Comparative Chromosome Painting in Mammals: Human and the Indian Muntjac (Muntiacus muntjak vaginalis)

We have used human chromosome-specific painting probes forin situhybridization on Indian muntjac (Muntiacus muntjak vaginalis,2n= 6, 7) metaphase chromosomes to identify the homologous chromosome regions of the entire human chromosome set. Chromosome rearrangements that have been involved in the karyotype evolution of these two species belonging to different mammalian orders were reconstructed based on hybridization patterns. Although, compared to human chromosomes, the karyotype of the Indian muntjac seems to be highly rearranged, we could identify a limited number of highly conserved homologous chromosome regions for each of the human chromosome-specific probes. We identified 48 homologous autosomal chromosome segments, which is in the range of the numbers found in other artiodactyls and carnivores recently analyzed by chromosome painting. The results demonstrate that the reshuffling of the muntjac karyotype is mostly due to fusions of huge blocks of entire chromosomes. This is in accordance with previous chromosome painting analyses between various Muntjac species and contrasts the findings for some other mammals (e.g., gibbons, mice) that show exceptional chromosome reshuffling due to multiple reciprocal translocation events.     

Mouse chromosome-specific painting probes generated from microdissected chromosomes

Using degenerate primer amplification of chromosomes microdissected from banded cytogenetic preparations, we constructed both whole chromosome painting probes for mouse Chromosomes (Chrs) 1, 2, 3, and 11 and a centromere probe that strongly paints most mouse centromeres. We also amplified a Robertsonian translocation chromosome microdissected from unstained preparations to construct a painting probe for Chrs 9 and 19. The chromosomes probes uniformly painted the respective chromosomes of origin. We demonstrated the utility of the Chr 11 probe in aberration analysis by staining mutants that we had previously identified as containing a Chr 11 translocation, and in some mutant cell lines we observed chromosome rearrangements not previously detected in stained cytogenetic preparations. The technology of microdissection and amplification applies to all mouse chromosomes or to specific subchromosomal regions and will be useful in mouse genetics, in aberration analysis, and for chromosome identification.     

New insights into the karyotypic relationships of Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis)

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.     

利用微分离黑麦1R染色体的体外扩增产物进行染色体着染研究

首先对显微分离出的黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡ＿ＰＣＲ）。经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后,再利用１Ｒ染色体的第二轮扩增产物、黑麦基因组ＤＮＡ、ｒＤＮＡ基因为探针,与其根尖细胞中期分裂相进行染色体原位杂交,发现微分离的１Ｒ染色体体外扩增产物中包含大量的非该染色体特异性重复序列,而其信息量却较黑麦总基因组少;当以适量的黑麦基因组ＤＮＡ进行封阻时,微分离染色体的体外扩增产物成功地被重新定位在中期分裂相的一对１Ｒ染色体上,说明微分离１Ｒ染色体的ＰＣＲ扩增产物中的确包含了该染色体特异性的片段。此外,以从１Ｒ染色体微克隆文库中筛选出的一单、低拷贝序列和一高度重复序列分别为探针,染色体原位杂交检测发现,这一高度重复序列可能为端粒相关序列;而单、低拷贝序列却未检测到杂交信号。这些结果从不同侧面反映出染色体着染技术是证实微分离、微切割染色体的真实来源及筛选染色体特异性探针的有利工具。建立了可供参考的植物染色体着染实验体系,为染色体微克隆技术在植物中的进一步应用提供了便利。     

Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae)

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.     

Sex chromosomes in the spiny eel (Mastacembelus aculeatus) revealed by mitotic and meiotic analysis

Lower vertebrates like fish exhibit tremendous diversity in sex determination. There are wide interplays between environment-dependent sex differentiation ranging from natural hermaphroditism to sex reversal and genetic sex determination. Diverse systems of male and female heterogamety coexist in fish and sex chromosomes are rarely distinguishable in morphology. Here we show that the spiny eel ((Mastacembelus aculeatus) of the Perciformes, has evolved highly heteromorphic X and Y chromosomes. The metacentric X and Y chromosomes are the largest among 24 homologous pairs, differ from each other in size and morphology, and become distinct after C-banding because of conspicuous heterochromatin blocks which exhibit alternate distribution around the centromeric region. Chromosome painting using probes from the microdissected X chromosome revealed sequence homology between X and Y. During the pachytene stage of meiosis the X and Y form a bivalent. However, their synapsis is delayed which is particularly evident in one terminus. Therefore, the X and Y have resulted from a pericentric inversion in the Y. We conclude that M. aculeatus represents an example of a highly advanced stage of sex chromosome evolution in fish.     

