Animal Cytogenetics and Comparative Mapping. Manuscript received 2 March 2004; accepted in revised form for publication by T. Haaf 18 July 2004.

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1 Animal Cytogenetics and Comparative Mapping Cytogenet Genome Res 108: (2005) DOI: / New insights into the karyotypic relationships of Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis) J. Chi, a,d B. Fu, b W. Nie, a J. Wang, a A.S. Graphodatsky c and F. Yang a,b a Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming (P.R. China); b Centre for Veterinary Science, Cambridge (UK); c Institute of Cytology and Genetics, Novosibirsk (Russia); d Graduate School of the Chinese Academy of Sciences, Beijing (P.R. China) Manuscript received 2 March 2004; accepted in revised form for publication by T. Haaf 18 July Abstract. To investigate the karyotypic relationships between Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis), a complete set of Chinese muntjac chromosome-specific painting probes has been assigned to G-banded chromosomes of these three species. Sixteen autosomal probes (i.e. 6 10, 12 22) of the Chinese muntjac each delineated one pair of conserved segments in the forest musk deer and gayal, respectively. The remaining six autosomal probes (1 5, and 11) each delineated two to five pairs of conserved segments. 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 interspecific homologous segments show a high degree of conservation in G-banding patterns. Eleven chromosome fissions and five chromosome fusions differentiate the karyotypes of Chinese muntjac and forest musk deer; twelve chromosome fissions and six fusions are required to convert the Chinese muntjac karyotype to that of gayal; one chromosome fission 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 for the Cervidae and Bovidae. Our results substantiate that chromosomes 1 5 and 11 of Chinese muntjac originated through exclusive centromere-to-telomere fusions of ancestral acrocentric chromosomes. Copyright 2005 S. Karger AG, Basel Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis) belong to the same order Artiodactyla but different families (Gao, 1963; Groves et al., 1995; Tian et al., 1998). The genus Muntiacus is famous for its high degree of interspecific karyotype diversity Supported by grants from the National Natural Science Foundation (P.R. China) to F.Y., and PCB, and RFBR research grants and INTAS Grant to A.S.G. Request reprints from: Dr. Fengtang Yang Key Laboratory of Cellular and Molecular Evolution Kunming Institute of Zoology, The Chinese Academy of Sciences Kunming, Yunnan, (P.R. China) telephone: ; fax: kcb@mail.kiz.ac.cn or fy@mole.bio.cam.ac.uk and the radical karyotypic reorganization through tandem fusion (Shi et al., 1980; Yang et al., 1995; Yang, 1998; Wang and Lan, 2000). The Chinese muntjac has a 2n = 46 karyotype (the highest diploid number so far found in Muntiacus), which could have originated from a 2n = 70 karyotype via 12 tandem fusions (Neitzel, 1987; Fontana and Rubini, 1990; Yang et al., 1997c; Yang, 1998). Musk deer are widely distributed in the mountain areas of Asia, from Siberia, to south Asia (Wang et al., 1993; Groves et al., 1995). The musk deer exhibits a mixture of bovid and cervid characters and its taxonomic placement remains a topic of hot debates (Hassanin and Douzery, 2003). The genus Moschus has traditionally been placed in the subfamily Moschinae within the family Cervidae (Simpson, 1945; Wang et al., 1993; Groves et al., 1995), but there are strong arguments that Mos- ABC Fax karger@karger.ch S. Karger AG, Basel /05/ $22.00/0 Accessible online at:

2 chus should be placed in its own family, Moschidae, within the superfamily Cervoidea (Groves and Grubb, 1987; Su et al., 1999, 2001). The most recent molecular phylogeny surprisingly places Moschus as the sister group of bovids rather than cervids (Hassanin and Douzery, 2003). The gayals (Bos frontalis), which have undergone only about two hundred years domestication, are mainly distributed in the narrow valley of Dulong river in western Yunnan, China and its adjacent region in northern Burma. Thus they also are named Dulong cattle (Nie et al., 1995). For its morphological similarity with the gaur (Bos gaurus), many zoologists regarded the gayal as the domestic gaur, and until 1968 Walker classified the gayal as a species in the genus Bos (Walker et al., 1968). The gayal s origin is still not clear. It had been taken as the offspring of urus and cattle or the hybrid of urus and zebu (Lan et al., 1993). The forest musk deer and gayal, although belonging to different families, have the same diploid number 2n = 58 (Guo et al., 1988). Up to now the karyotypic relationships among the Chinese muntjac, musk deer and gayal remain undefined. Chromosome painting is a rapid and accurate way to establish the genome-wide homology among different species and its increasing application in comparative cytogenetics has provided new insights into karyotype evolution and aided the transfer of gene-mapping information from map-rich species to the map-poor species (Yang et al., 1995; Chowdhary et al., 1998; O Brien et al., 1999). The rapid progress in the bovine genome project has made the cattle genome an ideal reference for comparative genomic and cytogenetic studies (Larkin et al., 2003; In this study, we establish the comparative chromosome maps of Chinese muntjac, forest musk deer and gayal by cross-species chromosome painting with Chinese muntjac chromosome-specific painting probes and G band comparison. These maps have been integrated with the previously established homology maps among Chinese muntjac-indian muntjac (Yang et al., 1997b), Indian muntjac-human (Yang et al., 1997a), human-cattle (Hayes, 1995; Chowdhary et al., 1996; Hayes et al., 2000), and deer-cattle (Bonnet et al., 2001; Slate et al., 2002). Our results shed new insights into karyotypic relationships of the three artiodactyl species. Materials and methods Cell culture, metaphase preparation and G banding Fibroblast cell lines from the Chinese muntjac (KCB91001), forest musk deer (KCB86002) and gayal (KCB20007S) were obtained from the Kunming Cell Bank of the Chinese Academy of Sciences, Kunming, Yunnan , P.R. China. Chromosome preparations were made following conventional procedures. G banding followed the classical trypsin/giemsa staining procedure (Seabright, 1972). Fluorescence in situ hybridization (FISH) The set of chromosome-specific painting probes for the Chinese muntjac used in this study was made previously from flow-sorted chromosomes (Yang et al., 1997c) by degenerate oligonucleotide primed-pcr (DOP-PCR, Telenius et al., 1992), with the exception of Chinese muntjac chromosome 19 probe, which is made from a batch of newly flow-sorted chromosomes. Both single-color and multicolor FISH were performed following Yang et al. (1997b). For each hybridization about 50 ng labeled DNA from each Chinese muntjac chromosome were added. Sequential G banding and multicolor FISH were performed as previously described (Yang et al., 2004). Images were captured and processed following Yang et al. (1999) and Nie et al. (2002). Results Assigning the Chinese muntjac probes to G-banded chromosomes The set of Chinese muntjac probes used in our experiment has previously been assigned to DAPI-banded chromosomes of Chinese muntjac (Yang et al., 1997c). But apart from the first five autosomes (1 5) and the sex chromosomes that display well-defined DAPI-banding patterns, the other 17 autosomes remain poorly defined. To facilitate future comparative cytogenetic studies, we assigned, for the first time, this set of probes to high-resolution G-banded chromosomes of the Chinese muntjac by sequential G banding and multicolor FISH (Fig. 1a, b). As demonstrated in Yang et al. (1997c), most Chinese muntjac chromosome-specific probes showed cross-hybridization to centromeric regions of other Chinese muntjac chromosomes. The cross-hybridization signals, however, facilitate the determination of centromeric positions. Figure 1c shows a typical high-resolution G-banded karyotype of Chinese muntjac, defined by sequential G banding and multicolor chromosome painting. Painting forest musk deer chromosomes with Chinese muntjac probes The G-banded karyotype of the forest musk deer (M. berezovskii) (Fig. 2) is identical to that of the Siberian musk deer (Moschus moschiferus, 2n = 58, Graphodatsky, 1989). All Chinese muntjac probes were hybridized onto G-banded metaphases of the forest musk deer. In total, they detected 33 conserved autosomal segments in the genome of forest musk deer. The Chinese muntjac probes specific for chromosomes 6 10 and each detected one segment; chromosomes 5 and 11 probes each revealed 2 segments; chromosomes 1, 3 and 4 probes each painted 3 segments; chromosome 2 probe highlighted 4 segments. The X chromosome probe hybridized onto the whole X chromosome of the forest musk deer, but no specific signals were detected with the Y chromosome probe. The distribution patterns of all the conserved segments detected by chromosome painting were summarized on a G-banded karyotype of the forest musk deer (Fig. 2). Painting gayal chromosomes with Chinese muntjac probes As reported previously (Nie et al., 1995), the gayal has a 2n = 58 karyotype. Figure 3 shows a G-banded karyotype of the gayal, which has, to our best knowledge, not been reported previously. The 22 Chinese muntjac autosomal painting probes detected 34 conserved segments in the genome of gayal. The painting results of all probes on gayal metaphases are similar to those on forest musk deer metaphases, with the exception of Chinese muntjac chromosome 2 probe, which painted five homologous segments in the genome of gayal but four in forest musk deer (Fig. 3). Cytogenet Genome Res 108: (2005) 311

