Repetitive DNAs and shrink genomes: A chromosomal analysis in nine Columbidae species (Aves, Columbiformes)

Rafael Kretschmer Thays Duarte de Oliveira Ivanete de Oliveira Furo Fabio Augusto Oliveira Silva Ricardo José Gunski Analía del Valle Garnero Marcelo de Bello Cioffi Edivaldo Herculano Corrêa de Oliveira Thales Renato Ochotorena de Freitas About the authors

Abstract

An extensive karyotype variation is found among species belonging to the Columbidae family of birds (Columbiformes), both in diploid number and chromosomal morphology. Although clusters of repetitive DNA sequences play an important role in chromosomal instability, and therefore in chromosomal rearrangements, little is known about their distribution and amount in avian genomes. The aim of this study was to analyze the distribution of 11 distinct microsatellite sequences, as well as clusters of 18S rDNA, in nine different Columbidae species, correlating their distribution with the occurrence of chromosomal rearrangements. We found 2n values ranging from 76 to 86 and nine out of 11 microsatellite sequences showed distinct hybridization signals among the analyzed species. The accumulation of microsatellite repeats was found preferentially in the centromeric region of macro and microchromosomes, and in the W chromosome. Additionally, pair 2 showed the accumulation of several microsatellites in different combinations and locations in the distinct species, suggesting the occurrence of intrachromosomal rearrangements, as well as a possible fission of this pair in Geotrygon species. Therefore, although birds have a smaller amount of repetitive sequences when compared to other Tetrapoda, these seem to play an important role in the karyotype evolution of these species.

Keywords:
Birds; FISH; microsatellites; sex chromosomes; chromosomal rearrangements

Introduction

Columbiformes is one of the most easily recognized bird orders in the world, with more than 300 species and traditionally divided into two families: Columbidae (pigeons and doves) and Raphidae (Pereira et al., 2007Pereira SL, Johnson KP, Clayton DH and Baker AJ (2007) Mitochondrial and nuclear DNA sequences support a cretaceous origin of Columbiformes and a dispersal driven radiation in the paleogene. Syst Biol 56:656-672.). Three large clades are supported on Columbiformes, referred to as A, B, and C by Pereira et al. (2007)Pereira SL, Johnson KP, Clayton DH and Baker AJ (2007) Mitochondrial and nuclear DNA sequences support a cretaceous origin of Columbiformes and a dispersal driven radiation in the paleogene. Syst Biol 56:656-672., based on mitochondrial and nuclear DNA data. Clade A is subdivided into two well-supported subclasses: one referring exclusively to America genera and the other includes pigeons and turtle doves from the Old and New Worlds. Clade B groups only New World pigeon species and Clade C includes many genera found in Africa, Asia, Australia, the East Indies, and New Zealand.

Cytogenetic studies based mainly on conventional staining have shown an interesting variation in diploid number, which ranges from 76 to 86 (Takagi and Sasaki, 1974Takagi N and Sasaki M (1974) A phylogenetic study of bird karyotypes. Chromosoma 46:91-120.; de Lucca and de Aguiar, 1976de Lucca EJ and de Aguiar MLR (1976) Chromosomal evolution in Columbiformes (Aves). Caryologia 29:59-68.; de Lucca, 1984de Lucca EJ (1984) Chromosomal evolution of South American Columbiformes (Aves). Genetica 62:177-185.). Other aspects of their karyotypical organization remain unknown, although the observed variation in chromosome morphology suggests the occurrence of intra- and interchromosomal rearrangements (de Lucca, 1984de Lucca EJ (1984) Chromosomal evolution of South American Columbiformes (Aves). Genetica 62:177-185.).

There is evidence supporting that some groups of vertebrates with a high metabolic demand have smaller cells, and as consequence, smaller genomes (Szarski, 1983Szarski H (1983) Cell size and the concept of wasteful and frugal evolutionary strategies. J Theor Biol 105:201-209.). In accordance with this hypothesis, the relationship between flying and the reduced genome size of birds, bats and possibly pterosaurs, has been interpreted as an evidence that the high energetic demand of flying exerted selective pressures for small cells and small genomes (Hughes and Hughes, 1995Hughes AL and Hughes MK (1995) Small genomes for better flyers. Nature 377:391.; Organ and Shedlock, 2009Organ CL and Shedlock AM (2009) Palaeogenomics of pterosaurs and the evolution of small genome size in flying vertebrates. Biol Lett 5:47-50.; Zhang and Edwards, 2012Zhang Q and Edwards SV (2012) The evolution of intron size in amniotes: a role for powered flight? Genome Biol Evol 4:1033-1043.). Conformingly, birds have the lowest average genome sizes among Tetrapoda (Andrews et al., 2009Andrews CB, Mackenzie SA and Gregory TR (2009) Genome size and wing parameters in passerine birds. Proc R Soc B 276:55-61.) while bats show the smallest genomes when compared to most Mammalian species (Smith and Gregory, 2009Smith JDL and Gregory TR (2009) The genome sizes of megabats (Chiroptera: Pteropodidae) are remarkably constrained. Biol Lett 5:347-351.). In addition, humming birds have the smallest genomes among birds, probably associated with their intense necessity of energy to hover during flight (Gregory et al., 2009Gregory TR, Andrews CB, McGuire JA and Witt CC (2009) The smallest avian genomes are found in hummingbirds. Proc R Soc B 276:3753-3757.).

