Research Article |
Corresponding author: Cristian Araya-Jaime ( carayaj_uls@yahoo.es ) Academic editor: Natalia Golub
© 2017 Cristian Araya-Jaime, Natalia Lam, Irma Vila Pinto, Marco A. Méndez, Patricia Iturra.
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:
Araya-Jaime C, Lam N, Pinto IV, Méndez MA, Iturra P (2017) Chromosomal organization of four classes of repetitive DNA sequences in killifish Orestias ascotanensis Parenti, 1984 (Cyprinodontiformes, Cyprinodontidae). Comparative Cytogenetics 11(3): 463-475. https://doi.org/10.3897/compcytogen.v11i3.11729
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Orestias Valenciennes, 1839 is a genus of freshwater fish endemic to the South American Altiplano. Cytogenetic studies of these species have focused on conventional karyotyping. The aim of this study was to use classical and molecular cytogenetic methods to identify the constitutive heterochromatin distribution and chromosome organization of four classes of repetitive DNA sequences (histone H3 DNA, U2 snRNA, 18S rDNA and 5S rDNA) in the chromosomes of O. ascotanensis Parenti, 1984, an endemic species restricted to the Salar de Ascotán in the Chilean Altiplano. All individuals analyzed had a diploid number of 48 chromosomes. C-banding identified constitutive heterochromatin mainly in the pericentromeric region of most chromosomes, especially a GC-rich heterochromatic block of the short arm of pair 3. FISH assay with an 18S probe confirmed the location of the NOR in pair 3 and revealed that the minor rDNA cluster occurs interstitially on the long arm of pair 2. Dual FISH identified a single block of U2 snDNA sequences in the pericentromeric regions of a subtelocentric chromosome pair, while histone H3 sites were observed as small signals scattered in throughout the all chromosomes. This work represents the first effort to document the physical organization of the repetitive fraction of the Orestias genome. These data will improve our understanding of the chromosomal evolution of a genus facing serious conservation problems.
Orestias , molecular cytogenetics, multigene families
Cytogenetic analysis is a useful tool for describing evolutionary patterns and the histories of closely-related species or species complexes. Orestias Valenciennes, 1839 is a genus of freshwater fish endemic to the South American Altiplano. The genus includes 45 species, grouped into four complexes: O. cuvieri, O. mulleri, O. gilsoni and O. agassii (
The most commonly-used approaches for comparative cytogenetic analysis of fish include characterizing the distribution and composition of constitutive heterochromatin and fluorescence in situ hybridization (FISH) mapping of molecular landmarks such as 18S and 5S ribosomal DNA. New markers of repeated elements such as histone H1, H3 and H4 genes and the U2 snRNA gene have recently been incorporated into these studies (
Orestias ascotanensis Parenti, 1984 is an endemic species restricted to the small isolated freshwater springs of the Salar de Ascotán. This fish is on the Chilean Endangered Species List (MINSEGPRES, 2008). Major threats to conservation of this species include global climate change and intense regional mining activity. Both situations contribute to a gradual lowering of the water level in the springs, potentially making the salinity of the water incompatible with life for these populations (
The aim of this study was to identify for the first time the constitutive heterochromatin distribution and chromosome organization of four classes of repetitive DNAs (histone H3 DNA, U2 snRNA and 18S and 5S rDNA) in the chromosomes of O. ascotanensis. This data will shed light on the physical organization of the repetitive fraction of the genome of O. ascotanensis, a species endemic to the Chilean Altiplano that is facing serious conservation problems. In addition, application of these cytogenetic tools will allow for comparisons among Orestias species, facilitating the identification of genomic modifications underlying the chromosomal variations observed in these species.
Eight O. ascotanensis individuals, 3 male and 5 female, were obtained from Salar de Ascotán (21°31'S 68°15'W), Region de Antofagasta, Chile, under Scientific Collection Permit Number 1103 issued by SERNAPESCA. The fish were transported to the laboratory and maintained alive in aquaria until processing. Mitotic chromosomes were obtained from kidney cell suspensions according to a modified version of the protocol established by
The constitutive heterochromatin (HC) distribution pattern was visualized according to a modified version of the protocol established by
U2 snRNA, 5S rDNA, 18S rDNA and histone H3 DNA probes were obtained from the genomic DNA of O. ascotanensis. DNA was collected from a piece of fin tissue with the Wizard Genomic DNA Purification Kit (Promega) according to manufacturer instructions, using previously-described primers (Table
Primers used to PCR amplification for gene fragments 5S rDNA, 18S rDNA, U2 snRNA and Histone H3.
Gene | Primers sequences | References |
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5S rDNA | 5SA 5’-TCAACCAACCACAAAGACATTGGCAC-3’ | Pendás et al. 1994 |
5SB 5’-TAGACTTCTGGGTGGCCAAAGGAATCA-3’ | ||
18S rDNA | 18SF 5’-GTAGTCATATGCTTGTCTC-3’ |
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18SR 5’-TCCGCAGGTTCACCTACGGA-3’ | ||
U2snRNA | U2 F 5’-ATCGCTTCTCGGCCTTATG-3’ |
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U2 R 5’-TCCCGGCGGTACTGCAATA-3’ | ||
Histone H3 | H3F 5’- ATATCCTTRGGCATRATRGTGAC-3’ |
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H3R 5’- ATGGCTCGTACCAAGCAGACVGC-3’ |
All O. ascotanensis individuals analyzed had a diploid number of 48 chromosomes, consistent with the chromosome formula defined by
Dual FISH detected 18S and 5S rDNA probes on different chromosome pairs (Fig.
Karyotype of O. ascotanensis (female), 2n=48. A Giemsa-stained karyotype and CMA3 positive bands (box) B C-banded somatic metaphase C metaphases counterstained with DAPI after FISH treatment using 5S and 18S rDNA probes D metaphases counterstained with U2 snDNA/histone H3 DNA probes. The arrows show a block of HC on the short arm of pair 3. Bar = 10µm.
Previous cytogenetic studies involving the seven species of O. agassii complex of the Chilean Altiplano were limited to characterizing the chromosome number and morphology of the species. The diploid number has been reported to vary between 48 and 55 chromosomes and the fundamental number of chromosome arms (FN) between 54 and 56 (
Characterization of the repetitive fraction of the genome is a useful tool for identifying recent genomic changes during the evolutionary process as well as possible hotspots associated with chromosomal rearrangements (
In O. ascotanensis, the C-band regions were found mainly in the pericentromeric regions, unlike other Cyprinodontiformes that have been studied. CMA3 also revealed that the conspicuous blocks of HC found in the short arm of pair 3 have a higher proportion of GC bases than previously-analyzed fish. Moreover, the presence of 18S rDNA sequences in this chromosome arm defines this pair as the carrier of the NOR. An association between 18S and 28S rDNA sequences and heterochromatin has been found in other fish, such as salmonids (
The single 18S rDNA sequence-bearing chromosome pair in O. ascotanensis (Fig.
Data on the physical location of U2 snRNA sites in fish is also scarce. Two general configurations are recognized: (I) clustered on a single pair of chromosomes, as in the present case and (II) scattered throughout the genome (
The finding of scattered histone H3 sites distributed throughout the O. ascotanensis chromosomes diverges strongly from data reported for other fish, such as Characiformes (
To understand the relationship of these repeated genomic elements to the chromosomal evolution of these fish and to historical changes in the fishes’ environment, further studies are needed to physically map the repetitive DNA in other Orestias representatives. These findings would enhance our understanding of native wildlife species facing serious conservation problems.
This research work was financed by projects Fondecyt 1080390 and Fondecyt 1140543.