESD exists in Daphnia, the two sexes were suggested to be genetically identical, based on a 1972 study on Daphnia magna (2n=20) that used three allozyme markers. This study cannot, however, rule out an X0 system, as all three markers may be located on autosomes. Motivated by the life cycle similarities of Daphnia and aphids, and the absence of karyotype information for Daphnia males, we tested for GSD (homomorphic sex chromosomes and X0) systems in D. magna using a whole-genome approach by comparing males and females of three genotypes. Our results confirm the absence of haploid chromosomes or haploid genomic regions in D. magna males as well as the absence of sex-linked genomic regions and sex-specific single-nucleotide polymorphisms. Within the limitations of the three studied populations here and the methods used, we suggest that our results make the possibility of genetic differences among sexes in the widely used Daphnia model system very unlikely.

Identification of genomic differences between the sexes and sex-specific molecular markers in Monostroma angicava (Ulvophyceae).

There is little information available regarding genomic differences between sexes in ulvophycean green algae. The detection of these differences will enable the development of sex-discriminating molecular markers, which are useful for algae showing little apparent difference between sexes. In this study, we identified male- and female-specific DNA sequences in the ulvophycean marine green alga Monostroma angicava, which has a dioicous heteromorphic haplo-diplontic life cycle, via next-generation sequencing. Fluorescence in situ hybridization (FISH) showed that signals for the sex-specific sequences exist only in the nuclei of the corresponding sex, confirming the specificity of the sequences. Sex-specific molecular markers that targeted these sequences successfully distinguished the sex of gametophytes even in geographically distant populations, indicating that the sex-specific sequences are universal. These results consistently suggest that male and female gametophytes of M. angicava are genetically different, implying that sex may be determined genetically in this alga.

Regain of sex determination system and sexual reproduction ability in a synthetic octoploid male fish.

Polyploids in vertebrates are generally associated with unisexual reproduction, but the direct consequences of polyploidy on sex determination system and reproduction mode remain unknown. Here, we synthesized a group of artificial octoploids between unisexual gynogenetic hexaploid Carassius gibelio and sexual tetraploid Carassius auratus. The synthetic octoploids were revealed to have more than 200 chromosomes, in which 50 chromosomes including the X/Y sex determination system were identified to transfer from sexual tetraploid C. auratus into the unisexual gynogenetic hexaploid C. gibelio. Significantly, a few synthetic octoploid males were found to be fertile, and one octoploid male was confirmed to regain sexual reproduction ability, which exhibits characteristics that are the same to sexual reproduction tetraploid males, such as 1:1 sex ratio occurrence, meiosis completion and euploid sperm formation in spermatogenesis, as well as normal embryo development and gene expression pattern during embryogenesis. Therefore, the current finding provides a unique case to explore the effect of sex determination system incorporation on reproduction mode transition from unisexual gynogenesis to sexual reproduction along with genome synthesis of recurrent polyploidy in vertebrates.

Methods for identifying and interpreting sex-linked SNP markers and carrying out sex assignment: application to thornback ray (Raja clavata).

Sex-determining modes remain unknown in numerous species, notably in fishes, in which a variety of modalities have been reported. Additionally, noninvasive individual sexing is problematic for species without external sex attributes or for early life stages, requiring cytogenetic or molecular analyses when sex chromosomes or sex-linked markers have been characterized. Genomics now provide a means to achieve this. Here, we review common sex-determination systems and corresponding statistical methods for identifying sex-linked genetic markers and their use for sex assignment, focusing on single nucleotide polymorphism (SNP) markers derived from reduced representation sequencing methods. We demonstrate the dependence of expected sex assignment error on the number of sex-linked SNPs and minor allele frequency. The application of three methods was made here: (a) identification of heterozygote excess in one sex, (b) FST outlier analysis between the two sexes and (c) neuronal net modelling. These methods were applied to a large SNP data set (4604 SNPs) for 1680 thornback rays (Raja clavata). Using method (a), nineteen putative sex-linked SNPs were identified. Comparison with the reference genome of a related species (Amblyraja radiata) indicated that all 19 SNPs are probably located on the same chromosome. These results suggest that thornback ray has a XX/XY sex-determination system. Method (b) identified eight SNPs probably located on different chromosomes. Method (a) led to the lowest sex assignment error among the three methods (4.2% error for females and 3.7% for males).

