Corresponding author: Roberto Ferreira Artoni ( firstname.lastname@example.org )
Academic editor: Inna Kuznetsova
© 2017 Patrícia Barbosa, Eliza Viola Leal, Maelin da Silva, Mara Cristina de Almeida, Orlando Moreira-Filho, Roberto Ferreira Artoni.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Barbosa P, Leal AV, Silva M, Almeida MC, Moreira-Filho O, Artoni RF (2017) Variability and evolutionary implications of repetitive DNA dynamics in genome of Astyanax scabripinnis (Teleostei, Characidae). Comparative Cytogenetics 11(1): 143-162. https://doi.org/10.3897/CompCytogen.v11i1.11149
DNA sequences of multiple copies help in understanding evolutionary mechanisms, genomic structures and karyotype differentiation. The current study investigates the organization and distribution of different repetitive DNA in the standard complement and B chromosomes in Astyanax scabripinnis (Jenyns, 1842) chromosomes from three allopatric populations in Campos do Jordão region, São Paulo State, Brazil. The location of microsatellite sequences showed different chromosome distribution between Lavrinha Farm Stream (LFS) and Lake of Pedalinho (LP) populations. However, the karyotype of these populations basically followed the pattern of dispersed distribution in the A complement, conspicuous in telomeric/interstitial regions and preferential accumulation in the B chromosome. The B chromosome showed heterogeneous location of microsatellite probes CA, CAC and GA. The H3 and H4 histone genes were isolated from the total genome of the species and then the chromosomal mapping was performed by fluorescence in situ hybridization (FISH). The FISH signals showed high similarity for the probes H3 and H4 mapping in genomes of the populations analyzed. The sequences (GATA)n revealed a sex-specific trend between the chromosomal location in males and females at (LFS) and (LP) populations. Although species that comprise the Astyanax scabripinnis complex do not have morphologically differentiated sex chromosomes, the preferential GATA location – sex-associated – may represent a sex chromosome in differentiation.
Microsatellites chromosomic mapping, sex-specific GATAn sequence location, B chromosomes microsatellites accumulation
DNA sequences of multiple copies help in understanding evolutionary mechanisms, genomic structures and karyotype differentiation, including in fishes. Astyanax scabripinnis (Jenyns, 1842) is a freshwater fish species of the family Characidae (
Astyanax scabripinnis is a model in evolutionary studies due to the frequent presence of B chromosomes in some populations (see
Some functional genes found in the DNA of eukaryotes may have simple copies and unique sequences, whereas other genes have repetitive nature when they are found in more than one copy (
The sequences of repetitive DNA with tandem distribution are classified as satellites, microsatellites and minisatellites according to the degree of repetition (
Other repetitive DNAs with great evolutionary and functional interest are the Bkm (banded krait minor satellite) satellite sequences, which are found in the heterogametic sex of most vertebrates (
The microsatellite chromosomic mapping is widely used in evolutionary studies, including in Astyanax (
The aim of this study is to investigate patterns of organization and distribution of different repetitive DNAs, in the standard complement and B chromosomes in Astyanax scabripinnis.
Fifty six specimens of Astyanax scabripinnis (18 females and 38 males) were analyzed from three different locations in the Campos do Jordão region, State of São Paulo, Brazil, collected with permission from Ministério do Meio Ambiente, Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis, Instituto Chico Mendes de Conservação da Biodiversidade – ICMBio MMA / IBAMA / SISBIO, number 15115-1: Lavrinha Farm Stream (LFS) (22°40'49.5"S; 45°23'31.9"W), Lake of Pedalinho (LP) (22°43'02.8"S; 45°33'91.9"W) and Ribeirão das Perdizes (RP) (22°44'35.3"S; 45°34'11.6"W). For cytogenetic analyses, the specimens were anesthetized with benzocain 0.01% and dissected, and the mitotic chromosomes were obtained from kidney tissue using the technique described by
The genomic DNA extraction was carried out by the cetyl trimethyl ammonium bromide method (
Primer sequences employed.
