Comparative Cytogenetics Analysis Among Peckoltia Species (Siluriformes, Loricariidae): Insights on Karyotype Evolution and Biogeography in the Amazon Region

Peckoltia is widely distributed genus in the Amazon and Orinoco basins and the Guiana Shield, containing 18 valid species, and distinct morphotypes still needing description in the scientific literature due to its great taxonomic complexity. This study performed a comparative chromosomal analysis of two undescribed Peckoltia species (Peckoltia sp. 3 Jarumã and Peckoltia sp. 4 Caripetuba) from the Brazilian Amazon using conventional chromosome bands methods and in situ localization of the repetitive DNA (5S and 18S rRNA and U1 snRNA genes and telomeric sequences). Both species presented 2n = 52 but differed in their karyotype formula, probably due to inversions or translocations. The nucleolus organizer regions (NORs) showed distal location on a probably homeologous submetacentric pair in both species, besides an extra signal in a subtelocentric chromosome in Peckoltia sp. 4 Caripetuba. Heterochromatin occurred in large blocks, with different distributions in the species. The mapping of the 18S and 5S rDNA, and U1 snDNA showed differences in locations and number of sites. No interstitial telomeric sites were detected using the (TTAGGG)n probes. Despite 2n conservationism in Peckoltia species, the results showed variation in karyotype formulas, chromosomal bands, and locations of repetitive sites, demonstrating great chromosomal diversity. A proposal for Peckoltia karyotype evolution was inferred in this study based on the diversity of location and number of chromosomal markers analyzed. A comparative analysis with other Peckoltia karyotypes described in the literature, their biogeography patterns, and molecular phylogeny led to the hypothesis that the derived karyotype was raised in the left bank of the Amazon River.

Karyotype, C-band and NOR phenotype of Anatolian endemic fish Squalius anatolicus (Bogutskaya, 1997) (Teleostei, Leuciscidae)

The karyotype and distribution of constitutive heterochromatin and nucleolus organizer regions (NORs) of Anatolian leuciscine endemic to Lake Beysehir, Squalius anatolicus (Bogutskaya, 1997) were analyzed respectively using conventional Giemsa-staining, C-banding and Ag-impregnation. Diploid chromosome number was 2n = 50 and karyotype consisted of 7 pairs of metacentric, 13 pairs of submetacentric, 5 pairs of subtelo- to acrocentric chromosomes, NF value equaled 90. Heteromorphic elements indicating sex chromosomes were not detected. C-banding revealed clear pericentromeric constitutive heterochromatin blocks in several chromosomes. Ag-impregnation revealed the size heteromorphism of NORs that covered almost the entire short arms of the middle-sized submetacentric chromosome pair. The karyotype pattern and simple NOR phenotype of S. anatolicus are nearly identical with that found not only in Squalius species analyzed to date but also in many other representatives of the Eurasian leuciscine cyprinids, which indicates remarkable chromosome stasis in this leuciscid lineage.

Evolutionary pattern of karyotypes and meiosis in pholcid spiders (Araneae: Pholcidae): implications for reconstructing chromosome evolution of araneomorph spiders