Dataset on the production of predominantly male tilapia progeny using two malawian tilapias, Oreochromis karongae and Oreochromis shiranus.

A dataset is presented of an experiment that was conducted to compare the proportions of males obtained from hormonal sex reversed pure strain of Oreochromis shiranus and from interspecific hybridization of O.karongae male x O. shiranus female. Part of the data in the dataset were published in a journal article https://doi.org/10.1016/j.aqrep.2020.100274. The data were generated from four SETs of treatments of an experiment that was conducted at the National Aquaculture Center, in Malawi. The first SET of treatment comprised hybrids from interspecific crossing of O.karongae male and O. shiranus female in a pond based breeding hapa 1. SETs 2 and 4 were for fry from hapa spawned pure cross of O.shiranus males and females in a pond based breeding hapa 2. SET 3 comprised fry from pure cross of O. shiranus males and females under controlled temperature (27°C) in an indoor re-circulatory hatchery (Tables 1 to 4). During the first part of the experiment, the fry was raised for 28 days in tanks at three replicates and was fed a fry formulated feed containing 38% crude protein, three times a day. However, for SETs 2 and 3, the feed contained 17α-methyl testosterone at 60mg/kg of feed. The second part of the experiment involved growth performance testing of fry in each SET. The growth experiment was conducted in rearing hapas that were inserted in a common pond for a period of 70 days. The body weight data were collected every 14 days (Tables 1 to 4). On the 70th day, the proportions of males and females in the four SETs was determined using Aceto-carmine staining method (Table 5). The pictures of the stained male and female gonads under compound microscope are presented in the published journal article at https://doi.org/10.1016/j.aqrep.2020.100274. Dataset presented in this paper is for body weights of fish from day 0 to day 70 and proportion of males and females in each SET on the 70th day of the experiment (Tables 1, 2, 3, 4 and 5). The data were analyzed using SPSS version 20.0. Chi-square goodness of fit test was used to investigate if the observed sex ratios significantly deviated from the expected sex ratios at 5% level of significance. The differences among fish body weights were determined using Analysis of Variance and significantly different means were separated using Duncan's multiple range test at the 5% test level of significance. These data are useful for various stakeholders that are interested in sexing juvenile tilapia and for scientists that conduct tilapia sex reversal experiments. This data can also guide hatchery managers during commercial production of all male tilapias which grow faster than those in the mixed sex tilapia culture.

Identification of male-specific SNP markers and development of PCR-based genetic sex identification technique in crucifix crab (Charybdis feriatus) with implication of an XX/XY sex determination system.

In this study, we first identified male-specific SNP markers using restriction site-associated DNA sequencing, and further developed a PCR-based sex identification technique for Charybdis feriatus. A total of 296.96 million clean reads were obtained, with 114.95 and 182.01 million from females and males. After assembly and alignment, 10 SNP markers were identified being heterozygous in males but homozygous in females. Five markers were further confirmed to be male-specific in a large number of individuals. Moreover, two male-specific sense primers and a common antisense primer were designed, using which, a PCR-based genetic sex identification method was successfully developed and used to identify the sex of 103 individuals, with a result of 49 females and 54 males. The presence of male-specific SNP markers suggests an XX/XY sex determination system for C. feriatus. These findings should be helpful for better understanding sex determination mechanism, and drafting artificial breeding program in crustaceans.

High-Density Genetic Linkage Maps Provide Novel Insights Into ZW/ZZ Sex Determination System and Growth Performance in Mud Crab (Scylla paramamosain).

