Gene Diversity of Chimpanzee ABO BloodGroup Genes Elucidated from
Intron 6 Sequences
T. Kitano, R. Noda, K. Sumiyama, R. E. Ferrell, and N. Saitou

The human and nonhuman primate ABO blood group gene shows relatively large
numbers of nucleotide differences around the exon 7 region. In this study we determined intron 6 sequences for 9 alleles of common chimpanzee and for 3 allelesof bonobo to estimate nucleotide diversities among them. Sequence length polymorphisms are observed in this region as a repeat appears one to five times. Froma phylogenetic network of intron 6 sequences of ABO blood group genes for humans, common chimpanzee, and bonobo, parallel substitutions and/or some kindsof convergent events are predicted in the chimpanzee lineage. We also estimatednucleotide diversities for common chimpanzee and bonobo ABO blood group
genes; these values were 0.219% and 0.208%, respectively.

From the Laboratory of Evolutionary Genetics, National Institute of Genetics, Mishima, 411-8540 Japan ( Kitano, Noda, Sumiyama, and Saitou) and the Departmentof Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania ( Ferrell). K. Sumiyama is currently at the Department of Biology, Yale University, New
Haven, Connecticutt. This study was partially supported by grants in aid for scientific studies from the Ministry of Education, Science, Sport, and Culture to N.S.
We thank Ingrid Jakobsen for her comments on an earlier version of this article. Address correspondence to
N. Saitou at the address above or e-mail: nsaitou@genes.nig.ac.jp. This paper was delivered at a symposium entitled ‘‘Genetic Diversity and Evolution’’ sponsored by the American Genetic Association at thePennsylvania State University, University Park, PA,
USA, June 12–13, 1999.
 2000 The American Genetic Association 91:211–214

The human ABO blood group was discovered by Karl Landsteiner in 1900, and its
mode of inheritance as multiple alleles ata single genetic locus was established byBernstein 25 years later (Crow 1993). The
chemical basis of ABO blood group specificities was shown to be carbohydrate
structures of glycoproteins and glycolipids through studies performed in the
1950s and early 1960s [see Yamamoto(1995) for a review]. Based on this finding, ABO alleles A and B were considered
to code glycosyltransferases, whichtransfer GalNAc and galactose, respectively. Yamamoto et al. (1990a) determined the cDNA sequences of three common alleles A1, B, and O, and Yamamoto
et al. (1995) determined the genomic organization of the gene. Critical sites forthe distinction between A and B activitiesof the glycosyltransferases have also
been identified ( Yamamoto et al. 1990a,b,
1991, 1992, 1995). These sites are locatedin the exon 7 region, and these regions ofmany other nonhuman ABO blood group
genes were sequenced (e.g., Kermarrec etal. 1999; Kominato et al. 1992; Martinko etal. 1993). Saitou and Yamamoto (1997)
compared nucleotide sequences of primate ABO blood group genes, and relatively large numbers of nucleotide differences were found among them. These
differences were unusually large for human allelic divergence under neutral evolution. In this study we determine and analyze the nucleotide sequences of intron
6 for two chimpanzee species to compare

and elucidate the gene diversity among
the ABO blood group genes.

Materials and Methods
Sequencing of Genomic DNA
Genomic DNAs of nine common chimpanzees (Pan troglodytes) and three bonobo(Pan paniscus) were used. PCR reaction
was performed using 1⫻ Gene Taq Universal Buffer (Mg2⫹ free) ( Nippon Gene), 1.5mM MgCl2, 0.2 mM dNTP, 10 pmol of upper
primer (SN-12; 5⬘-ACCCCCAGCCAAAGGTGCTGACA-3⬘ or SN-13; 5⬘-ACCCTGCCAGCTCCATGTGAC-3⬘) and lower primer (SN-10; 5⬘CGATGCCGTTGGCCTGGTC-3⬘), and 1 unitof AmpliTaq Gold (Perkin-Elmer). Amplification was carried out in DNA GeneAmp
PCR System 2400 (Perkin-Elmer) with thefollowing temperature parameters: 10 min
at 95⬚C followed by 40 cycles of 95⬚C for
30 s, 65⬚C for 15 s, and 72⬚C for 1 min. PCR
products were purified using MicroSpin
Columns S-300 HR (Pharmacia Biotech).
At first, PCR products were sequenced byusing the direct sequencing method. Thenheterogeneous PCR products were cloned
in the TA cloning vectors pCRII ( Invitrogen). DNA sequencing was performed on
double-stranded plasmid DNA and PCR
products using the Dye Terminator Cycle
Sequencing Kit and ABI prism 377 DNA sequencer (Perkin-Elmer). A progressive sequencing strategy was carried out with design of further primers to both strands ofthe DNA.

