Nucleolar organizer regions and a new chromosome number for Rhea americana (Aves: Rheiformes)

Nucleolar organizer regions and a new chromosome number for Rhea americana (Aves: Rheiformes)

Ricardo José Gunski¹ and Miriam Luz Giannoni²

¹Departamento de Genética, Facultad de Ciencias Exactas, Químicas y Naturales, Universidad Nacional de Misiones, 3300 Posadas, Misiones, Argentina. Send correspondence to R.J.G.

²Centro Universitário de São José do Rio Preto, UNIRP, 15025-400 São José do Rio Preto, SP, Brasil.

ABSTRACT

Sequential banding analysis (Giemsa-C-banding-Ag NOR) of chromosomes of the common rhea (Rhea americana) was performed. Metaphases were obtained by peripheral blood lymphocyte culture and monolayer embryo cell culture. The diploid chromosome number was 80, different from the 2n = 82 in previous reports. Macrochromosome pairs 1, 2 and 5 were submetacentric and pair 3, subacrocentric. The 4th pair was acrocentric and all of the microchromosomes appeared to be acrocentric, with the exception of a clearly metacentric pair which was fully heterochromatic. The Z was slightly larger than the W, both being acrocentric and C-band negative. Nucleolar organizer regions were observed in the secondary constriction of a microchromosome pair. Correct identification of the NOR-bearing pair was possible only by sequential analyses, Giemsa staining followed by the Ag-NOR technique.

INTRODUCTION

Few bird species have been investigated for nucleolar organizer regions (NORs), although variations have been observed, for example, in various species of the order Gruiformes (Nishida and Sasaki, 1980) and of the families Columbidae (Gunski et al., 1995) and Tinamidae (Garnero, 1996), in which NORs are found in a single microchromo-some pair. Other birds have microchromosomes with cistrons for nucleolar organization, including species of the order Strigiformes (Biederman et al., 1980; Sasaki et al., 1981), Coturnix coturnix japonica and Phoenicopterus ruber (Funaki et al., 1975). Rocha and Lucca (1988) observed NOR in the satellites of the 9th chromosome pair in Pitangus sulphuratus and Guira guira, and in a variable number of microchromosomes and on the short arm of the 3rd pair in Mimus saturninus. The only chromosome banding investigations (G, C, and Q banding) carried out on birds of the subclass Ratitae are those of Takagi and Sasaki (1974) and Ansari et al. (1988), who studied the macrochromosome patterns, including the sex pair, and demonstrated the euchromatic nature of chromosome W.

R. americana, the largest bird on the American continent, currently on the list of endangered species, is commonly called ema in Brazil and ñandú in Argentina and has been studied only in a few investigations involving a reduced number of specimens (Takagi et al., 1972; Takagi and Sasaki, 1974; Shoffner, 1974; Ansari et al., 1988).

MATERIAL AND METHODS

The study was conducted on 19 specimens of R. americana (7 males and 12 females) consisting of adults, young individuals and embryos, from different regions of Brazil (São Paulo and Rio Grande do Sul States and Brasília, DF). Metaphases for chromosome analysis were obtained by peripheral blood lymphocyte culture and monolayer embryo cell culture described by Moorhead et al. (1960) and Freshney (1990), respectively, with modifications. NOR bands were obtained by the technique of Howell and Black (1980), modified, and C bands by the technique of Sumner (1972), performed in sequence on Giemsa-stained slides, without previous destaining, according to the technique described by Giannoni et al. (1991).

RESULTS AND DISCUSSION

The karyotype of R. americana (Figure 1) presented a diploid number of 80 chromosomes. Among the macrochromosomes, pairs 1, 2 and 5 were found to be submetacentric and pair 3 subacrocentric (Figure 1), as confirmed by C banding (Figure 2A,B). Takagi et al. (1972) and Ansari et al. (1988) have described this pair as acrocentric with a minuscule short arm. The 4th pair was acrocentric and all the microchromosomes appeared to be acrocentric, with the notable exception of a clearly metacentric pair (Figure 1, arrows), which was fully heterochromatic.

