High-Density Comparative BAC Mapping in the Black Muntjac

Genomics 87 (2006) 608–615 www.elsevier.com/locate/ygeno

High-density comparative BAC mapping in the black muntjac (Muntiacus crinifrons): Molecular cytogenetic dissection of the origin of MCR 1p+4 in the X1X2Y1Y2Y3 sex chromosome system

Ling Huang a,b, Jianxiang Chi a, Jinhuan Wang a, Wenhui Nie a, ⁎ Weiting Su a, Fengtang Yang a,c,

a Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, People's Republic of China b The Graduate School of the Chinese Academy of Sciences, Beijing 100039, People's Republic of China c Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK Received 8 September 2005; accepted 16 December 2005 Available online 26 January 2006

Abstract

The black muntjac (Muntiacus crinifrons,2n =8♀/9♂) is a critically endangered mammalian species that is confined to a narrow region of southeastern China. Male black muntjacs have an astonishing X1X2Y1Y2Y3 sex chromosome system, unparalleled in eutherian mammals, involving approximately half of the entire genome. A high-resolution comparative map between the black muntjac (M. crinifrons) and the Chinese muntjac (M. reevesi, 2n = 46) has been constructed based on the chromosomal localization of 304 clones from a genomic BAC (bacterial artificial chromosome) library of the Indian muntjac (M. muntjak vaginalis,2n =6♀/7♂). In addition to validating the chromosomal homologies between M. reevesi and M. crinifrons defined previously by chromosome painting, the comparative BAC map demonstrates that all tandem fusions that have occurred in the karyotypic evolution of M. crinifrons are centromere–telomere fusions. The map also allows for a more detailed reconstruction of the chromosomal rearrangements leading to this unique and complex sex chromosome system. Furthermore, we have identified 46 BAC clones that could be used to study the molecular evolution of the unique sex chromosomes of the male black muntjacs. © 2005 Elsevier Inc. All rights reserved.

Keywords: BAC mapping; Chromosomal rearrangements; Muntiacus crinifrons; Tandem fusion; Sex chromosome system

Asian muntjacs (genus Muntiacus, family Cervidae) provide fusion, and centromere–telomere fusion [9]. Comparative powerful models for studying karyotypic evolution and chromosome painting [14,17] and mapping of centromeric or speciation because of their radical and rapid karyotypic telomeric repetitive DNA sequences [10–13] indicated that diversification during the past 2 million years [1–4]. A tandem most, if not all, of the tandem fusions that have occurred during fusion hypothesis has been proposed to explain the karyotypic the karyotypic evolution of the extant muntjacs from a differences between Chinese muntjac (M. reevesi,MRE, hypothetical 2n = 70 ancestral karyotype were centromere– 2n = 46) [5] and Indian muntjac (M. muntjak vaginalis, telomere fusions [16,17,20]. This view is supported by 2n =6♀/7♂) [6,7]. This hypothesis is supported by both architecture analysis of the fusion point [21] and comparative classical cytogenetic [8,9] and molecular cytogenetic studies bacterial artificial chromosome (BAC) mapping in M. reevesi [3,10–22]. Three types of tandem fusions could have occurred and M. m. vaginalis [22]. depending on the orientations of ancestral chromosomes The black muntjac (M. crinifrons, MCR), endemic to a involved: centromere–centromere fusion, telomere–telomere narrow region of the southeastern China [23],hasa karyotype of 2n =8♀/9♂ [24,25]. Previous chromosome painting studies [3,17,18] demonstrated that despite the ⁎ Corresponding author. Fax: +86 871 5191823. diploid chromosome number of M. crinifrons being close E-mail addresses: [email protected], [email protected] (F. Yang). to that of M. m. vaginalis, the genomic organization of the