211

Table 1. Variant positions of ABO blood group genes in intron 6
Nucleotide position

Sequenceab

1111222222224555667777788889900
2448957457888999057480234501330122
5299041219017564914435738804582779

Repeata

Exon 7b

Common chimpanzee (P. troglodytes)
CGATACCCCCTCACCTTTAACGGGATAGTCAGAG
c-1
CGATACCCCCTCACCTTTAATGGGATAGTCAGAG
c-2
CGATACCCCCTCACCTTTAACGGGACAGTCAGAG
c-3
CGATACCCCCTCACCTTTAACGGGATAGTCAGAG
c-4
CGATACCCCCTCACCTTTAACGGGATAGCCAGAG
c-5
CGATACCCCCTCAACTTTAACGGGATAGCCAGAG
c-6
CGATACCCCCTCACCTTTAACGGGATAGCCAGAG
c-7
CGATACCCCCTCACCCTCGACGAGACAGTCAGAG
c-8
CGATACCCCCTCACCCTCGACGAGACAGTCCGAG
c-9

5
5
5
4
4
4
3
3
3

II
IV
V
I, II
III
III
III
V
V

Bonobo (P. paniscus)
CGATACTCC---ACCTTCAACGGGACAGCCAGAG
b-1
CGATACCCCCTCACCTTCAACGGGACAGCCAGAG
b-2
CGTTATCCCCTCACCTTCAACGGGATAACCAGAG
b-3

3
3
1

bI
bII
bIII

Human (H. sapiens)
TTATGCCCCCTCACTTTCGGCAGGGCGGCTAGGAh-A
TTATGCCCCCTCGCTTTCGGCGGGGCGGCCAAAG
h-B
TGAAGCCTGCTCACCTCCGACGGAGCGGCCAGGA
h-0

3
3
3

A
B
0

Number of copies of repeat in intron 6 (see Figure 1).
Allele type of exon 7 (Sumiyama et al., unpublished data).

Sequence Analyses
CLUSTAL W version 1.6 ( Thompson et al.
1994) was used for constructing the multiple alignments. Phylogenetic networks
were constructed following the procedureof Bandelt (1994) and Saitou and Yamamoto (1997). The nucdiv program of the
ODEN package ( Ina 1994) was used for estimation of nucleotide diversities.

Results and Discussion
Comparison of Sequences
We determined nine alleles for common
chimpanzee and three alleles for bonobo
of intron 6 sequences of ABO blood group
genes ( DDBJ/EMBL/GenBank international
nucleotide sequence database accession
numbers AB031235–AB031246). Table 1

Figure 1. Sequence comparison around the repeat region (sites 160–240) of intron
6 for human and chimpanzee ABO blood group genes. Hyphens and asterisks designate gap and invariant site, respectively. Equal signs surrounded by angled brackets indicate the repeat unit of eight nucleotides.

212 The Journal of Heredity 2000:91(3)shows the variant sites of ABO bloodgroup genes in intron 6. Three human sequences (AF016622–AF016624; Olsson and
Chester 1998) are also shown. O’hUigin etal. (1997) sequenced the intron 6 region
for humans, chimpanzees, and gorillas but
those were partial sequences. Therefore
those data are not included in this table.
One bonobo gene ( b-1) had a gap (279–281) of three nucleotides. Sequence lengths
were 1036–1068 bp. These sequence length
differences were caused by different numbers of repeats of the sequence TGGGCTCG. Figure 1 shows a sequence comparison of repeat regions (160–240) of ABO
blood group genes in intron 6. There are
one to five copies of the repeat.
The Phylogenetic Network of Hominoid
ABO Blood Group Genes
Figure 2 shows the phylogenetic networkof intron 6 sequences of ABO blood group
genes for humans, common chimpanzee,
and bonobo. This phylogenetic network
was constructed from the sequence dataof Table 1. Four sites (25, 90, 748, 800) divide genes of humans and chimpanzees.
O’hUigin et al. (1997) presented partial sequences (sites 1–398 and 837–1068) for gorilla, and we determined the branching
point of gorilla genes using the consensus
sequence of two gorilla B genes. From this
phylogenetic network, the branching pointof gorilla B genes was located between hu-

Figure 2. The phylogenetic network of intron 6 sequences for human and two
chimpanzee ABO blood group genes. Numbers are nucleotide positions responsible
for corresponding edges and edge lengths are proportional to the number of nucleotide differences. Because sites 173–204 include gaps by repeat units, these sites
were not used for this phylogenetic network. Alleles are marked by circles, andblack, white, vertical stripes, and slant stripes designate 1, 3, 4, and 5 repeats inintron 6 (see also Figure 1), respectively. A gap on the branch connecting to b-1
means a gap (279–281) of three nucleotides. Abbreviations of alleles are the sameas in Table 1.

