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. 2016 Feb 16;8(3):519–527. doi: 10.1093/gbe/evw022

No Distinction of Orthology/Paralogy between Human and Chimpanzee Rh Blood Group Genes

Takashi Kitano 1,2,*, Choong-Gon Kim 1,3, Antoine Blancher 4, Naruya Saitou 1
PMCID: PMC4824203  PMID: 26872772

Abstract

On human (Homo sapiens) chromosome 1, there is a tandem duplication encompassing Rh blood group genes (Hosa_RHD and Hosa_RHCE). This duplication occurred in the common ancestor of humans, chimpanzees (Pan troglodytes), and gorillas, after splitting from their common ancestor with orangutans. Although several studies have been conducted on ape Rh blood group genes, the clear genome structures of the gene clusters remain unknown. Here, we determined the genome structure of the gene cluster of chimpanzee Rh genes by sequencing five BAC (Bacterial Artificial Chromosome) clones derived from chimpanzees. We characterized three complete loci (Patr_RHα, Patr_RHβ, and Patr_RHγ). In the Patr_RHβ locus, a short version of the gene, which lacked the middle part containing exons 4–8, was observed. The Patr_RHα and Patr_RHβ genes were located on the locations corresponding to Hosa_RHD and Hosa_RHCE, respectively, and Patr_RHγ was in the immediate vicinity of Patr_RHβ. Sequence comparisons revealed high sequence similarity between Patr_RHβ and Hosa_RHCE, while the chimpanzee Rh gene closest to Hosa_RHD was not Patr_RHα but rather Patr_RHγ. The results suggest that rearrangements and gene conversions frequently occurred between these genes and that the classic orthology/paralogy dichotomy no longer holds between human and chimpanzee Rh blood group genes.

Keywords: gene duplication, phylogeny, rearrangement

Introduction

The Rh blood group is one of the major blood group systems in humans, and bears important practical considerations in blood transfusions and clinical medicine. The nucleotide sequences of the Rh blood group genes were determined independently by Chérif-Zahar et al. (1990) and Avent et al. (1990). This blood group system was shown to be composed of two closely linked RHD and RHCE loci (Mouro et al. 1993), as predicted by Tippett (1986). RHD and RHCE loci are located on chromosome 1p34–p36 in humans (Ruddle et al. 1972; Chérif-Zahar et al. 1991), and individuals are classified in practice as Rh positive and Rh negative according to the presence or absence of the D antigen. Nucleotide sequences of the Rh blood group genes in nonhuman primates have also been reported (Salvignol et al. 1994, 1995; Mouro et al. 1994). Genomic DNA analyses by Southern blot have shown that chimpanzees (Pan troglodytes) possesses three Rh loci (Salvignol et al. 1993, 1994). Gorillas carry two Rh loci (Apoil et al. 1999), whereas orangutans, gibbons, Old World monkeys, and New World monkeys carry a single Rh locus (Blancher et al. 1992). This suggests that the Rh blood group genes were duplicated in the common ancestor of humans, chimpanzees, and gorillas, after splitting from their common ancestor with orangutans.

Suto et al. (2000) found that the two human Rh loci have opposite orientations by using fiber-FISH (Fluorescence in situ Hybridization) analysis, a useful approach for analyzing genome sequences containing duplicated genes. Wagner and Flegel (2000) went on to report that the 3′ ends of these genes face each other and are separated by an ∼30-kb segment that contains the TMEM50A (transmembrane protein 50A) gene. In addition, Suto et al. (2003) later discovered the existence of two-Rh-gene and four-Rh-gene types in chimpanzees, again using fiber-FISH analysis. The two-Rh-gene type is arranged as Rh (5′→3′) – Rh (3′←5′), with the distance between the two genes about 50-kb longer than that in humans, while the arrangement of the four-Rh-gene type is Rh (5′→3′) – Rh (3′←5′) – Rh (3′←5′) – Rh (3′←5′) within a region spanning about 300 kb. The fact that the Rh blood group genes have been duplicated in humans, chimpanzees, and gorillas suggests that their evolutionary history is complex (Kitano and Saitou 1999). As no authoritative chimpanzee reference genome is yet available in Ensembl, GenBank Map Viewer, or UCSC genome browser (supplementary fig. S1B, Supplementary Material online) with which to examine the gene cluster of chimpanzee Rh blood group genes, we examined an ∼400-kb sequence region of the chimpanzee genome to carry out detailed analyses on the evolution of this gene cluster and its constituent genes.