Mud crab, Scylla paramamosain is one of the most important crustacean species in global aquaculture. To determine the genetic basis of sex and growth-related traits in S. paramamosain, a high-density genetic linkage map with 16,701 single nucleotide polymorphisms (SNPs) was constructed using SLAF-seq and a full-sib family. The consensus map has 49 linkage groups, spanning 5,996.66 cM with an average marker-interval of 0.81 cM. A total of 516 SNP markers, including 8 female-specific SNPs segregated in two quantitative trait loci (QTLs) for phenotypic sex were located on LG32. The presence of female-specific SNP markers only on female linkage map, their segregation patterns and lower female: male recombination rate strongly suggest the conformation of a ZW/ZZ sex determination system in S. paramamosain. The QTLs of most (90%) growth-related traits were found within a small interval (25.18-33.74 cM) on LG46, highlighting the potential involvement of LG46 in growth. Four markers on LG46 were significantly associated with 10-16 growth-related traits. BW was only associated with marker 3846. Based on the annotation of transcriptome data, 11 and 2 candidate genes were identified within the QTL regions of sex and growth-related traits, respectively. The newly constructed high-density genetic linkage map with sex-specific SNPs, and the identified QTLs of sex- and growth-related traits serve as a valuable genetic resource and solid foundation for marker-assisted selection and genetic improvement of crustaceans.

Female-specific SNP markers provide insights into a WZ/ZZ sex determination system for mud crabs Scylla paramamosain, S. tranquebarica and S. serrata with a rapid method for genetic sex identification.

BACKGROUND: Mud crabs, Scylla spp., are commercially important large-size marine crustaceans in the Indo-West Pacific region. As females have the higher growth rate and economic value, the production of all female stocks is extremely essential in aquaculture. However, the sex determination mechanism is still unclear. Development of sex-specific genetic markers based on next-generation sequencing proved to be an effective tool for discovering sex determination system in various animals. RESULTS: Restriction-site associated DNA sequencing (RAD-seq) was employed to isolate sex-specific SNP markers for S. paramamosain. A total of 335.6 million raw reads were obtained from 20 individuals, of which 204.7 million were from 10 females and 130.9 million from 10 males. After sequence assembly and female-male comparison, 20 SNP markers were identified to be sex-specific. Furthermore, ten SNPs in a short sequence (285 bp) were confirmed heterozygous in females and homozygous in males in a large population by PCR amplification and sequencing. Subsequently, a female-specific primer was successfully designed according to the female-specific nucleotide which could amplify an expected band from females but not from males. Thus, a rapid and effective method for molecular sexing in S. paramamosain was developed, meanwhile, this method could successfully identify the sex of S. tranquebarica and S. serrata. Finally, nine and four female-specific SNP markers were detected in S. tranquebarica and S. serrata, respectively. CONCLUSIONS: Sex-specific SNP markers were firstly identified in crab species and showed female heterogamety and male homogamety, which provided strong genetic evidence for a WZ/ZZ sex determination system in mud crabs S. paramamosain, S. tranquebarica and S. serrata. These findings will lay a solid foundation for the study of sex determination mechanism, sex chromosome evolution, and the development of mono-sex population in crustaceans.

QTL Mapping and Marker Identification for Sex-Determining: Indicating XY Sex Determination System in the Swimming Crab (Portunus trituberculatus).

Sex determination is an important area of research, which has always had an intriguing aspect in evolutionary and developmental biology. Quantitative trait locus (QTL) mapping for sex will be helpful in clarifying the sex determination system. In this study, the sex QTL mapping of the swimming crab (Portunus trituberculatus) was performed based on a high-density linkage map, and a highly significant QTL specifically mapped on a single linkage group (LG) was firstly identified (LG24, LOD > 14). Twenty markers in the QTL region showed significant associations with sex by association analysis, of which heterogametic genotypes in males supported the XY sex determination mechanism. Two sex-specific markers at the family level were identified via segregation distortion analysis, which were known to be the most closely linked to the sex of P. trituberculatus. Based on sex marker sequences (Marker3840, Marker20320, and Marker10494), three potential sex-related genes were identified, and the quantitative real-time PCR results suggested that these genes were important in spermatogenesis or sex characteristics in males. Our results will contribute to the fine-mapping of sex-determining genes and clarify the sex determination mechanism of P. trituberculatus.