Table 1 Table 1 (continued) Summary of BAC clones mapped on the chromosomes of Muntiacus reevesi MCR MRE Clone (MRE) and Muntiacus crinifrons (MCR) (1p+4)q 3c 04D5, 05A3, 06A3, 06F1, 09A6, 53B13, MCR MRE Clone 54O19, 54P19, 55C13, 55B8, 56A4 1p 17 01H6, 04D9, 06G5, 07A9, 09E3, 10F2, (1p+4)q 3d 01B8, 01B10, 01E5, 06C3, 07B11, 09A7, 09B1, 09B2, 54P15, 54P20, 55C8, 56A19, 58B18, 58C16 09E11, 09G3, 09H4, 09H6, 54N16, 1p 8 06D3, 06E3, 09A2, 09D5, 09E5, 53A19, 56B10, 58B17 55B4, 55C12, 56A11, 58C9 1q 18 01B9, 01F10, 04D6, 06A1, 06A4, 09E2, 56D15, 58C17 Yq Yq 07D6 1q 5a 06A6, 06D6, 06E2, 07B7, 09B7, 56B11, 58A4, 58C5, 58C7 Boldface indicates those BACs shown in Fig. 2. 1q 19 01F6, 06H3, 07B8, 09A5, 54O2, 55C7, 55O2, 58B24 1q 9 06B3, 07D12, 07E12, 09E9, 10F4, 53A9, 53B8, 55B13, 55B16, 58B19, 58C6 1q 16 09E1, 09F8, 54A11, 55A8, 55B17 two species differs considerably. More interestingly, the male 1q 21 01A2, 01H3, 06G4, 07A8, 09G12, 10E4, 10E7, 10E12, 54B22 M. crinifrons has a sex chromosome system of 1q 6 01E7, 06A2, 06C7, 06D8, 06F2, 07A10, 09D2, X1X2Y1Y2Y3 that comprises approximately half of the 09E2, 09E4, 54M16, 55A8, 56E11 entire genome [25]. Subsequent chromosome painting studies 1q 5b 01B12, 01D6, 04B9, 06F7, 07E5, 09D6, 54F23, have shown that the X1, X2, Y1, Y2, and Y3 correspond to 56E3, 57A12 chromosomes X+4, 1, Y, 1p+4, and 1q of M. crinifrons, 2p 7 06C10, 07B12, 07C8, 07C12, 54M14, 55A2, 55A3, 56E5, 56E21, 58B5 respectively [3,18]. Such a complex sex chromosome system, 2q 10 04F9, 05A1, 06B6, 06C5, 06C11, 09C7, 09D4, which originated within the past 0.5 million years [4],is 09F1, 09G4, 09G8, 09H8, 10E5, 56A3, 56D4, unparalleled in other eutherian mammals so far studied. 56D14, 56D21, 58B1, 58B8, 58B14 However, due to the inability of chromosome painting to 2q 11a 01B3, 06D10, 53A4, 53A5, 53A6, 53A7, 53A10, resolve intrachromosomal rearrangements, the orientations of 53B7, 55A7 2q 11b 05A2, 06E8, 10E11, 53A3 conserved segments as well as the detailed rearrangements 2q 15 06B12, 07E7, 54B12, 58B21, 58C4 leading to the unique Y2 (MCR 1p+4) in male M. crinifrons 2q 13 01A1, 01B1, 04H2, 06C4, 09A1, 09B6, 09F4, 10F8, 56E1 remain a puzzle [18]. 2q 14 04B2, 06A5, 09D12, 10E8, 56A16, 58A5, 58B22 Here we have developed a comparative BAC map between 2q 2b 04G2, 06G6, 09A9, 09A10, 09B9, 09F3, 54C1, 56E7 M. crinifrons and M. reevesi based on the chromosomal 2q 2c 04D11, 05A5, 06B2, 06H5, 07C7, 07D3, 56D18 2q 2d 01D11, 01E3, 06E11, 06G10, 07B5, 07C4, 07D9, 10F5 assignment of 304 BACs from the M. m. vaginalis library. The 3q 2a 01A8, 01D5, 07C6, 09B10, 09C9, 54J24, 55A6, 56A5 map refines the segmental homologies between M. reevesi and 3q 20 01A4, 01A11, 06B7, 06C8, 06D5, 07B1, 07D10, 