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Cytogenetic characterization of alpaca (Lama pacos, fam. Camelidae) prometaphase chromosomes Article in Cytogenetic and Genome Research · February 2006 DOI: 10.1159/000095234 · Source: PubMed
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Original Article Cytogenet Genome Res 115:138–144 (2006) DOI: 10.1159/000095234
Cytogenetic characterization of alpaca (Lama pacos, fam. Camelidae) prometaphase chromosomes D. Di Berardino a D. Nicodemo a G. Coppola b A.W. King b L. Ramunno a G.F. Cosenza a L. Iannuzzi c G.P. Di Meo c G. Balmus d, e J. Rubes f a
Department of Animal Science and Food Inspection, University of Naples ‘Federico II’, Portici, Naples (Italy) Department of Biomedical Sciences, University of Guelph, Guelph, Ontario (Canada) c National Research Council (CNR), ISPAAM, Laboratory of Animal Cytogenetics and Gene Mapping, Naples (Italy) d Department of Clinical Veterinary Medicine, Centre for Veterinary Science, University of Cambridge, Cambridge (UK) e Faculty of Veterinary Medicine, Iasi (Romania); f Veterinary Research Institute, Brno (Czech Republic) b
Manuscript received 1 February 2006; accepted in revised form for publication by M. Schmid, 24 March 2006.
Abstract. The present study provides specific cytogenetic information on prometaphase chromosomes of the alpaca (Lama pacos, fam. Camelidae, 2n = 74) that forms a basis for future work on karyotype standardization and gene mapping of the species, as well as for comparative studies and future genetic improvement programs within the family Camelidae. Based on the centromeric index (CI) measurements, alpaca chromosomes have been classified into four groups: group A, subtelocentrics, from pair 1 to 10; group B, telocentrics, from pair 11 to 20; group C, submetacentrics, from pair 21 to 29; group D, metacentrics, from pair 30 to 36 plus sex chromosomes. For each chromosome pair, the following data are provided: relative chromosome
length, centromeric index, conventional Giemsa staining, sequential QFQ/C-banding, GTG- and RBG-banding patterns with corresponding ideograms, RBA-banding and sequential RBA/silver staining for NOR localization. The overall number of RBG-bands revealed was 391. Nucleolus organizer-bearing chromosomes were identified as pairs 6, 28, 31, 32, 33 and 34. Comparative ZOO-FISH analysis with camel (Camelus dromedarius) X and Y painting probes was also carried out to validate X-Y chromosome identification of alpaca and to confirm close homologies between the sex chromosomes of these two species.
The alpaca (Lama pacos) belongs to the family Camelidae, suborder Tylopoda, which includes the vicuna (Lama vicugna), llama (Lama glama), guanaco (Lama guanicoe) and the two camels, bactrian (Camelus bactrianus) and dromedary (Camelus dromedarius).
Early cytogenetic investigations reported for the alpaca a diploid number of 72 (Capanna and Civitelli, 1965; Hungerford and Snyder, 1966) vs. 74 (Hsu and Benirschke, 1967). Other cytogenetic studies on the members of this family found a diploid number of 74 in guanaco, bactrian and dromedary (Taylor et al., 1968) as well as in vicuna and bactrian (Koulischer et al., 1971); they also indicated extensive similarities in the karyotypes of these species, thus suggesting that all members of the family, including the alpaca, would have the same basic karyotype. However, discrepancies were found in the proportion of the various types of chromosomes, including the sex chromosomes. These karyotypes, in fact, are composed of a gradually decreasing row of mostly bi-armed elements that makes it difficult to delimit one morphological type of chromosome from another (meta-/submeta-/subtelo-/telocentrics). As a result,
This study was financially supported by the RAIZ Finalized Project no. RZ 282 from the Ministry of Agriculture MiPAF of Rome, Italy and by the project MZE 0002716201 from the Grant Agency of the Ministry of Agriculture of the Czech Republic. Request reprints from Dino Di Berardino Department of Animal Science and Food Inspection University of Naples ‘Federico II’ IT–80055 Portici (Italy) telephone: +39 081-25 39 265; fax: +39 081-776 28 86 e-mail:
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the Fundamental Number (NF) has been estimated, by different authors, between 86 and 112. For example, Taylor and colleagues (1968) reported 5 distinct pairs of submetacentric autosomes and 31 acrocentrics, the X chromosome as metacentric and the Y could not be identified; Koulischer and collaborators (1971) reported 7 pairs of meta- and submetacentrics, 29 pairs of acrocentrics, the X and the Y chromosomes were both indicated as metacentrics. Later, Bunch et al. (1985) published the first GTG-banding pattern in chromosomes of llama, guanaco and bactrian; they also reported similarities in the G-banding pattern, but indicated 3 pairs of submetacentrics and 33 pairs of acrocentrics, the X chromosome as the largest submetacentric and the Y as the smallest acrocentric. These reports all agree with the diploid number of 74 but disagree with the relative numbers and proportions of the various types of chromosomes, including the X and the Y. In order to contribute to the clarification of the chromosomal constitution within the family Camelidae, we investigated the alpaca species (Lama pacos), which – so far – has not been cytogenetically studied in detail, despite the increasing worldwide economical interest in the peculiar characteristics of its wool (extremely fine, unallergic