Research

Gorilla genome structural variation reveals

evolutionary parallelisms with chimpanzee
Mario Ventura,1,2 Claudia R. Catacchio,1,2 Can Alkan,1,3 Tomas Marques-Bonet,1,4
Saba Sajjadian,1 Tina A. Graves,5 Fereydoun Hormozdiari,6 Arcadi Navarro,4,7,8
Maika Malig,1 Carl Baker,1 Choli Lee,1 Emily H. Turner,1 Lin Chen,1 Jeffrey M. Kidd,1,9
Nicoletta Archidiacono,2 Jay Shendure,1 Richard K. Wilson,5 and Evan E. Eichler1,3,10
1
Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington 98195, USA; 2Department
of Genetics and Microbiology, University of Bari, Bari 70126, Italy; 3Howard Hughes Medical Institute, Seattle, Washington
98195, USA; 4IBE, Institut de Biologia Evolutiva (UPF-CSIC), Universitat Pompeu Fabra, Barcelona 08003, Catalonia, Spain;
5
Washington University Genome Sequencing Center, School of Medicine, St. Louis, Missouri 63108, USA; 6School of Computing
Science, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada; 7Population Genomics Node (GNV8), National Institute
for Bioinformatics (INB), Barcelona 08003, Catalonia, Spain; 8Institució Catalana de Recerca i Estudis Avancxats (ICREA) and Universitat
Pompeu Fabra, Barcelona 08003, Catalonia, Spain; 9Department of Genetics, Stanford University, Stanford, California 94305, USA

Structural variation has played an important role in the evolutionary restructuring of human and great ape genomes.
Recent analyses have suggested that the genomes of chimpanzee and human have been particularly enriched for this form
of genetic variation. Here, we set out to assess the extent of structural variation in the gorilla lineage by generating 10-fold
genomic sequence coverage from a western lowland gorilla and integrating these data into a physical and cytogenetic
framework of structural variation. We discovered and validated over 7665 structural changes within the gorilla lineage,
including sequence resolution of inversions, deletions, duplications, and mobile element insertions. A comparison with
human and other ape genomes shows that the gorilla genome has been subjected to the highest rate of segmental du-
plication. We show that both the gorilla and chimpanzee genomes have experienced independent yet convergent patterns
of structural mutation that have not occurred in humans, including the formation of subtelomeric heterochromatic caps,
the hyperexpansion of segmental duplications, and bursts of retroviral integrations. Our analysis suggests that the
chimpanzee and gorilla genomes are structurally more derived than either orangutan or human genomes.
[Supplemental material is available for this article.]

The nature of the genetic differences between humans and other whole-genome shotgun sequence data sets (Marques-Bonet et al.
great apes has fascinated scientists since the discovery of DNA in 2009b). Studies of structural variation, however, are complicated
the 1950s (Sarich and Wilson 1973; Yunis and Prakash 1982; by difficulties in detecting and accurately resolving the sequence
Goodman et al. 1989). The genetic relationship and phylogeny of structure of these regions. In this study, we set out to systematically
humans and great apes is well established, based primarily on investigate the pattern of structural variation in the gorilla genome,
studies of single nucleotide variation (Koop et al. 1986; Enard and combining capillary-based clone sequencing and next-generation
Paabo 2004). A surprising finding has been the extent of larger genome sequencing in conjunction with detailed cytogenetic char-
forms of structural variation among hominid genomes well below acterization and experimental validation. We present a comprehen-
the limit of cytogenetic resolution (Locke et al. 2003; Fortna et al. sive overview of inversions, deletions, segmental duplications, and
2004; Cheng et al. 2005; Bailey and Eichler 2006; Gibbs et al. 2007; retrotranspositions within the gorilla genome. Comparisons with
Marques-Bonet et al. 2009a). Interestingly, the hominid genomes humans and other apes reveal that parallel and independent muta-
appear to be enriched with respect to structural variation, but the tional processes have more dramatically restructured chimpanzee
extent to which this has impacted each of the major lineages is and gorilla genomes when compared with other hominid genomes.
not yet completely known. To date, three hominid genomes have
been sequenced and assembled to the working draft stage using
capillary-based approaches [human (The International Human Results
Genome Sequencing Consortium 2001, 2004), chimpanzee (The In order to investigate the gorilla’s pattern of genome structural
Chimpanzee Sequencing and Analysis Consortium 2005), and variation, we undertook a three-pronged approach. First, we tested
orangutan (Locke et al. 2011)]. Projects are underway to sequence 788 human BAC clones by fluorescence in situ hybridization (FISH),
additional apes including the bonobo, gorilla, and gibbon. Many comparing the probe order on human and gorilla chromosomal
of these remaining ape genomes will be sequenced and assembled metaphases, thus providing a refined cytogenetic framework of large-
using a combination of next-generation sequencing and capillary scale and intermediate-sized rearrangement events (Supplemental
Note). Next, we completely end sequenced 176,880 BAC clones
10
Corresponding author. (http://www.genome.gov/Pages/Research/Sequencing/BACLibrary/
E-mail eee@gs.washington.edu.
Article published online before print. Article, supplemental material, and pub- primateProposal.pdf) from a gorilla BAC library (CH277) and map-
lication date are at http://www.genome.org/cgi/doi/10.1101/gr.124461.111. ped them to the human reference genome [NCBI build 35 (NCBI35)]

