Comparison of GTG-banded karyotypes and microsatellite sequences in some species of the Bovidae and Cervidae families *

Comparison of GTG-banded cattle, sheep, goat, and fallow deer chromosomes showed conformity of G-banding patterns for chromosomes of fallow deer. In Bovidae species, autosomes were shown that were involved in centric fusion, giving a metacentric pair in fallow deer. Twenty-two microsatellite markers were amplifi ed with the use of bovine specifi c primers in the investigated species. The results of cross-species amplifi cation showed that microsatellites BM143, CSSM016 and TGLA53 seem to be conservative across all investigated ruminant species. Sequencing confi rmed short tandem repeat motives of the TGLA53 marker among selected Bovidae and Cervidae species. About 60% of microsatellite loci were successfully amplifi ed with the use of bovine primers in sheep and red deer species, 50% in goat, 55% in western roe deer, and 36% in fallow deer. This suggests that, on the DNA level, the genomes of red deer and sheep are more closely related to the bovine genome than genomes of the other studied ruminants.


INTRODUCTION
Comparative studies of the genomes of different animal species are based mainly on the phenomenon of genetic conservation.This concerns chromosome banding patterns (Iannuzzi andDi Meo, 1991, 1995;Ansari et al., 1999), nucleotide sequences (e.g., microsatellite sequences) (de Gortari et al., 1997) and groups of linked or syntenic genes that often have the same relationships even in taxonomically distant species (Slate et al., 2002).
Comparison of karyotypes after differential staining of chromosomes using GTG, RBA, RBG and QFQ techniques reveals conservation of chromosome banding patterns (Ansari et al., 1999;Słota et al., 2001).Identifi cation of homologous chromosomes or their fragments from different animal species is most often conducted within systematic units, providing further evidence that evolutionary relatedness is paralleled by karyotype similarity (Iannuzzi and Di Meo, 1995;Słota et al., 2001).
A large number of papers also reported conservation on the DNA level between different mammalian species, based on anonymous DNA markers such as microsatellite loci (Moor et al., 1991;de Gortari et al., 1997;Slate et al., 1998).Microsatellite markers have been effi ciently used in genome mapping projects, pedigree determination, and population genetics in humans and animals.Cross-species utilization of microsatellite loci enables the construction of comparative maps between related species (O'Brien et al., 1993).These tandemly repeated, highly polymorphic, single locus DNA sequences are well distributed over genomes (Tauz, 1989) and are readily adaptable to the polymerase chain reaction method in terms of simultaneously amplifying a number of markers in one reaction (Ziegle et al., 1992).The aim of the presented study was to compare metaphase chromosome G-banded patterns and DNA microsatellite markers of some species from the Bovidae and Cervidae families.

Laboratory techniques
Metaphase chromosome preparations obtained after routine in vitro lymphocyte culture were analysed.The GTG differential staining technique was used for precise identifi cation of chromosome pairs (Wang and Fedoroff, 1972).
The cattle, sheep, and goat karyotypes were arranged based on the G-banding standards developed by Ansari et al. (1999) and Di Berardino et al. (2001).Because there is no international standard for the G-banding pattern of Dama dama, the karyogram for this species was arranged based on chromosome morphology, chromosome size and G-band homology following the guidelines given by Rubini et al. (1990).A comparative karyogram was then made by comparing the G-band patterns on the chromosomes of the cattle, sheep, goats and fallow deer and homology was identifi ed between them.
DNA of the studied animals, derived from blood or hair roots, was amplifi ed using bovine specifi c primers for 22 microsatellite markers (Table 1) in two PCR reactions of the multiplex type.Eleven markers (BM1824, BM2113, ETH10, ETH225, ETH3, INRA23, SPS115, TGLA122, TGLA126, TGLA227 and TGLA53) were amplifi ed with the use of Stock Marks for Cattle PCR-typing Kit (PE Applied Biosystems, Foster City, CA) with PCR conditions as described in the kit protocol.For the markers forming the additional panel for cattle parentage verifi cation used by the authors, PCR multiplex was earlier elaborated to amplify bovine genomic DNA.PCR products were subjected to vertical electrophoresis in 4% denaturing polyacrylamide gel on a Genetic Analyser (ABI PRISM 377).The allele sizes in base pairs (bp) were determined after processing of raw data using the software packages GENESCAN 2.0 and GENOTYPER 2.1 (Applied Biosystems).A single-band PCR product was considered to be a conserved microsatellite only if the observed band showed a distinctive 'stutter' pattern and was close to the size range of the observed cattle product.As an example of conservation of a DNA sequence, some of the STR alleles were sequenced to determine the structure and conservative region of tandem repeats among four species (cattle, fallow deer, sheep and goat).Namely, a new set of TGLA53 primers was designed to obtain longer PCR products for the sequencing reaction (around 400 bp).Direct sequencing of TGLA53 PCR products was performed from both strands using Big Dye Terminator Chemistry (Applied Biosystems).Only repeat regions were considered in the comparison of sequencing reads between species.