|Chromosomal markers||Primer sequence (5’-3’)||Reference|
|18S rDNA F||GTAGTCATATGCTTGTCTC||
|18S rDNA R||TCCGCAGGTTCACCTACGGA||
|5S rDNA F||TACGCCCGATCTCGTCCGATC||
|5S rDNA R||CAGGCTGGTATGGCCGTAAGC||
|As51 F||GGTCAAAAAGTCGAAAAA||Mestriner et al. 1999|
|As51 R||GTACCAATGGTAGACCAA||Mestriner et al. 1999|
|H3 R||ATATCCTTRGGCAT RATRGTGAC||
The nucleotide sequencing of the clones and DNA fragments was performed in an automatic sequencer ABI 3130x1, using o Kit Big Dye (Applied Biosystems) following manufacturer instructions. The sequences were aligned in Clustal W program (
Seven different types of repetitive probes were used as chromosomal markers: 18S rDNA; 5S rDNA; As51 satellite DNA; H3 and H4 histone genes; the general vertebrate telomere sequence minisatellite (TTAGGG)n; and the (GATA)n sequence. The As51 satellite DNA, 5S rDNA and H4 histone gene probes was labelled with Biotin-11-dUTP (Roche Applied Science), whereas 18S rDNA, H3 histone gene, (GATA)n, and (TTAGGG) n probes were labelled with digoxigenin by nick translation.
Oligonucleotide probes containing microsatellite sequences (CA)15, (CAC)10, (CAG)10, (CAT)10, (GA)15, (GAA)10, (GAG)10 and (GC)15 were directly labeled with Cy5 during synthesis by Sigma (St. Louis, MO, USA), as described by
For each FISH assay 30 cells were analyzed. The FISH was performed using the protocol of
The diploid number verified for Astyanax scabripinnis was 2n = 50 chromosomes in the three analyzed populations. The fundamental number (FN) was equal to 88 and karyotype formula composed by 6 m + 22 sm + 10 st + 12 a. Some specimens showed B chromosomes in 100% of the analyzed cells (2n = 51) (Suppl. material
The amplification of the H3 and H4 histone genes from the total genome of the specimens was performed. The product of the PCR generated bands with approximately 400 base pair (bp), when it was analyzed in gel electrophoresis (Suppl. material
The H3 and H4 histones were co-located in two chromosomal pairs: metacentric pair No. 2 and subtelocentric pair No. 16 in the three populations (LFS, LP, RP). The sites of chromosome metacentric pair 2 were proximal, whereas the signal were terminal in other chromosomal pairs (Figs
Karyotype of the three Astyanax scabripinnis populations (a, b Lavrinha Farm Stream – LSF); (c, d Lake of Pedalinho – LP); (e, f Ribeirão das Perdizes – RP). In a, c, e are visualized signals of 18S (red) and 5S (green) with rDNA probes and in b, d, f H3 (red) and H4 (green) with histones probes, after double Fluorescence in situ Hybridization (FISH), respectively. Scales bar: 10 µm.
The probe of the minisatellite sequence (TTAGGG)n was uniformly located in the telomeres of all chromosomes at the studied populations, including B chromosome. No interstitial signal (ITS) was found (data not shown) (Suppl. material
The (GATA)n sequence was hybridized in males and females of the three populations (Fig.
Males and females from the (LP) population did not show significant differences regarding the signal of the (GATA)n probe. Females showed three main signals: the homologues of metacentric chromosome pair No. 2, in the proximal region, without heterochromatin; and one of the homologues of pair 24 a in terminal region of the long arm, with evidences of heterochromatin (Fig.
The male specimens from the (RP) population showed the pair of subtelocentric chromosomes 15 and 16 as their main signal (Fig.