Comparative chromosome painting defines the high rate of karyotype changes between pigs and bovids

Human and sheep chromosome-specific probes were used to construct comparative painting maps between the pig (Suiformes), cattle and sheep (Bovidae), and humans. Various yet unknown translocations were observed that would assist in a more complete reconstruction of homology maps of these species. The number of homologous segments that can be identified with sheep probes in the pig karyotype exceeds that described previously by chromosome painting between two non-primate mammals belonging to the same order. Sheep probes painted 62 segments on pig autosomes and delineated not only translocations, but also 9 inversions. All inversions were paracentric and indicate that these rearrangements may be characteristic for chromosomal changes in suiforms. Hybridizations of all sheep painting probes to cattle chromosomes confirmed the chromosome conservation in bovids. In addition, we observed a small translocation that was previously postulated from linkage mapping data, but was not yet described by physical mapping. The chromosome painting data are complemented with a map of available comparative gene mapping data between pig and sheep genomes. A detailed table listing the comparative gene mapping data between pig and cattle genomes is provided. The reanalysis of the pig karyotype with a new generation of human paint probes provides an update of the human/pig comparative genome map and demonstrates two new chromosome homologies. Seven conserved segments not yet identified by chromosome painting are also reported. Received: 2 October 2000 / Accepted: 15 January 2001     

Zoo-FISH with microdissected arm specific paints for HSA2, 5, 6, 16, and 19 refines known homology with pig and horse chromosomes

Microdissected arm specific paints (ASPs) for human (HSA) chromosomes (Chrs) 2, 5, 6, 16, and 19 were used as probes on pig (SSC) and horse (ECA) metaphase chromosomes. Regions homologous to individual human arms were delineated in the two species studied. Of the ten ASPs used, HSA6 and 16 ASPs showed complete synteny conservation of individual arms as single blocks/arms both in pig and horse. A similar trend was, in general, also observed for HSA19 ASPs. However, contrary to these observations, synteny conservation of individual arms of HSA2 and HSA5 was not observed in pig and horse. The arm specific painting data, coupled with the available gene mapping data, showed that, although HSA2 corresponded to two arms/chromosomes each in pig and horse, the breakpoint of this synteny in humans was not located at the centromere, but at HSA2q13 band. Similarly, arm specific paints for HSA5 showed that of the two blocks/chromosomes painted in pig and horse, one corresponded to HSA5q13-pter, the other to HSA5q13-qter. The findings suggest that 5q13 band may also be an evolutionary break point, similar to the one detected on HSA2q13. The microdissected human arm specific painting probes used in the present work provide more accurate and refined comparative information on pig and horse chromosomes than that available through the use of human whole chromosome specific paints. Received: 1 June 1997 / Accepted: 5 September 1997     

Chromosome painting between human and lorisiform prosimians: evidence for the HSA 7/16 synteny in the primate ancestral karyotype

Multidirectional chromosome painting with probes derived from flow-sorted chromosomes of humans (Homo sapiens, HSA, 2n = 46) and galagos (Galago moholi, GMO, 2n = 38) allowed us to map evolutionarily conserved chromosomal segments among humans, galagos, and slow lorises (Nycticebus coucang, NCO, 2n = 50). In total, the 22 human autosomal painting probes detected 40 homologous chromosomal segments in the slow loris genome. The genome of the slow loris contains 16 sytenic associations of human homologues. The ancient syntenic associations of human chromosomes such as HSA 3/21, 7/16, 12/22 (twice), and 14/15, reported in most mammalian species, were also present in the slow loris genome. Six associations (HSA 1a/19a, 2a/12a, 6a/14b, 7a/12c, 9/15b, and 10a/19b) were shared by the slow loris and galago. Five associations (HSA 1b/6b, 4a/5a, 11b/15a, 12b/19b, and 15b/16b) were unique to the slow loris. In contrast, 30 homologous chromosome segments were identified in the slow loris genome when using galago chromosome painting probes. The data showed that the karyotypic differences between these two species were mainly due to Robertsonian translocations. Reverse painting, using galago painting probes onto human chromosomes, confirmed most of the chromosome homologies between humans and galagos established previously, and documented the HSA 7/16 association in galagos, which was not reported previously. The presence of the HSA 7/16 association in the slow loris and galago suggests that the 7/16 association is an ancestral synteny for primates. Based on our results and the published homology maps between humans and other primate species, we propose an ancestral karyotype (2n = 60) for lorisiform primates.     