Materials and Methods

Genomic DNA Sequencing

Five chimpanzee BAC (Bacterial Artificial Chromosome) clones for the Rh blood group genes (PTB-081F20, PTB-149J04, RP43-004E10, RP43-024B01, and RP43-026A23) were screened by BLAST searches using BAC-end sequences (Fujiyama et al. 2002). The PTB and RP43 BAC clone libraries are from the chimpanzee individuals “Gon” and “Donald,” respectively. Gon is P. troglodytes verus (http://www.shigen.nig.ac.jp/pgdb2/greatApe.html?individualId=338), while Donald, a second-generation captive, is a hybrid between troglodytes and the verus subspecies (Prado-Martinez et al. 2013). Nucleotide sequences were determined primarily using the shotgun method following Watanabe et al. (2004). The Phred/Phrap software package (Ewing et al. 1998) was used for base calling and assembly and to obtain quality scores for assembled data. Editing to identify all low-quality bases and to check the correct assembly on the basis of linking information was performed using Consed (Gordon et al. 1998). Finishing was then performed by primer walking and Polymerase Chain Reaction (PCR)-coupled primer walking. We completely sequenced (finished) the two BAC clones (RP43-004E10 and RP43-026A23), while the remaining three (PTB-081F20, PTB-149J04, and RP43-024B01) were retained as ordered piece (i.e., phase 2) sequences.

Haplotypes of Exon 7 Sequences

We sequenced the region surrounding exon 7 (∼860 bp) from 12 chimpanzees (P. troglodytes verus), 3 bonobos (P. paniscus), 3 gorillas (Gorilla gorilla), 1 orangutan (Pongo pygmaeus), and 2 siamangs (Symphalangus syndactylus). Five (“Akina,” “Aluku,” “Chiko,” “Kanao,” and “Mikota”) of the 12 chimpanzees were the same individuals used in the Suto et al. (2003) fiber-FISH analysis. Two primers on intron 6 (forward: 5′-TTT GCA TAT GTG TCC ACA TCT G-3′) and on intron 7 (reverse: 5′-GCC AAC AAA TAT TCA CCG AAG C-3′) were used for each PCR experiment and sequencing. KOD DNA polymerase (TOYOBO), which has 3′→5′ exonuclease activity, was used for all PCR experiments to ensure high fidelity. Following PCR, one extension reaction (10 min, 72 °C) using Ex Taq (TaKaRa) was carried out with one nucleotide addition for TA cloning. PCR products were confirmed by 1.5% agarose gel electrophoresis, purified using Micro Spin Columns (Amersham Biosciences), and inserted into a vector using the TOPO TA Cloning Kit (Invitrogen). At least two clones for each sequence type from each individual were sequenced using a BigDye Terminator Cycle Sequencing Kit and an ABI PRISM 3100 DNA Genetic Analyzer (Applied Biosystems).

To confirm the presence of Patr_RHβS in each individual, two primers on exon 3 (forward: 5′-GTG CTG GTG GAG GTG ACA GCT TTA-3′) and intron 8 (reverse: 5′-CAT CCA CCT CCT GCT TAG GGA TAC C-3′), able to amplify the ∼650-bp product from Patr_RHβS, were used. This primer set cannot amplify products from complete Rh blood group genes obtained by ordinary PCR because the expected size reaches ∼28 kbp.