07D11, M. crinifrons established previously by comparative painting, 53A23, 58C1 demonstrating that the tandem fusions underpinning karyo- 3q 4b 01H4, 04D3, 04D4, 05A8, 06F3, 07C5, 07E11, typic evolution in M. crinifrons are exclusive centromere– 09D7, 53A1, 54N5, 55A4, 56A15, 56D20, 56E8, 58C13 3q 4c 01E6, 07A11, 07D7, 07E8, 09B5, 57A7 telomere fusions. The results have also enabled the recon- 3q 1b 01A3, 01B4, 01B11, 01F12, 04C1, 04H1, 06H9, 07D1, structionofthepathwayleadingtotheM. crinifrons 09G5, 10E2, 54E8 chromosome 1p+4 (i.e., Y2), which, in a sense, represents a 3q 1c 01D9, 07B9, 07C10, 09G2, 10E3, 54L3, 55A1, 58A1, neo-Y chromosome. 58A2, 58B2 (X+4)X X 01A7, 01D10, 07D6 (X+4)q 3b 06B8, 06D1, 07B3, 07C11, 09D7, 54A21 Results and discussion (X+4)q 1a 01H12, 04A7, 06A7, 53A21, 58C21 (X+4)q 3a 06D9, 09B9, 09G6, 53A17, 53B17, 54B3, 56B1 Previous cytogenetic studies on muntjacs have predomi- (X+4)q 4a 05A6, 06B5, 06G7, 07B10, 09C12, nantly focused on the M. reevesi and M. m. vaginalis because of 53A16, 53P15, 58A16, 58B23 the extreme karyotypic differences (2n =46inM. reevesi while (X+4)q 22 04C6, 06D2, 06D11, 09A12, 09B11, 09B12 (X+4)q 12 06B4, 07E9, 07E10 2n = 6/7 in M. m. vaginalis) and wide availability of (X+4)q 3c 04D5, 05A3, 06A3, 06F1, 09A6, 53B13, 54O19, 54P19, experimental materials. In contrast, the other muntjac species 55C13, 55B8, 56A4 with equally interesting karyotypes [3,18] have been poorly (X+4)q 3d 01B8, 01B10, 01E5, 06C3, 07B11, 09A7, 09B1, studied. Here, 304 BAC clones containing unique DNA 09B2, 09E11, 09G3, 09H4, 09H6, 54N16, 55B4, 55C12, sequences derived from an M. m. vaginalis library have been 56A11, 58C9 (1p+4)p 17b 07A9, 54P15, 56A19, 55C8, 10F2 assigned comparatively onto the chromosomes of M. crinifrons (1p+4)p 22a 06D11 and M. reevesi (Table 1). The chromosomal localizations of all (1p+4)p 4a 05A6, 06B5, 06G7, 07B10, 09C12, 53A16, BACs are summarized on a high-resolution idiogram of M. 53P15, 58A16, 58B23 reevesi (Fig. 1). A comparative BAC map between M. crinifrons (1p+4)p 3a 06D9, 09G6, 53A17, 09B9, 53B17, 54B3, 56B1 and M. reevesi was constructed based on the chromosomal (1p+4)p 1a 01H12, 04A7, 06A7, 53A21, 58C21 (1p+4)p 8 06D3, 06E3, 09A2, 09D5, 09E5, 53A19, 56B10, 58B17 localizations of these BACs in both species (Fig. 2), which (1p+4)q 3b 06B8, 06D1, 07B3, 07C11, 09D7, 54A21 provides valuable information on the karyotypic evolution of M. (1p+4)q 17a 01H6, 04D9, 06G5, 09E3, 54P20, 58B18, 58C16 crinifrons. Of the BACs mapped so far in M. crinifrons, 194 (1p+4)q 22b 04C6, 06D2, 09A12, 09B11, 09B12 have also been mapped previously to M. reevesi and M. m. (1p+4)q 12 06B4, 07E9, 07E10 vaginalis [22]. 610 L. Huang et al. / Genomics 87 (2006) 608–615

