Short Communication |
Corresponding author: Silvana Melo ( silvanamelo90@gmail.com ) Academic editor: Nina Bulatova
© 2017 Silvana Melo, Ricardo Utsunomia, Manolo Penitente, Patrícia Elda Sobrinho-Scudeler, Fábio Porto-Foresti, Claudio Oliveira, Fausto Foresti, Jorge Abdala Dergam.
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:
Melo S, Utsunomia R, Penitente M, Elda Sobrinho-Scudeler P, Porto-Foresti F, Oliveira C, Foresti F, Dergam J (2017) B chromosome dynamics in Prochilodus costatus (Teleostei, Characiformes) and comparisons with supernumerary chromosome system in other Prochilodus species. Comparative Cytogenetics 11(2): 393-403. https://doi.org/10.3897/CompCytogen.v11i2.12784
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Within the genus Prochilodus Agassiz, 1829, five species are known to carry B chromosomes, i.e. chromosomes beyond the usual diploid number that have been traditionally considered as accessory for the genome. Chromosome microdissection and mapping of repetitive DNA sequences are effective tools to assess the DNA content and allow a better understanding about the origin and composition of these elements in an array of species. In this study, a novel characterization of B chromosomes in Prochilodus costatus Valenciennes, 1850 (2n=54) was reported for the first time and their sequence complementarity with the supernumerary chromosomes observed in Prochilodus lineatus (Valenciennes, 1836) and Prochilodus argenteus Agassiz, 1829 was investigated. The hybridization patterns obtained with chromosome painting using the micro B probe of P. costatus and the satDNA SATH1 mapping made it possible to assume homology of sequences between the B chromosomes of these congeneric species. Our results suggest that the origin of B chromosomes in the genus Prochilodus is a phylogenetically old event.
additional chromosomes, genome, Prochilodontidae
Supernumerary or B chromosomes are dispensable genomic elements found in approximately 15% of eukaryotes (
Prochilodus is the most species-rich genus within family Prochilodontidae and its species exhibit a well-preserved karyotypic macrostructure, a diploid number of 54 chromosomes and karyotypic formula of 40m + 14sm (
In a previous study,
Prochilodus costatus Valenciennes, 1850 is an endemic species of the São Francisco River basin and previous cytogenetic analyses did not reveal the presence of B chromosomes in this species (
Prochilodus costatus specimens (N=23) were collected in three distinct sites along São Francisco River basin (Table
B chromosome frequency and Mitotic Instability index (MI) of somatic cells in P. costatus.
Sample | Locality | Geographic coordinates | Number of B per cell | MB | N | MI | |
---|---|---|---|---|---|---|---|
0B | 1B | ||||||
JD5480 | São Francisco River – Iguatama | 20°09'50"S, 45°43'08"W | 12 | – | 0B | 12 | 0 |
JD5481 | São Francisco River – Iguatama | 20°09'50"S, 45°43'08"W | 29 | 1 | 0B | 30 | 0.01 |
JD5482 | São Francisco River – Iguatama | 20°09'50"S, 45°43'08"W | 28 | 2 | 0B | 30 | 0.01 |
JD5483 | São Francisco River – Iguatama | 20°09'50"S, 45°43'08"W | – | 30 | 1B | 30 | 0 |
JD5486 | São Francisco River – Iguatama | 20°09'50"S, 45°43'08"W | 15 | – | 0B | 15 | 0 |
JD5490 | São Francisco River – Iguatama | 20°09'50"S, 45°43'08"W | 27 | 3 | 0B | 30 | 0.01 |
JD5497 | São Francisco River – Iguatama | 20°09'50"S, 45°43'08"W | 29 | 1 | 0B | 30 | 0.01 |
JD5502 | São Francisco River – Iguatama | 20°09'50"S, 45°43'08"W | 17 | 1 | 0B | 18 | 0.11 |
JD5604 | São Francisco River – Iguatama | 20°09'50"S, 45°43'08"W | 27 | 3 | 0B | 30 | 0.01 |
JD5605 | São Francisco River – Iguatama | 20°09'50"S, 45°43'08"W | 27 | 3 | 0B | 30 | 0.01 |
JD5619 | São Francisco River – Iguatama | 20°09'50"S, 45°43'08"W | 30 | – | 0B | 30 | 0 |
JD5620 | São Francisco River – Iguatama | 20°09'50"S, 45°43'08"W | 16 | 1 | 0B | 17 | 0.01 |
JD5517 | Pandeiros River – Januária | 15°23'28"S, 44°53'37"W | 30 | – | 0B | 30 | 0 |
JD5531 | Pandeiros River – Januária | 15°23'28"S, 44°53'37"W | 27 | 3 | 0B | 30 | 0.01 |
JD5562 | São Francisco River – Três Marias Dam | 18°08'47"S, 45°13'37"W | 25 | 5 | 0B | 30 | 0.1 |
JD5563 | São Francisco River – Três Marias Dam | 18°08'47"S, 45°13'37"W | 11 | – | 0B | 11 | 0 |
JD5565 | São Francisco River – Três Marias Dam | 18°08'47"S, 45°13'37"W | 15 | 0B | 15 | 0 | |
JD5566 | São Francisco River – Três Marias Dam | 18°08'47"S, 45°13'37"W | 15 | 1 | 0B | 16 | 0.04 |