比较染色体涂色显示小熊猫(Ailurus fulgens)具有高度保守的核型

以狗的整条染色体特异探针,通过比较染色体涂色（Comparative Chromosome Painting）,建立了小熊猫和狗的比较染色体图谱。狗的38条常染色体探针在小熊猫染色体上共检出71个同源片段。其中狗的18条常染色体每一条在小熊猫染色全上各有1个同源片段,其余的20条常染色体每一条在小熊猫染色体上各有2至5个同源片段。广泛的染色体结构重排造成了小熊猫与狗的核型差异：至少需要经过28次断裂,49次融合,4次倒位才能将狗的核型（2n=78）“转变”为小熊猫的核型（2n=36）。结合已发表的狗与家猫的比较染色体图谱,我们推测：小熊猫与家猫之间共存在26个同源片段,二者的核型之间显示了较高的同源性。通过比较分析狗的染色体同源片段在小熊猫与家猫染色体上的分布和排列,可以看出：4次染色体易拉,2次倒位造成了小熊猫与家猫的核型差异。我们的工作进一步证实了利用基因组高度重排的物种（如：狗）的染色体特异探针与核型保守的物种（如：家猫、水貂、小熊猫）进行比较染色体涂色研究,不但可以准确快速地鉴别物种进化过程中所发生的染色体间的结构重排,而且还可揭示染色体内的结构重组。     

Dog chromosome-specific paints reveal evolutionary inter- and intrachromosomal rearrangements in the American mink and human

Forty chromosome-specific paint probes of the domestic dog (Canis familiaris, 2n = 78) were used to delineate conserved segments on metaphase chromosomes of the American mink (Mustela vison, 2n = 30) by fluorescence in situ hybridisation. Half of the 38 canine autosomal probes each painted one pair of homologous segments in a diploid mink metaphase, whereas the other 19 dog probes each painted from two to five pairs of discrete segments. In total, 38 canine autosomal paints highlighted 71 pairs of conserved segments in the mink. These painting results allow us to establish a complete comparative chromosome map between the American mink and domestic dog. This map demonstrates that extensive chromosome rearrangements differentiate the karyotypes of the dog and American mink. The 38 dog autosomes could be reconstructed from the 14 autosomes of the American mink through at least 47 fissions, 25 chromosome fusions, and six inversions. Furthermore, comparison of the current dog/mink map with the published human/dog map discloses 23 cryptic intrachromosomal rearrangements in 10 regions of conserved synteny in the human and American mink genomes and thus further refined the human/mink comparative genome map.     

Delimiting the Origin of a B Chromosome by FISH Mapping,Chromosome Painting and DNA Sequence Analysis in Astyanax paranae (Teleostei,Characiformes)

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.     

PCR in situ followed by microdissection allows whole chromosome painting probes to be made from single microdissected chromosomes

Whole-chromosome painting probes (WCPs) and chromosome-arm painting probes (CAPs) are an integral part of the cytogenetic analysis of chromosome abnormalities. While these are routinely made by chromosome microdissection, multiple copies of the dissected region have been necessary to achieve a library sufficiently complex to provide adequate painting. Performing multiple dissections of chromosomes or chromosome regions is time consuming and occasionally impossible, such as when working with species whose banded karyotype is not well defined. We have developed a method whereby chromosome paints can be reliably generated by dissecting single chromosomes. The technique consists of performing degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) in situ on the chromosomes, prior to dissection. Enough amplification occurs to enable a single dissected chromosome to be used to create a painting probe sufficiently complex for use in fluorescence in situ hybridization (FISH). The amplification products remain localized on the chromosomes; this allows region-specific chromosome paints to be made. We detail this novel technique and show whole-chromosome, arm-specific, and contiguous region-specific probes for human and rat, each created from single dissected fragments of chromatin. Received: 14 January 1999 / Accepted: 28 January 1999     

Comparative molecular cytogenetic studies in the order Carnivora: mapping chromosomal rearrangements onto the phylogenetic tree