Fig. 1. Summary of the chromosomal localizations of 304 clones from a genomic BAC library of M. m. vaginalis on a high-resolution G-banded idiogram of M. reevesi. (Note: the clones in boldface are those BACs added in this article).

Comparative BAC mapping of M. crinifrons and M. reevesi 10, 11, 15, 13, 14, and 2b–2d are joined together in tandem on identifies centromere–telomere fusions as the drivers of MCR 2 [18]. BAC clones 06C5, 06D10, 06B12, 09B6, and genome reorganization of M. crinifrons 09D12 locate to the proximal regions of MRE 10, 11, 15, 13, and 14, respectively, and 07D3 locates on the middle of MRE 2c High-density comparative BAC map between M. crinifrons (Fig. 3E). From the assignments of six clones on MCR 2 (Fig. and M. reevesi has enabled us to define the orientation of tandem 3F), we can deduce that these six homologous segments have fusions during the karyotypic evolution of M. crinifrons (Figs. 1 maintained the same orientations in M. crinifrons and thus they and 2). For example, clone 06F2 (Fig. 3A, green) locates on the must have been joined together by centromere-telomere fusions distal region of MRE 6q, clone 09D2 (red) also maps to MRE 6q (Fig. 2). As shown in M. m. vaginalis [22], all tandem fusions but its location is proximal to the former (i.e., close to the that have occurred in the karyotypic evolution of M. crinifrons centromere); this orientation has been retained on MCR 1 and 1q are exclusive centromere–telomere fusions, suggesting an (Fig. 3B). As another example, homologous segments of MRE inherent capacity of muntjac genomes to undergo this type of .Hage l eois8 20)608 (2006) 87 Genomics / al. et Huang L. – 615