mans and chimpanzee. Saitou and Yamamoto (1997) proposed that B-type allelesevolved independently on human and gorilla lineages. Our result for intron 6 sequences is compatible with theirs.
There is one big rectangle among thehuman sequences of this phylogenetic
network. Sites 1027 and 1029 divide A/O
alleles from the B allele. Because thesesites are contiguous, there is a possibilityof recombination in this region among
these alleles.
Four small parallelograms were createdfor chimpanzee sequences. Therefore parallel substitutions and/or some kinds ofconvergent events (recombination) must
be assumed. For example, c-8, c-9, and c-3
are identical in exon 7 (Sumiyama et al.,unpublished data), but there are four nucleotide differences between c-8 and c-3 inintron 6. In these site differences, sitechanges 551 and 574 are embedded in parallelograms. Because these two sites arecontiguous, they may have been transferred between alleles by recombination.
As mentioned above, chimpanzee sequences have variations of repeat number(see Figure 1). It is clear that ABO bloodgroup genes for humans and gorilla havethree repeats from O’hUigin et al.’s (1997)
and Olsson and Chester’s (1998) data.
Therefore the common ancestor of humans, chimpanzees, and gorillas probably
had three repeats. After the speciation ofhumans and chimpanzees, the number of
repeats has changed in the chimpanzee
lineage.
The Phylogenetic Relationship Among
Chimpanzee ABO Blood Group Genes
In the phylogenetic network of intron 6 sequences of ABO blood group genes for humans, common chimpanzee, and bonobo( Figure 2), genes of common chimpanzeeand bonobo do not form a clear treelike
structure. Therefore we deduced two possible trees for common chimpanzee andbonobo ( Figure 3). The tree of Figure 3A
is deduced based on species relationships. In this tree, genes for common
chimpanzee and bonobo form different
clusters defined by sites 835 and 574, respectively. Three genes of common chimpanzee (c-5, c-6, and c-7) form a cluster.
The numbers of nucleotide changes andrepeat changes in this tree were 16 and 4,
respectively.
The tree of Figure 3B is the maximum
parsimony tree. This tree was constructedby using the branch and bound method of
PAUP version 3.1.1 ( Illinois Natural History Survey). Numbers of nucleotide chang-

Figure 3. Two possible trees (A and B) deduced from the phylogenetic network of intron 6 sequences ( Figure2) of chimpanzee ABO blood group genes. Numbers on each branch denote the nucleotide positions in which
substitutions occurred. Numbers with asterisks signify nucleotide position in which parallel substitutions occurred.
Branches with equal signs and angled bracket designate change in the number of repeats.

es and repeat changes in this tree were 15
and 3, respectively. From the parsimony
principle, tree ( B) is more parsimonious
than tree (A). However, we have to assume
ancestral polymorphism among common
chimpanzee and bonobo in tree ( B).
Therefore, we believe that tree (A) is
more plausible. In this tree, the number of
repeats first increased from three to five,then decreased from five to four, and finally decreased from four to three. In theb-3 gene, repeats decreased from three toone. On the other hand, three genes ofcommon chimpanzee (c-3, c-8, and c-9)
have a deletion of 9 bp in exon 7 ( Kermarrec et al. 1999; Sumiyama et al., unpublished data). It is difficult to imagine
that all the other common chimpanzee
ABO genes evolved from a gene having adeletion of 9 bp in exon 7. Therefore it ispossible that the c-3 gene is a recombinant
between a gene with 9 bp deletion and another common allele (probably a type I or
II gene).
Rates of Nucleotide Substitution and
Nucleotide Diversities in Chimpanzee
ABO Blood Group Genes
To estimate rates of nucleotide substitution (␭) of intron 6 sequences of hominoid
ABO blood group genes, we estimated thesingle lineage number of nucleotide substitutions per site (K) for each species byapplying Ishida et al.’s (1995) method. The
number of nucleotide substitutions in thecommon chimpanzee lineage ( Figure 3A)
is 3.65. The number of nucleotide substi-