RESULTS
Karyotype analyses were performed in cattle, sheep, goats, and fallow deer and the compared karyograms are presented in Figure 1.

DISCUSSION
The fi rst comparative study in the Bovidae family showed band homology on the chromosomes of cattle, sheep and goats (Evans et al., 1973).These fi ndings were confi rmed by Iannuzzi and Di Meo (1995), who identifi ed autosome pairs with a homologous pattern of G-and R-bands in these three species.These authors also performed detailed analyses of the X heterosome in cattle, water buffaloes, sheep and goats, and based on the analogies identifi ed, suggested possible rearrangements of this chromosome in the evolutionary process (Iannuzzi and Di Meo, 1995).
It is assumed that in the course of evolution, the number of chromosomes was reduced as a result of Robertsonian translocations of acrocentric chromosomes.These suggestions were confi rmed by studies of polymorphic forms of karyotype in wild Ovis species, in which different diploid numbers of chromosomes were observed: 2n=58 (Ovis vignei), 2n=56 (Ovis ammon), 2n=54 (Ovis dalli, Ovis musimon, Ovis orientalis), and 2n=52 (Ovis nivicola) (Bunch and Nadler, 1980).
Karyotype studies of different species of the Cervidae family (elk, roe deer, red deer, sika deer, and fallow deer) living in the wild, conducted by Gustavsson and Sundt (1968), concerned routinely stained metaphase chromosomes, which were classifi ed according to size and morphology.For the Dama dama species, the 68,XY or 68,XX karyotype as well as the number of arms of autosomal chromosomes (68) were determined.Among the autosomes, one pair of long metacentric and 32 pairs of acrocentrics were identifi ed.Concerning sex chromosomes, X was identified as the acrocentric chromosome and Y as a small submetacentric.
In the next step of determining the karyotype of fallow deer, the following differential staining techniques were used: GTG, with 350 G-bands obtained on metaphase chromosomes (Rubini et al., 1990) and RBA, with 527 bands obtained on prometaphase chromosomes (Lioi et al., 1994).
In our analyses, the GTG-banding karyotype of fallow deer, used for comparison with G-banding patterns on the metaphase chromosomes of cattle, sheep, and goats, revealed 450 bands and helped to pinpoint homologous chromosomes in the compared species, indicating that a level of 450 bands is sufficient for comparative studies.
A remarkable homology of most autosomes of fallow and roe deer (Capreolus capreolus) was revealed by comparison of the G-banded karyotypes (Rubini et al., 1990).According to these authors, the metacentric pair in the fallow deer retains the same band patterns as the two acrocentric pairs in the roe deer, while the X chromosomes of the roe deer differ as a result of pericentric inversion.
A comparison of R-banded chromosomes of Vietnamese sika deer (Cervus nippon pseudaxis, 2n=66) with bovine R-banded chromosomes was described by Bonnet et al. (2001).Next, the probes for twenty-nine Texas nomenclature type I markers for each cattle autosome, sixteen other type I and fourteen microsatellite markers were used in FISH technique on sika deer chromosomes.A complete correspondence between sika deer and cattle chromosomes was established, however, autosome pair 7 of sika deer presented the most complex rearrangement as compared with cattle chromosomes.
According to our fi ndings based on G-banding patterns, metacentric chromosomes in the fallow deer karyotype involve chromosomes having the following homology: p arm -cattle pair 19, sheep 11 and goat 19 and q arm -cattle pair 18, sheep 14 and goat pair 18.Comparison between G-banding patterns on cattle, sheep, goats and fallow deer chromosomes confi rmed the chromosome homology in the Bovidae family described by Iannuzzi and Di Meo (1995).
To investigate the karyotype relationships between Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis), Chi et al. ( 2005) assigned a complete set of Chinese muntjac chromosome-specifi c painting probes to G-banded chromosomes of these three species.In total, the 22 autosomal painting probes of Chinese muntjac delineated 33 and 34 conserved chromosomal segments in the genomes of forest musk deer and gayal, respectively.The combined analysis of comparative chromosome painting and G-band comparison reveals that most interspecifi c homologous segments show a high degree of conservation in G-banding patterns.Eleven chromosome fi ssions and fi ve chromosome fusions differentiate the karyotypes of Chinese muntjac and forest musk deer, twelve chromosome fi ssions and six fusions are required to convert the Chinese muntjac karyotype to that of gayal, one chromosome fi ssion and one fusion separate the forest musk deer and gayal.The musk deer has retained a highly conserved karyotype that closely resembles the proposed ancestral pecoran karyotype but shares none of the rearrangements characteristic of the Cervidae and Bovidae.
