Corresponding author: Patricia Pasquali Parise-Maltempi ( firstname.lastname@example.org )
Academic editor: Eugene Krysanov
© 2017 Keteryne Rodrigues da Silva, Sandra Mariotto, Liano Centofante, Patricia Pasquali Parise-Maltempi.
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: da Silva KR, Mariotto S, Centofante L, Parise-Maltempi PP (2017) Chromosome mapping of a Tc1-like transposon in species of the catfish Ancistrus. Comparative Cytogenetics 11(1): 65-79. https://doi.org/10.3897/CompCytogen.v11i1.10519
The Tc1 mariner element is widely distributed among organisms and have been already described in different species of fish. The genus Ancistrus (Kner, 1854) has 68 nominal species and is part of an interesting taxonomic and cytogenetic group, as well as presenting a variation of chromosome number, ranging from 2n=34 to 54 chromosomes, and the existence of simple and multiple sex chromosome system and the occurrence of chromosomal polymorphisms involving chromosomes that carry the nucleolus organizer region. In this study, a repetitive element by restriction enzyme, from Ancistrus sp.1 “Flecha” was isolated, which showed similarity with a transposable element Tc1-mariner. Its chromosomal location is distributed in heterochromatic regions and along the chromosomal arms of all specimens covered in this study, confirming the pattern dispersed of this element found in other studies carried out with other species. Thus, this result reinforces the hypothesis that the sequence AnDraI is really a dispersed element isolated. As this isolated sequence showed the same pattern in all species which have different sex chromosomes systems, including in all sex chromosomes, we could know that it is not involved in sex chromosome differentiation.
Repetitive DNA, enzyme digestion, chromosomal mapping, transposable elements, in situ fluorescence hybridization
The genome of eukaryotes consists mostly of large amounts of repetitive DNA, which has been associated with several functions in the genome, as can be seen in the review carried out by
Basically, the repetitive sequences are represented by tandem repeats, as satellite DNA, minisatellite, and microsatellite repeats or dispersed along the DNA as retrotransposons and transposons (
When transposed, if the transposition occurs within promoter regions, introns or untranslated regions, it can affect the expression of this gene (
Based on the similarity between the sequences and phylogenetic analysis of the transposase, the transposable elements can be classified in ten families: Tc1/mariner, haT, P element, MuDR/Fokdback, Cacta, PiggyBac, Pif/Harbinger, Merlin, Transib and Banshee (
This element, belonging to a superfamily of transposons, presents 1000 up to 2000 bp (
The genus Ancistrus (Kner, 1854) is one of the most diverse of tribe Ancistrinae, popularly known as “cascudos”, and currently has 68 nominal species (
Thus, taking into account the karyotype diversity of Ancistrus, including different sex chromosome systems, location of nucleolus organizer regions (NOR) and number of chromosomes, this study aimed to isolate repetitive sequences that could help in better understanding of the karyotype organization of the Ancistrus species.
The species of Ancistrus covered in this study were collected in the Flecha river, Creek Currupira, Pari and Sangradouro in the Paraguay river basin (Table
Collection site and number of species collected.
|Specie||Collection site||Number of collected species|
|Ancistrus sp1 “Flecha”||15°58'7"S 57°19'7"W||18 F – 6 M|
|Ancistrus sp “Currupira”||15°7'59"S 56°49'47"W||19 F – 23 M|
|Ancistrus sp “Pari”||15°36'6"S 56°12'19"W||7 F – 12 M|
|Ancistrus sp “Sangradouro”||16°4'25"S 57°40'31.1"W||5 F – 4 M|
The chromosome preparations were made from the kidney of specimens collected following the methodology described by
C-bands were detected according to
The extraction of genomic DNA was performed from liver and fin of the specimens collected, basically following the protocol phenol / chloroform / isoamyl alcohol by
The amplified and purified DNA by treatment with ExoSAP enzyme (USB) was sequenced by the method of
The recombinant clones were subjected to PCR (Polymerase Chain Reaction) for amplification using the universal primers M13 F - GTA AAA CGA CGG CCA G and M13 R - CAG GAA ACA GCT ATG AC under the following conditions: denaturation at 95 °C for 3 minutes, 34 cycles of denaturation at 95 °C for 30 seconds, annealing at 50 for 1 minute, 72 °C extension for 2 minutes and elongation at 72 °C for 5 minutes.
After sequencing, the divergent primers were designed KD7116F – TCA CAA CAC ACG TTT GTG GA and KD7116R – AGA GCA GGC TTT GAA TCG G manually, which was synthesized by SIGMA company. Subsequently, the amplification of the sequence with the primer KD7116-1 also in other possible different species from other populations was performed following conditions: denaturation at 94 for 5 minutes, 30 cycles of denaturation at 94 °C for 30 seconds, annealing at 58 °C for 1 minute 72 °C extension for 1 min and elongation at 72 °C for 7 minutes.
In situ hybridization was performed following the protocol by
The analysis of constitutive heterochromatin by C-banding was performed to characterize all species karyotypes covered in this study. Ancistrus sp.1 “Flecha” has 2n=44 chromosomes and no heterochromatin block or sex chromosome system was shown (Fig.
