Corresponding author: Érica Alves Serrano ( email@example.com )
Academic editor: Petr Rab
© 2017 Érica Alves Serrano, Ricardo Utsunomia, Patrícia Sobrinho Scudeller, 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: Serrano ÉA, Utsunomia R, Scudeller PS, Oliveira C, Foresti F (2017) Origin of B chromosomes in Characidium alipioi (Characiformes, Crenuchidae) and its relationship with supernumerary chromosomes in other Characidium species. Comparative Cytogenetics 11(1): 81-95. https://doi.org/10.3897/CompCytogen.v11i1.10886
B chromosomes are apparently dispensable components found in the genomes of many species that are mainly composed of repetitive DNA sequences. Among the numerous questions concerning B chromosomes, the origin of these elements has been widely studied. To date, supernumerary chromosomes have been identified in approximately 60 species of fish, including species of the genus Characidium Reinhardt, 1867 in which these elements appear to have independently originated. In this study, we used molecular cytogenetic techniques to investigate the origin of B chromosomes in a population of Characidium alipioi Travassos, 1955 and determine their relationship with the extra chromosomes of other species of the genus. The results showed that the B chromosomes of C. alipioi had an intraspecific origin, apparently originated independently in relation to the B chromosomes of C. gomesi Travassos, 1956 C. pterostictum Gomes, 1947 and C. oiticicai Travassos, 1967, since they do not share specific DNA sequences, as well as their possible ancestral chromosomes and belong to different phylogenetic clades. The shared sequences between the supernumerary chromosomes and the autosommal sm pair indicate the origin of these chromosomes.
Microdissection, Chromosome painting, FISH, B chromosomes
B or supernumerary chromosomes are extra genomic elements in addition to the standard chromosomal set (A) and are found in approximately 15% of eukaryotic organisms (
A significant evolutionary feature of B chromosomes is the accumulation of repetitive DNA sequences (
Among genomes of fish species bearing B chromosomes, the genus Characidium Reinhardt, 1867 exhibits interesting cytogenetic features; the B chromosomes probably originated independently in the different species of this group, whereas the heteromorphic ZZ/ZW sex chromosomes seem to have originated once in the genus. Therefore, the B chromosomes of C. oiticicai Travassos, 1967 originated interspecifically, whereas in C. gomesi Travassos, 1956 and C. pterostictum Gomes, 1947, these elements have an intraspecific origin from the sex chromosomes (
A total of 19 C. alipioi samples were analyzed (9 females and 10 males) from the Ribeirao Grande river, Paraíba do Sul River Basin, Pindamonhangaba, São Paulo (22°49'00.3"S 45°25'23.7") (Table
B chromosome polymorphisms in Characidium alipioi. Samples: number of males (M) and females (F) analyzed. Prevalence: the total and sex-specific percentage of individuals carrying B chromosomes.
|Samples||Samples number with up to:||Prevalence|
To perform the cytogenetic preparations, the animals were anesthetized and dissected, and mitotic chromosome preparations were obtained following the protocol of
DNA extraction was performed using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s instructions.
Amplification and partial sequencing of cytochrome oxidase c subunit 1 (COI) and cytochrome b (Cyt B) was performed to identify the specimens. The primers used were as follows: Cyt BL 14841 (5’-CCA TCC CAA ATC ACT GCA TGA TGA AA-3 ‘) and Cyt BH 15915b (5’-AAC CTC TCT CGA GCT GAT TACAAG AC -3’) (
Chromosome microdissection was performed in an Eppendorf TransferMan NK2 micromanipulator coupled with a Zeiss Axiovert 100 microscope. For chromosome painting, ten B chromosomes were microdissected from the cytogenetic preparations of the samples from each species (C. alipioi, C. gomesi and C. oiticicai) carrying one extra chromosome. The probes for C. alipioi, C. gomesi and C. oiticicai denoted CaB, CgB and CoB, respectively.