1640 Genome Research 21:1640–1649 Ó 2011 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/11; www.genome.org
www.genome.org
 Structural variation in great apes

to generate a clone-based framework of the gorilla genome (Eichler 14 representative BAC clones corresponding to the classical gorilla-
and DeJong 2002). This approach defined potential rearrangements human breakpoints and completely sequenced them using capillary-
based on discordant end sequence placements. Last, we obtained based sequencing methods (Table 2). Detailed breakpoint analyses
blood DNA from Kwan, a middle-aged silverback gorilla, and gener- (Fig. 1; Supplemental Figs. S1–S5) showed SDs (translocation 5;17
ated 9.6-fold effective sequence coverage using massively parallel and inversion 7) or common repeat elements (Alus and LINEs) at or in
Illumina sequencing. While this sequence coverage means that each close proximity to all breakpoints. Due to the abundance of these
base is represented on average nine to 10 times, the paired-end se- elements in the hominid genome and the small number of se-
quences flanking a portion of the insert that is not sequenced means quenced sites, this enrichment is not significant. In most cases, the
a larger fraction of the genome is spanned by anchored mate pairs repetitive sequences were not homologous, suggesting that mecha-
(34-fold). These data were used to identify regions of copy number nisms other than nonallelic homologous recombination were re-
variation based on sequence read-depth and paired-end mapping sponsible for these evolutionary rearrangements. None of these
revealing smaller forms of structural variation including mobile ele- rearrangements disrupted unique genes.
ment insertions (>300 bp) using end sequence profiling approaches The BAC read-pair analysis predicted an unusually large
(Tuzun et al. 2005; Hormozdiari et al. 2009; Hormozdiari et al. 2010). number of putative inversions and translocations, which is in-
The experimental and molecular data were integrated (Table 1; Sup- consistent with previous chromosomal analyses and our own cy-
plemental Note), allowing us to correctly reclassify events that par- togenetic framework. We selected a subset of these events (six pu-
ticularly distinguished translocations from duplicative transposition tative translocations and 14 inversions) (Supplemental Table S3) for
events and inversions from segmental duplications (SDs). For further investigation. For each rearrangement, if the predicted
example, translocations could be distinguished from duplicative translocations and inversions were bona fide, we would expect a
transpositions because read-depth and array comparative genomic change in the order of flanking probes (inversions) or a change in
hybridization (arrayCGH) predicted copy number changes of a chromosomal location (translocation) when comparing human and
segment of DNA but with no evidence of chromosomal rear- gorilla. For each of these breakpoints, we selected gorilla BAC clones
rangement using cytogenetic markers. In those cases where we spanning the putative rearrangement breakpoints, as well as go-
were able to completely sequence the corresponding BAC clone, rilla BAC clones located distally and proximally to each breakpoint,
the breakpoints could be resolved at the single base pair level. We and tested their order between human and gorilla. In all cases, no
summarize the pattern of gorilla genome structural variation from change in the order of flanking unique sequences was observed.
the perspective of size and class, and then compare our findings These FISH results suggested the presence of duplicated sequences at
with human and other great ape genomes. new locations in the gorilla genome.
We completely sequenced 20 of the corresponding BAC clones
(3.2 Mb of finished capillary-based sequence) to resolve the structure
Large-scale rearrangements and duplicative transpositions of these selected loci (Supplemental Table S3). In each case, we con-
Yunis and Prakash originally reported 11 large-scale cytogenetic firmed duplicative transpositions and gorilla-specific juxtapositions
differences between human and gorilla (eight pericentric inversions, of SDs as opposed to inversions and translocations. For example,
one paracentric inversion, one translocation, and one fusion) (Yunis BLAST sequence similarity searches of the sequenced gorilla BACs
and Prakash 1982). Using FISH and data from other studies from chromosome 5 (AC23944) (Fig. 1B) indicate that this portion of
(Dutrillaux 1980; Yunis and Prakash 1982; Montefalcone et al. 1999; the gorilla genome consists of a mosaic of four diverse SDs originating
Muller et al. 2000, 2003; Carbone et al. 2002; Eder et al. 2003; Locke from different locations on human chromosome 5. The Miropeats
et al. 2003; Misceo et al. 2003, 2005; Ventura et al. 2003, 2004; analysis (Parsons 1995) shows the extent of each duplicated segment
Cardone et al. 2006, 2007; Stanyon et al. 2008), we refined all clas- (ranging in length from 9 to 86 kb). Sequence read-depth analysis
sical evolutionary breakpoints that distinguish the human and gorilla among the various species [whole-genome shotgun sequence de-
karyotypes (Supplemental Table S1). It should be noted that cytoge- tection (WSSD) tracks; see Supplemental Note] suggests that different
netics typically only resolves megabase pair-level variation, while segments have been duplicated at different time points during evo-
FISH analyses utilizing overlapping probes can be used to resolve lution. We conclude that this particular architecture is unique to
events ;50 kb in size, depending on the complexity of the region. gorilla originating from a series of duplicative transpositions to gorilla
Using gorilla BAC end-sequence (BES) data mapped against the hu- chromosome 5p13.2. We note that none of the 20 sequenced regions
man genome (NCBI35), we identified 424 putative chromosomal from BACs were collinear with the human genome. These regions
rearrangements (Supplemental Table S2; Supplemental Note) in- carry, on average, three reconfigured or newly interspersed duplica-
cluding six of the 11 original classical rearrangements. The re- tions with an average length of 29 kb. We estimate 79% map inter-
maining five were confirmed by FISH but mapped to large and stitially within gorilla chromosomes, with another 21% mapping to
nearly identical SDs that could not be traversed by BES. We selected subtelomeric or pericentromeric regions. These data reveal extensive

Table 1. Gorilla sequence and FISH resources

Average Average
# read insert
Resource Technology Reads/clones length size (bp) Data release

WGS libraries Illumina 1,619,928,596 36 244 SRA: SRP002878

BAC clones Sanger finished 34 750 161,627 GenBank accessions
(see Table 2; Supplemental Table S3)
BAC end sequence (BES) Sanger paired end 353,761 776 160,447 Trace Archive
BAC clones FISH 788 NA 170,000 http://www.biologia.uniba.it/primates/

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Table 2. BAC sequenced chromosomal breakpoints

Human Gorilla Repeat elements

Rearrangement Breakpoint Clone Accession Size (bp) Location by BES mapping mapping at/near breakpoint Class