Fig. 2. Comparative BAC map between M. reevesi and M. crinifrons (MCR), demonstrating the orientation of conserved segments on MCR chromosomes. 611 612 L. Huang et al. / Genomics 87 (2006) 608–615

Fig. 3. FISH examples with clone DNA probes map onto the chromosomes of M. reevesi (MRE) and M. crinifrons (MCR). (A and B) Clone 09D2 (red) and clone 06F2 (green) map onto the middle and distal region of MRE 6 (A) and its homologous segments on MCR 1 and 1q (B). (C and D) Clone 07D6 (yellow) gave signals on the distal region of MRE X and Y (C) as well as MCR X and Y (D). (E and F) Clones 06C5 (red), 06D10 (green), 06B12 (red), 09B6 (green), 09D12 (red), and 07D3 (green) locate to the proximal region of MRE 10, 11, 15, 13, and 14 and the middle of MRE 2c, respectively (E), as well as their homologous segments on MCR 2 (F).

rearrangement. Further comparative sequencing of the tandem centric chromosomes of a hypothetical 2n = 70 ancestral fusion points will provide new insights into the molecular karyotype by repeated tandem fusions [16,22,26]. The chro- mechanism underlying such exclusive tandem fusion events. mosome paints from MRE 1–5 and 11 each correspond to one to three homologous segments in the genome of M. crinifrons High-resolution comparative BAC map defines the [18], but the detailed subchromosomal correspondence remains subchromosomal homologies between MRE 1–5, 11, 17, undefined. The comparative BAC map has enabled the and 22 and their homologous segments in M. crinifrons unambiguous establishment of such correspondence (Fig. 2). For example, MRE 5 detected two homologous segments on In addition to defining the orientations of the evolutionarily MCR 1q1.3–q1.4 and 1q4.1–q4.4 [18]. Clones 09B7 and 07B7 conserved segments, high-resolution comparative BAC map- locate on MRE 5a, clones 01B12 and 07E5 map to MRE 5b ping allows us to distinguish both inter- and intrachromosomal ([22], Fig. 1). In M. crinifrons, clones 09B7 and 07B7 locate to rearrangements. MRE 1–5 and 11 have been shown to be MCR 1q1.3–q1.4, while clones 01B12 and 07E5 map to MCR derived respectively from two to four nonhomologous acro- 1q4.2–q4.4. Our results thus confirm that MRE 5a is L. Huang et al. / Genomics 87 (2006) 608–615 613 homologous to the MCR 1q1.3–q1.4 while MRE 5b is establishment of the orientations and subchromosomal homol- homologous to the MCR 1q4.1–q4.4 (Fig. 2). ogies of the conserved segments in M. crinifrons allows us to Moreover, MRE 17 and 22 have each been conserved as one propose a three-step model for the evolution of the unique MCR single segment in all muntjac species so far studied with the 1p+4 (Y2) (Fig. 4). First, a translocation of MCR 1p (equivalent exception of the male M. crinifrons, in which they each to MRE 17+8) to MCR 4 (equivalent to MRE 3d+3c+12+22+4a correspond to two segments on MCR 1p+4, “MRE 17a” and +3a+1a+3b) produced the preform of MCR 1p+4 (Fig. 4a). “MRE 22a” on q1.3–q1.5 and “MRE 17b” and “MRE 22b” on Second, a pericentric inversion with breakpoints in the distal p2.5 [18]. Our results help to resolve the subchromosomal region of “MRE 17” and proximal region of “MRE 22” resulted correspondence between MRE 17 and 22 and their homologous in the intermediate form of MCR 1p+4 (Fig. 4b). Last, a second segments on MRE 1p+4. For instance, three clones (09E3, pericentric inversion with breakpoints at the putative fusion 06G5, and 07A9) map to MRE 17 (Fig. 1), with two clones points within the syntenic segment associations of “MRE 3b/1a” (09E3, 06G5) mapping to the q1.3–q1.4 region of MCR 1p+4 and “MRE 8/17a” led to the formation of the extant MCR 1p+4 and 07A9 to the p2.5 region of MCR 1p+4. We can thus (Fig. 4c). Hence a combination of chromosomal fissions, conclude that the “MRE 17a” on the long arm of MCR 1p+4 is fusions, and inversions most likely has contributed to the origin homologous to the proximal region of MRE 17 (Fig. 2), while of MCR 1p+4 from 1p and 4. the “MRE 17b” on the short arm of MCR 1p+4 is homologous The origin and evolution of the Y chromosome in higher to the distal region of MRE 17. Similarly, six clones (06D11, eukaryotic organisms have attracted the interest of researchers 04C6, 06D2, 09A12, 09B12, and 09B11) have been assigned to for almost a century. Although several hypotheses have been MRE 22 ([22], Fig. 1). Among them, only one clone 06D11 proposed [27–31], the tests of such hypotheses remain a great maps to the q2.5 region on MCR 1p+4 and five clones (004C6, challenge. Except for a few species in Drosophila [32,33] and 006D2, 09A12, 09B12, and 09B11) map to the q1.4–q1.5 plants [34], the Y chromosome in most extant higher eukaryotic region on MCR 1p+4, demonstrating that “MRE 22b” on the species differentiated from its autosomal progenitor hundreds of short arm of MCR 1p+4 is actually homologous to the proximal millions of years ago, making it difficult to trace the regions of MRE 22q. To maintain consistency with the evolutionary history. In the male M. crinifrons, despite the nomenclature [22] of the hypothetical ancestral segments (i.e., great structural differences between 1p+4 (Y2) and its counter- the proximal part being designated as “a”, while the more distal parts (X+4 (X1) and 1 (X2)), they, together with MCR 1q (Y3) part are “b”, “c”, …), we renamed “MRE 22a” previously and the real muntjac Y (Y1), form a pentavalent at the male defined by chromosome painting [18] as MRE 22b, while meiotic prophase [25], with 1p (X2p) pairing with the top one- “MRE 22b” is MRE 22a (Figs. 1, 2, and 4c). third of MCR 1p+4 (Y2), (X+4)q (i.e., chromosome 4) pairing with the other two-thirds, and the Y (Y1) pairing with the real X Comparative BAC mapping allows the reconstruction of (i.e., the short arm of X+4) [18,25]. Since no inversion loop is complex chromosomal rearrangements leading to MCR 1p+4 evident even at the early stages of synaptonemal complex (Y2) in male M. crinifrons formation [25], a large segment on MCR 1p+4 (from MRE 22a to MRE 17a) must have been undergoing heterosynapsis and is Our BAC map demonstrates that the male-specific metacen- thus devoid of recombination [18]. MCR 1p+4 originated in tric MCR 1p+4 (i.e., Y2) consists of 12 conserved segments male M. crinifrons within the past 0.5 million years [4]; it could homologous to seven MRE chromosomes (Fig. 2). The thus be regarded as a “neo-Y” chromosome. It becomes highly tempting to test if the lack of recombination would lead to a gradual accumulation of mutations and eventually degradation of the entire 1p+4 as happened during the origination of Y chromosomes in higher eukaryotic organisms [27–31]. The 46 M. m. vaginalis BAC clones that map to the nonrecombination region of MCR 1p+4 (Table 1) are quite suitable for such study as of the molecular evolution of this neo-Y, since the sequences of genes contained in these M. m. vaginalis BACs can be utilized to design primers and amplify the corresponding genes from other muntjacs, in particular, the orthologs from the flow- sorted MCR 1p+4, X+4, and 1. The preliminary results of our ongoing research indicate that the genes on the nonrecombination region of MCR 1p+4 have already differentiated from their corresponding genes in other muntjac species (Dr. Wen Wang, personal communication). In summary, we have constructed a high-density comparative BAC map between M. reevesi and M. crinifrons. This map allows Fig. 4. A three-step model for the reconstruction of extant MCR 1p+4 (Y2) from 1p and 4 in male M. crinifrons. The breakpoints (BR) are marked with red (1) a more accurate delineation of the orientation and subchro- arrows. 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