Repetitive DNAs represent an important proportion of the genome in eukaryotes, being composed by sequences in tandem (satellites, minisatellites and microsatellites) and transposable elements (transposons and retrotransposons) (Charlesworth et al., 1994Charlesworth B, Snlegowski P and Stephan W (1994) The evolutionary dynamics of repetitive DNA in eukaryotes. Nature 371:215-220.; López-Flores and Garrido-Ramos, 2012López-Flores I and Garrido-Ramos MA (2012) The repetitive DNA content of eukaryotic genomes. Genome Dyn 7:1-28.). These repetitive sequences play an important role in genome evolution in eukaryotes (Biémont and Vieira, 2006López-Flores I and Garrido-Ramos MA (2012) The repetitive DNA content of eukaryotic genomes. Genome Dyn 7:1-28.). For example, it was proposed that the genome evolution in mammals has been driven by chromosomal rearrangements in fragile sites, composed by in tandem repetitive sequences (Ruiz-Herrera et al., 2006Ruiz-Herrera A, Castresana J and Robinson TJ (2006) Is mammalian chromosomal evolution driven by regions of genome fragility? Genome Biol 7:R115.). In addition, transposable elements can also influence the occurrence of chromosomal rearrangements by inducing chromosomal breakage (Biémont and Vieira, 2006Biémont C and Vieira C (2006) Genetics: Junk DNA as an evolutionary force. Nature 443:521-524.).

An important class of repetitive sequences is formed by the microsatellites, small sequences (1–6 base pairs) repeated in tandem and dispersed through the genome. Mono-, di-, tri-, and tetranucleotide repetitions are the most common types of microsatellites (Ellegren, 2004Ellegren H (2004) Microsatellites: simple sequences with complex evolution. Nat Rev Genet 5:435-445.). Mutation rates in these sequences are 10-100,000 folds higher than the mean of other genome regions, making them important markers for genetic variability studies of natural and captive populations (Gemayel et al., 2010Gemayel R, Vinces MD, Legendre M and Verstrepen KJ (2010) Variable tandem repeats accelerate evolution of coding and regulatory sequences. Annu Rev Genet 44:445-477.). Cytogenetic mapping of these sequences has also contributed to a better comprehension of sex chromosome evolution and chromosomal differentiation, and have been extensively analyzed in fishes (Cioffi and Bertollo, 2012Cioffi MB and Bertollo LAC (2012) Chromosomal distribution and evolution of repetitive DNAs in fish. Genome Dyn 7:197-221.). In general, repetitive sequences accumulate preferentially in centromeric and heterochromatic regions, as observed in many fishes (Cioffi et al., 2012Cioffi MB, Kejnovsky E, Marquioni V, Poltronieri J, Molina WF, Diniz D and Bertollo LAC (2012) The key role of repeated DNAs in sex chromosome evolution in two fish species with ZW sex chromosome system. Mol Cytogenet 5:28.), lizards (Pokorná et al., 2011Pokorná M, Kratochvíl L and Kejnovsky E (2011) Microsatellite distribution on sex chromosomes at different stages of heteromorphism and heterochromatinization in two lizard species (Squamata: Eublepharidae: Coleonyx elegans and Lacertidae: Eremias velox). BMC Genet 12:90.) and plant species (Kejnovsky et al., 2013Kejnovsky E, Michalovova M, Steflova P, Kejnovska I, Manzano S, Hobza R, Kubat Z, Kovarik J, Jamilena M and Vyskot B (2013) Expansion of microsatellites on evolutionary young Y chromosome. PLoS One 8:e45519.). However, little is known about the dynamic of repetitive sequences in birds. In sauropsids (reptiles and birds), many microsatellites have been intensely amplified in sex chromosomes Y/W in seven species (six reptiles and Gallus gallus), associated to the differentiation and heterochromatinization of these chromosomes (Matsubara et al., 2015Matsubara K, O’Meally D, Azad B, Georges A, Sarre SD, Graves JAM, Matsuda Y and Ezaz T (2015) Amplification of microsatellite repeat motifs is associated with the evolutionary differentiation and heterochromatinization of sex chromosomes in Sauropsida. Chromosoma 125:111-123.).