We have made a set of chromosome-specific painting probes for the American mink by degenerate oligonucleotide primed-PCR (DOP-PCR) amplification of flow-sorted chromosomes. The painting probes were used to delimit homologous chromosomal segments among human, red fox, dog, cat and eight species of the family Mustelidae, including the European mink, steppe and forest polecats, least weasel, mountain weasel, Japanese sable, striped polecat, and badger. Based on the results of chromosome painting and G-banding, comparative maps between these species have been established. The integrated map demonstrates a high level of karyotype conservation among mustelid species. Comparative analysis of the conserved chromosomal segments among mustelids and outgroup species revealed 18 putative ancestral autosomal segments that probably represent the ancestral chromosomes, or chromosome arms, in the karyotype of the most recent ancestor of the family Mustelidae. The proposed 2n = 38 ancestral Mustelidae karyotype appears to have been retained in some modern mustelids, e.g., Martes, Lutra, Ictonyx, and Vormela. The derivation of the mustelid karyotypes from the putative ancestral state resulted from centric fusions, fissions, the addition of heterochromatic arms, and occasional pericentric inversions. Our results confirm many of the evolutionary conclusions suggested by other data and strengthen the topology of the carnivore phylogenetic tree through the inclusion of genome-wide chromosome rearrangements.     

Complete homology maps of the rabbit (Oryctolagus cuniculus) and human by reciprocal chromosome painting

Fluorescence in situ hybridization (FISH) was used to construct a homology map to analyse the extent of evolutionary conservation of chromosome segments between human and rabbit (Oryctolagus cuniculus, 2n = 44). Chromosome-specific probes were established by bivariate fluorescence activated flow sorting followed by degenerate oligonucleotide-primed PCR (DOP-PCR). Painting of rabbit probes to human chromosomes and vice versa allowed a detailed analysis of the homology between these species. All rabbit chromosome paints, except for the Y paint, hybridized to human chromosomes. All human chromosome paints, except for the Y paint, hybridized to rabbit chromosomes. The results obtained revealed extensive genome conservation between the two species. Rabbit chromosomes 12, 19 and X were found to be completely homologous to human chromosomes 6, 17 and X, respectively. All other human chromosomes were homologous to two or sometimes three rabbit chromosomes. Many conserved chromosome segments found previously in other mammals (e.g. cat, pig, cattle, Indian muntjac) were also found to be conserved in rabbit chromosomes.     

A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps

Cross-species reciprocal chromosome painting was used to delineate homologous chromosomal segments between domestic dog, red fox, and human. Whole sets of chromosome-specific painting probes for the red fox and dog were made by PCR amplification of flow-sorted chromosomes from established cell cultures. Based on their hybridization patterns, a complete comparative chromosome map of the three species has been built. Thirty-nine of the 44 synteny groups from the published radiation hybrid map and 33 of the 40 linkage groups in the linkage map of the dog have been assigned to specific chromosomes by fluorescence in situ hybridization and PCR-based genotyping. Each canine chromosome has at least one DNA marker assigned to it. The human-canid map shows that the canid karyotypes are among the most extensively rearranged karyotypes in mammals. Twenty-two human autosomal paints delineated 73 homologous regions on 38 canine autosomes, while paints from 38 dog autosomes detected 90 homologous segments in the human genome. Of the 22 human autosomes, only the syntenies of three chromosomes (14, 20, and 21) have been maintained intact in the canid genome. The dog-fox map and DAPI banding comparison demonstrate that the remarkable karyotype differences between fox (2n = 34 + 0-8 Bs) and dog (2n = 78) are due to 26 chromosomal fusion events and 4 fission events. It is proposed that the more easily karyotyped fox chromosomes can be used as a common reference and control system for future gene mapping in the DogMap project and CGH analysis of canine tumor DNA.     