Background Despite progress in genomic analysis of spiders, their chromosome evolution is not satisfactorily understood. Most information on spider chromosomes concerns the most diversified clade, entelegyne araneomorphs. Other clades are far less studied. Our study focused on haplogyne araneomorphs, which are remarkable for their unusual sex chromosome systems and for the co-evolution of sex chromosomes and nucleolus organizer regions (NORs); some haplogynes exhibit holokinetic chromosomes. To trace the karyotype evolution of haplogynes on the family level, we analysed the number and morphology of chromosomes, sex chromosomes, NORs, and meiosis in pholcids, which are among the most diverse haplogyne families. The evolution of spider NORs is largely unknown. Results Our study is based on an extensive set of species representing all major pholcid clades. Pholcids exhibit a low 2n and predominance of biarmed chromosomes, which are typical haplogyne features. Sex chromosomes and NOR patterns of pholcids are diversified. We revealed six sex chromosome systems in pholcids (X0, XY, X1X20, X1X2X30, X1X2Y, and X1X2X3X4Y). The number of NOR loci ranges from one to nine. In some clades, NORs are also found on sex chromosomes. Conclusions The evolution of cytogenetic characters was largely derived from character mapping on a recently published molecular phylogeny of the family. Based on an extensive set of species and mapping of their characters, numerous conclusions regarding the karyotype evolution of pholcids and spiders can be drawn. Our results suggest frequent autosome–autosome and autosome–sex chromosome rearrangements during pholcid evolution. Such events have previously been attributed to the reproductive isolation of species. The peculiar X1X2Y system is probably ancestral for haplogynes. Chromosomes of the X1X2Y system differ considerably in their pattern of evolution. In some pholcid clades, the X1X2Y system has transformed into the X1X20 or XY systems, and subsequently into the X0 system. The X1X2X30 system of Smeringopus pallidus probably arose from the X1X20 system by an X chromosome fission. The X1X2X3X4Y system of Kambiwa probably evolved from the X1X2Y system by integration of a chromosome pair. Nucleolus organizer regions have frequently expanded on sex chromosomes, most probably by ectopic recombination. Our data suggest the involvement of sex chromosome-linked NORs in achiasmatic pairing.

Targeted Enrichment of rRNA Gene Tandem Arrays for Ultra-Long Sequencing by Selective Restriction Endonuclease Digestion

Large regions of nearly identical repeats, such as the 45S ribosomal RNA (rRNA) genes of Nucleolus Organizer Regions (NORs), can account for major gaps in sequenced genomes. To assemble these regions, ultra-long sequencing reads that span multiple repeats have the potential to reveal sets of repeats that collectively have sufficient sequence variation to unambiguously define that interval and recognize overlapping reads. Because individual repetitive loci typically represent a small proportion of the genome, methods to enrich for the regions of interest are desirable. Here we describe a simple method that achieves greater than tenfold enrichment of Arabidopsis thaliana 45S rRNA gene sequences among ultra-long Oxford Nanopore Technology sequencing reads. This method employs agarose-embedded genomic DNA that is subjected to restriction endonucleases digestion using a cocktail of enzymes predicted to be non-cutters of rRNA genes. Most of the genome is digested into small fragments that diffuse out of the agar plugs, whereas rRNA gene arrays are retained. In principle, the approach can also be adapted for sequencing other repetitive loci for which gaps exist in a reference genome.

Cytogenetic Analysis of Panaqolus tankei Cramer & Sousa, 2016 (Siluriformes, Loricariidae), an Ornamental Fish Endemic to Xingu River, Brazil

Despite conservation of the diploid number, a huge diversity in karyotype formulae is found in the Ancistrini tribe (Loricariidae, Hypostominae). However, the lack of cytogenetic data for many groups impairs a comprehensive understanding of the chromosomal relationships and the impact of chromosomal changes on their evolutionary history. Here, we present for the first time the karyotype of Panaqolus tankei Cramer & Sousa, 2016. We focused on the chromosomal characterization, using conventional and molecular cytogenetic techniques to unravel the evolutionary trends of this tribe. P. tankei, as most species of its sister group Pterygoplichthini, also possessess a conserved diploid number of 52 chromosomes. We observed heterochromatin regions in the centromeres of many chromosomes; pairs 5 and 6 presented interstitial heterochromatin regions, whereas pairs 23 and 24 showed extensive heterochromatin regions in their q arms. In situ localization of 18S rDNA showed hybridization signals correlating with the nucleolus organizer regions, which are located in the q arms of pair 5. However, the 5S rDNA was detected in the centromeric and terminal regions of the q arms of pair 8. (TTAGGG)n hybridized only in the terminal regions of all chromosomes. Microsatellite in situ localization showed divergent patterns, (GA)15 repeated sequences were restricted to the terminal regions of some chromosomes, whereas (AC)15 and (GT)15 showed a scattered hybridization pattern throughout the genome. Intraspecific comparative genomic hybridization was performed on the chromosomes of P. tankei to verify the existence of sex-specific regions. The results revealed only a limited number of overlapping hybridization signals, coinciding with the heterochromatin in centromeric regions without any sex-specific signals in both males and females. Our study provides a karyotype description of P. tankei, highlighting extensive differences in the karyotype formula, the heterochromatin regions, and sites of 5S and 18S rDNA, as compared with data available for the genus.