3 Fig. 1. (a) Simultaneous hybridization of six chromosome-specific Chinese muntjac painting probes (13, 14, 15, 16, 20 and 22) onto a Chinese muntjac metaphase. (b) The same G-banded metaphase as shown in a before FISH. (c) High-resolution G-banded karyotype of a male Chinese muntjac prepared from b. (d, e) Simultaneous hybridization of Chinese muntjac 16 (red) and 21 (green) probes onto metaphases of forest musk deer (d) and gayal (e). 312 Cytogenet Genome Res 108: (2005)

4 Fig. 2. G-banded karyotype of a male forest musk deer. Chromosome numbers are indicated below the chromosomes, and the homologous Chinese muntjac segments revealed after hybridization with Chinese muntjac chromosome-specific painting probes are indicated to the right of each forest musk deer chromosome. Fig. 3. G-banded karyotype of a male gayal. Chromosome numbers are indicated below the chromosomes, and the homologous Chinese muntjac segments revealed after hybridization with Chinese muntjac chromosome-specific painting probes are indicated to the right of each gayal chromosome. Cytogenet Genome Res 108: (2005) 313

5 Fig. 4. A comparative map of Chinese muntjac (MRE) chromosomes 1 5 and 11 and their corresponding chromosomes in forest musk deer (MBE, left) and gayal (BFR, right) based on FISH results and G-banding comparison. Chromosome numbers are indicated below or beside the chromosomes. Homologous segments are linked by lines. The map shows that all tandem fusions that occurred during the evolution of Chinese muntjac chromosomes 1 5 and 11 are of centromere-totelomere type. Comparative chromosome map of the Chinese muntjac, forest musk deer and gayal Except for the chromosomes 1 5 and 11 probes, all other Chinese muntjac autosomal probes each revealed one pair of homologous segments in both forest musk deer and gayal. To further define the evolutionary chromosomal rearrangements, we constructed a comparative map of the Chinese muntjac chromosomes 1 5 and 11 with their corresponding chromosomes in the forest musk deer and gayal, based on a combined analysis of the painting and banding homologies (Fig. 4). Discussion The comparative map of Chinese muntjac, forest musk deer and gayal and its integration with human, bovid and cervid comparative maps For the first time, a genome-wide comparative chromosome map including the Chinese muntjac, forest musk deer, and gayal has been established based on painting homologies. The comparative map suggests that it requires at least eleven chromosome breaks and five fusions to transform the karyotype of the Chinese muntjac to that of the forest musk deer. Similarly, it takes at least twelve chromosome breaks and six fusions to convert the Chinese muntjac karyotype to that of the gayal. Although forest musk deer and gayal have the same diploid number (2n = 58), at least one chromosome fusion (between musk deer chromosome 2 and proximal 12q) and one chromosome break (in musk deer chromosome 12) are required to transform the forest musk deer karyotype to that of gayal. Using the chromosomes of Indian muntjac, Chinese muntjac, cattle and human as references, our maps can be integrated with the previously established homology maps between bovids, cervids and human, including Chinese muntjac-indian muntjac (Yang et al., 1997b), Indian muntjac-human (Frönicke et al., 1997; Yang et al., 1997a), human-cattle (Hayes, 1995; Chowdhary et al., 1996; Hayes et al., 2000, ISCNDB, 2001), human-cattle-muntjac (Ferguson-Smith et al., 1998; Yang, 1998), and cattle-deer maps (Bonnet et al., 2001; Slate et al., 2002). This integration has enabled the establishment of genome-wide chromosomal correspondence among the domestic cattle, gayal, musk deer, Indian muntjac, Chinese muntjac, human, and deer linkage maps and their proposed ancestor (Table 1). It is noteworthy that cattle chromosomes 2 and 28 are homologous to the gayal chromosome 1q and 1p, respectively. Only one Robertsonian translocation (involving cattle chromosomes 2 and 28) differentiates the karyotypes of the domestic cattle (2n = 60) and gayal (2n = 58). The same 2;28 translocation has been reported for Bos gaurus (2n = 58, Gallagher and Womack, 1992), suggesting that the gayal and gaur may have identical karyotypes and that the gayals could have originated from domesticated gaurs. The musk deer has a highly conserved karyotype Combined FISH and G band comparison analyses demonstrate that most conserved chromosomal segments among the three artiodactyl species display highly conserved G banding patterns. In particular, the homologous segments between the forest musk deer and gayal show almost identical G banding patterns, thus supporting the notion that the musk deer and bovid are karyotypically closely related (Graphodatsky, 1989). 314 Cytogenet Genome Res 108: (2005)

