Research Article |
Corresponding author: Patricia Elda Sobrinho Scudeler ( patyelda@yahoo.com.br ) Academic editor: Alicja Boroń
© 2015 Patricia Elda Sobrinho Scudeler, Débora Diniz, Adriane Pinto Wasko, Claudio Oliveira, Fausto Foresti.
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:
Scudeler PES, Diniz D, Wasko AP, Oliveira C, Foresti F (2015) Whole chromosome painting of B chromosomes of the red-eye tetra Moenkhausia sanctaefilomenae (Teleostei, Characidae). Comparative Cytogenetics 9(4): 661-669. https://doi.org/10.3897/CompCytogen.v9i4.5460
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B chromosomes are dispensable genomic elements found in different groups of animals and plants. In the present study, a whole chromosome probe was generated from a specific heterochromatic B chromosome occurring in cells of the characidae fish Moenkhausia sanctaefilomenae (Steindachner, 1907). The chromosome painting probes were used in fluorescence in situ hybridization (FISH) experiments for the assessment of metaphase chromosomes obtained from individuals from three populations of M. sanctaefilomenae. The results revealed that DNA sequences were shared between a specific B chromosome and many chromosomes of the A complement in all populations analyzed, suggesting a possible intra-specific origin of these B chromosomes. However, no hybridization signals were observed in other B chromosomes found in the same individuals, implying a possible independent origin of B chromosome variants in this species. FISH experiments using 18S rDNA probes revealed the presence of non-active ribosomal genes in some B chromosomes and in some chromosomes of the A complement, suggesting that at least two types of B chromosomes had an independent origin. The role of heterochromatic segments and ribosomal sequences in the origin of B chromosomes were discussed.
Chromosomal evolution, Chromosome microdissection, Chromosome painting, Genome organization, Repetitive DNA
Supernumerary or B chromosomes are defined as nonhomologous extra genomic elements that actively recombine with chromosomes of the A complement (
The red-eye tetra M. sanctaefilomenae (Steindachner, 1907) provides a good model for studying B chromosomes in fishes. This species possesses (i) from 0 to 8 B chromosomes (with numbers varying intra- and inter-individually); (ii) B chromosomes with a remarkable polymorphism, morphology and structure (as evidenced by C-banding, with either euchromatic or partially or fully heterochromatic Bs); (iii) putative sex-related B chromosomes (
We collected 30 individuals (21 males and 9 females) of M. sanctaefilomenae from Araquá stream 22°44.83'S and 48°28.5'W (DDM), 19 individuals (10 males and 9 females) from Mané-Teixeira stream 22°45.78'S and 48°15.71'W (DDM) and 6 individuals (3 males and 3 females) from Olaria stream 21°9.18'S and 50°3.06'W (DDM), all tributaries of the Tietê River, São Paulo, Brazil. Vouchers were deposited in the fish collection of Laboratório de Biologia e Genética de Peixes, UNESP, Botucatu (LBP 18986/18987/18988, LBP18983 and LBP18982, respectively). The samples were collected in accordance with the Brazilian environmental protection legislation (collection permission MMA/IBAMA/SISBIO - No 3245). The procedures for sampling, maintenance and analysis of the specimens were performed in compliance with the Brazilian College of Animal Experimentation (COBEA) and approved by Bioscience Institute/UNESP Ethics Committee on use of animals (CEUA) (protocol 405).
The mitotic chromosomes were obtained from kidney and gill tissues using the technique described by
Microdissection of the B chromosome which are easily identified after GC-specific triple fluorochrome staining (CMA3/DA/DAPI), was performed using an Eppendorf Transfer Man NK2 micromanipulator coupled to a Zeiss Axiovert 100 microscope. Ten B chromosomes, previously identified as heterochromatic B chromosomes using the CMA3/DA/DAPI staining protocol, were microdissected and transferred into a microcentrifuge tube containing a DOP-PCR mix solution. The probes, here referred to as MsB, were obtained by PCR (Polymerase Chain Reaction) using a DOP primers according to
For FISH experiments, the chromosomes were treated according to the procedures described by
Hybridization with the MsB probe on metaphase chromosomes of individuals of the Araquá stream resulted in the complete painting of one B chromosome (Fig.
Karyotypes of Moenkhausia sanctaefilomenae from the Araquá (a), Mané Teixeira (b) and Olaria (c) streams, arranged from chromosomes after double-FISH. Chromosome painting with MsB-probe (red) and 18S rDNA (green). Bar =10 µm.
Individuals from the Mané Teixeira stream showed hybridization signals in the pericentromeric region of 12 chromosome pairs in the A complement and hybridization signals in only one of the homologous of pairs 15 and 22 with the MsB probe. Signals with the 18S rDNA probe were identified in pairs 7, 10, 14, 15 and 17 (Fig.
Hybridization with the MsB probe in the individuals collected in the Olaria stream showed signals in the pericentromeric region of chromosome pair 15 in the A complement. Notwithstanding, in some cases, a single chromosome of a pair was stained, as observed in pairs 2 and 5 in (Fig.
The B chromosomes observed in the populations of M. sanctaefilomenae were all very small and characterized as B microchromosomes by
Our results of chromosome painting with the DNA probes obtained from the B heterochromatic chromosome from the Araquá Stream population (here referred to as MsB) showed that specific hybridization signals were observed in the pericentromeric region of many chromosomes of the A complement and in only one type of B chromosome. In M. sanctaefilomenae, those pericentromeric regions are heterochromatic (
The presence of B chromosomes with no signals of hybridization with the specific B chromosome probe pointed to an independent origin of these elements. Alternatively, it is possible to suppose that: (i) in some B chromosomes, the number of the MsB sequences were too small to be detectable by the technique used here; (ii) the loss of some specific chromosome segments during the dynamic process of modification occurred in the independent evolution of different B chromosome lineages; (iii) the B chromosomes that did not stain with the MsB probe may represent B chromosomes derived from different heterochromatic regions; and (iv) the B chromosomes that did not stain with the MsB probe may represent chromosomes recently originated from euchromatic segments.
Sharing of repetitive DNA sequences between B chromosomes and the elements of the A complement is considered a common feature in the chromosomes of different organisms, such as in the mammals Vulpes vulpes (Linnaeus, 1758) (
FISH with 18S probes revealed high variability in the M. sanctaefilomenae samples studied here, involving seven chromosome pairs (Fig.
The presence of ribosomal sites in B chromosomes of M. sanctaefilomenae suggests that these chromosomes had 18S sequences. Nevertheless, as they are not silver-stained, they may not correspond to active NORs (
According to
The authors are grateful to FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for their financial support and to Renato Devidé for the logistic support including sample collection and sharing cytogenetic technique protocols.