The five sequence data sets for “Clint” available in GenBank (NW_003481311.1, NW_003481846.1, NW_003460690.1, NW_003456511.1, and NW_003460689.1) were used.

Sequence Analyses

Multiple alignments were performed using ClustalW (Thompson et al. 1994) implemented in the MEGA5 software (Tamura et al. 2011). To select substitution models, the Find Best DNA/Protein Models (ML) option in the MEGA5 software package was used for nucleotide sequence data. Phylogenetic trees were inferred by using the maximum likelihood method (Felsenstein 1981) with nearest-neighbor-interchange heuristic search from the initial neighbor-joining tree (Saitou and Nei 1987). Phylogenetic networks were constructed using the Neighbor-Net method (Bryant and Moulton 2004) with p-distance implemented in the SplitsTree4 software (Huson and Bryant 2006). The number of synonymous and nonsynonymous substitutions (p-distance) was estimated following Nei and Gojobori (1986).

Results and Discussion

Genome Sequence of the Chimpanzee Rh Blood Group Genes

BAC clones containing Rh genes were characterized from the two chimpanzees Gon and Donald. By analyzing the BAC sequences, we reconstructed the three chromosomes (RP43-1, RP43-2, and PTB-1) shown in figure 1. Chromosomes RP43-1 and RP43-2 were characterized in a single individual (Donald), and differ by 1,403 of the 167,165 bp (0.008). RP43-1 consists of two completely sequenced BAC clones (RP43-026A23 and RP43-004E10) that are strictly identical in their overlapping region (3,801 bp), which encompasses the 3′ end of TMEM50A. The BAC clone (RP43-024B01) defines chromosome RP43-2. PTB-1 consists of two BAC clones (PTB-149J04 and PTB-081F20) which overlap from a “Rhesus box” (Wagner and Flegel 2000; a DNA segment of ∼9 kb that contains a nearly complete remnant of a transposon-like human element, THE-1B) on the 5′ end of Patr_RHα3 to exon 1 of Patr_RHβS3. These two BAC clones were derived from the same individual (Gon) and differ only by four nucleotide changes and two length differences consisting of 4- and 2-base repeats throughout the 137,844-bp overlap region. Because the nucleotide difference between these clones (4/137,844 = 2.9 × 10−5) is too small to assume they originate from different chromosomes of the same individual, we conclude that they most probably derive from the same chromosome, and that the observed differences were most likely caused by mutations during DNA replication in bacterial cells.

Fig. 1.—

Fig. 1.—

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Genome structure of the gene cluster of chimpanzee Rh blood group genes. Three chromosomes (RP43-1, RP43-2, and PTB-1) and three loci (Patr_RHα, Patr_RHβ, and Patr_RHγ) are shown. Vertical bars with numbers 1–10 show the exons for each Rh gene. Black boxes denote the SYF2, C1orf63, TMEM50A, and TMEM57 genes. Gray boxes represent Rhesus boxes, and an arrow in a box indicates its direction. The gap indicated by broken lines in PTB-1 is a large deletion that produced a short version of the Rh gene (Patr_RHβS3) in chimpanzees. Light gray bars below the genome structure show the BAC clones for the sequencing. The genome structure of the human Rh blood group genes is provided for reference at the bottom of the figure. A scale bar (kb) is shown between the chimpanzee and human Rh blood group genes.