Recently, distinct hybridization patterns of microsatellite sequences have been demonstrated in species of two different orders of birds (de Oliveira et al., 2017Furo IO, Kretschmer R, dos Santos MS, Carvalho CAL, Gunski RJ, O’Brien PCM, Ferguson-Smith MA, Cioffi MB and de Oliveira EHC (2017) Chromosomal mapping of repetitive DNAs in Myiopsitta monachus and Amazona aestiva (Psittaciformes, Psittacidae: Psittaciformes), with emphasis on the sex chromosomes. Cytogenet Genome Res 151:151-160.; Furo et al., 2017Furo IO, Kretschmer R, dos Santos MS, Carvalho CAL, Gunski RJ, O’Brien PCM, Ferguson-Smith MA, Cioffi MB and de Oliveira EHC (2017) Chromosomal mapping of repetitive DNAs in Myiopsitta monachus and Amazona aestiva (Psittaciformes, Psittacidae: Psittaciformes), with emphasis on the sex chromosomes. Cytogenet Genome Res 151:151-160.). In Piciformes, a large accumulation of 10 sequences was observed on autosomes and especially on the Z sex chromosome in three woodpecker species (Picidae). The Z chromosome corresponds to the larger element of their karyotype due to the accumulation of such sequences, which increased its size (de Oliveira et al., 2017de Oliveira TD, Kretschmer R, Bertocchi NA, Degrandi TM, de Oliveira EHC, Cioffi MB, Garnero ADV and Gunski RJ (2017) Genomic organization of repetitive DNA in woodpeckers (Aves, Piciformes): Implications for karyotype and ZW sex chromosome differentiation. PLoS One 12:e0169987.). On the other hand, in Myiopsitta monachus (Psittaciformes, Psittacidae) these sequences accumulated preferentially in the W sex chromosome, which has the same size of the Z chromosome, unlike most Neognathae bird species (Furo et al., 2017Furo IO, Kretschmer R, dos Santos MS, Carvalho CAL, Gunski RJ, O’Brien PCM, Ferguson-Smith MA, Cioffi MB and de Oliveira EHC (2017) Chromosomal mapping of repetitive DNAs in Myiopsitta monachus and Amazona aestiva (Psittaciformes, Psittacidae: Psittaciformes), with emphasis on the sex chromosomes. Cytogenet Genome Res 151:151-160.). These two examples show that the analysis and mapping of repetitive sequences in the genome of avian species may contribute for a better understanding of the processes underlying sex chromosomes differentiation and karyotype evolution.

Thus, the analysis of microsatellite sequences in groups of birds showing chromosomal variation both in diploid number and chromosomal morphology, such as Columbiformes, may bring important information concerning their karyotypical evolution. In this study, we report the chromosomal mapping of different repetitive sequences, including 18S rDNA clusters and 11 different microsatellite sequences in Columbidae species in order to verify the role of these sequences in their karyotypical diversity. The results suggest that, despite their lower amount in the genome, repetitive DNAs seem to play an important role in the karyotype evolution of these species.

Material and Methods

Specimens and chromosome preparations

Nine species of Columbidae family were analyzed in this study. Individuals were collected in their natural habitat, except for G. montana and G. violacea, which were collected from captivity (Table 1). Experiments followed protocols approved by the Ethics Committee on the Use of Animals (CEUA - Universidade Federal do Pampa, 026/2012, and permission number SISBIO 33860-1 and 44173-1).

Table 1
Information concerning the individual samples used for this study.

Chromosomes were obtained from fibroblast cultures, according to Sasaki et al. (1968)Sasaki M, Ikeuchi T and Maino S (1968) A feather pulp culture for avian chromosomes with notes on the chromosomes of the peafowl and the ostrich. Experientia 24:1923-1929. or from bone marrow, following Garnero and Gunski (2000)Garnero AV and Gunski RJ (2000) Comparative analysis of the karyotype of Nothura maculosa and Rynchotus rufescens (Aves: Tinamidae). A case of chromosomal polymorphism. Nucleus 43:64-70.. Both techniques included exposition to colcemid (1 h, 37 ºC), hypotonic treatment (0.075 M KCl, 15 min, 37 ºC) and fixation with methanol/acetic acid (3:1).