Figure 1
- Metaphase and karyotype of a female Rhea americana 2n = 80.

Figure 2
- Metaphase plate of Rhea americana showing sequential (A) Giemsa-stained, (B) C-banding and (C) Ag-NOR banding. Arrow, Demonstrates the presence of positive centromeric C-banding in each component of the NOR-carrier microchromosome pair.

The sex pair was acrocentric, with the Z slightly larger than the W, and both C-band negative (Figure 2A,B). Silver staining of the nucleolar organizer regions was observed in the secondary constriction of a microchromosome pair. The correct identification of the NOR-bearing pair was only possible by Giemsa staining followed by the Ag-NOR technique (Figure 3A,B). There was a small but clearly visible difference in the size of the chromosomes in this pair. Rocha and Lucca (1988) observed asymmetry of NOR labeling between the sister chromatids of one of the homologous chromosomes, a fact that was also observed in the present study (Figure 3B). Sequential Giemsa-Ag-NOR staining revealed that the NOR-bearing chromosomes may be associated (Figure 4A,B).

Figure 3
- Metaphase plate of Rhea americana showing sequential Giemsa-stained (A) and Ag-NOR banding (B). The nucleolar organizer region (NOR)-carrier microchromosomes are shown by arrows.

Figure 4
- Metaphase plate of Rhea americana showing sequential Giemsa-stained (A) and Ag-NOR banding (B) staining with the associated NOR-bearing microchromosomes (arrows).

Sequential Giemsa-C banding-Ag-NOR analysis (Figure 2A,B,C) demonstrated the presence of a positive centromeric C band (B) in each component of the NOR-bearing pair. As a result of the identification of this chromosome pair, the diploid number detected for R. americana was 2n = 80, FN = 95. This result differs from those of Takagi et al. (1972), Takagi and Sasaki (1974), Shoffner (1974) and Ansari et al. (1988), who reported 2n = 82. This variation in chromosome number may be related to the pair identified here for the first time since, in view of its small size and marked secondary constriction interstitially present on the long arm, each member of the pair may be observed as two microchromosomes and not simply as one in preparations submitted to standard staining. Sequential analysis of the same metaphase by standard staining and banding techniques proved to be a highly useful methodology for chromosome identification, especially with respect to the smaller chromosomes.

ACKNOWLEDGMENTS

We wish to thank Departamento de Melhoramento Genético Animal, FCAVJ, UNESP and J.M.B. Duarte, M.E.G. Moro, A.S. Fenocchio and the technicians P.A. Toste and J.A. Boer. Research supported by Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, UNESP and CNPq. Publication supported by FAPESP.

RESUMO

Foram efetuadas análises seqüenciais de bandeamento cromossômico (Giemsa-banda-C-AgNOR) em material da espécie Rhea americana (ema) com o objetivo de identificar os cromossomos portadores de regiões organizadoras de nucléolos e confirmar o cariótipo desta espécie. As metáfases foram obtidas de culturas de leucócitos e de células de embrião. O número diplóide de cromossomos, determinado pela análise de metáfases oriundas de 19 espécimes, foi de 80 (2n = 80, NF = 95), o que difere da literatura. Os pares de macrocromossomos números 1, 2 e 5 eram submetacêntricos e o par 3 era sub-acrocêntrico, confirmado pelo bandeamento C. O par 4 era acrocêntrico, bem como todos os microcromossomos, com exceção de um metacêntrico inteiramente heterocromático. O cromossomo Z era ligeiramente maior que o W, sendo ambos acrocêntricos e banda-C negativos. A região organizadora de nucléolos foi observada na constrição secundária de um par de microcromossomos. A correta identificação do par portador da NOR só foi possível com a utilização da análise seqüencial de coloração pelo Giemsa seguida da aplicação da técnica Ag-NOR.

(Received December 12, 1996)

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Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  June 1998

History

• Received
  12 Dec 1996