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Genome-Wide Comparative Chromosome Map between Human and the Forrest’s Pika (Ochotona forresti) Established by Cross-Species Chromosome Painting: Further Support for the Glires Hypothesis

Comparative genomic data for ochotonids (pikas) are important for understanding the karyotype evolution of lagomorphs. Here, we have established the first genome-wide chromosomal homologies between human and the Forrest’s pika (Ochotona forresti, 2n = 54) by cross-species chromosome painting. Integration of our map with the published comparative chromosome map between human and rabbit has enabled the establishment of an indirect homology map between Forrest’s pika and domestic rabbit (Oryctolagus cuniculus, 2n = 44), representing the 2 Lagomorpha families: Leporidae and Ochotonidae. Our results demonstrate that (1) most of the proposed ancestral eutherian syntenies or syntenic associations have been retained in the O. forresti genome; (2) the HSA1/10p association, one of the 2 signature rearrangements that were proposed to support the grouping of the orders Lagomorpha and Rodentia into a monophyletic clade called Glires, is also present in the O. forresti genome; and (3) Robertsonian translocations have contributed to the karyotype differences between O. forresti and O. cuniculus.

42 man (HSA) homologous chromosomal segments in domestic rabbit [Korstanje et al., 1999] and squirrels [Richard et al., 2003;Stanyon et al., 2003;Li et al., 2004Li et al., , 2006 has led to the suggestion that the HSA1/10p association and, to a lesser extent, the HSA9/11 association may represent the cytogenetic signature characterizing the clade Glires [Stanyon et al., 2003;Li et al., 2004]. Nevertheless, the presence of such signature rearrangements in Ochotonidae remains to be established.
Comparative cytogenetic studies have made a great contribution to our understanding of karyotype evolution of species within the families Leporidae and Ochotonidae, respectively [Hsu and Benirschke, 1971;Stock, 1976;Capanna et al., 1991;Ivanitskaya, 1991;Robinson et al., 2002;Robinson, 2006 and literature cited therein]. However, earlier cross-family comparison based on G-banding has only revealed a few conserved chromosomal segments between representative species from Leporidae and Ochotonidae, leading to the suggestion that tandem fusion could be responsible for the familylevel karyotype divergence in Lagomorpha [Stock, 1976].
Cross-species chromosome painting is the most robust method for establishing genome-wide comparative chromosome maps in mammals. Such comparative chromosome maps, usually using human chromosomes as the reference, have enabled the reconstruction of ancestral eutherian genome organization and landscape chromosomal rearrangements accompanying the radiation of major phylogenetic lineages [see Ferguson-Smith and Trifonov, 2007 for review]. Up to now, the comparative chromosome maps between human and representative species of the extant 18 eutherian orders (including one Lagomorpha species, Oryctolagus cuniculus ) [Korstanje et al., 1999] have been reported [see Ferguson-Smith and Trifonov, 2007 for review;Nie et al., 2008]. Nevertheless, genome-wide comparative chromosome maps between human and representative species of Ochotonidae, as well as between representative species of Leporidae and Ochotonidae have not been reported hitherto.
In this study, we have established the first genomewide chromosomal homology map between human and Forrest's pika ( Ochotona forresti , OFO, 2n = 54) by crossspecies chromosome painting with human chromosomespecific painting probes. Such a map sheds further insight into the ancestral lagomorph karyotype and chromosomal rearrangements underlying the karyotype divergence between Leporidae and Ochotonidae and provides further support for the Glires hypothesis.

Metaphase Preparations and DAPI-Banding
The O. forresti fibroblast cell line was derived from skin biopsies of a male Forrest's pika collected from Gaoligong Mountain, Yunnan, China. Metaphase preparations were made following conventional methods as previously described [Yang et al., 2003a]. The O. forresti chromosomes were karyotyped based on inverted DAPI-banding patterns that are similar to the G-banding patterns.

Discussion
Cross-species chromosome painting with human chromosome-specific painting probes has allowed us to construct the first genome-wide comparative chromosome map between human and O. forresti , a representative species of the pika family. An integrated analysis of our comparative chromosome map and the previously published maps [Korstanje et al., 1999;Robinson et al., 2002] provides new insight into the comparative genome organization of the Lagomorpha.

Further Cytogenetic Evidence for the Superorder Glires
Previous comparative analyses have indicated that HSA1/10p and HSA9/11 (to a lesser extent) associations are considered to be the cytogenetic signatures characterizing the clade Glires [Stanyon et al., 2003;Li et al., 2004]. Our results show that the HSA1/10p association appears to be present in the O. forresti chromosome 1. Thus, the existence of HSA1/10p association in the pika genome provides further cytogenetic evidence for the Glires hypothesis. However, the HSA1/10p association is absent in the genomes of Pedetes capensis (Pedetidae), Sicista betulina (Dipodidae), and Castor fiber (Castoridae) [Graphodatsky et al., 2008], it seems likely that this association has been disrupted in their common ancestor. Interestingly, HSA1/10 is also present in all Perissodactyla species for which the Zoo-FISH data are available [Yang et al., 2003a[Yang et al., , 2004Trifonov et al., 2008] and in the Malayan flying lemur (Galeopterus variegatus) [Nie et al., 2008]. However, reciprocal chromosome painting data show that the HSA1/10 association detected in both Perissodactyla species and the Malayan flying lemur came from the segments homologous to HSA1 and 10q [Yang et al., 2003a[Yang et al., , 2004Nie et al., 2008;Trifonov et al., 2008]. Therefore, the HSA1/10p association is indeed a cytogenetic signature that supports the grouping of orders Lagomorpha and Rodentia into the superorder Glires.
Although HSA9/11 association is found in domestic rabbit [Korstanje et al., 1999;Chantry-Darmon et al., 2005], it is absent in the genomes of other lagomorphs so far investigated. The karyotypic relationships within the leporids have been examined by chromosome painting using O. cuniculus chromosome-specific probes [Robinson et al., 2002]. The results showed that O. cuniculus chromosome 1 (homologous to HSA9/11) has broken up in the leporid lineage (i.e. homologous to HSA9 and 11, respectively) [Robinson et al., 2002]. Moreover, HSA9/11 association is also absent in the genome of O. forresti in our study. Nevertheless, comparative chromosome maps between human and representative species of the Rodentia [Richard et al., 2003;Stanyon et al., 2003;Li et al., 2004Li et al., , 2006Graphodatsky et al., 2008] have suggested that the HSA9/11 association could be regarded as a component of the ancestral karyotype of the Rodentia [Graphodatsky et al., 2008]. Considering that the family Ochotonidae consists of about 30 species, comparative chromosome painting data from more representative pika species, in particular those with lower diploid numbers, are needed to further validate if the HSA9/11 association could represent the shared derived cytogenetic character of the superorder Glires.