We characterized three Rh loci (Patr_RHα, Patr_RHβ, and Patr_RHγ) in the gene cluster of chimpanzee Rh blood group genes (fig. 1). The Patr_RHα locus was located between C1orf63 and TMEM50A, while Patr_RHβ and Patr_RHγ loci were located between TMEM50A and TMEM57. In the Patr_RHβ locus, a short version of the gene, which lacked the middle part containing exons 4–8, was observed in PTB-1; we refer to this as Patr_RHβS (Patr_RHβS3). In the human genome sequence data (GRCh38.p2, Release 107), Hosa_RHD (NG_007494.1) and Hosa_RHCE (NG_009208.3) loci are located between C1orf63 and TMEM50A and between TMEM50A and TMEM57, respectively. Thus, the location of the Patr_RHα locus corresponds to that of Hosa_RHD, while Patr_RHβ (and Patr_RHγ) has the same location as Hosa_RHCE. Two Rhesus boxes are located between the 5′ and 3′ ends of the Patr_RHα locus, similar to Hosa_RHD. In addition, chimpanzees were found to have another Rhesus box, between Patr_RHβ and Patr_RHγ.

As mentioned before, Suto et al. (2003) reported the existence of two-Rh-gene and four-Rh-gene types in chimpanzees. It is likely that PTB-1 (fig. 1) is a two-Rh-gene type. Because Suto et al. used two genomic DNA probes that contained introns 3 and 7 in the fiber-FISH analysis, they did not detect Patr_RHβS, but instead observed that the two-Rh-gene type of chimpanzees have a longer interval (∼120 kb) than in humans and gorillas. Another interesting difference between our results is that although they did not observe any three-Rh-gene types from five chimpanzee individuals, we did discovered one: RP43-1 (fig. 1). This incongruity could be caused by subspecies differences. All samples examined by Suto et al. were from P. troglodytes verus; in contrast, Donald, in whom the three-Rh-gene type was detected, is a hybrid between troglodytes and the verus subspecies (Prado-Martinez et al. 2013). Some studies have reported that some functional genes are highly diversified among subspecies of chimpanzees because of region-specific natural selection (Hvilsom et al. 2008; Groeneveld et al. 2012; Hayakawa et al. 2012; Ferreira et al. 2013). The uniqueness of RP43-1 could derive from the troglodytes subspecies.

Sequence Differences between Human and Chimpanzee Rh Blood Group Genes

To examine the overall sequence differences between the chimpanzee Rh blood group genes, we constructed a phylogenetic tree using genome sequences (∼62 kb in length from start to stop codons) of five chimpanzee Rh blood group genes (Patr_RHα1, Patr_RHα3, Patr_RHβ1, Patr_RHγ1, and Patr_RHγ2; fig. 1) and the human genes Hosa_RHD and Hosa_RHCE (fig. 2). The GTR + G + I model was selected as the best by the model selection option in MEGA5. Initially, Patr_RHα1 and Patr_RHα3, and Patr_RHγ1 and Patr_RHγ2 formed two clusters, both with bootstrap values of 100%. Moreover, Hosa_RHD and Hosa_RHCE inexplicably formed a cluster with a bootstrap value of 100%. When we constructed a phylogenetic network (supplementary fig. S2A, Supplementary Material online), an alternative branch supporting a Patr_RHβ1–Hosa_RHCE cluster and an alternative branch supporting a Patr_RHγ1–Patr_RHγ2–Hosa_RHD cluster were observed. These alternative findings demonstrate the advantages of using phylogenetic networks that can show second-best alternative clustering using reticulations. Patr_RHβ and Hosa_RHCE might be true orthologs because they share the same genomic locations (fig. 1). In contrast, the genomic locations of Patr_RHγ and Hosa_RHD differ. Although Patr_RHα is located on the genomic location corresponding to Hosa_RHD’s, no branching signal supports their clustering, even in the phylogenetic network. Supplementary figure S2B, Supplementary Material online, shows a phylogenetic network of amino acid sequences using the same genes as supplementary figure S2A, Supplementary Material online. Similar phylogenetic relationships were observed in this phylogenetic network. These inconsistencies between phylogenetic relationships and their genomic locations are likely caused by sequence rearrangements such as gene conversions and recombination, as suggested by Kitano and Saitou (1999).