Inv chr7 7q arm CH277-505P01 AC243004.1 36,298 chr7:76489360-102048010 7q15; 7q22 VIIq Seg Dup Inversion
CH277-325N15a AC242656.3 95,809 chr7:76558315-102048039 7q15; 7q22 VIIq
Inv chr8 8q arm CH277-11L17 AC242655.3 180,892 chr8:31006213-86005534 8p12; 8q21 VIIIq L1P3 (LINE) Inversion
CH277-481C13 AC242627.3 158,706 chr8:31148163-85832627 8p12; 8q21 VIIIq
CH277-402K23 AC242595.2 102,724 chr8:31148078-85914766 8p12; 8q21 VIIIq
CH277-401D02 AC243002.1 55,127 chr8:31287889-86049917 8p12; 8q21 VIIIq
Inv chr10 10p arm CH277-125A6a AC241241.3 198,809 chr10:27573884-80780850 10p12 Xp, Xq L1m4 (LINE)/AluSc Inversion
CH277-103D21a AC241522.2 188,246 chr10:27675315-80631965 10p12 Xp, Xq
Inv chr12 12q arm CH277-205P14 AC240968.2 146,531 chr12:21041953-63546183 12p12; 12q14 XIIp AluSx/L1M5 (LINE) Inversion
CH277-242I19 AC240954.2 116,473 chr12:21163982-63679105 12p12; 12q14 XIIp
Inv chr18 18q arm CH277-230I8 AC239638.4 59,640 chr18:211564-16874356 18pter; 18q11 XVIIIq AluSq2 Inversion
CH277-492F03 AC243003.2 36,580 chr18:177407-16836303 18pter; 18q11 XVIIIq
CH277-545D07 AC243178.1 187,650 chr18:287777-16800762 18pter; 18q11 XVIIIq
t5:17 5q arm CH277-159N16a AC240953.2 169,000 chr5:79817957-80044726 5q14 XVIIp Seg Dup Translocation
chr17:16517994-16524015 17p11

All positions are relative to NCB135.

a
Clone duplicated in human, chimpanzee, gorilla, and orangutan.
 Structural variation in great apes

duplicative transposition in the gorilla

genome creating a complex pattern of SDs
unique to this lineage (see below).

Deletions
We initially detected 79 large deletions
(>50 kb) compared with human using
a combination of interspecies arrayCGH,
BES mapping, and depletion in sequence
read-depth. Of these events, 89% (70/
79) were confirmed experimentally by
arrayCGH and/or FISH (Supplemental
Table S4). Based on human genome an-
notation, these regions contained 61
genes that were either completely (38) or
partially (23) deleted in Kwan (Supple-
mental Table S4). We examined 52 of
these regions by FISH and found that 62%
(32/52) of these apparent deletions cor-
respond to regions of duplication in the
human where the gorilla simply showed
reduced copy number. Only 16 of the re-
gions contained unique hominid genomic
regions that had been completely deleted
in the gorilla lineage (Supplemental Fig.
S6; Supplemental Table S4). In order to
detect smaller deletions in the gorilla ge-
nome, we searched for clusters of discor-
dant read pairs based on mapping gorilla
sequence (Supplemental Note) against the
human reference genome (Tuzun et al.
2005; Hormozdiari et al. 2009). We exper-
imentally validated 1820 deletion inter-
vals (6.7 Mb) using a customized micro-
array (Supplemental Note). This included
580 partial and 13 complete gene de-
letions (Supplemental Table S5; Supple-
mental Note). Many of the completely
deleted genes belong to well-known
gene families, including olfactory recep-
tors (OR10K1, OR5L2, OR5D16, OR1M1,
and OR7G2), keratin-associated proteins
(KRTAP13-3 and KRTAP13-4) and HLA
genes [HCP5 (HLA complex P5) and HCG26
(HLA complex group 26)]. One particularly
intriguing deletion included SELV (seleno-
protein V), thought to be important in the
metabolism of dietary selenium, implicated
in cancer prevention, immune function,
aging, male reproduction, and other phys-
iological processes (Kryukov et al. 2003).
Among the partial gene deletions, 36 span
more than a single exon—the largest be-
ing DNAH14 with 45 deleted exons. In
total, we discovered and validated 1863
deletion intervals in the gorilla genome
corresponding to 12.69 Mb.

Mobile elements
We initially excluded regions with >80%
repeat content from our deletion analysis Figure 1. (Legend on next page)