tutions in the bonobo lineage ( Figure 3A)
is 2.67. The number of nucleotide substitutions in the human lineage ( Figure 2) is7.33. Therefore K values of common chimpanzee, bonobo, and human lineages are
estimated to be 0.00353, 0.00258, and
0.00711 by Jukes and Cantor’s (1969)
method. Divergence times (T) between humans and chimpanzee and between common chimpanzee and bonobo are thought
to be 5.5 ( Kumar and Hedges 1998) and2.5 ( Horai et al. 1992) million years ago,
respectively. Thus ␭ values of common
chimpanzee, bonobo, and human lineages
are estimated to be 1.41 ⫻ 10⫺9, 1.03 ⫻10⫺9, and 1.29 ⫻ 10⫺9 by the formula ␭ ⫽K/T, respectively.
Li and Graur (1991) estimated ␭ values
in the 5⬘ and 3⬘ untranslated regions andat fourfold degenerate sites of protein-coding genes, based on comparisons between
humans and rodents for 16 genes. Our estimated values were similar to those forthe 5⬘ and 3⬘ untranslated regions (averages are 1.96 ⫻ 10⫺9 and 2.10 ⫻ 10⫺9, respectively). In the case of human ABO
blood group genes, three alleles (i.e., A, B,
and O alleles) have been observed, but it
is known that chimpanzees have two alleles (i.e., A and O alleles). We do notknow the actual phenotypes of the chimpanzee ABO blood group genes used inthis study. Nevertheless, the ␭ value of thecommon chimpanzee lineage is higher
than that of the human lineage.
We computed nucleotide diversities ofcommon chimpanzee and bonobo ABO

Kitano et al • Chimpanzee ABO Blood Group Genes 213

Table 2. The list of chimpanzees and their
genotypes for ABO blood group gene intron 6
Individuala1999. Comparison of allele O sequences of the humanand non-human primate ABO system. Immunogenetics
49:517–526.

Samplesize

Kominato Y, McNeill PD, Yamamoto M, Russell M, Hakomori S, and Yamamoto F, 1992. Animal histo-bloodgroup ABO genes. Biochem Biophys Res Commun 189:
154–164.

Genotype

Common chimpanzee (P. troglodytes)
CH-46
c-5/c-7
CH-90
c-2/-aCH-206c-3/c-8
CH-220c-3/c-8
CH-235c-6/c-6
CH-75
c-4/c-4
CH-83
c-1/c-4
CH-80
c-1/c-9
CH-76
c-4/c-4
CH-86
c-1/c-1
Bonobo (P. paniscus)
BO-3
BO-4
BO-5
BO-8

Table 3. Nucleotide diversities and rates ofnucleotide substitution in chimpanzee ABO bloodgroup genes

b-2/b-2b-1/b-1b-2/b-3b-2/b-3

The chimpanzee CH-90 is heterozygous, but another allele could not be determined.

blood group gene intron 6 sequences. Table 2 shows the genotype of the ABO
blood group genes for each individual.
The chimpanzee CH-90 has a heterozygous
genotype, but another allele could not be
determined. Table 3 shows the comparison of nucleotide diversities. Nucleotide
diversities of common chimpanzee andbonobo were 0.219% and 0.208%, respectively.
Nachman et al. (1998) analyzed nucleotide diversity at X-linked loci in humans
and compared those with previous studies(e.g., Li and Sadler 1991) of nucleotide diversity in humans. To compare directlywith variation at autosomal loci they used
standardized values, and they concluded
that the average level of nucleotide diversity in humans for silent and noncoding
DNA is 0.1%, and the highest levels of nucleotide diversity at any locus is about 2.5
times the average for all loci. Therefore,
nucleotide diversity among alleles ofchimpanzee ABO blood group genes ishigher than that of most human genes.
In this study we determined common
chimpanzee and bonobo ABO blood groupgene intron 6 sequences. We observed sequence length polymorphisms, as a repeat
appears one to five times in this intron 6
sequences. From a phylogenetic networkof intron 6 sequences of ABO blood group
genes for humans, common chimpanzee,

214 The Journal of Heredity 2000:91(3)Number of sites Nucleotide
compared ( bp) diversity (%)

Common chimpanzee (P. troglodytes)
19
1052
0.219
Bonobo (P. paniscus)
8
1033

0.208

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and bonobo, parallel substitutions and/or
some kinds of convergent events are predicted in the chimpanzee lineage. We also
estimated nucleotide diversities among alleles of chimpanzee ABO blood group
genes to be about 0.2%. This value is higher than that of most human genes. Wise etal. (1997) compared nuclear and mitochondrial gene diversities of humans andchimpanzees. They proposed that chimpanzees appear to have more mitochondrial gene diversity than humans, whereas
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sequence data for chimpanzee nuclear
genes to allow comparison with humannucleotide diversity. Therefore we needmore nuclear sequence data for chimpanzees to estimate nucleotide diversity reliably.

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Corresponding Editor: Masatoshi Nei