Chromosome probes and FISH experiments

18S rDNA fragments were amplified by PCR using primers NS1 5’-GTA GTC ATA TGC TTG TCT C-3’ and NS8 5’-TCC GCA GGT TCA CCT ACG GA-3’ and nuclear DNA of Ocyurus chrysurus (Perciformes: Lutjanidae) (White et al., 1990White TJ, Bruns T, Lee S and Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Shinsky JJ and White TJ (eds) PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, pp 315-322.). Subsequently, fragments were labeled with digoxigenin by nick translation (Roche) and detected with anti-digoxigenin-rhodamine, following the manufacturer’s instructions. Preparation of slides, hybridization and washes were performed according to Daniels and Delany (2003)Daniels LM and Delany ME (2003) Molecular and cytogenetic organization of the5S ribosomal DNA array in chicken (Gallus gallus). Chromosome Res 11:305-317..

FISH experiments using microsatellite probes were done according to Kubat et al. (2008)Kubat Z, Hobza R, Vyskot B and Kejnovsky E (2008) Microsatellite accumulation in the Y chromosome in Silene latifolia. Genome 51:350-356.. Oligonucleotides (CA)15, (CAA)10, (CAC)10, (CAG)10, (CAT)10, (CG)15, (CGG)10, (GA)15, (GAA)10, (GAG)10 and (TA)15, directly labeled with Cy3 at the 5terminal were obtained from SIGMA. After denaturation, probes were applied on the slides and incubated for 16 h at 37 ºC in a humid chamber. Next, slides were washed twice in 2xSSC, twice in 1xSSC, and in PBS (phosphate buffered saline), and then dehydrated in an ascending ethanol series (70, 90 and 100%).

At least 30 metaphase spreads were analyzed to confirm the 2n, karyotype structure and FISH results. Images were captured using a Zeiss Imager Z2, coupled with the software Axiovison 4.8 (Zeiss, Germany). The chromosomes were classified as metacentric (m), submetacentric (sm), telocentric (t) or acrocentric (a) according to their arm ratios (Guerra, 1986Guerra MS (1986) Reviewing the chromosome nomenclature of Levan et al. Rev Bras Genet 9:741-743.).

Results

Diploid number and chromosomal morphology of the species analyzed are described in Table 2. Figures 1 and 2 show the karyotypes in conventional staining. We found a morphological variation in the Z chromosome of L. verreauxi, which corresponded to a submetacentric or acrocentric element (Figure 1). Additionally, pair 3 also showed morphological variation in G. montana as telocentric and acrocentric (Figure 2b).

Table 2
Diploid number and chromosomal morphology of the nine Columbidae species included in this study.
Figure 1
Partial karyotype showing the largest autosomal pairs and ZW sex chromosomes of three Leptotila verreauxi individuals analyzed by conventional Giemsa-staining: (a) male with a submetacentric and acrocentric Z chromosomes; (b) female with submetacentric Z and W chromosomes, (c) female with an acrocentric Z and a submetacentric W chromosome. Sex chromosomes are boxed. Bar = 5 μm.
Figure 2
Partial karyotype showing the largest autosomal pairs and ZW sex chromosomes of eight Columbidae analyzed by conventional Giemsa-staining: (a) Zenaida auriculata, male; (b) Geotrygon montana, male; (c) Geotrygon violacea, female; (d), Columba livia, female; (e) Patagioenas cayennensis, male; (f) Columbina talpacoti, female; (g) Columbina passerina, male; (h) Columbina picui, male. Sex chromosomes are boxed. Bar = 5 μm.

18S rDNA probes hybridized onto microchromosomes in the nine species analyzed here. In Z. auriculata, G. montana, G. violacea, L. verreauxi, P. cayennensis, C. livia, C. talpacoti and C. passerina this sequences were detected in only one microchromosome pair, however, in C. picui these probes revealed the presence of clusters in three pairs of microchromosomes. Examples of 18S rDNA hybridization in the Columbidae are shown in Figure 3.

Figure 3
Representative examples of FISH experiments using 18S rDNA probes in Columbidae species. (a) L. verreauxi; (b) Z. auriculata; (c) C. livia; (d) C. picui. The arrows point to the hybridization signals. Bar = 5 μm.

Chromosome mapping of microsatellite sequences

Of the nine species analyzed, only C. picui showed no hybridization signals for the microsatellite sequences used. In this species, we performed the hybridizations with chromosomal preparations obtained from two distinct protocols, fibroblasts and direct culture of bone marrow and obtained the same negative result. The other species showed an exclusive pattern of distribution for at least some of the microsatellite sequences used (Table 3). In general, these sequences were preferentially accumulated in the centromeric region of some macrochromosome pairs, in microchromosomes and in the W chromosome. There was no evident signal in the Z chromosome of any species. In addition, pair 2 showed an interesting accumulation of some sequences, of which the position varied in some species – a single band in the short arms in Z. auriculata, C. passerina and C. talpacoti, a single band in the long arms in L. verreauxi, G. montana and P. cayennensis, and two bands (GA15) in the short arms in P. cayennensis. The highest number of sequences was found in L. verreauxi (Figure 4). Representative experiments of other species are shown in Figure 5.