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due to difficulties in validating these calls by arrayCGH. However, Table 3. Mobile element comparison among hominid genomes
such deletions likely correspond to mobile element insertions in
Humana Humanb Chimpanzee Gorilla Orangutan
the human genome that occurred since the two lineages diverged.
We specifically searched for the aforementioned events by identify-
Alu 584 7082 2340 4272 250
ing deletions where the corresponding locus in human was com- L1 52 1814 1979 299 5000
posed largely of a particular common repeat. We predicted 2481 Alu SVA 14 970 400 325 1800
(887 kb), 1861 L1 (5.59 Mb), 663 SVA (1.17 Mb), 524 LTR (1.27 Mb), PTERV ND ND 275 263 ND
and 110 HERV (409 kb) insertions in human when compared with
Gorilla-specific mobile elements detected by VariationHunter (see text for
gorilla (Hormozdiari et al. 2010). A subset of these were full length,
details); orangutan-specific elements (Locke et al. 2011). Mobile element
including 2372 Alu ($275 bp) and 564 L1 ($5.8 kb) elements. Of prediction is based on comparison with human genome (NCBI35). (ND)
these mobile elements, 37% (2066/5639) mapped within 1650 genes not detected
a
(Supplemental Table S6). We performed the reciprocal analysis by Human mobile elements (Venter vs. reference genome) (Xing et al. 2009).
b
searching for retrotranspositions specifically within the gorilla line- Human-specific mobile elements compared with chimpanzee (The
Chimpanzee Sequencing and Analysis Consortium 2005).
age. We detected the insertion breakpoints of various classes of active
mobile elements (Alu, L1, SVA, and LTR) as well as nonhuman pri-
mate-specific endogenous retroviruses (PTERV1 and PTERV2) (Yohn
et al. 2005) and predicted a total of 263 PTERV1, 4272 Alu, 325 SVA, (Bailey et al. 2002; Alkan et al. 2009; Marques-Bonet et al. 2009a). We
113 LTR, and 299 full-length L1 insertions (Supplemental Table S7). detected 99 Mb of SDs (>20 kb in length and >95% identity) (Cheng
In order to estimate the rate of false positives, we tested 30 full-length et al. 2005; Marques-Bonet et al. 2009a). We validated the duplica-
gorilla Alu retrotransposons by PCR analysis of gorilla and human tions by interspecies arrayCGH, discovering 68 complete or partial
DNA (Supplemental Note). Of the events, 90% (27/30) validated as gene duplications in the gorilla (Supplemental Note). Although
fixed insertions (Supplemental Fig. S7), with the remaining three most of the duplications are shared with other hominids (Fig. 3;
being polymorphic. Consistent with recent analyses of human and Supplemental Note), we note an apparent excess of gorilla-specific
other ape genomes, these results predict an acceleration of SVA ret- duplications when compared with human, chimpanzee, or orang-
rotransposition in the chimpanzee-human ancestral lineage with a utan. Comparing the SD maps of five primate genomes, we assigned
more recent surge of Alu retrotransposons in the human branch shared and lineage-specific SDs (Fig. 3B) and computed a genomic
(Table 3). While we found no evidence of PTERV2 integrations in duplication rate along each branch under a maximum likelihood
gorilla, we did identify 263 full-length integrations of PTERV1—an model, which assumes 20% homoplasy. The addition of gorilla du-
endogenous retrovirus initially discovered in chimpanzee (Yohn et al. plication data into a maximum likelihood framework suggests an
2005). A comparison with a previously developed integration map of excess of SD in the human-African great ape ancestor that is larger
chimpanzee revealed that 99.6% of these integrations are non- than previously reported (Marques-Bonet et al. 2009a), with an
orthologous, mapping to different locations in the gorilla and chim- estimated rate four- to fivefold higher when compared with the
panzee genomes (Fig. 2). Both experimental and sequence analyses human or chimpanzee branches. Surprisingly, the gorilla-specific
confirm that this mobile element is completely absent from human branch is also significantly accelerated compared with the human
and orangutan genomes. This provides strong support that PTERV1 (approximately two to four times), but less so when compared with
arose from an exogenous source that retrotransposed independently the common ancestral branch of humans and chimpanzees.
in both gorilla and chimpanzee lineages <6 million years ago. This difference becomes more dramatic in the gorilla when
adjusting for copy number (Fig. 3A), indicating that several se-
quences have expanded more prominently. We tested and validated
Segmental duplications by FISH (Supplemental Table S8) 11 gorilla-specific duplications
We developed an SD map of the gorilla genome based on detecting showing the highest copy number increase (11–45 copies). Nine of
regions with excess sequence read-depth as described previously these 11 regions contain genes completely and/or partially ex-
panded specifically in the gorilla lineage
(e.g., NDUFA8—unknown function, LRPA-
Figure 1. Sequencing of large-scale gorilla chromosome rearrangements. (A) Sequence characteriza- P1—low-density lipoprotein receptor-re-
tion of a gorilla inversion on chromosome 12 (Egozcue and Chiarelli 1967; Miller et al. 1974). A schematic lated protein, DOK7—downstream from
of the 42.5 Mb inversion (top panel ideogram) is refined by paired-end BES and confirmed by FISH (bottom
tyrosine kinase 7, HGF—activator pre-
right panel). A gorilla BAC clone (CH277-205P14) corresponding to the long-arm breakpoint is com-
pletely sequenced (AC240968). Miropeats analysis (Parsons 1995) compares two regions on human proprotein, LETM1—leucine zipper-EF-
chromosome 12 (middle panel; blue and orange) with the gorilla sequence. (Repeat elements, LINEs, hand containing transmembrane, and
SINEs, LTRs, SDs, and genes are annotated based on the human genome.) (Bottom left panel) ClustalW FGFR3—fibroblast growth factor receptor
alignment pinpoints an AluSx element (purple) at the precise breakpoint. (B) Sequence characterization of 3 isoform 2). Most of these expansions
a duplicative transposition region in gorilla. (Top panel) A region on chromosome 5p13 near the retinoic
acid-induced 14 gene (RAI14) has acquired at least four SDs in the gorilla lineage. (Middle panel) Se- map to the termini of gorilla chromo-
quencing of gorilla BAC clone CH277-50D8 (AC239444.4) and Miropeats analysis show SDs ranging in somes indicating that the subtelomeric
size from 10 to 28 kb along chromosome 5. Read-depth analyses (WSSD tracks) predict that these gorilla regions of gorilla have become increas-
duplicative transpositions are focused on a more ancient duplication carrying the spinal muscular atrophy ingly complex as a result of duplicative
type 4 gene (PSMA4), with flanking duplications becoming increasingly lineage-specific. (Lower panel)
FISH analysis with the clone as a probe shows multiple signals. Unique probes proximal and distal to the transpositions (see above). In this regard,
region indicate no change in the order between human and gorilla. Duplicative transpositions have oc- the most expanded gorilla-specific dupli-
curred without a large-scale chromosomal rearrangement. (C ) Duplicative transposition at translocation cation (n = 45 copies) maps within 10 kb
fusion point. (Top panel) Gorilla chromosome XVII arose as a lineage-specific fusion between chromosome of the evolutionary fusion point that led
5 and 17 (Stankiewicz et al. 2001). Cloning and sequencing of the breakpoint (CH277-159N16,
AC240953) show the presence of a >85 kb complex gorilla-specific duplication block at the breakpoint.
to the formation of human chromosome
The duplication block is a mosaic composed of at least five distinct SDs originating primarily from chro- 2 (chr2: 114145970-114215607). We also
mosome 5 (see Supplemental Note for more detail). compared the copy number of shared

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Figure 2. Endogenous retroviral integration map. A comparison of chimpanzee (n = 275, blue) and gorilla (n = 265, red) PTERV1 sites of integration
based on mapping to the human genome. None of the map positions in the two genomes are orthologous except one (indicated in green and corre-
sponding to gorilla chr2: 143467521-143467682; chimpanzee chr2: 143467889-143468851). The endogenous retroviral element is absent in human
and orangutan genomes and appears to have expanded largely independently in the two lineages after they diverged.

