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Research ArticlesEvolutionary and Biomedical Insights from the Rhesus Macaque GenomeRhesus Macaque Genome Sequencing and Analysis Consortium: *
The rhesus macaque (Macaca mulatta) is an abundant primate species that diverged from the ancestors of Homo sapiens about 25 million years ago. Because they are genetically and physiologically similar to humans, rhesus monkeys are the most widely used nonhuman primate in basic and applied biomedical research. We determined the genome sequence of an Indian-origin Macaca mulatta female and compared the data with chimpanzees and humans to reveal the structure of ancestral primate genomes and to identify evidence for positive selection and lineage-specific expansions and contractions of gene families. A comparison of sequences from individual animals was used to investigate their underlying genetic diversity. The complete description of the macaque genome blueprint enhances the utility of this animal model for biomedical research and improves our understanding of the basic biology of the species.
1 Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX 77030, USA.
* To whom correspondence should be addressed. Richard A. Gibbs, E-mail: agibbs{at}bcm.edu Rhesus macaques (Macaca mulatta) (1) are one of the most frequently encountered and thoroughly studied of all nonhuman primates (table S1.1). They have a broad geographic distribution that reaches from Afghanistan and India across Asia to the Chinese shore of the Pacific Ocean. As an Old World monkey (superfamily Cercopithecoidea, family Cercopithecidae), this species is closely related to humans and shares a last common ancestor from about 25 million years ago (Mya) (2). The two species often live in close association, and macaques exhibit complex and intensely social behavioral repertoires. The relationship between humans and macaques is even more important because biomedical research has come to depend on these primates as animal models. Compared with rodents, which are separated from humans by more than 70 million years (2, 3), macaques exhibit greater similarity to human physiology, neurobiology, and susceptibility to infectious and metabolic diseases. Critical progress in biomedicine attributed to macaques includes the identification of the "rhesus factor" blood groups and advances in neuroanatomy and neurophysiology. Most important, their response to infectious agents related to human pathogens, including simian immunodeficiency virus and influenza, has made macaques the preferred model for vaccine development. Lesser-known contributions of these animals include their early use in the U.S. space program—a rhesus monkey was launched into space more than a dozen years before any chimpanzee. The cynomolgus macaque (M. fascicularis), pigtailed macaque (M. nemestrina), and Japanese macaque (M. fuscata) have all contributed to research, but the rhesus macaque has been used most widely. Taxonomists recognize six M. mulatta subspecies (1), which differ substantially in their geographical range, body size, and a variety of morphological, physiological, and behavioral characteristics. North American research colonies include animals representing both Indian and Chinese subspecies, although India ended the exportation of these animals in the 1970s. With the advent of whole-genome sequencing, a highly accurate human genome sequence and a draft of the chimpanzee genome have been generated and compared. The chimpanzee shared a common ancestor with humans approximately 6 Mya (4, 5), and the major impact of the chimpanzee genome sequence data has been in their direct comparison with data from the human genome. However, the chimpanzee data have major limitations. First, because the alignable sequence is only 1 to 2% different from that of the human, there is no informative "signal" to distinguish conserved elements from the overall high background level of conservation. This is exacerbated by the fact that the chimpanzee genome was an incomplete draft, containing sequence errors that could potentially mask true divergence. Second, the differences that are found between humans and chimpanzees are difficult to assign as specific to either the chimpanzee or the human. As a result, the chimpanzee analyses have on their own provided relatively few answers to the fundamental question of the nature of the specific molecular changes that make us human.
By contrast, the genome of the rhesus macaque has diverged farther from our own, with an average human-macaque sequence identity of
We examined the basic elements of the rhesus macaque genome and undertook reconstruction of the major changes in the human-chimpanzee–rhesus macaque (HCR) trio. The regions of the genome that were duplicated in macaque were then identified and correlated with other genome features. Individual macaque genes were studied, and the orthologous genes in the HCR trio were aligned to reveal evidence for the action of selection on individual loci. Additional animals from other populations were also sampled by DNA sequencingtostudy their genetic diversity. Throughout, complementary methods were applied and the different results combined in order to represent the most complete picture of macaque biology. For a visual representation of some of the insights gained from the genome and more information about the importance of the macaque as a model organism, see the poster in this issue (6).