Table 3
Hybridization of 11 microsatellite sequences in nine Columbidae species.
Figure 4
Metaphases of a female Leptotila verreauxi in experiments of FISH using nine different microsatellite sequences (a-i). Chromosomes were counterstained with DAPI (blue) and probes were directly Cy3 (red) labeled. Microsatellite sequences are indicated on the bottom left of each figure. Sex chromosomes are indicated in each metaphase. Bar = 5 μm.
Figure 5
Representative examples of FISH experiments using microsatellite sequences in six Columbidae species (a-f). Probes were directly labeled with Cy3 (red), while chromosomes were counterstained with DAPI (blue). Microsatellite sequences are indicated on the bottom left of each figure. Sex chromosomes are indicated in each metaphase. ZAU: Zenaida auriculata (a); GMO: Geotrygon montana (b); GVI: Geotrygon violacea (c); CLI: Columba livia (d); PCA: Patagioenas cayennensis (e); CPA: Columbina passerina (f). Sex chromosomes are indicated in each metaphase. Bar = 5 μm.

Discussion

Corroborating previous studies (Takagi and Sasaki, 1974Takagi N and Sasaki M (1974) A phylogenetic study of bird karyotypes. Chromosoma 46:91-120.; de Lucca and de Aguiar, 1976de Lucca EJ and de Aguiar MLR (1976) Chromosomal evolution in Columbiformes (Aves). Caryologia 29:59-68.; de Lucca, 1984de Lucca EJ (1984) Chromosomal evolution of South American Columbiformes (Aves). Genetica 62:177-185.) we observed a variation in the 2n number of the Columbidae species analyzed, ranging from 76 (Z. auriculata, C. picui, C. passerina, P. cayennensis and C. talpacoti) to 86 (G. violacea and G. montana) L. verreauxi and C. livia showed an intermediate 2n (78 and 80, respectively). Among the species, the karyotype of G. violacea was described for the first time, showing that this species has a karyotype very similar to another species of this genus, G. montana, both in terms of chromosome morphology and in the diploid number.

In birds, it is accepted that the presence of one pair of microchromosomes bearing 18S rDNA clusters is the ancestral state, considering that this is the condition observed in basal groups, such as Ratites and Galloanserae (Ladjali-Mohammedi et al., 1999Ladjali-Mohammedi K, Bitgood JJ, Tixier-Boichard M and Ponce de Leon FA (1999) International System for Standardized Avian Karyotypes (ISSAK): Standardized banded karyotypes of the domestic fowl (Gallus domesticus). Cytogenet Cell Genet 86:271-276.; Nishida-Umehara et al., 2007Nishida-Umehara C, Tsuda Y, Ishijima J, Ando J, Fujiwara A, Matsuda Y and Griffin DK (2007) The molecular basis of chromosome orthologies and sex chromosomal differentiation in palaeognathous birds. Chromosome Res 15:721-734.), and also in many species belonging to more derived groups, such as some Passeriformes and Accipitriformes (Tagliarini et al., 2011Tagliarini MM, O’Brien PCM, Ferguson-Smith MA and de Oliveira EHC (2011) Maintenance of syntenic groups between Cathartidae and Gallus gallus indicates symplesiomorphic karyotypes in new world vultures. Genet Mol Biol 34:80-83.; dos Santos et al., 2015dos Santos MS, Kretschmer R, Silva FA, Ledesma MA, O’Brien PC, Ferguson-Smith MA, Garnero ADV, de Oliveira EHC and Gunski RJ (2015) Intrachromosomal rearrangements in two representatives of the genus Saltator (Thraupidae, Passeriformes) and the occurrence of heteromorphic Z chromosomes. Genetica 143:535-543.). This characteristic seems to be conserved also in Columbiformes, since, with the exception of Columbina picui, which showed three pairs of microchromosomes bearing 18S rDNA clusters, the other eight species analyzed presented only one microchromosome pair bearing these clusters, including two other Columbina species. One of the most accepted causes of this variation, even among phylogenetically related species, is the transposition or translocation of these sequences (Nishida et al., 2008Nishida C, Ishijima J, Kosaka A, Tanabe H, Habermann FA, Griffin DK and Matsuda Y (2008) Characterization of chromosome structures of Falconinae (Falconidae, Falconiformes, Aves) by chromosome painting and delineation of chromosome rearrangements during their differentiation. Chromosome Res 16:171-181.; Kretschmer et al., 2014Kretschmer R, Gunski RJ, Garnero ADV, Furo IO, O’Brien PCM, Ferguson-Smith MA and de Oliveira EHC (2014) Molecular cytogenetic characterization of multiple intrachromosomal rearrangements in two representatives of the genus Turdus (Turdidae, Passeriformes). PLoS One 9:e103338.).