duplications among humans, chimpanzees, and gorillas searching selected three large-insert BAC clones corresponding to the cap re-
for regions of hyperexpansion (>500 copies) in one lineage when gions of both gorilla and chimpanzee and subjected these to capil-
compared with the other two (Supplemental Table S9). Only in the lary-based sequence and assembly. The sequence analysis shows
gorilla and chimpanzee genomes were such hyperexpanded SDs dramatic differences in the organization of heterochromatic caps
identified with expansions of 1000–1500 copies mapping primarily between the two species. While both possess tracts of pCht satellite
to acrocentric, pericentromeric, subtelomeric, and subterminal cap sequence ranging in size from 10 to 50 kb, chromosome 2 SDs are
regions of African ape chromosomes (Supplemental Table S9). predominant in the chimpanzee cap, whereas chromosome 10 du-
plications define the cap organization in gorilla (Fig. 4B). These
Subterminal caps duplications appear to have expanded in concert with the satellite
One of the most striking karyotypic differences between humans sequence creating a higher-order tandem array structure of several
and African apes is the presence of subterminal heterochromatic hundred copies in each species. Since subterminal heterochromatic
caps at the ends of ape chromosomes (Fig. 4A). Evident by G blocks have also been reported among the lesser apes (Wijayanto
banding (Yunis and Prakash 1982) and post-denaturation DAPI et al. 2005), we tested pCht satellite probes on gibbon metaphase
staining, these regions have been classified as subterminal hetero- chromosomes and observed no hybridization signal above back-
chromatin found exclusively among gorillas (80/96 chromosomes), ground (data not shown). Combined, these data strongly suggest
the common chimpanzee (Pan troglodytes) (42/96 chromosomes), that the heterochromatic caps have evolved independently in both
and bonobo (Pan paniscus) (42/96 chromosomes). They are thought chimpanzee and gorilla and possibly all ape species.
to be composed primarily of a 32-bp satellite repeat sequence
(pCht7/13 sequence) arrayed in tandem (Royle et al. 1994). In
addition, it is known that the formation of the subterminal cap
Discussion
in chimpanzee was accompanied by the hyperexpansion of Structural variation has been extensive and episodic during human-
SDs, which map near the human chromosome 2 fusion point great ape evolution. In this study, we identified and validated >7665
(113997859-114024033, NCBI35) (Fan et al. 2002; Cheng et al. (Table 4) structural variant events in the gorilla when compared
2005). In gorilla, we find no evidence of an association of chromo- with human. It is important to note that our analysis is based
some 2 sequences with the heterochromatic caps, but rather our primarily on a single gorilla genome (Kwan). Consistent with other
copy number and FISH analysis suggests that a segment of chro- studies, we expect 10%–30% of these variants to be polymorphic
mosome 10 (19557646-19564636, NCBI35) is one of the primary (i.e., not fixed within the gorilla lineage) (Chen and Li 2001;
components of the gorilla cap (Fig. 4A). To confirm these results, we Ebersberger et al. 2002; Marques-Bonet et al. 2009a). Nevertheless,

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tated genetic isolation of emerging species during evolution (White

1978). We caution, however, that we cannot accurately estimate the
time of such SDs with respect to hominid speciation events, so such
correlations remain speculative. Thus, a reasonable line of inquiry
going forward will be to compare the extent of genetic diversity in an
unbiased fashion within each great ape lineage and compare these
with divergence estimates between species.
In this study, we document hundreds of duplicative trans-
position differences between human and gorilla that alter the
structure of duplication blocks between the two lineages. Most of
these structural differences are opaque to standard whole-genome
shotgun sequence assembly methods or would be incorrectly clas-
sified without integration into a higher-level cytogenetic framework.
Our structural variation analysis also suggests that the genomes of
chimpanzee and gorilla have experienced several independent ge-
nomic rearrangements that did not occur during the evolution of the
orangutan and human. The African ape genomes have been bom-
barded by retroviral integrations that entered the germline after the
two lineages diverged. Neither orangutan nor human genomes carry
these retroelements (Yohn et al. 2005) and the fact that fine mapping
of the integration sites are largely nonorthologous argues for ancient
parallel infections (Kaiser et al. 2007). Gorilla and chimpanzee have
independently acquired subtelomeric heterochromatin caps, and
this chromosome feature has been associated with the hyperex-
pansion of different SDs in the two lineages. Our molecular analyses
suggest that these events occurred independently and in parallel
Figure 3. Segmental duplication distributions. (A) SDs (>20 kb) were
early during the evolution of the Pan and Gorilla lineages, adding
classified as lineage-specific or shared based on a three-way comparison of
human, chimpanzee, and gorilla genomes. The inclusion of gorilla sug- many new megabase pairs of DNA that altered the chromosome and
gests that most SDs are shared among humans and African apes but not chromatin architecture of these two species compared with all other
with Asian apes (Supplemental Note; Marques-Bonet et al. 2009a). primates. We propose that the orangutan and human genomes
Numbers are in megabase pairs; all SDs were validated by interspecies
represent the hominid archetype, while the African ape genomes are
arrayCGH; (*) megabase pairs adjusted for copy number. (B) Using par-
simony, we assigned the number of megabase pairs to different terminal more structurally derived with respect to these properties.
and ancestral branches in the human-ape phylogeny. The copy-number-
corrected megabase pairs are shown (bold type) and a calculated rate of
megabase pairs/million years (in brackets) is estimated. A simple maxi- Methods
mum-likelihood ratio test showed a dramatic SD burst in the African ape
ancestor and in the common ancestor of humans and chimpanzees. The FISH
gorilla lineage-specific rate is greater than any other hominid. BAC and human fosmids clones (n = 1022) were used as probes to
develop a comparative cytogenetic framework and to test rearrange-
ments specific to the gorilla lineage. Ancestral state was determined
we find that the gorilla branch shows a significant increase in based on comparison with other primate species. Metaphases from
the rate of SD when compared with human or chimpanzee ( p < nonhuman primates were obtained from lymphoblastoid or fibro-
5.6 3 10 7), being more similar to the rate predicted in the human- blast cell lines of the following species: common chimpanzee (Pan
African ape ancestor. Notably, our estimated Homo-Pan ancestral troglodytes, PTR); gorilla (Gorilla gorilla, GGO), and Borneo orangutan
rate of duplication appears higher than rates estimated for the (Pongo pygmaeus pygmaeus, PPY) as representative of great apes; and
chimpanzee and human terminal branches. In general, our data rhesus monkey (Macaca mulatta, MMU, Cercopithecinae) as repre-
support a model where SD activity slowed after hominid speciation sentative of Old World Monkeys. FISH experiments were essentially
events in all lineages, with this deceleration being the least evident performed as previously described (Ventura et al. 2003).
for gorilla. This slowdown is supported by the observation that the
sequence identity spectrum of SDs in humans peaks at 99.2% (Bailey Gorilla genome sequencing
and Eichler 2006) and the finding of few large-scale SD differences Peripheral blood DNA was isolated from a male silverback gorilla,
between human and Neandertal, which separated <1 million years Kwan (Studbook #1107, b. 02/03/1989), housed at the Lincoln
ago (Green et al. 2010). The basis for deceleration is unknown, but it Park Zoo. Paired-end whole-genome sequence data were generated
is possible that extensive differences in the SD architecture facili- on an Illumina Genome Analyzer II using a modified protocol (see