The results obtained after comparison of the Bovidae and Cervidae families belonging to the suborder Ruminantia suggest that genetic conservatism is a phenomenon also frequently observed between larger systematic units than the family.
The ability to use PCR across species depends on the conservation of priming sites.Point mutations within the priming sites result in poor or no amplifi cation (Moor et al., 1995).Despite synteny existing between cattle and other ruminants for numerous investigated loci (see Table 2) (information from INRA BovMap and arkdb Deerbase), these loci were not amplifi ed with bovine-specifi c primers on DNA of ruminant species other than cattle.In our study about 60% of micro-  satellite loci were successfully amplifi ed with the use of bovine primers in sheep and red deer species, being comparable to such a study in sheep performed by de Gortari et al. (1997) and much higher than the results obtained in red deer by Kuhn et al. (1996) (around 50% loci typed).Slate et al. (1998) obtained a substantially higher yield of microsatellite amplifi cation in sheep and two deer species (above 70%) after extensive optimization of PCR conditions with the use of bovine primers for both species.In case of goat, our amplifi cation reached 50% of bovine microsatellite loci and was lower than in the study of Pepin et al. (1995) (60% of loci typed).For other investigated ruminant species we reached a 55% amplifi cation yield of bovine microsatellites in western roe deer and only 36% in fallow deer.
The PCR amplifi cation yield of bovine microsatellite loci among different species of Ruminantia determined by conservation of priming sites and the presence of microsatellite alleles with the same size between species illustrate to some extent the level of their divergence in relation to the cattle genome.The presence of PCR products for BM143, CSSM016 and TGLA53 in all ruminant species shows that genomic regions containing these loci may be conservative across these species; however, the sequence of these regions needs to be verifi ed to confi rm specifi city not only of the primers but also of the amplifi ed region (Sun and Kirkpatrick, 1996).Stronger evidence for the conservation of the studied microsatellite markers may be the presence of alleles with the same size common for different species.In our study, the greatest number of loci with alleles equal in size to the cattle microsatellite alleles was detected in red deer.This may suggest that the genomes of these two ruminant species are most closely related than other investigated ruminants.In this regard sheep and western roe deer take the second position in phylogenetic divergence to the cattle genome and fallow deer is most distantly related.However, these fi ndings are slightly inconsistent with syntenic data, where the number of microsatellite loci with known synteny in other ruminants, the same as bovine loci, is greater for sheep (16 loci) than red deer (11 loci).As mentioned above, this bias is caused by mismatches in primer binding sites during cross-species amplifi cation.For example, Slate et al. (1998) pointed to close relationships between cattle, red deer and sheep genomes based on a similar proportion of markers yielding a product of cross-amplifi cation in all three species; however, as pointed out in the work of Sun and Kirkpatrick (1996), such results must be confi rmed through sequencing data.In this term sequencing of the repeat region of microsatellite TGLA53 revealed two conserved blocks of (AC) 5 and (CA) 3 motive among selected Bovidae and Cervidae species.No differences in the repeat structure between cattle and fallow deer species are consistent with the same fragment size of the investigated TGLA53 allele in both species.Smaller allele sizes observed in sheep and goat result from the shorter repeat region of the sheep and goat TGLA53 allele.

CONCLUSIONS
Cytogenetic comparative analyses make it possible to identify chromosome markers common to species belonging to different families, for example cattle, sheep, goats and fallow deer, based on chromosome homologies for these species.These homologies could be useful in evolutionary research and in diagnostics of chromosome abnormalities found in wild species, because our knowledge about their karyotypes and banding patterns is insuffi cient.
Different amplifi cation yields for microsatellite loci among the investigated ruminant species and also the presence or absence of microsatellite alleles with the same size common to different species are associated with the different genomic organization in particular species and thus, with different levels of conservatism of particular genomic regions.This affects not only the conservatism level of primer binding sites but also the sequence of the amplifi ed region.

Figure 2 .
Figure 2. Sequence alignment of TGLA53 alleles among selected Bovidae and Cervidae species

Table 1 .
PCR results for 22 microsatellite markers among Bovidae and Cervidae species Species

Table 2 .
Chromosomal location of 22 microsatellite markers in investigated species (information from INRA BovMap and arkdb Deerbase) Species