Considering the microsatellite sequences, we applied eleven different oligonucleotide probes, which gave different results for each species, demonstrating that the analysis of these repetitive sequences may represent an important chromosome marker in evolutionary and phylogenetic studies in birds. Only one species, C. picui, did not show a signal for any of the sequences used. A possible explanation is that microsatellites have a characteristic mutational behavior, with rates that are 10 to 100,000 times higher than the average mutation rates in other parts of the genome (Gemayel et al., 2010Gemayel R, Vinces MD, Legendre M and Verstrepen KJ (2010) Variable tandem repeats accelerate evolution of coding and regulatory sequences. Annu Rev Genet 44:445-477.). Therefore, a microsatellite sequence can expand (addition of repeat units) or contract (deletion of repeat units) (López-Flores and Garrido-Ramos, 2012López-Flores I and Garrido-Ramos MA (2012) The repetitive DNA content of eukaryotic genomes. Genome Dyn 7:1-28.). It is possible that contraction of the microsatellites sequences occurred in C. picui, so the probes used were not complementary to the new sequence, considering the limitations inherent to FISH techniques, which needs at least 2–5 kb to be visible.

Accumulation of microsatellites in pair 2 was observed in practically all species, (the exceptions were C. livia and C. picui), although in different positions (Figure 6), probably due to intrachromosomal rearrangements, such as inversions, which are very frequent among birds (Warren et al., 2010Warren WC, Clayton DF, Ellegren H, Arnold AP, Hillier LW, Künstner A, Searle S, White S, Vilella AJ, Fairley S, et al. (2010) The genome of a songbird. Nature 464:757-762.; Kretschmer et al., 2014Kretschmer R, Gunski RJ, Garnero ADV, Furo IO, O’Brien PCM, Ferguson-Smith MA and de Oliveira EHC (2014) Molecular cytogenetic characterization of multiple intrachromosomal rearrangements in two representatives of the genus Turdus (Turdidae, Passeriformes). PLoS One 9:e103338., 2015Kretschmer R, de Oliveira EHC, dos Santos MS, Furo IO, O’Brien PCM, Ferguson-Smith MA, Garnero ADV and Gunski RJ (2015) Chromosome mapping of the large elaenia (Elaenia spectabilis): evidence for a cytogenetic signature for passeriform birds? Biol J Linn Soc 115:391-398.; dos Santos 2015dos Santos MS, Kretschmer R, Silva FA, Ledesma MA, O’Brien PC, Ferguson-Smith MA, Garnero ADV, de Oliveira EHC and Gunski RJ (2015) Intrachromosomal rearrangements in two representatives of the genus Saltator (Thraupidae, Passeriformes) and the occurrence of heteromorphic Z chromosomes. Genetica 143:535-543., 2017Furo IO, Kretschmer R, dos Santos MS, Carvalho CAL, Gunski RJ, O’Brien PCM, Ferguson-Smith MA, Cioffi MB and de Oliveira EHC (2017) Chromosomal mapping of repetitive DNAs in Myiopsitta monachus and Amazona aestiva (Psittaciformes, Psittacidae: Psittaciformes), with emphasis on the sex chromosomes. Cytogenet Genome Res 151:151-160.). Interestingly, while (GGA)10 produced signals in pair 2 of Zenaida auriculata, this sequence did not produce any signal in the two species of the genus Geotrygon. Instead, the sequence (GA)15 hybridized in pair 2 of G. montana and G. violacea. In the remaining species, a higher number of sequences accumulated in pair 2: L. verreauxi [(CA)15, (GA)15, (GAA)10, (CAC)10, (CGG)10 and (GAG)10]; P. cayennensis [(CA)15, (GA)15, (GAA)10 and (GAG)10]; C. talpacoti [(CA)15, (GA)15, (GAA)10 and (CAC)10], and; C. passerina [(CA)15, (GA)15, (GAA)10 and (CAC)10].

Figura 6
Distribution and localization of microsatellite sequences in chromosome 2 of seven Columbidae species: ZAU (Zenaida auriculata), LVE (Leptotila verreauxi), PCA (Patagioenas cayennensis), GVI (Geotrygon violacea), GMO (Geotrygon montana), CTA (Columbina talpacoti) and CPA (Columbina passerina).

From a phylogenetic point of view, the occurrence of the same sequences found in the same position in pair 2 of different species could be a reflection of a common origin, as for example the sequences (CA)15, (GA)15, (GAA)10 and (CAC)10 in the species L. verreauxi, C. talpacoti, and C. passerina, and the three first ones in P. cayennensis. Furthermore, a more detailed analysis of these sequences in pair 2 of Columbidae species revealed that this pair is very informative about the karyotypical evolution in this group.