To generate a draft genome sequence for the rhesus macaque, whole-genome shotgun sequences were assembled. The bulk of the sequencing used DNA from a single M. mulatta female, whereas DNA from an unrelated male was used to construct a bacterial artificial chromosome (BAC) library to provide BAC end sequences and to aid in selective finishing. We used several whole-genome shotgun libraries with different insert sizes ( To produce an optimal representation of the genome, the three intermediate assemblies were merged (Fig. 2). Melding the assemblies involved mapping the Atlas–whole-genome shotgun and PCAP data to the Celera Assembler output, which had longer contiguity than the other two data sets at this stage of the process. There was little difference between assemblies at the sequence contig level, at which robust sequence alignments guide the reconstructions, so we focused our attention instead on contigs that were joined into scaffolds. Additional pairs of Celera Assembler scaffolds were joined based on their mapping to the other two macaque assemblies. Analysis of the output showed that this composite assembly was superior to any of its components (table S2.4).
During assembly, a comparison with the human genome sequence [National Center for Biotechnology Information (NCBI) accession code bld35] identified a small number (<100) of obvious inconsistencies, such as improper joins of different chromosomes. These scaffolds were therefore split at the misassembly point. The human map was also used to help place large merged scaffolds onto the macaque chromosomes (8, 9) [the chromosome numbering of Rogers et al. (8) was used] at the highest level of the assembly process. Given that the human data were only used to split scaffolds and that de novo macaque assemblies were always given precedence over the mapping to the human genome in the macaque assembly merging and chromosome assignment process, the final product should not be regarded as a "humanized assembly."
The total length of the combined genome assembly was approximately 2.87 Gb (Table 1). This incorporated
Selected sequence finishing. The rhesus macaque genome assembly is a draft DNA sequence, and it contains many gaps. A higher data quality with greater contiguity was desired at several genomic regions that attracted additional interest. In these cases, individual BAC clones were isolated, and data quality was improved by sequence "finishing." Many of these BACs were in regions of pronounced genome duplication, whereas others were gene-rich. All finished BACs, their gene content, and their genome coordinates are listed in table S2.6.
General organization and content. The macaque genome is organized into 20 autosomes and the XY sex chromosomes. With the exception of 48 breakpoints (Fig. 1)—including three fusions, one fission, and breakpoints induced by inversions that are each detectable through chromosome staining, by radiation hybrid mapping, or by comparative linkage mapping—there is a superficial similarity between the macaque and human chromosomes (8–11). Several chromosomes in the macaque are also more acrocentric than their human counterparts, but many from the two species are difficult to distinguish. Nucleotide sequences that aligned between the human and rhesus average 93.54% identity. If, however, small insertions and deletions are included in the calculation, identity is reduced to 90.76%. Considering regions that are difficult to align, such as lineage-specific interspersed repeat elements, would further decrease the level of computed identity. Moreover, evolutionary distances exhibit local fluctuations, as in other mammals (3), and less divergence was observed in chromosome X (94.26% identity of aligned bases). The GC-content of the rhesus in aligned bases was not notably lower than that of the human (40.71% versus 40.74%).
Gene content. A human-centric approach was used to generate new macaque gene sets (table S3.1 and fig. S3.1). These sets include (i) Ensembl (12) gene models based primarily on the alignment of the human Uniprot and RefSeq resources with the current assembly to define the overall gene model, followed by the introduction of the macaque-specific sequences (mainly as lineage-specific paralogs) in that framework; (ii) Gnomen (NCBI) models that include the consideration of the available (
Overall repetitive landscape. Repeat elements account for
Cytogenetically visible rearrangements. The most notable genomic differences among the HCR trio are the presence of cytogenetically visible rearrangements. The human and chimpanzee karyotypes are distinguishable by one chromosome fusion and nine cytogenetically visible pericentric inversions (16); with the use of the macaque as an outgroup, all of these breakpoints (except those induced by two inversions) have now been characterized at the DNA sequence level (17). Analysis of genomic sequence confirms that 14 breakpoints, corresponding to seven inversions, occurred in the chimpanzee lineage, as indicated in Fig. 1. (Five of the inversions are summarized in table S4.1.) The pericentric inversions of human chromosomes 1 and 18 and the fusion creating human chromosome 2 are specific to the human. Comparison of the reconstructed human-chimpanzee ancestral genome and the rhesus genome reveals 43 breakpoints on the microscopic scale (Figs. 1 and 3).