Figure 4. Subterminal heterochromatic cap architecture in chimpanzee and gorilla. (A) Different FISH hybridization patterns using human fosmid
probes (ABC8_40868200_C16 and ABC8_40925900_F12) corresponding to one hyperexpanded SD in gorilla (chr10: 19530349-19564732) and one in
chimpanzee (chr2: 113978394-114020431). Extracted metaphase chromosomes (top panel) and cohybridization experiments (lower panel) in chimpanzee
and gorilla reveal differences in the composition of the heterochromatic cap in each species. (B) Complete sequence analysis of three large-insert BAC clones
sampled from gorilla and chimpanzee genomes confirm large-scale differences in the sequence organization. pCht satellite sequence (purple) interdigitates
between different SDs (color bars) depending on the species. These SDs have expanded in copy from 500 to 1000 copies (y-axis represents copy number
count based on read-depth) in chimpanzee (blue) and gorilla (red). This architecture has emerged in a species-specific fashion in conjunction with the
evolution of the subterminal heterochromatic satellite.

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Figure 4. (Legend on previous page)

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Ventura et al.

Table 4. Summary of gorilla genome structural variation tested loci not embedded within other repetitive elements or SDs to
facilitate reliable primer design.
SV type # Intervals # Base pairs

Duplications Data access

All 1,258 87,103,094
GGO specific 88 6,813,344 Gorilla sequence data (western lowland) have been deposited into
Deletions the SRA under accession SRP002878.
$50 kb 44 6,298,067 We designed two oligonucleotide microarrays GEO acces-
<50 kb 1,820 6,290,005 sion number: GSE27072; samples: GSM665036, GSM665334,
Mobile elementsa GSM665336, GSM665992, GSM665993, GSM667894, and
Alu 4,274 1,325,597
GSM668114; and platforms GGO 2.1 custom: GPL11674 and Human
L1 299 872,642
LTR 123 125,879 2.1 standard: GPL9684.
SVA 325 450,450 A subset of clones was selected for complete insert sequencing:
PTERV1 263 2,033,779 AC243004.1, AC242656.3, AC242655.3, AC242627.3, AC242595.2,
Large chromosomal rearrangements AC243002.1, AC241241.3, AC241522.2, AC240968.2, AC240954.2,
Fusion 1 NA AC239638.4, AC243003.2, AC243178.1, AC240953.2, AC239379.3,
Inversions 9b NA
AC239356.2, AC239357.3, AC239280.2, AC239360.3, AC239282.3,
Translocation 1 NA
Duplicative transpositions 418c NA AC239362.3, AC239380.3, AC239639.1, AC239363.3, AC239796.3,
AC239381.3, AC239444.4, AC239382.3, AC239359.3, AC239358.3,
Alu calls include FRAM element (2); and LTR includes THE1 (11). The AC239361.3 AC239281.3, AC239393.3, AC239640.2.
number of base pairs is estimated with the length of the consensus se-
quence of the predicted retrotransposon subclass.
a
b
Only full-length sequence considered. Acknowledgments
Five of nine detected by BEM.
c
Forty-seven of 424 validated by FISH, with 20/47 validated by sequence We thank Peter Sudmant and Tonia Brown for valuable comments in
(Supplemental Table S3). the preparation of this manuscript. We are grateful to Lisa Faust,
Mollie Herget, and the staff of the Lincoln Park Zoo for the gorilla
material used in this study. We are also indebted to the large genome
Supplemental Note). Sequence data have been deposited into the sequencing centers for early access to BAC end sequence data for
SRA under accession SRP002878. targeted analysis of structural rearrangements. This work was sup-
ported, in part, by an NIH grant HG002385 to E.E.E. and NIH grant
BAC sequencing U54 HG003079 to R.K.W. Financial support was provided by the
End sequences from a gorilla BAC library (CH277) were retrieved Spanish Ministry of Science and Innovation (Grant BFU2009-13409-
from the NCBI Trace Archive and mapped to the human reference C02-02) and the Spanish National Institute for Bioinformatics (INB,
genome (NCBI35) to identify and clone rearrangement break- www.inab.org) to A.N. T.M-B. is supported by a Ramon y Cajal pro-
points as described previously (Newman et al. 2005). A subset of gram (MICINN-RYC) and the European FP7 (MC/StGr 2010). Re-
clones was selected for complete insert sequencing using capillary sources for exploring the gorilla genome structural variation online
sequencing methods (McPherson et al. 2001) in order to obtain are available at University of Washington Genome Sciences (http://
high quality finished sequence within duplicated regions. Rear- gorillaparalogy.gs.washington.edu). E.E.E. is an investigator of the
rangements were visualized using Miropeats (Parsons 1995) and Howard Hughes Medical Institute.
previously described in-house visualization tools (Kidd et al.
2010).
References
Structural variation discovery Alkan C, Kidd JM, Marques-Bonet T, Aksay G, Antonacci F, Hormozdiari F,
Gorilla sequence reads were aligned to the human reference ge- Kitzman JO, Baker C, Malig M, Mutlu O, et al. 2009. Personalized copy
number and segmental duplication maps using next-generation
nome using the mrFAST and mrsFAST mapping algorithms (Alkan sequencing. Nat Genet 41: 1061–1067.
et al. 2009; Hach et al. 2010). Deletions and mobile element in- Bailey JA, Eichler EE. 2006. Primate segmental duplications: Crucibles of
sertions were detected using VariationHunter (Hormozdiari et al. evolution, diversity, and disease. Natl Rev 7: 552–564.
2009; Hormozdiari et al. 2010), while SDs (>20 kb) were detected Bailey JA, Gu Z, Clark RA, Reinert K, Samonte RV, Schwartz S, Adams MD,
Myers EW, Li PW, Eichler EE. 2002. Recent segmental duplications in the
and copy number quantified using measures of read-depth (see human genome. Science 297: 1003–1007.
Supplemental Note; Alkan et al. 2009). Carbone L, Ventura M, Tempesta S, Rocchi M, Archidiacono N. 2002.
Evolutionary history of chromosome 10 in primates. Chromosoma 111:
267–272.
ArrayCGH Cardone MF, Alonso A, Pazienza M, Ventura M, Montemurro G, Carbone L,
We designed two oligonucleotide microarrays (n = 385,000) targeted de Jong PJ, Stanyon R, D’Addabbo P, Archidiacono N, et al. 2006.
Independent centromere formation in a capricious, gene-free domain of
to regions of gorilla deletions and duplications and performed cross- chromosome 13q21 in Old World monkeys and pigs. Genome Biol 7:
species arrayCGH as previously described (GEO accession numbers: R91. doi: 10.1186/gb-2006-7-10-r91.
GSE27072; samples: GSM665036, GSM665334, GSM665336, Cardone MF, Lomiento M, Teti MG, Misceo D, Roberto R, Capozzi O,
GSM665992, GSM665993, GSM667894, and GSM668114; and D’Addabbo P, Ventura M, Rocchi M, Archidiacono N. 2007.
Evolutionary history of chromosome 11 featuring four distinct
platforms GGO 2.1 custom: GPL11674 and Human 2.1 standard: centromere repositioning events in Catarrhini. Genomics 90: 35–43.
GPL9684). Chen FC, Li WH. 2001. Genomic divergences between humans and other
hominoids and the effective population size of the common ancestor of
humans and chimpanzees. Am J Hum Genet 68: 444–456.
PCR Cheng Z, Ventura M, She X, Khaitovich P, Graves T, Osoegawa K, Church D,
DeJong P, Wilson RK, Paabo S, et al. 2005. A genome-wide comparison
Thirty PCR assays were designed to test the specificity and poly- of recent chimpanzee and human segmental duplications. Nature 437:
morphism of predicted Alu insertions in the gorilla genome. We only 88–93.