6 Table 1. Chromosomal correspondence between human, cattle, gayal, musk deer, sika deer, Chinese muntjac, the putative pecoran ancestor and deer linkage group Bos taurus a Bos frontalis Moschus berezovskii Pecoran ancestor b Deer linkage group b Cervus nippon pseudaxis c Muntiacus reevesi Homo sapiens a A2 19, 31 26, 7 7, 18 3, q 2 B2 8, 33 12, 18 3 (two sites)1p, 2q A D C2 3, 22 27, 22 4, 1 1q, 12, F 6, 17 16, 23 16, E q, 19p B1 16, 29 15, 32 19, 11 8p, H1 26, 28 17, 31 14, 3 6q G q, 14, C , 9q I J p, H q L , K N1 5 2q 20 4q, 12, M 4 1p 2 16q, 19q N2 5 2p O , P , Q R S T q, 16p U q V , p 12 U , 10q W X X X X X X X X a b c ISCNBD, Slate et al., Bonnet et al., This view is apparently supported by our painting results. Of the six associations of adjacent Chinese muntjac (MRE) chromosome homologous segments found in the gayal (i.e. MRE1/ 4, 2/3, 3/14, 7/18, 11/19, and 16/21), five are present in the genome of the musk deer (MRE1/4, 3/14, 7/18, 11/19 and 16/ 21). Only the MRE2/3 association is unique for the gayal and gaur. More interestingly, the five shared associations (MRE1/4, 3/14, 7/18, 11/19, and 16/21) are absent in all cervids studied so far (Yang et al., 1997a, b, c; Yang, 1998) which at a first glance suggests that these five shared associations could represent cytogenetic signatures that link Moschidae with Bovidae. But as shown in Table 1, four of these five shared syntenic associations each correspond to one ancestral mammalian synteny (MRE1/4 = HSA12/22, MRE3/14 = HSA6q, MRE7/18 = HSA3/21 and MRE16/21 = HSA4) rather than reflecting shared derived synteny. They are therefore not phylogenetically informative and only suggest that both Bovidae and Moschidae have conserved karyotypes. Comparative mapping studies have allowed a more detailed reconstruction of the karyotype phylogeny of the pecoran ruminants that includes Antilocapridae, Cervidae, Moschidae and Bovidae (Nowak, 1999). It has been proposed that the pecoran ancestor had a 2n = 58 ancestral karyotype (Slate et al., 2002). The cervids, including muntjacs, have a 2n = 70 ancestral karyotype (Neitzel, 1987; Fontana and Rubini, 1990; Yang et al., 1997c; Yang, 1998) which could be derived from the 2n = 58 ancestral karyotype via six fissions, one fusion, one inversion and one translocation, while the bovids had a 2n = 60 ancestral karyotype which originated via one fission (that produced the equivalents of Bos taurus chromosomes 26 and 28). Intriguingly, the forest musk deer has undergone none of the above mentioned rearrangements characteristic for the Cervidae and Bovidae lineages and has retained a conserved karyotype that probably resembles the ancestral pecoran karyotype. Evolution of Chinese muntjac chromosomes 1 5 and 11 through exclusive centromere-to-telomere tandem fusion Recent comparative molecular cytogenetic studies demonstrate that the Chinese muntjac karyotype has originated from a 2n = 70 ancestral karyotype via 12 tandem fusions that produced the current chromosomes 1 5 and 11 of Chinese muntjac (Yang et al., 1997c; Yang, 1998). The establishment of a comparative map of the Chinese muntjac chromosomes 1 5, 11 and their homologues in the forest musk deer and gayal (Fig. 4) provides further test for this hypothesis, since this map allows for the more accurate determination of the orientation of corresponding conserved segments. For instance, according to comparative chromosome painting, Chinese muntjac chromosome 5 corresponds to the acrocentric chromosomes 15 and 27 of the musk deer, but G-banding alignment of the homologous Cytogenet Genome Res 108: (2005) 315

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