Microdissected DNA from each species was placed into a tube containing 9 µL of DNase-free ultrapure water and amplified using the GenomePlex Single Cell Whole Genome Amplification Kit (wga4 Sigma) (
Probes for 18S and 5S rDNA, U2 snDNA and histone H3 genes were obtained using PCR from the C. alipioi genome with previously described primers. Sequences for 5S and 18S rDNA were amplified using the primers 5SA (5’-TCA ACC AAC CAC AAA GAC ATT GGC AC-3’) and 5SB (5’-TAG ACT TCT GGG TGG CCA AAG GAA TCA-3’) (
For the FISH experiments, the prehybridization conditions were performed according to procedures described by
Analysis of mitochondrial DNA was performed in order to make a correct identification of the specimens, besides that, the position of the clades could be informative to discuss aspects of the origin of the B chromosomes in the genus. The average distance analysis of the COI and CytB sequences obtained in this study and other species taken from GenBank showed high similarity between the C. alipioi sequences of Santa Bárbara do Tugúrio-MG and the specimens analyzed here (Suppl. materials
The analyzed C. alipioi individuals showed diploid chromosome number 2n = 50 and karyotype composed of 32 m + 18 sm with heteromorphic ZZ/ZW sex chromosomes, which was similar to the findings in other species of the genus (Figure
Chromosome painting performed in the C. alipioi chromosomes with the CaB probe showed signals on the entire length of the B chromosomes and the pericentromeric region of the sm chromosome pair nº 19 of the A complement (Figure
FISH experiments on the C. alipioi chromosome preparations using a 18S rDNA probe revealed sites of this gene in the terminal position of autosomal pair No. 18, whereas the 5S rDNA was mapped in the pericentromeric region of chromosome pair No. 20 (Figure
Karyotypes of Characidium alipioi arranged from mitotic metaphases after FISH with repetitive DNA probes. a 18S and 5S rDNA probes b histone H3 probe, and c U2 snRNA probe. Bar =10 µm.
FISH with probes containing the microsatellite sequences (CA)15 and (GA)15 in the C. alipioi chromosomal preparations showed similar patterns of distribution with preferential accumulation in the terminal regions of the chromosomes, except for the W and B chromosomes (Figure
The occurrence of B chromosomes in Characidium alipioi genome was revealed for the first time in this study, particularly since the population of C. alipioi of Santa Bárbara do Tugúrio-MG analyzed by
The individuals of the same species and location included in this study were analyzed in a previous study by
Chromosome painting using the B chromosomes of C. alipioi (CAB) as the probe indicated shared sequences between the A and B complements and, more specifically, the pericentromeric region of a sm chromosome pair (pair No. 19). This result probably reflects a relationship between these chromosomes and intraspecific origin of the supernumerary chromosomes in genome of this species and that pair No. 19 is likely the chromosome of its origin. Previous studies have identified the origin of B chromosomes from the A complement of the host species (
The intraspecific origin of B chromosomes has been reported in other species of the genus Characidium, namely C. pterostictum, C. gomesi and Characidium sp. aff. C. vidalli (
Hybridizations with microsatellite DNA sequences demonstrated the presence of these repetitive elements in B chromosomes. (GA)15 and (CA)15 are both dispersed with conspicuous blocks in the terminal regions of the A chromosomes and are less abundant in the supernumerary chromosomes. Moreover, a clear accumulation was observed with respect to the (GAG)10 sequence. Notably, the hybridization with the (GAG)10 probe revealed a preferential accumulation in B and W chromosomes in C. alipioi. Similarly, microsatellites were detected in the B chromosomes of maize (
Our present results extend the knowledge of the structure and composition of B chromosomes between representatives of the Characidium genus, particularly in C. alipioi. In addition, the shared sequences between the A and B chromosomes of this species suggests an intraspecific origin of these chromosomes that is independent from the B chromosomes of other congeneric species. These observations reinforce the idea that this fish group is an interesting model to study the origin and structure of B chromosomes.
This study was funded by the State of São Paulo Research Foundation (FAPESP), National Council for Research and Development (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).