1648 Genome Research

www.genome.org
 Structural variation in great apes

The Chimpanzee Sequencing and Analysis Consortium. 2005. Initial segmental duplications in the genome of the African great ape ancestor.
sequence of the chimpanzee genome and comparison with the human Nature 457: 877–881.
genome. Nature 437: 69–87. Marques-Bonet T, Ryder OA, Eichler EE. 2009b. Sequencing primate genomes:
Dutrillaux B. 1980. Chromosomal evolution of the great apes and man. What have we learned? Annu Rev Genomics Hum Genet 10: 355–386.
J Reprod Fertil Suppl Suppl 28: 105–111. McPherson JD, Marra M, Hillier L, Waterston RH, Chinwalla A, Wallis J,
Ebersberger I, Metzler D, Schwarz C, Paabo S. 2002. Genomewide Sekhon M, Wylie K, Mardis ER, Wilson RK, et al. 2001. A physical map of
comparison of DNA sequences between humans and chimpanzees. Am the human genome. Nature 409: 934–941.
J Hum Genet 70: 1490–1497. Miller DA, Firschein IL, Dev VG, Tantravahi R, Miller OJ. 1974. The gorilla
Eder V, Ventura M, Ianigro M, Teti M, Rocchi M, Archidiacono N. 2003. karyotype: Chromosome lengths and polymorphisms. Cytogenet Cell
Chromosome 6 phylogeny in primates and centromere repositioning. Genet 13: 536–550.
Mol Biol Evol 20: 1506–1512. Misceo D, Ventura M, Eder V, Rocchi M, Archidiacono N. 2003. Human
Egozcue J, Chiarelli B. 1967. The idiogram of the lowland gorilla (Gorilla chromosome 16 conservation in primates. Chromosome Res 11: 323–
gorilla gorilla). Folia Primatol (Basel) 5: 237–240. 326.
Eichler EE, DeJong PJ. 2002. Biomedical applications and studies of Misceo D, Cardone MF, Carbone L, D’Addabbo P, de Jong PJ, Rocchi M,
molecular evolution: A proposal for a primate genomic library resource. Archidiacono N. 2005. Evolutionary history of chromosome 20. Mol Biol
Genome Res 12: 673–678. Evol 22: 360–366.
Enard W, Paabo S. 2004. Comparative primate genomics. Annu Rev Genomics Montefalcone G, Tempesta S, Rocchi M, Archidiacono N. 1999. Centromere
Hum Genet 5: 351–378. repositioning. Genome Res 9: 1184–1188.
Fan Y, Linardopoulou E, Friedman C, Williams E, Trask BJ. 2002. Genomic Muller S, Stanyon R, Finelli P, Archidiacono N, Wienberg J. 2000. Molecular
structure and evolution of the ancestral chromosome fusion site in cytogenetic dissection of human chromosomes 3 and 21 evolution. Proc
2q13-2q14.1 and paralogous regions on other human chromosomes. Natl Acad Sci 97: 206–211.
Genome Res 12: 1651–1662. Muller S, Hollatz M, Wienberg J. 2003. Chromosomal phylogeny and
Fortna A, Kim Y, MacLaren E, Marshall K, Hahn G, Meltesen L, Brenton M, evolution of gibbons (Hylobatidae). Hum Genet 113: 493–501.
Hink R, Burgers S, Hernandez-Boussard T, et al. 2004. Lineage-specific Newman TL, Tuzun E, Morrison VA, Hayden KE, Ventura M, McGrath SD,
gene duplication and loss in human and great ape evolution. PLoS Biol 2: Rocchi M, Eichler EE. 2005. A genome-wide survey of structural
E207. doi: 10.1371/journal.pbio.0020207. variation between human and chimpanzee. Genome Res 15: 1344–1356.
Gibbs RA, Rogers J, Katze MG, Bumgarner R, Weinstock GM, Mardis ER, Parsons JD. 1995. Miropeats: Graphical DNA sequence comparisons.
Remington KA, Strausberg RL, Venter JC, Wilson RK, et al. 2007. Comput Appl Biosci 11: 615–619.
Evolutionary and biomedical insights from the rhesus macaque Royle NJ, Baird DM, Jeffreys AJ. 1994. A subterminal satellite located
genome. Science 316: 222–234. adjacent to telomeres in chimpanzees is absent from the human
Goodman M, Koop BF, Czelusniak J, Fitch DH, Tagle DA, Slightom JL. 1989. genome. Nat Genet 6: 52–56.
Molecular phylogeny of the family of apes and humans. Genome 31: Sarich VM, Wilson AC. 1973. Generation time and genomic evolution in
316–335. primates. Science 179: 1144–1147.
Green RE, Krause J, Briggs AW, Maricic T, Stenzel U, Kircher M, Patterson N, Stankiewicz P, Park SS, Inoue K, Lupski JR. 2001. The evolutionary chromosome
Li H, Zhai W, Fritz MH, et al. 2010. A draft sequence of the Neandertal translocation 4;19 in Gorilla gorilla is associated with microduplication of
genome. Science 328: 710–722. the chromosome fragment syntenic to sequences surrounding the human