Fig. 2.—

Fig. 2.—

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A maximum likelihood tree based on the GTR model constructed from genomic sequences for human and chimpanzee Rh blood group genes of figure 1. Orangutan (Popy_RH) sequence is used as an outgroup. A discrete gamma distribution was used to model evolutionary rate differences among sites (8 categories; +G, parameter = 0.4117). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 55.4082% sites). A scale bar representing nucleotide substitutions per site is shown. Bootstrap values (1,000 replications) are shown on each branch. Black, gray, and horizontal-striped circles indicate Patr_RHα, Patr_RHβ, and Patr_RHγ, respectively. Black boxes indicate Hosa_RHD and Hosa_RHCE.

To analyze the distributions of phylogenetically informative sites, we first searched for sites that differed between the Hosa_RHD and Hosa_RHCE genomic sequences, and found 307 of them. Then, we checked whether each of the five chimpanzee genes (Patr_RHα1, Patr_RHα3, Patr_RHβ1, Patr_RHγ1, and Patr_RHγ3) had identical nucleotide sites with Hosa_RHD or Hosa_RHCE, and categorized these locations as shared sites. Furthermore, we defined sets of more than three contiguous shared sites as shared blocks (fig. 3). For example, sites 2178, 2180, 2184, 2193, 2203, and 2210 are shared sites between Patr_RHα1 and Hosa_RHCE, and they comprise one shared block between Patr_RHα1 and Hosa_RHCE. Patr_RHα1 had 137 shared sites with Hosa_RHD and 172 with Hosa_RHCE, and Patr_RHα3 had 132 shared sites with Hosa_RHD and 177 with Hosa_RHCE. In terms of shared blocks, Patr_RHα1 had 16 with Hosa_RHD and 19 with Hosa_RHCE, and Patr_RHα3 had 17 with Hosa_RHD and 24 with Hosa_RHCE. There is no clear distribution bias of shared sites and blocks between Patr_RHα1/Patr_RHα3 and Hosa_RHD or between it and Hosa_RHCE. Thus, it is not clear whether Patr_RHα1/Patr_RHα3 is phylogenetically homologous to Hosa_RHD or to Hosa_RHCE. Patr_RHβ1 had 209 shared sites and 31 shared blocks with Hosa_RHCE, respectively, about 2 times as many and 2.8 times as many as with Hosa_RHD (which had 100 shared sites and 11 shared blocks). The nucleotide sequence similarity between Patr_RHβ1 and Hosa_RHCE is also observed in the phylogenetic network (supplementary fig. S2A, Supplementary Material online) as the alternative clustering. Furthermore, Patr_RHβ and Hosa_RHCE share the same genomic locations (fig. 1). Thus, one can assume that Patr_RHβ and Hosa_RHCE are orthologous genes, and that some sequence rearrangements such as gene conversions and recombination have occurred on some genomic regions of Patr_RHβ and/or Hosa_RHCE. Patr_RHγ1 had 228 shared sites and 28 shared blocks with Hosa_RHD, respectively, about 2.8 times as many and 5.6 times as many as with Hosa_RHCE (which had 81 shared sites and 5 shared blocks). Similarly, Patr_RHγ2 had 230 shared sites and 24 shared blocks with Hosa_RHD, respectively, about 2.9 times as many and 8 times as many as with Hosa_RHCE (which had 79 shared sites and 3 shared blocks). The nucleotide sequence similarities between Patr_RHγ1/Patr_RHγ2 and Hosa_RHD are also observed in the phylogenetic network (supplementary fig. S2A, Supplementary Material online). However, the genomic location of Patr_RHγ1 and Patr_RHγ2 differs from that of Hosa_RHD. One interpretation is that in the chimpanzee lineage, a gene that had been located between C1orf63 and TMEM50A moved to the current Patr_RHγ genomic location and became Patr_RHγ. Alternatively, Hosa_RHD moved to a location between C1orf63 and TMEM50A in the human lineage. When a phylogenetic tree was constructed by adding two genome sequences of the RH genes obtained from the gorilla draft genome data (supplementary fig. S1C, Supplementary Material online), the two gorilla genes formed a cluster with a bootstrap value of 99% and the two-gorilla-gene cluster formed a cluster with Patr_RHβ with a bootstrap value of 93% (supplementary fig. S3, Supplementary Material online). From the parsimonious standpoint, the former, that is a gene that had been located between C1orf63 and TMEM50A moved to the current Patr_RHγ genomic location and became Patr_RHγ in the chimpanzee lineage, might be possible. Since the two gorilla sequences are incomplete because of the draft genome data, it calls for further investigation. In either case, the result suggests the volatility of the genomic location of the genes in the region.

