FISH mapping of Indian muntjac BACs. Three Indian muntjac BACs (whose sequences correspond to accession numbers (a) [GenBank:AC154146], (b) [GenBank:AC152355], and (c) [GenBank:AC154920]) were mapped by FISH to metaphase spreads prepared from an Indian muntjac fibroblast cell line. Hybridization is seen at: (a) interstitial positions on chromosomes 1 (arrows), 3, and 3+X, as well as the centromere of chromosome 3 (the signals on 3 and 3+X are indicated with arrowheads); (b) an interstitial position on chromosome 1 (arrows) and at the neck of chromosome 3+X (arrowhead); and (c) multiple sites on various Indian muntjac chromosomes (arrowheads). FISH composite images generated from merging the DAPI (blue) and Spectrum Orange (red) channels (left) and inverted DAPI banding images (right) are provided in each case. Based on further studies (see text for details), the origins of the analyzed BACs were ultimately found to be on chromosome 1 in the case of (a, b), but not yet established in the case of (c); further, the analyzed clones were found to contain the indicated ancestral chromosome fusion sites (IMFS1, IMFS3, and IMFS5, respectively; Table 1).

Self-self comparative sequence analysis of an Indian muntjac chromosome fusion site. A 60 kb sequence within IMFS1 was compared to itself using PipMaker [63]. (a) Pip plot reveals the putative chromosome fusion site, which consists of a stretch of telomeric repeats (TTAGGG)_n (blue), and then a large segment of centromeric satellite I (green); note that the latter has extensive amounts of self-self aligning sequences (reflecting satellite I monomers with high sequence identity). Also highlighted are additional features of interest: satellite IV (yellow) and a short stretch of (TTCGGG)_n (purple). (b) Dot plot of the same 60 kb region shown in (a). Expanded view reveals the periodic nature of the satellite I monomers.

Long-range organization of chromosome fusion sites in Indian muntjac. The content and organization of the seven generated Indian muntjac sequences (black lines) is depicted. The positions of (TTAGGG)_n (blue), (TTCGGG)_n (purple), satellite I (green), and satellite IV (yellow) blocks as well as duplicated segments (brown and beige) are indicated. Generated orthologous Chinese muntjac sequences are shown in gray (for IMFS1, IMFS3, and IMFS4 only). The junction is defined as the point where the (TTAGGG)_n telomeric repeats are fused with satellite I repeats (red dashed line). The bracketed area of IMFS1 indicates the region depicted in Figure 2; the bracketed area of IMFS7 indicates the region matching TGS400 [19].

Synteny relationships between IMFS sequences and corresponding human, cow, dog, and mouse genome sequences. Each generated Indian muntjac sequence (IMFS1-IMFS7) is depicted and represented by a different color; the vertical hatch marks on each sequence indicate identity with other IMFS sequences and are colored to correspond with those IMFS sequences. Telomere (TTAGGG)_n repeats are indicated with black arrows per their orientation; also indicated are centromeric satellite sequences (both with a black caret symbol and shaded blue). Tracks below each IMFS depict regions of synteny with the indicated genome (H, human; C, cow; D, dog; and M, mouse) as determined by BLAST-based alignments, with the chromosome location in the respective genome indicated in each case.

FISH-based characterization of an ancestral chromosome fusion site in the Indian muntjac genome. (a) Sequence IMFS3 (Table 1) was derived from three overlapping Indian muntjac BACs (BACs a-c), and contains the features expected of an ancestral chromosome fusion site (Figure 3). Each of the BACs was mapped by FISH to metaphase chromosomes from Indian muntjac cells. A probe (red circle) derived from the end of the middle clone (BAC a) was used to isolate an overlapping Indian muntjac BAC (BAC b) as well as two orthologous Chinese muntjac BACs (BACs d and e); the latter were sequenced, confirmed to be orthologous to the telomeric end of IMFS3, and designated CMTel3 (Table 2). (b) FISH studies were performed on the five clones (BACs a-e) depicted in (a). Indian muntjac BAC [GenBank:AC152355] (BAC a) hybridized to an interstitial position on chromosome 1 and at the neck of chromosome 3+X (shown in Figure 1b). The FISH composite image (upper row, far left) generated by merging the DAPI (blue), Spectrum Orange (red), and Spectrum Green (green) channels shows BAC [GenBank:AC197641] (BAC b; in red) hybridizing to an interstitial site on Indian muntjac chromosome 1 (arrow) and BAC [GenBank:AC166188] (BAC c; in green) hybridizing to the latter position as well as other centromeric and interstitial sites (arrowhead), reflecting its high content of satellite I and duplicated segments; an enlarged view of chromosome 1 (upper row, far right) confirms the site of co-hybridization of these two BACs. The gray scale channel images (upper row, middle) corresponding to the enlarged view of chromosome 1 show the hybridization pattern of each BAC separately. A similar FISH study with Chinese muntjac chromosomes is shown on the bottom row, with Chinese muntjac BAC [GenBank:AC196603] (BAC d; in green) and BAC [GenBank:AC198815] (BAC e; in red) co-hybridizing to a telomeric position on a pair of chromosomes (arrowheads). The two gray scale channel images show the hybridization pattern of each BAC separately. Synteny analysis suggests that the site of hybridization of these two BACs is Chinese muntjac chromosome 22 (see text).

Evolutionary history of Indian muntjac chromosome fusion sites. A proposed model is shown tracing the evolutionary history of Indian muntjac IMFS1 and IMFS3 as well as the orthologous Chinese muntjac sequences CMTel1 and CMTel3. The hypothetical ancestral muntjac genome contained as many as 70 chromosomes, a small subset of which is shown on the left. There is evidence [18,39] that Chinese muntjac chromosome 3 was derived from three fusion events involving ancestral chromosomes 3a-3d. Ancestral chromosomes 3d, 3c, 12, and 22 appeared to have fused in head-to-tail fusions to form the distal end of Indian muntjac chromosome 1p; note that ancestral chromosomes 3a and 3b fused with other chromosomes and are present elsewhere in the Indian muntjac genome [18,39]. The chromosome fusion sites on Indian muntjac chromosomes contain telomeric repeats adjacent to satellite I sequences (Figures 2 and 3), consistent with sequential head-to-tail fusions of telocentric chromosomes. During these events the telomeric centromere of the 'head' chromosome (containing telomeric repeats and satellite II and IV sequences) becomes lost, and satellite I sequences become fused with telomeric repeats from the 'tail' chromosome.