Chromosomal Instability and Origin of B Chromosomes in the Amazonian Glass Tetra Moenkhausia oligolepis (Günther, 1864) (Characiformes, Characidae)

B chromosomes occur in different species of the small characid fishes of the genus <i>Moenkhausia.</i> These supernumerary elements, that do not recombine with chromosomes of the standard A complement and follow their own evolutionary mechanism vary in number, morphology, and distribution. Here, we show karyotypic data of individuals of 2 populations of <i>Moenkhausia oligolepis</i> of the Brazilian Amazon (Pedro Correia and Taboquinha streams, Tocantins river basin), both with a diploid number of 50 chromosomes and karyotypic formula of 10m + 32sm + 8a. In addition to the normal complement, we also observed the occurrence of B chromosomes in the 2 populations with intra- and interindividual variation ranging from 0 to 10 Bs, independent of sex. The C-banding pattern evidenced heterochromatic blocks located mainly in the pericentromeric region of the chromosomes, while the B chromosomes appeared euchromatic. Silver-stained nucleolus organizer regions were identified in multiples sites, and some of these blocks were positive when stained with chromomycin A_3. The karyotype analysis and the application of whole-chromosome painting in populations of <i>M. oligolepis</i> reinforce the conservation of the basal diploid number for the genus, as well as the evolutionary tendency in these fishes to carry B chromosomes. Both populations turned out to be in different stages of stability and expansion of their B chromosomes. We further suggest that the origin of these chromosomes is due to the formation of isochromosomes. Here, we identified a pair of complement A chromosomes involved in this process.

Comparative cytogenetic of six species of Amazonian Peacock bass (Cichla, Cichlinae): intrachromosomal variations and genetic introgression among sympatric species

Cytogenetic data for the genus Cichla Bloch et Schneider, 1801 are still very limited, with only four karyotype descriptions to date. The sum of the available cytogenetic information for Cichla species, points to a maintenance of the diploid number of 48 acrocentric chromosomes, considered a typical ancestral feature in cichlids. In the current study, we performed molecular and classical cytogenetic analyses of the karyotype organization of six species of Cichla, the earliest-diverging genus of Neotropical cichlids. We cytogenetically analysed Cichla kelberi Kullander et Ferreira, 2006, Cichla monoculus Agassiz, 1831, Cichla piquiti Kullander et Ferreira, 2006, Cichla temensis Humboldt, 1821, Cichla vazzoleri Kullander et Ferreira, 2006 and Cichla pinima Kullander et Ferreira, 2006, including three individuals that showed mixed morphological characteristics, likely from different species, suggesting they were hybrid individuals. All individuals analysed showed 2n = 48 acrocentric chromosomes, with centromeric heterochromatic blocks on all chromosomes and a terminal heterochromatic region on the q arm of the 2nd pair. Mapping 18S rDNA gave hybridization signals, correlated with the nucleolus organizer regions, on the 2nd pair for all analyzed individuals. However, we found distinct patterns for 5S rDNA: interstitially at the proximal position on 6th pair of four species (C. kelberi, C. pinima, C. piquiti and C. vazzoleri), and on the distal of the 4th pair in two (C. monoculus and C. temensis). Accordingly, we present here new data for the genus and discuss the evolutionary trends in the karyotype of this group of fish. In addition, we provide data that supports the occurrence of hybrid individuals in the Uatumã River region, mainly based on 5S rDNA mapping.