Karyotype of Ancistrus sp.1 “Flecha” using C-banding. A Conventional staining- showing the karyotype of Ancistrus sp.1 “Flecha” with 2n = 44 chromosomes, including a pair of microchromosomes B, C is C-band showing that the chromosomes of Ancistrus is not rich in heterochromatic regions in female and male. Bar = 10 µm.
Karyotype after C band. Ancistrus sp. “Currupira” has 2n=44 chromosomes and showed heterochromatin blocks mainly at centromeric regions and pair 13 (A), Ancistrus sp. “Pari” has 2n=42 chromosomes with heterochromatin blocks along centromeric regions and a big block in pair 15 (B) and Ancistrus sp. “Sangradouro” has 2n=42 chromosomes, a karyotype similar to Ancistrus sp. “Pari” but the big heterochromatin block is in pair 6 (C). Bar = 10 µm.
After isolation of repetitive sequences with restriction enzymes using Ancistrus sp.1 “Flecha” DNA, it was possible to observe the formation of a band of approximately 700 bp. The product from this band was then cloned, resulting in 34 recombinant clones, from which one, named AnDraI was used in this study. The sequence of this clone had 618 bp and 44.34% of GC base pair. According with the databases consulted: Blast 2.0 RepeatMasker and Censor, the sequence obtained showed greater than 86% identity with the type of transposon Mariner/Tc1 of Xenopus tropicalis (Gray, 1864). In the analysis performed for possible coding regions, an ORF region of frame 3+ 188 bp (87-275) was found. As conserved domain, a region of approximately 180 nucleotides which corresponds to HTH_Tnp_Tc3_2_Transposase was found. By submitting this sequence in the protein data bank (Blastx), similarities were found with transposases of several species, including, Rana pipiens (Schreber, 1782), Xenopus tropicalis, Dicentrarchus labrax (Linnaeus, 1758), Salmo salar (Linnaeus, 1758), and Cyprinus carpio (Linnaeus, 1758) (Fig.
It was realized an amplification of AnDraI element by polymerase chain reaction in the genome of other specimens of other populations: Ancistrus sp. “Currupira” from Currupira Creek, Ancistrus sp. “Pari” from Pari Creek and Ancistrus sp. “Sangradouro” from Sangradouro Creek. Both, males and females, of this species showed the same length of fragments (not shown).
Chromosomal in situ hybridization performed on Ancistrus sp.1 “Flecha” revealed signals throughout all the chromosomes preferentially located in pericentromeric regions, with no difference between males and females (Fig.
In situ fluorescence hybridization using AnDraI as probe in Ancistrus sp.1 “Flecha”: A female B male. The arrows show some examples of pericentromeric and spread signals. Bar = 10 µm.
Cross-Fish conducted in individuals from other localities also showed results similar to that found in Ancistrus sp.1 “Flecha” (Fig.
In situ fluorescence hybridization using AnDraI as probe in Ancistrus chromosomes: A Female of Currupira creek B Male of Currupira creek C Female of Pari creek D Male of Pari creek E Female of Sangradouro creek e F Male of Sangradouro creek. The arrows show some examples of pericentromeric and spread signals. Bar = 10 µm.
The transposable element Tc1, first identified in invertebrates as Caenorhabditis elegans (Maupas, 1900), have around 1600 bp and share similar structures, known as Terminal inversed regions (TIRs) that show a sequence of 5 to 6 bp identical in/or near the highly conserved ends (CAGTG/CAGTC) (
The Tc1-like element isolated in this work from Ancistrus sp.1 “Flecha” appears to share a very similar structure to TcMar-Tc1 of Xenopus (Siluriana) tropicalis, which is 532 bp, equivalent to 86.1%. This element appears widely distributed throughout all the chromosomes of all specimens of Ancistrus covered in this study, with some specific concentrations in pericentromeric regions. These specimens belong to distinct localities with a high geographic distance and isolated among themselves, with different karyotypes which show number chromosomes ranging from 2n=42 to 44 and species with no sex chromosomes system and others with XX/XY or ZZ/ZW sex chromosomes. Despite of the known cytogenetic differences among the Ancistrus group it can be said that, at least among the specimens analyzed, the AnDraI element presents the same homogeneous pattern of distribution and no correlation can be made regarding the karyotypes differences neither the origin and differentiation of sex chromosomes of this group involving its isolated sequence. However, it can be inferred that, despite the low number of populations analyzed it is possible to consider that the AnDra I element is present in the genome of the Ancistrus genus. Although the Siluriformes group have a scientific and economic importance, their systematic and taxonomy are still highly problematic and, in this context, studies involving repetitive sequences, which are showed to be important cytogenetic markers, may help uncover the evolutionary history of the group.
Repetitive sequences may be present in centromeres and telomeres of eukaryotic chromosomes which are rich regions in heterochromatin, as well as regions over the interstitial chromosomal arms (
Results found in the literature about the genomic organization of transposons suggest that these elements are differently distributed in distinct groups of fish (