The As51 satellite DNA occurs in acrocentric chromosomes of the three populations: pairs 22 and 23 in the (LFS) and (LP) populations, pair 20 in the (RP) population and also in the chromosome B (Fig.
B chromosome of Astyanax scabripinnis showing the chromatin and localization of DNA sequences by FISH.
The oligonucleotide probes (CAC)10, (CA)15 and (GA)15 microsatellite sequences showed dispersed signal among the chromosomes of complement A in the three populations. In addition, the probes showed an accumulation in B chromosomes of individuals of the (LFS) population (Fig.
Fluorescence in situ Hybridization in metaphases of the three Astyanax scabripinnis populations (a, b, c Lavrinha Farm Stream – LSF); (d, e, f Lake of Pedalinho – LP); (g, h, i Ribeirão das Perdizes - RP) with microsatellites probes. Scales bar: 10 µm.
Although the diploid number (2n) verified for Astyanax scabripinnis species complex indicates a variation of 46, 48 or 50 chromosomes (
The karyotype description for the (LP) population was performed here for the first time. The population showed standard diploid number and karyotype formula, with multiple NORs. The karyotype of the (RP) population corroborates data obtained by Vicente et al. (1996) regarding the presence of B chromosome. However, differences in the location of NORs within this population were also observed, since they were exclusively located in the short arm of pair 10 sm in previous studies. Probably, these trends are mediated by the association of additional sites of NORs with LINE retrotransposons – among other possible transposable elements (TEs) – and with repetitive sequences such as As51 (
Astyanax scabripinnis forms small isolated populations in streams and it facilitates the establishment of chromosomal changes that lead to polymorphism which may explain karyotype variability among individuals and populations (see Moreira-Filho et al. 2004 for review). The higher amount in the number of ribosomal gene copies and in the transposition may originate natural polymorphisms, e.g. in Gymnotus carapo Linnaeus, 1758, Apteronotus albifrons (Linnaeus, 1766), Sternopygus macrurus (Bloch & Schneider, 1801), Eigenmannia virescens (Valenciennes, 1836) and Eigenmannia sp., in which only one of the homologues carries the NOR (
The number of 5S rDNA sites varied between 4 and 5 in the populations, wherein the proximal signal located in pair 2 m is a feature conserved among them. Similar results were described by
Data on the location of histone genes are still rare. According to
The location of interstitial telomeric sites (ITS) is an important tool that helps to tell the evolutionary history of a group (
Furthermore, an accumulation of repetitive sequences in the chromosome 2 of the three studied populations and also in the chromosome 16 of the (LP) population was evident. Piscor and Parisi-Maltempi (2016) proposed evolutionary pathways for microsatellites in the genome of Astyanax. The authors propose that the association of 5S rDNA-GATA can stabilize the structure of DNA and act as ‘hot spots’ for chromosomal recombination. Our data contrast with the Piscor and Parisi-Maltempi (2016) and show that GATA sequences are not restricted to co-location with the 5S rDNA and may occur with sex-specific location in A. scabripinnis.
The location of (GATA)n repetitive sequences revealed a slightly dispersed pattern in chromosomes of A. scabripinnis, but with preferential accumulation in terminal and interstitial regions of the three studied populations. Dispersed signal of GATA sequences in fishes are verified by FISH in Solea senegalensis Kaup, 1858 (
The presence of B chromosome in A. scabripinnis confirmed the heterochromatic pattern found in different populations (see
The mapping of microsatellites in the chromosomes may help in investigating the chromatin nature. Similarly, to data found in chromosomes of A. scabripinnis (LP), there might be similar patterns among different species, such as the dinucleotide (CA)15, which is found at the centromeric region of chromosome W in lizards Eremias velox Eremchenko & Panfilov, 1999 (
Thus, the study brings up the required association condition and co-location of different group of repetitive DNAs expressed and non-encoding. Although these sequences may be related to the evolution of genome and karyotype, they still suggest a possible relation with sex determination in the species.
The author PB received a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This study received financial support from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).