Submicroscopic rearrangements. Previous analyses [reviewed in (14)] have indicated that primate genomes harbor more structural differences than visible by cytogenetic staining. Analysis of these events is complicated by two issues: the draft state of the genomes and the presence of extensive segmental duplications. We analyzed these structural rearrangements by using the distance between orthologous blocks in each species to infer the ancestral genome structure and determine where rearrangements occurred on the phylogenetic tree. We excluded events smaller than 10 kilobase pairs (kbp), which are mostly due to retroposon insertions, and focused on cytogenetically undetectable breakpoints induced by insertions, deletions, inversions, and complex rearrangements of sizes between 10 kbp and 4 Mbp. Data were combined from inversion detection and ancestral reconstructions by the contiguous ancestral regions method (18) and gap detection by the genomic triangulation method (19), which further integrates data from genomic sequence comparisons (20) and comparative maps (8, 9, 21). The analysis revealed more than 1000 rearrangement-induced breakpoints through the HCR lineages, of which 820 occur between rhesus and the reconstructed human-chimpanzee ancestor (Fig. 3 and fig. S4.1). Each chromosome therefore constitutes a complex mosaic, with multiple changes introduced to orthologous counterparts. When rhesus macaque is compared with the human-chimpanzee ancestor, the X chromosome exhibits three times more rearrangements per megabase than the autosomes. This is both statistically significant and consistent with a slightly more than threefold difference observed in the human lineage following the branching off of chimpanzee (19). Given that a slower rate of variability at the single-nucleotide level in the X chromosome compared with autosomes has been interpreted as support for speciation models, this difference is worthy of further investigation (22).
Genomic Duplications. Segmental duplication of genomic regions and the genes they contain are well known in mammals and are postulated to drive fundamental processes, including the birth of new genes and the subsequent expansion of gene families (23). To discover duplications in the macaque genome, we used a battery of different complementary approaches. Two of these, whole-genome assembly comparison (24) and BLASTZ (25) analysis of segmental duplications, depended directly on the assembly. We used a third method, whole-genome shotgun sequence detection (26), that calculated depth of coverage of the raw shotgun sequence reads relative to the assembly. A fourth procedure was created on the basis of BAC end sequence reads combined with BACs that were directly mapped by means of the pooled genomic indexing method (21). The common interspersed repeat families were not considered in any of these analyses.
The first two approaches identified approximately 35.0 Mb of a recently duplicated sequence in the macaque assembly. A further
The pooled genomic indexing and BAC end sequence read methods suggested slightly higher levels of overall duplication, on the basis of fluorescence in situ hybridization analysis of randomly selected large-insert BAC clones (28). However, this estimate was still less than the 4.8% recently estimated for the baboon genome (28). Overall, we consider 2.3% to be the lower bound of duplicated genomic DNA in the macaque genome. As with the human and chimpanzee, the analysis of the macaque assembly revealed an enrichment of segmental duplications near gaps, centromeres, and telomeres (14, 29). The study also identified segmental duplications that contain genes of high biological significance. For example, the CCL3L1-CCL4 gene region [for which copy-number variation in humans is correlated with susceptibility to HIV infection (30)], cytochrome P450 (associated with toxicity response), KRAB-C2H2 zinc finger (a developmental regulatory transcription factor), olfactory receptor (smell), human leukocyte antigen (HLA), and other immune and autoantigen gene families were all observed in regions of genome duplication. Expansion of gene families. Two approaches were used to study gene family structure directly within the draft genome sequence: (i) a statistical approach, based on a likelihood model of gene gain and loss across the mammalian tree (31) and (ii) hybridization of whole genomic DNA to cDNA arrays [a variation of array-based comparative genomic hybridization (array CGH)] to observe changes in gene content directly (32). The results are shown in Tables 3 and 4.
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All authors with their contributions and affiliations appear at the end of this paper. 
93%. 