For instance, the presence of (GA)15 in pair 2 of Geotrygon species, which is telocentric in this species but submetacentric in most of the other ones, suggests the occurrence of a chromosomal rearrangement, such as an inversion or fission in this pair. However, if we consider that the 2n of Geotrygon is higher than that for the other species (2n=86), with pair 2 being slightly smaller (Figure 1), it seems that fission is the most probable rearrangement to have occurred in this genus. Moreover, the sequence (GA)15 hybridized in two different bands in the long arms of pair 2 in P. cayennensis, probably due to an inversion, which fragmented the block of repetitive sequences in two distinct ones. Similarly, the variation in the position of these repetitive sequences blocks in chromosome 2 – 2p in C. passerina and C. talpacoti, while 2q in L. verreauxi, G. montana, G. violacea, P. cayennensis – adds evidence for the occurrence of intrachromosomal rearrangements. A possible approach to test this hypothesis is the use of whole-chromosome probes of a species in which the syntenic group corresponding to GGA1 is found fragmented, such as Leucopternis albicollis (Falconiformes, Accipitridae), in which GGA2 corresponds to three different pairs (de Oliveira et al., 2010de Oliveira EHC, Tagliarini MM, Rissino JD, Pieczarka JC, Nagamachi CY, O’Brien PC and Ferguson-Smith MA (2010) Reciprocal chromosome painting between white hawk (Leucopternis albicollis) and chicken reveals extensive fusions and fissions during karyotype evolution of Accipitridae (Aves, Falconiformes). Chromosome Res 18:349-355.).

The importance of repetitive sequences in chromosomal instability has been proposed by some authors (e.g. Ruiz-Herrera et al., 2006Ruiz-Herrera A, Castresana J and Robinson TJ (2006) Is mammalian chromosomal evolution driven by regions of genome fragility? Genome Biol 7:R115.). For example, the molecular characterization of evolutionary breakpoints in the genome of humans, primates and mouse has demonstrated that the genomic reorganizations mainly occur in regions with duplications or with some type of repetitive sequences, such as the dinucleotide (TA)n, or close to these regions (Kehrer-Sawatzki et al., 2005Kehrer-Sawatzki H, Sandig CA, Goidts V and Hameister H (2005) Breakpoint analysis of the pericentric inversion between chimpanzee chromosome 10 and the homologous chromosome 12 in humans. Cytogenet Genome Res 108:91-97.; Fan et al., 2002Fan Y, Linardopoulou E, Friedman C, Williams E and Trask BJ (2002) Genomic structure and evolution of the ancestral chromosome fusion site in 2q13-2q14.1 and paralogous regions on other human chromosomes. Genome Res 12:1651-1662.; Kehrer-Sawatzki et al., 2002Kehrer-Sawatzki H, Schreiner B, Tanzer S, Platzer M, Muller S and Hameister H (2002) Molecular characterization of the pericentric inversion that causes differences between chimpanzee chromosome 19 and human chromosome 17. Am J Hum Genet 71:375-388.; Locke et al., 2003Locke DP, Archidiacono N, Misceo D, Cardone MF, Deschamps S, Roe B, Rocchi M and Eichler EE (2003) Refinement of a chimpanzee pericentric inversion breakpoint to a segmental duplication cluster. Genome Biol 4:R50.). Although there is no single sequence responsible for the chromosomal instability, it is known that common fragile sites are enriched with A/T sequences and have the potential to form secondary structures (Schwartz et al., 2006Schwartz M, Zlotorynski E and Kerem B (2006) The molecular basis of common and rare fragile sites. Cancer Lett 232:13-26.; Glover, 2006Glover TW (2006) Common fragile sites. Cancer Lett 232:4-12.). These features may affect the DNA replication and lead to chromosomal instability (Ruiz-Herrera et al., 2006Ruiz-Herrera A, Castresana J and Robinson TJ (2006) Is mammalian chromosomal evolution driven by regions of genome fragility? Genome Biol 7:R115.). Interestingly, the dinucleotide (TA)15 did not produce any positive signals in our studies, revealing a possible characteristic intrinsic to the genome of birds. Although the absence of signals may reflect not only the inexistence of clusters of this sequence, it may instead represent a lower number of repetitions, considering the limitations inherent to FISH techniques, which needs at least 2–5 kb to be visible. This lower number of repetitions may be related to the small size of the genome of birds, at the expense of loss of repetitive sequences (Hughes and Hughes, 1995Hughes AL and Hughes MK (1995) Small genomes for better flyers. Nature 377:391.; Organ and Shedlock, 2009Organ CL and Shedlock AM (2009) Palaeogenomics of pterosaurs and the evolution of small genome size in flying vertebrates. Biol Lett 5:47-50.; Zhang and Edwards, 2012Zhang Q and Edwards SV (2012) The evolution of intron size in amniotes: a role for powered flight? Genome Biol Evol 4:1033-1043.).