and naturally stained). In the present study we provide, for each chromosomal pair, the following cytogenetic data: relative chromosome length (RCL), centromeric index (CI), conventional Giemsa staining, sequential QFQ/C-banding for the distribution of centromeric heterochromatin, GTG- and RBG-banding patterns with corresponding ideograms, RBA-banding and sequential RBA/silver staining for NOR localization and identification. Comparative ZOO-FISH analysis with camel (Camelus dromedarius) X-Y painting probes was also performed to validate X-Y chromosome identification of alpaca and to demonstrate the close homologies between the sex chromosomes of the two species. Material and methods Peripheral blood cultures from sixteen (eight males and eight females) clinically healthy adult alpacas belonging to the Huacaya breed, reared in central Italy were performed. After 48 h of growth, cells were synchronized overnight with methotrexate (0.20 g/ml, f.c.). Cell block was released 14 h later by washing cells twice and recovering cells in fresh medium containing thymidine (50 g/ml, f.c.) for GTG-banding or with BrdU (30 g/ml) + H33258 (50 g/ml) and Heparine (5 g/ml) for RBG-banding, for subsequent 5–6 h, including a 20-min treatment with colcemid (0.05 g/ml). Sequential Giemsa conventional/GTG-banding was performed on fifty conventional Giemsa-stained metaphases which were photographed using the Leica Qwin software for measuring centromeric index (CI) and relative chromosome length (RCL), and subsequently destained with ethanol and air dried. The GTG-banding was carried out the next day. The best ten sequentially stained metaphases were used for determining the mean values of CI and RCL. Sequential QFQ/C-banding, GTG-banding, RBG-H banding, sequential RBA/NORs and FISH-technique were as reported in Lin et al. (1977), Di Berardino et al. (1980, 1985), Ronne (1984) and Yang et al. (1997), respectively.
X and Y chromosome-specific probes from camel (Camelus dromedarius) were prepared by DOP-PCR of X and Y flow-sorted chromosomes, as described in Yang et al. (1997). The nomenclature used for karyotyping was that proposed by Levan et al. (1964), while the centromeric index (CI) was calculated as the ratio between the length of the short arm ! 100/total chromosome length. Accordingly, chromosomes were classified as metacentric (CI) = 0.50–0.38; submetacentric (CI) = 0.37–0.26; subtelocentric (CI) = 0.25–0.13 and telocentric (CI) = 0.12–0. The relative chromosome length (RCL) was calculated as the ratio between the length of the chromosome ! 100 over the overall length of all the chromosomes. All measurements were made by using the Leica Qwin software on ten metaphase plates which have been first stained with Giemsa and then GTG-banded.
Results and discussion
To facilitate karyotyping of the species, the chromosomes have been subdivided into four groups, according to the criteria reported by Levan et al. (1964) and to the centromeric index (CI) as follows (Fig. 1): group A, subtelocentric, from pairs no.1 to no. 10 with CI varying from 0.13 to 0.16; group B, telocentric, from pairs 11 to 20 with CI varying from 0 to 0.12; group C, submetacentric, from pairs 21 to 29 with CI varying from 0.26 to 0.32; group D, metacentric, from pair 30 to 36 with CI varying from 0.38 to 0.44, including the X and Y chromosomes with CI of 0.40 and 0.50, respectively. Within each group, chromosomes were ranked according to decreasing chromosome length. The conventional Giemsa staining shows a remarkable variation in the size of the short arms, probably due to pronounced HC-polymorphism, as demonstrated by the sequential QFQ/C-banding (Fig. 1). This variation makes the conventional karyotyping of alpaca quite unreliable and ambiguous. C-bands (Fig. 1) were quite large in pairs 1–3 and 9 of the A group; in pairs 13 and 16 of the B group; in pairs 22, 26, 27 and 28 of the C group; in pairs 31, 33, 34, 35 and 36 of the D group; on the contrary, they were almost negligible in chromosomes 18 and 19 of the B group. Dimorphism in C-band size between the two homologues was quite evident in several pairs, such as chromosomes 4 to 10 of the A group, chromosomes 11 to 15 of the B group, chromosomes 23 to 25 of the C group. C-bands were almost negligible in chromosomes 18 and 19 of the B group while they were very pronounced in pair 16, 28 and 31. The X chromosome showed a very small amount of C-band material, while the Y chromosome was mostly heterochromatic. The C-band analysis revealed a pronounced variation of the centromeric heterochromatin among individual pairs and, more important, between homologous chromosomes, up to the disappearance of entire short arms on one of the homologues. Such a pronounced variation in the C-band size could be the result of extensive events of addition/deletion of constitutive heterochromatin which, at least partly, could explain the discrepancy between different authors in determining the NF in camelid species, even though the occurrence of pericentric inversions could not be excluded a priori. On the basis of these findings, the interindividual
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Fig. 1. Cytogenetic characterization of Lama pacos chromosomes: for each individual pair the following features are reported (starting from the left): chromosome number (N), relative chromosome length (RCL), centromeric index (CI), sequential QFQ/C-banding, GTG-banding and ideogram G, RBG-banding and ideogram R, RBA-banding, sequential RBA/silver staining.