CT4639 | São Francisco River – Três Marias Dam | 18°08'47"S, 45°13'37"W | 21 | 6 | 0B | 27 | 0.01 |
CT4640 | São Francisco River – Três Marias Dam | 18°08'47"S, 45°13'37"W | 23 | 1 | 0B | 24 | 0.04 |
CT4644 | São Francisco River – Três Marias Dam | 18°08'47"S, 45°13'37"W | 10 | – | 0B | 10 | 0 |
CT4645 | São Francisco River – Três Marias Dam | 18°08'47"S, 45°13'37"W | 10 | – | 0B | 10 | 0 |
CT4647 | São Francisco River – Três Marias Dam | 18°08'47"S, 45°13'37"W | 10 | – | 0B | 10 | 0 |
XMI=0,0292 |
Before analysis, the animals were anesthetized and euthanized with a 300 mg L-1 clove oil aqueous solution (
Because the B chromosome frequency was variable among cells within the same individual, an analysis of mitotic instability causing this variation was performed. For this purpose, we used a mitotic instability index previously developed in a migratory locust (
Microdissection was performed in an Eppendorf TransferMan NK2 micromanipulator attached to a Zeiss Axiovert 100 microscope. Ten B chromosomes were microdissected from the same P. costatus specimen (JD5483) carrying one B chromosome. The microdissected DNAs were placed in 9 μl of DNase-free ultrapure water and then fragmented and amplified using the GenomePlex Single Cell Whole Genome Amplification Kit (wga4-Sigma) (
Considering that SATH1 satDNA was described to occur on the B chromosomes of P. lineatus (
Fluorescent in situ hybridization (FISH) was performed under high stringency conditions using the method described by
The analyzed samples of P. costatus showed the expected karyotypic macrostructure of 54 chromosomes (40m + 14sm) (Fig.
Metaphases of P. costatus with conventional staining (a) and C-banding (b). Arrowheads indicate the supernumerary chromosomes. Bar = 5 μm.
Mitotically unstable B chromosomes were observed in 14 of 23 P. costatus specimens analyzed, ranging from 0 to 1 B chromosome per cell, characterizing the sixth species within Prochilodus carrying these elements (Table
The Mitotic Instability calculation resulted in an average index of 0.0292 (Table
C-banding revealed small pericentromeric heterochromatic regions in all chromosomes (Fig.
Cross-species chromosome painting showed that the BPC probe hybridized on the B chromosomes of P. costatus, P. lineatus, and P. argenteus (Fig.
Chromosome painting with BPC probe on metaphases of P. costatus (a), P. argenteus (b) and P. lineatus. Arrowheads indicate the supernumerary chromosomes. Bar = 10 μm.
FISH experiments revealed large clusters of SATH1 satDNA in the pericentromeric regions of many A-chromosomes in the three studied species. Remarkably, the largest metacentric chromosome of P. lineatus exhibited a strong signal in the pericentromeric region, differently from P. costatus and P. argenteus (Fig.
Metaphases of P. costatus (a), P. argenteus (b) and P. lineatus hybridized with SATH1 probe. The asterisks indicate the first pair of metacentric chromosomes and the arrowheads indicate the supernumerary chromosomes. Bar = 10 μm.
Cytogenetic data show a conservative trend within the family Prochilodontidae, with a diploid number of 2n=54 biarmed chromosomes (
Only eight out of the 13 valid species of Prochilodus have been karyotyped and the presence of B chromosomes was reported for six species. In this sense, B chromosomes are present in most species, except for the P. vimboides and the trans-Andean clade. However, our results suggest that these chromosomes may be absent in some populations, or their low frequency may demand higher sampling efforts. The hybridization patterns of both SATH1 satDNA and chromosome painting with the B-specific probe suggested an old and intraspecific origin of B chromosomes within this genus.
This work was supported by the Companhia Energética de Minas Gerais (CEMIG) project GT455, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp).
Alignment of three SATH1 satDNA sequences from Prochilodus lineatus genome
Data type: molecular data
Explanation note: Alignment of three SATH1 satDNA sequences from Prochilodus lineatus genome (accession no. AF363731.1, AF363732.1 and AF363734.1). Designed primers are boxed, evidencing a 563bp amplicon size.
B chromosome microdissection process and SATH1 satDNA amplification checked in 2% agarose gel
Data type: molecular data
Explanation note: Amplification patterns of the B chromosome after WGA (B-probe lane) and the SATH1 satDNA (SATH1 lane) from P. costatus genome. L: 1 Kb Plus DNA Ladder.