Concerning sex chromosomes, it is widely accepted that the accumulation of repetitive sequences plays an important role in the differentiation of the element found exclusively in the heterogametic sex – W or Y (Matsubara et al., 2015Matsubara K, O’Meally D, Azad B, Georges A, Sarre SD, Graves JAM, Matsuda Y and Ezaz T (2015) Amplification of microsatellite repeat motifs is associated with the evolutionary differentiation and heterochromatinization of sex chromosomes in Sauropsida. Chromosoma 125:111-123.). For instance, none of the sequences produced any signals in the Z chromosome, while different sequences were found accumulated in the W chromosome of the three species of which we analyzed female individuals: C. livia [(CAA)10, (CGG)10, (GA)15 and (GAG)10]; G. violacea [(GA)15 and (GAG)10], and L. verreauxi [(CA)15, (CAA)10, (CGG)10, (CAC)10, (GAG)10, (GAA)10 and (GA)15]. Of these, two were also found in the W chromosome in Gallus gallus: sequences (GA)15 and (GAG)10 (Matsubara et al., 2015Matsubara K, O’Meally D, Azad B, Georges A, Sarre SD, Graves JAM, Matsuda Y and Ezaz T (2015) Amplification of microsatellite repeat motifs is associated with the evolutionary differentiation and heterochromatinization of sex chromosomes in Sauropsida. Chromosoma 125:111-123.). Interestingly, these two sequences were shared by the three Columbidae species, possibly denoting some type of ancestral state. In fact, microsatellites are considered early colonizers of sex chromosomes and the differential accumulation of the same class of repeats on the W chromosome of distinct species reflects the inherent dynamism of these sequences (Charlesworth et al., 2005Charlesworth D, Charlesworth B and Marais G (2005) Steps in the evolution of heteromorphic sex chromosomes. Heredity 95:118-128.).

In summary, this study demonstrated the ubiquitous presence of repetitive elements in the genome of several Columbidae species, highlighting their possible role in the chromosomal diversification within this group. In addition, our data reinforced the view that the existence of one pair of microchromosomes bearing 18S rDNA clusters is apparently an ancestral character retained in Columbidae, and that repetitive sequences did preferentially accumulate in the centromeric regions of macro and microchromosomes, as well as in the W chromosomes. Additionally, despite the fact that studies with repetitive sequences in birds are still incipient, the comparison of our data with the ones for Psittaciformes, Piciformes and Galliformes (Matsubara et al., 2015Matsubara K, O’Meally D, Azad B, Georges A, Sarre SD, Graves JAM, Matsuda Y and Ezaz T (2015) Amplification of microsatellite repeat motifs is associated with the evolutionary differentiation and heterochromatinization of sex chromosomes in Sauropsida. Chromosoma 125:111-123.; de Oliveira et al., 2017de Oliveira TD, Kretschmer R, Bertocchi NA, Degrandi TM, de Oliveira EHC, Cioffi MB, Garnero ADV and Gunski RJ (2017) Genomic organization of repetitive DNA in woodpeckers (Aves, Piciformes): Implications for karyotype and ZW sex chromosome differentiation. PLoS One 12:e0169987.; Furo et al., 2017Furo IO, Kretschmer R, dos Santos MS, Carvalho CAL, Gunski RJ, O’Brien PCM, Ferguson-Smith MA, Cioffi MB and de Oliveira EHC (2017) Chromosomal mapping of repetitive DNAs in Myiopsitta monachus and Amazona aestiva (Psittaciformes, Psittacidae: Psittaciformes), with emphasis on the sex chromosomes. Cytogenet Genome Res 151:151-160.) shows interesting variation in accumulation sites for some of them, reinforcing microsatellites as important markers for studies on karyotype evolution.

Acknowledgments

We are grateful to all colleagues from the Laboratório de Citogenética e Evolução of the Departamento de Genética of Universidade Federal do Rio Grande do Sul, Grupo de Pesquisa Diversidade Genética Animal da Universidade Federal do Pampa, Laboratório Cultura de Tecidos e Citogenética SAMAM do Instituto Evandro Chagas and CAPES for support at various stages of this research.

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  • Associate Editor: Yatiyo Yonenaga-Yassuda

Publication Dates

  • Publication in this collection
    19 Feb 2018
  • Date of issue
    Jan-Mar 2018

History

  • Received
    22 Feb 2017
  • Accepted
    16 Aug 2017
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