Fig. 3.—

Fig. 3.—

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Distributions of phylogenetically informative sites for the chimpanzee Rh blood group genes (Patr_RHα1, Patr_RHα3, Patr_RHβ1, Patr_RHγ1, and Patr_RHγ2). Sites that differ between Hosa_RHD and Hosa_RHCE in their genome sequences are shown. Black boxes with numbers indicated exons. When a nucleotide of the chimpanzee Rh gene is identical to the corresponding Hosa_RHD nucleotide, the site is shown as a red box, when identical to the corresponding Hosa_RHCE nucleotide, it is shown as a blue box.

Apoil and Blancher (2000) reported that the length difference in intron 4 between Hosa_RHD and Hosa_RHCE (longer) in humans caused by a 645-bp segment that includes Alu-Sx is also observed in chimpanzees. We therefore decided to analyze the distribution of Alu in the chimpanzee Rh blood group genes from the five BAC clones (supplementary table S1, Supplementary Material online). Our findings showed that none of the chimpanzee genes used in this study had the 645-bp segment including Alu-Sx in intron 4. However, this does not necessarily signify that the intron including Alu-Sx does not exist in other chimpanzees. Moreover, because we could not sequence the whole gene region of Patr_RHδ in this study, it is possible that Patr_RHδ has the 645-bp segment in which Alu-Sx is present in intron 4.

We confirmed that two partially sequenced genes (Patr_RHβ2 and Patr_RHγ3) formed respective clusters with other Patr_RHβ and Patr_RHγ genes (supplementary fig. S4A and B, Supplementary Material online). Because Patr_RHβS3 formed a cluster with Patr_RHβ1 in the phylogenetic network (supplementary fig. S4C, Supplementary Material online), Patr_RHβS might be a deletion type of Patr_RHβ, instead of a combined type consisting of a downstream region of Patr_RHβ and an upstream region of Patr_RHγ.