Hach F, Hormozdiari F, Alkan C, Birol I, Eichler EE, Sahinalp SC. 2010. proximal CMT1A-REP. Genome Res 11: 1205–1210.
mrsFAST: A cache-oblivious algorithm for short-read mapping. Nat Stanyon R, Rocchi M, Capozzi O, Roberto R, Misceo D, Ventura M, Cardone
Methods 7: 576–577. MF, Bigoni F, Archidiacono N. 2008. Primate chromosome evolution:
Hormozdiari F, Alkan C, Eichler EE, Sahinalp SC. 2009. Combinatorial Ancestral karyotypes, marker order, and neocentromeres. Chromosome
algorithms for structural variation detection in high-throughput Res 16: 17–39.
sequenced genomes. Genome Res 19: 1270–1278. Tuzun E, Sharp AJ, Bailey JA, Kaul R, Morrison VA, Pertz LM, Haugen E,
Hormozdiari F, Hajirasouliha I, Dao P, Hach F, Yorukoglu D, Alkan C, Eichler Hayden H, Albertson D, Pinkel D, et al. 2005. Fine-scale structural
EE, Sahinalp SC. 2010. Next-generation VariationHunter: variation of the human genome. Nat Genet 37: 727–732.
Combinatorial algorithms for transposon insertion discovery. Ventura M, Mudge JM, Palumbo V, Burn S, Blennow E, Pierluigi M, Giorda R,
Bioinformatics 26: i350–1357. doi: 10.1093/bioinformatics/btq216. Zuffardi O, Archidiacono N, Jackson MS, et al. 2003. Neocentromeres in
The International Human Genome Sequencing Consortium. 2001. Initial 15q24-26 map to duplicons which flanked an ancestral centromere in
sequencing and analysis of the human genome. Nature 409: 860–921. 15q25. Genome Res 13: 2059–2068.
The International Human Genome Sequencing Consortium. 2004. Finishing Ventura M, Weigl S, Carbone L, Cardone MF, Misceo D, Teti M, D’Addabbo P,
the euchromatic sequence of the human genome. Nature 431: 931–945. Wandall A, Bjorck E, de Jong PJ, et al. 2004. Recurrent sites for new
Kaiser SM, Malik HS, Emerman M. 2007. Restriction of an extinct retrovirus centromere seeding. Genome Res 14: 1696–1703.
by the human TRIM5alpha antiviral protein. Science 316: 1756–1758. White MJD . 1978. Modes of speciation. W.H. Freeman, New York.
Kidd JM, Sampas N, Antonacci F, Graves T, Fulton R, Hayden HS, Alkan C, Wijayanto H, Hirai Y, Kamanaka Y, Katho A, Sajuthi D, Hirai H. 2005.
Malig M, Ventura M, Giannuzzi G, et al. 2010. Characterization of Patterns of C-heterochromatin and telomeric DNA in two representative
missing human genome sequences and copy-number polymorphic groups of small apes, the genera Hylobates and Symphalangus.
insertions. Nat Methods 7: 365–371. Chromosome Res 13: 717–724.
Koop BF, Goodman M, Xu P, Chan K, Slightom JL. 1986. Primate eta-globin Xing J, Zhang Y, Han K, Salem AH, Sen SK, Huff CD, Zhou Q , Kirkness EF,
DNA sequences and man’s place among the great apes. Nature 319: 234– Levy S, Batzer MA, et al. 2009. Mobile elements create structural
238. variation: Analysis of a complete human genome. Genome Res 19: 1516–
Kryukov GV, Castellano S, Novoselov SV, Lobanov AV, Zehtab O, Guigo R, 1526.
Gladyshev VN. 2003. Characterization of mammalian selenoproteomes. Yohn CT, Jiang Z, McGrath SD, Hayden KE, Khaitovich P, Johnson ME,
Science 30: 1439–1443. Eichler MY, McPherson JD, Zhao S, Paabo S, et al. 2005. Lineage-specific
Locke DP, Archidiacono N, Misceo D, Cardone MF, Deschamps S, Roe B, expansions of retroviral insertions within the genomes of African great
Rocchi M, Eichler EE. 2003. Refinement of a chimpanzee pericentric apes but not humans and orangutans. PLoS Biol 3: e110. doi: 10.1371/
inversion breakpoint to a segmental duplication cluster. Genome Biol 4: journal.pbio.0030110.
R50. doi: 10.1186/gb-2003-4-8-r50. Yunis JJ, Prakash O. 1982. The origin of man: A chromosomal pictorial
Locke DP, Hillier LW, Warren WC, Worley KC, Nazareth LV, Muzny DM, legacy. Science 215: 1525–1530.
Yang SP, Wang Z, Chinwalla AT, Minx P, et al. 2011. Comparative and
demographic analysis of orangutan genomes. Nature 469: 529–533.
Marques-Bonet T, Kidd JM, Ventura M, Graves TA, Cheng Z, Hillier LW, Jiang
Z, Baker C, Malfavon-Borja R, Fulton LA, et al. 2009a. A burst of Received April 7, 2011; accepted in revised form June 13, 2011.

Genome Research 1649

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