IDENTIFIKASI KROMOSOM HOMOLOG MELALUI DETEKSI NUCLEOLUS ORGANIZER REGIONS DENGAN PEWARNAAN AgNO3 PADA TANAMAN BAWANG MERAH - IN PRESS -

Generally, the standard procedure for karyotype analysis of shallot is sorted by chromosome sizes. Therefore, the identification of homologous chromosomes is difficult without using a specific probe. Nucleolus Organizing Regions (NORs) can be used as a probe for precise identification of homologous chromosomes. However, the use of NORs for plant karyotyping in Indonesia is poorly investigated. In this study, shallot chromosomes were prepared using modified Carnoy’s solution II, fixed in Carnoy’s solution, and stained by using aceto-carmine and AgNO3 for detecting NORs. Chromosome images were analyzed by CHIAS IV. One locus NOR bearing chromosome pair was detected at metaphase and interphase, and it was located at short arms of subtelomeric chromosome number 6. NORs can be used as a probe for precise identification of homologous chromosomes in shallot. Therefore, this technique has the potential to be applied on species closely related to shallot and on other plant species.Keywords: AgNO3, chromosome condensation, NORs, shallot chromosome, shallot karyotype ABSTRAKProsedur kariotipe untuk bawang merah umumnya masih disusun berdasarkan ukuran kromosom, sehingga diperlukan suatu penanda yang dapat mengidentifikasi kromosom homolog secara presisi. Identifikasi kromosom homolog secara presisi menggunakan suatu penanda, khususnya deteksi Nucleolus Organizing Regions (NORs), yang di Indonesia masih jarang dilakukan. Penelitian ini bertujuan untuk membuat kariotipe dan mengidentifikasi kromosom homolog bawang merah melalui deteksi NORs menggunakan metode pewarnaan AgNO3. Proses fiksasi akar dilakukan dengan menggunakan modifikasi larutan Carnoy II, lalu difiksasi dengan larutan Carnoy, dan kromosom diwarnai dengan aceto-carmine dan larutan AgNO3 untuk mendeteksi NORs. Selanjutnya, citra kromosom dianalisis menggunakan CHIAS IV. Hasil penelitian menunjukkan bahwa terdapat sepasang NORs yang terdeteksi pada fase metafase dan interfase yang terletak pada bagian lengan pendek di kromosom subtelosentrik nomor 6. Hasil dari penelitian ini dapat dijadikan sebagai dasar di bidang sitogenetika bawang merah untuk mengidentifikasi kromosom homolog secara presisi menggunakan penanda NOR. Oleh karenanya, teknik ini dapat diaplikasikan pada spesies yang berdekatan dengan bawang merah dan komoditas tanaman lainnya.Kata Kunci: AgNO3, kariotipe bawang, kondensasi kromosom, kromosom bawang, NORs

Chromosome Dynamics Regulating Genomic Dispersion and Alteration of Nucleolus Organizer Regions (NORs)

The nucleolus organizer regions (NORs) demonstrate differences in genomic dispersion and transcriptional activity among all organisms. I postulate that such differences stem from distinct genomic structures and their interactions from chromosome observations using fluorescence in situ hybridization and silver nitrate staining methods. Examples in primates and Australian bulldog ants indicate that chromosomal features indeed play a significant role in determining the properties of NORs. In primates, rDNA arrays that are located on the short arm of acrocentrics frequently form reciprocal associations (“affinity”), but they lack such associations (“non-affinity”) with other repeat arrays—a binary molecular effect. These “rules” of affinity vs. non-affinity are extrapolated from the chromosomal configurations of meiotic prophase. In bulldog ants, genomic dispersions of rDNA loci expand much more widely following an increase in the number of acrocentric chromosomes formed by centric fission. Affinity appears to be a significantly greater force: associations likely form among rDNA and heterochromatin arrays of acrocentrics—thus, more acrocentrics bring about more rDNA loci. The specific interactions among NOR-related genome structures remain unclear and require further investigation. Here, I propose that there are limited and non-limited genomic dispersion systems that result from genomic affinity rules, inducing specific chromosomal configurations that are related to NORs.