Predicting the Haplotypes of the Chimpanzee Rh Blood Group Genes

To determine the haplotypes of the gene cluster of chimpanzee Rh blood group genes, we compared the region surrounding exon 7 (∼860 bp) between 15 chimpanzee individuals. This region was selected because exon 7 is the most divergent exon between the human Hosa_RHD and Hosa_RHCE loci (Westhoff and Wylie 1996; Innan 2003; Kitano et al. 2007). We sequenced 12 chimpanzees, 3 bonobos, 3 gorillas, 1 orangutan, and 2 siamangs. In addition, sequences from Gon (PTB-1), Donald (RP43-1 and RP43-2), and Clint (genome shotgun data in the database) were used. We then constructed a phylogenetic tree using the sequences for the region surrounding exon 7 obtained from humans and these nonhuman primate species (fig. 4). The Kimura 2-parameter (Kimura 1980) with a gamma distribution model was selected as the best by the model selection option in MEGA5. In chimpanzees and bonobos, four (Patr_RHα, Patr_RHβ, Patr_RHγ, and Patr_RHδ) and three (Papa_RHα, Papa_RHβ, and Papa_RHγ) clusters were observed, respectively. Patr_RHα1 of RP43-1 (fig. 1) did not form a cluster with other Patr_RHα genes in the phylogenetic tree, but did form a cluster with Patr_RHδ genes (fig. 4) with a bootstrap value of 93%. Two Patr_RHγ genes (Patr_RHγ1 of RP43-1 and Patr_RHγ2 of RP43-2; fig. 1) did not form a cluster with other Patr_RHγ genes, but did form a cluster with Patr_RHα genes (fig. 4). We assigned cluster names in the phylogenetic tree (fig. 4) by following Patr_RHα3 and Patr_RHγ3 on the PTB-1 chromosome (fig. 1), because the RP43-1 chromosome came from a hybrid individual. Thus, the cluster of eight chimpanzee sequences including Patr_RHα3 and four bonobo sequences was designated as Patr_RHα–Papa_RHα, and the cluster of five chimpanzee sequences including Patr_RHγ3 as Patr_RHγ. A cluster containing five bonobo sequences (Papa_RHγ1–5) was designated as Papa_RHγ, because although this cluster did not group together with Patr_RHγ, they were located close to each other in the phylogenetic tree. The cluster of five chimpanzee sequences including Patr_RHβ1 and Patr_RHβ2, and one bonobo sequence (Papa_RHβ1) was designated as Patr_RHβ–Papa_RHβ. Inexplicably, Patr_RHβ2 of RP43-2 (fig. 1) from the hybrid individual formed a cluster with Papa_RHβ1 rather than with other Patr_RHβ genes. A remaining chimpanzee cluster including Patr_RHα1 of RP43-1 was designated as Patr_RHδ. Assuming that four is the maximum number of loci in chimpanzees (Suto et al. 2003), the four clusters α, β, γ, and δ might correspond to the chimpanzee Rh loci. Three clusters (Papa_RHα, Papa_RHβ, and Papa_RHγ) for bonobos and two clusters (Gogo_RHα and Gogo_RHβ) for gorillas were found in the phylogenetic tree (fig. 4), suggesting the presence of three and two loci, respectively, for these species. These findings are compatible with the results of Suto et al. (2003).

Fig. 4.—

Fig. 4.—

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A maximum likelihood tree based on the Kimura 2 parameter model constructed using the region surrounding exon 7 obtained from humans and the listed primate species. Orangutan (Popy_RH) and siamang (Sysy_RH1 and Sysy_RH2) sequences are used as outgroups. A discrete gamma distribution is used to model evolutionary rate differences among sites (8 categories; +G, parameter = 0.2057). A scale bar representing nucleotide substitutions per site is shown. Bootstrap values (1,000 replications) are shown on each branch. Sequences obtained from genomic data in figure 1 are noted in parentheses.

From the results of the phylogenetic tree (fig. 4), we extrapolated the haplotypes of the Rh genes for various chimpanzee individuals (fig. 5). Individuals Gon, Mikota, Akina, Aluku, “Rimi,” “Journey,” and “Hitomi” had sequence types categorized only in Patr_RHα and Patr_RHγ. We also confirmed the presence of Patr_RHβS, a short version of Patr_RHβ lacking the middle part (exons 4–8) of the gene, in these individuals using PCR. Fiber-FISH analysis (Suto et al. 2003) has shown that Mikota, Akina, and Aluku have two-Rh-gene types; the genome sequence PTB-1 determined in this study suggested that this is also true for Gon. The genomic location of Patr_RHδ is still unclear, as its location beside Patr_RHγ appears arbitrary. The haplotype [α3 - S - γ3] (which corresponds to PTB-1 in fig. 1) is most probably present in double dose in Mikota (fig. 5). “Chiko” and “Mary” have the haplotype [α3 - S - γ3] as a single dose, but more complex haplotypes on their other chromosome, namely [α4 - β4 - γ6 - δ2] and [α5 - β6 - γ3 - δ3], respectively. The other individuals (Akina, Aluku, Rimi, Journey, and Hitomi) most likely have the haplotype [α3 - S - γ3] (which corresponds to PTB-1), and the Rh haplotypes of these individuals on other chromosomes were deduced without issue (fig. 5).