variability of the NF might be a quality of the karyotype of alpaca and, probably, other species of the family Camelidae. The correspondence between the GTG- and the RBG- or the RBA-banding patterns was quite evident and satisfactory for all the chromosomes. In some cases, the RBG banding pattern revealed more bands than the GTG, as in the case of chromosomes 2 and 6, due to the visualization of minor bands. A total of 391 RBG-bands, of which 102 on the
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short arms and 289 in the long arm, were visualized in this level of banding resolution. The last two columns of Fig. 1 report the results of the sequential RBA/silver staining technique to demonstrate the localization of silver grains on six pairs of chromosomes, which were identified as pairs no. 6 (A group), 28 (C group) and 31 to 34 (D group). Out of more than 50 silver stained metaphase plates from five different individuals, the majority of the cells (more than 30%) showed six NORs, always
(Fig. 1 continued next pages.)
located on the short arms. Previous studies revealed the presence of NORs in the secondary constrictions of the short arms or the satellite stalks of five (Mayr et al., 1985) or six (Bunch et al., 1985) chromosomes, but no chromosome identification was attempted. In the present study we demonstrated the presence of six pairs of NORs and, more importantly, identified the NO chromosomes of this species. To validate the alpaca sex chromosomes, camel X- and Y-painting probes obtained from flow-sorted Camelus
dromedarius chromosomes were cross-hybridized onto alpaca metaphase chromosomes (Fig. 2), confirming that the Y chromosome of alpaca (green) is the smallest metacentric element, whereas the X chromosome (red) is the largest metacentric. In addition, green spots were visualized on the tips of the Xp arm indicating a possible presence of Y-specific DNA sequences on the X chromosome, probably related to the pseudoautosomal region (PAR), although the reverse condition (X-specific red signals) was not observed
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on the Y telomeres. The possibility that part of the Y chromosome is translocated onto the X chromosome must also be considered; this aspect, however, requires further investigation. Cytogenetic studies of the genus Lama have been carried out mainly for examining clinical cases such as the XX sex reversal condition (Wilker et al., 1994; Drew et al., 1999), the X chromosome monosomy (Hinrichs et al., 1997) and XX/XY chimerism and freemartinism in a female (Hinrichs et al., 1999) so far. All these studies refer to the basic
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paper by Bunch et al. (1985), who did not make measurements of the centromeric index and, therefore, the chromosomes were all ranked based on decreasing chromosome length. They found the GTG-banding patterns of the bactrian, llama and guanaco quite identical to each other, thus suggesting that the amount of divergent evolution which has occurred in the family Camelidae has been accomplished, mainly, by single gene mutations or minor chromosomal rearrangements. However, a recent report on failure of hybridization between a female dromedary and a gua-
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Fig. 2. (a) Inverted DAPI banding; (b) ZOO-FISH of X(red)-Y(green) painting probes from Camelus dromedarius onto Lama pacos metaphase chromosomes. Notice the green signal on the tips of the ‘p’ arm of the X chromosome indicating the presence of Y sequences on the X.
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naco stallion (Skidmore et al., 1999) would indicate that, despite the extensive similarity in the diploid number and chromosome banding, sufficient genetic change has taken place to make the pairing of homologous chromosomes no longer possible. This finding points out the need to explore chromosome homologies within the family Camelidae by using more effective techniques, such as high resolution banding, cross-species fluorescent in situ hybridization (ZOO-FISH) with chromosome specific painting probes and/or chromosome markers, and gene mapping. In conclusion, the present study is the first report providing specific cytogenetic information on the chromosomal constitution of the alpaca (Lama pacos) including the centromeric index (CI), relative chromosome length (RCL), sequential QFQ/C-banding pattern, GTG-RBG and RBAbanding patterns and related ideograms, identification of six pairs of Ag-NOR bearing chromosomes. The X and Y
chromosome identification has been further validated by the ZOO-FISH analysis using the X- and Y-painting probes obtained from flow-sorted Camelus dromedarius chromosomes which also revealed the total homoeology between the sex chromosomes of the two species. These results can be used as a point of reference for subsequent and necessary work on karyotype standardization, clinical cytogenetics and gene mapping of the species, as well as for comparative analysis and future genetic improvement programs within the family Camelidae. Acknowledgements Special thanks are given to Mrs. Gabriella Bozzini (Italpaca, GR), Mrs. Antonella Gistri (La valle degli alpaca, SI), and to Mr. Gianni Berna (la Maridiana farm, PG) for the collection of the specimens.
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