A Comparative Study of Pygmy Hippopotamus (<Emphasis Type="Italic">Choeropsis liberiensis</Emphasis>) Karyotype by Cross-Species Chromosome Painting

The family Hippopotamidae is comprised of two genera with two living species, the common hippo (Hippopotamus amphibius) and the pygmy hippo (Choeropsis liberiensis). Unlike the common hippo, the karyotype of C. liberiensis has not yet been investigated via cross-species chromosome painting methods. We established chromosomal homologies between the pygmy hippo, pig, and cattle by fluorescence in situ hybridization using whole chromosome, arm-specific, region specific, and bacterial artificial chromosome (BAC) probes. Probes from the 18 pig autosomes painted 45 conserved chromosomal segments in the pygmy hippo genome. The pygmy hippo and cattle homology map was deduced from our hybridization results of painting probes to pygmy hippo chromosomes with a combination of previously published dromedary hybridization data. On the pygmy hippo and cattle homology map, 29 cattle autosomes revealed 39 conservative segments on pygmy hippo chromosomes. For a more detailed structural analysis of genome rearrangements and X chromosome structure, we used cattle region specific and BAC probes. Our report demonstrates that cattle probes are useful not only in comparative studies within Ruminantia, but also in more phylogenetically distant Artiodactyla species.     

人类性染色体特异ＤＮＡ对三种鱼类染色体的描绘

染色体描绘是研究基因组进化的强有力手段之一,用人Ｘ和Ｙ染色体文库特异ＤＮＡ为探针,对３种硬骨鱼类－刺鳅、黄鳝和斑马鱼的有丝分裂中期裂染色体进行了描绘研究,结果表明,在这３种鱼类的染色体组中都发现有人Ｘ染色体特DN的同源片段,它们散布在几对同源染色体中,但用人Ｙ染色体ＤＮ描绘这３种鱼类染色体时,则没有检测出可见的同源片段。同时对从低等脊椎动物到人类的Ｘ染色体进化过程进行了进一步探讨。     

The pattern of phylogenomic evolution of the Canidae

Canidae species fall into two categories with respect to their chromosome composition: those with high numbered largely acrocentric karyotypes and others with a low numbered principally metacentric karyotype. Those species with low numbered metacentric karyotypes are derived from multiple independent fusions of chromosome segments found as acrocentric chromosomes in the high numbered species. Extensive chromosome homology is apparent among acrocentric chromosome arms within Canidae species; however, little chromosome arm homology exists between Canidae species and those from other Carnivore families. Here we use Zoo-FISH (fluorescent in situ hybridization, also called chromosomal painting) probes from flow-sorted chromosomes of the Japanese raccoon dog (Nyctereutes procyonoides) to examine two phylogenetically divergent canids, the arctic fox (Alopex lagopus) and the crab-eating fox (Cerdocyon thous). The results affirm intra-canid chromosome homologies, also implicated by G-banding. In addition, painting probes from domestic cat (Felis catus), representative of the ancestral carnivore karyotype (ACK), and giant panda (Ailuropoda melanoleuca) were used to define primitive homologous segments apparent between canids and other carnivore families. Canid chromosomes seem unique among carnivores in that many canid chromosome arms are mosaics of two to four homology segments of the ACK chromosome arms. The mosaic pattern apparently preceded the divergence of modern canid species since conserved homology segments among different canid species are common, even though those segments are rearranged relative to the ancestral carnivore genome arrangement. The results indicate an ancestral episode of extensive centric fission leading to an ancestral canid genome organization that was subsequently reorganized by multiple chromosome fusion events in some but not all Canidae lineages.     

Towards the delineation of the ancestral eutherian genome organization: comparative genome maps of human and the African elephant (Loxodonta africana) generated by chromosome painting

This study presents a whole-genome comparison of human and a representative of the Afrotherian clade, the African elephant, generated by reciprocal Zoo-FISH. An analysis of Afrotheria genomes is of special interest, because recent DNA sequence comparisons identify them as the oldest placental mammalian clade. Complete sets of whole-chromosome specific painting probes for the African elephant and human were constructed by degenerate oligonucleotide-primed PCR amplification of flow-sorted chromosomes. Comparative genome maps are presented based on their hybridization patterns. These maps show that the elephant has a moderately rearranged chromosome complement when compared to humans. The human paint probes identified 53 evolutionary conserved segments on the 27 autosomal elephant chromosomes and the X chromosome. Reciprocal experiments with elephant probes delineated 68 conserved segments in the human genome. The comparison with a recent aardvark and elephant Zoo-FISH study delineates new chromosomal traits which link the two Afrotherian species phylogenetically. In the absence of any morphological evidence the chromosome painting data offer the first non-DNA sequence support for an Afrotherian clade. The comparative human and elephant genome maps provide new insights into the karyotype organization of the proto-afrotherian, the ancestor of extant placental mammals, which most probably consisted of 2n=46 chromosomes.