Fig. 5.—

Fig. 5.—

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Predicted haplotypes of the chimpanzee Rh blood group genes based on comparisons of the region surrounding exon 7 (∼860 bp) from 15 chimpanzee individuals. Patr_RHα, Patr_RHβ, Patr_RHγ, and Patr_RHδ are shown as α, β, γ, and δ, respectively, with the relevant variant number. Patr_RHβS is denoted by “S” when its presence in the individual was confirmed by PCR and by “s” when it was supposed but not confirmed through PCR experiments. Although the genome location of Patr_RHδ is unknown, it was placed beside Patr_RHγ for this analysis.

Synonymous and Nonsynonymous Substitutions in the Chimpanzee Rh Blood Group Genes

Both variations in the numbers of loci of the chimpanzee Rh blood group genes and frequent arrangements among the genes themselves may contribute to the extant diversity of chimpanzee Rh proteins. It has been suggested that the Rh blood group genes have experienced some type of positive selection (Kitano et al. 1998; Kitano and Saitou 1999; Innan 2003). Blood types were originally distinguished by different molecular structures on the cell surface of erythrocytes. Therefore, products of blood group genes may interact with other organisms such as bacteria and viruses to protect against them, leading to the possibility of positive selection on these genes. To attempt to detect positive selection, we estimated synonymous (dS) and nonsynonymous (dN) substitutions for each exon of the chimpanzee Rh blood group genes (fig. 6). Significant dN and dS differences were observed in exon 2 (higher dS than dN), exon 5 (no dS at all), and exon 7 (higher dN than dS). The latter was particularly notable: Sixteen sites of different amino acids were observed in exon 7. In exon 7 of humans, the D-specific motif of amino acid residues D-350, G-353, and A-354 is located on the sixth outer membrane loop of the RHD protein (Cartron and Agre 1993). These amino acid residues may be directly correlated with antigenic properties, because their position with respect to the membrane makes them accessible to antibodies (Blancher and Socha 1997). Thus, it is possible that this protein region may be involved in some kinds of host–parasite interactions. Interestingly, two (G-353 and A-354) of the D-specific motif was present in the Patr_RHβ locus, although the Patr_RHβ locus did not share the same genomic location as Hosa_RHCE. This finding also supports the theory of partial rearrangements frequently reoccurring in the chimpanzee Rh blood group genes.

Fig. 6.—

Fig. 6.—

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Distributions of nonsynonymous (dN) and synonymous (dS) substitutions for each exon of the chimpanzee Rh blood group genes. Proportions of dN and dS within each exon are indicated by grey and white bars with standard error bars, respectively. These standard errors were obtained using a bootstrap procedure (Nei and Kumer 2000). The dN and dS for exons 8, 9, and 10 were estimated together due to the short length of these exons.

Supplementary Material

Supplementary table S1 and figures S1–S4 are available at Genome Biology and Evolution online (http://www.gbe.oxfordjournals.org/).

Supplementary Data
supp_8_3_519__index.html (747B, html)

Acknowledgments

We are grateful to the Primate Park of Sanwa Kagaku Kennkyusho Co., Ltd. (Kumamoto, Japan) for providing chimpanzee samples. We thank Asao Fujiyama of the Comparative Genomics Laboratory, National Institute of Genetics for helping with preparation of BAC clones, and Yuji Kohara of the Genome Biology Laboratory, National Institute of Genetics for helping with sequencing. We thank Akiko Aoshima, Atsuko Ide, and Yoshimi Noaki for technical assistance. We thank anonymous reviewers and the editor for their valuable comments. This study was partially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, to N.S. and T.K., and a JSPS Grant-in-Aid for Scientific Research to T.K.

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