Identification of Sex-associated SNPs of Greater Amberjack (Seriola dumerili).

The sex determination systems of fish are highly diverse compared with those of mammals. Thus, performing investigations using nonmodel fish species helps to understand the highly diverse sex determination systems of fish. Because greater amberjack (Seriola dumerili) is one of the most important edible fish globally and knowledge of its sex determination system is economically important in the field of aquaculture, we are interested in the mechanisms of sex determination of Seriola species. In this study, we identified sex-associated SNPs of greater amberjack using SNP information of 10 males and 10 females by an association test. We determined that the sex-associated SNPs were on chromosome 12 and mainly covered with two scaffolds (about 7.1 Mbp). Genotypes of sex-associated SNPs indicated that females are the heterogametic sex (ZZ/ZW). Furthermore, we compared the genomic structure of greater amberjack with those of Japanese amberjack (Seriola quinqueradiata), California yellowtail (Seriola dorsalis), and medaka (Oryzias latipes). Whole-genome alignments and synteny analysis indicated that the sex determination system of greater amberjack is markedly different from that of medaka and implied that the sex determination system is conserved in the Seriola species.

Sex-specific markers developed by next-generation sequencing confirmed an XX/XY sex determination system in bighead carp (Hypophthalmichehys nobilis) and silver carp (Hypophthalmichthys molitrix).

Sex-specific markers are powerful tools for identifying sex-determination system in various animals. Bighead carp (Hypophthalmichehys nobilis) and silver carp (Hypophthalmichthys molitrix) are two of the most important edible fish in Asia, which have a long juvenility period that can lasts for 4-5 years. In this study, we found one sex-specific marker by next-generation sequencing together with bioinformatics analysis in bighead carp. The male-specific markers were used to perform molecular sexing in the progenies of artificial gynogenetic diploids and found all progenies (n = 160) were females. Meanwhile, around 1 : 1 sex ratio was observed in a total of 579 juvenile offspring from three other families. To further extend the male-specific region, we performed genome walking and got a male-specific sequence of 8,661 bp. Five pairs of primers were designed and could be used to efficiently distinguish males from females in bighead carp and silver carp. The development of these male-specific markers and results of their molecular sexing in different populations provide strong evidence for a sex determination system of female homogametry or male heterogametry (XX/XY) in bighead carp and silver carp. To the best of our knowledge, this is the first report of effective sex-specific markers in these two large carp species.

Genomic Organization of Repetitive DNAs and Differentiation of an XX/XY Sex Chromosome System in the Amazonian Puffer Fish, Colomesus asellus (Tetraodontiformes).

The genus Colomesus is the sole representative of the family Tetraodontidae in the Amazon region. Here, Colomesus asellus was analyzed using conventional and molecular cytogenetic protocols. Its diploid chromosome number is 2n = 46 with 12 meta-, 10 submeta-, 16 subtelo-, and 8 acrocentric chromosomes and a fundamental number of FN = 84. An XX/XY sex chromosome system was identified. Mapping of 18S rDNA correlated with the nucleolus organizer regions (Ag-NORs) in the short arms of the 2 X chromosomes in females and in the Y chromosome in males. C-banding revealed heterochromatin in the centromeric regions of all chromosomes, except for pair 3. Prominent sex chromosome-specific heterochromatin amplification was observed, covering the short arms of the Y chromosome almost entirely. FISH with telomeric and tropomyosin (tpm1) sequences, respectively, revealed terminal signals in all chromosomes. The analysis of extended DNA fibers confirmed the colocalization and the interspersed pattern of the telomeric and tpm1 sequences. Thus, this study highlights the remarkable evolutionary dynamism presented by the Amazonian puffer fish regarding the differentiation of a heteromorphic XY sex chromosome system and a particular sex-specific amplification of rDNA sites. This is the first record of such an association in the Tetraodontidae family.