Heredity (2012) 108, 37–41
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ORIGINAL ARTICLE
Intrachromosomal rearrangements in avian genome
evolution: evidence for regions prone to breakpoints
BM Skinner1 and DK Griffin
It is generally believed that the organization of avian genomes remains highly conserved in evolution as chromosome number
is constant and comparative chromosome painting demonstrated there to be very few interchromosomal rearrangements.
The recent sequencing of the zebra finch (Taeniopygia guttata) genome allowed an assessment of the number of
intrachromosomal rearrangements between it and the chicken (Gallus gallus) genome, revealing a surprisingly high number of
intrachromosomal rearrangements. With the publication of the turkey (Meleagris gallopavo) genome it has become possible to
describe intrachromosomal rearrangements between these three important avian species, gain insight into the direction of
evolutionary change and assess whether breakpoint regions are reused in birds. To this end, we aligned entire chromosomes
between chicken, turkey and zebra finch, identifying syntenic blocks of at least 250 kb. Potential optimal pathways of
rearrangements between each of the three genomes were determined, as was a potential Galliform ancestral organization. From
this, our data suggest that around one-third of chromosomal breakpoint regions may recur during avian evolution, with 10% of
breakpoints apparently recurring in different lineages. This agrees with our previous hypothesis that mechanisms of genome
evolution are driven by hotspots of non-allelic homologous recombination.
Heredity (2012) 108, 37–41; doi:10.1038/hdy.2011.99; published online 2 November 2011
Keywords: chromosome; evolution; birds; avian; breakpoint
INTRODUCTION
It has been long established that chromosome rearrangements between
species can cause or reinforce reproductive isolation (Noor et al., 2001;
Rieseberg, 2001; Delneri et al., 2003). Hybrids arising from parents
with subtly different karyotypes can be compromised in their ability to
reproduce and this can function as an evolutionary barrier that
ultimately leads to speciation. Despite this, molecular evolutionary
research often focuses on the role of DNA, RNA and proteins (Kimura
and Ohta, 1974), often disregarding whole chromosomes and homologous synteny blocks. It is yet crucial to describe accurately the
comparative molecular cytogenetics between key species as a forerunner for understanding the role of chromosome changes in the
evolution of particular phylogenetic groups. Most studies have performed this through the use of zoo-FISH with chromosome paints
and/or individual bacterial artificial chromosome (BAC) clones, generally focusing on mammalian genomes (for example, Wienberg,
2004). The precise locations of evolutionary breakpoint regions are
possible to determine by zoo-FISH with BACs; however, such studies
are extensive and laborious. The increasing opportunity to be able to
visualize assembled genomes through modern sequence analysis tools
and freely available online data sets make it possible to pinpoint
homologous synteny blocks and evolutionary breakpoint regions
precisely, quickly and cheaply (Larkin et al., 2009). Again however,
such studies have focused on mammals, and birds are relatively
understudied in this regard.
The defining characteristics of birds include feathers, having (or
having lost) the ability to fly, oviparity, nesting/brooding, high body
temperature, longevity, high blood glucose levels and a small genome
(one-third the size of mammals). Avian karyotypes are also characteristic with nearly two-thirds of birds having a diploid chromosome
number of around 80 and the vast majority having a large number of
microchromosomes (Christidis, 1990). Between 2004 and 2010 the
chicken genome was the only avian representative being completely
characterized and karyotyped (Hillier et al., 2004; Masabanda et al.,
2004), but this nevertheless has allowed cross-species chromosome
painting to numerous other birds (reviewed in Griffin et al., 2007) and
BAC mapping to build physical maps of a few others. These include
turkey, duck and zebra finch (Griffin et al., 2008; Skinner et al., 2009a;
Volker et al., 2010). From these studies, a number of key messages
emerge: first, chicken chromosomes 1–3 and 5–10+Z are representative of the ancestral pattern. Second, homoplasy is commonplace with
the ancestral chromosomes 4 and 10 fusing on at least three occasions
(in chicken, goose and African collared dove), fusions of the same
smaller macrochromosomes and fissions at the centromeres of chromosomes 1 and 2 occurring convergently in several separate lineages
(Griffin et al., 2007). Third, whole-chromosome blocks represent
ancient conservation of synteny, with chromosomes 1–5+Z being
present and intact in turtles (Matsuda et al., 2005) and chromosome
4 similarly intact in humans (Chowdhary and Raudsepp, 2000; Hillier
et al., 2004). Finally, despite interchromosomal conservation, intrachromosomal changes are common; for example, 114 tentative
rearrangements, both inversions and translocations were noted
between chicken and zebra finch (Volker et al., 2010). The overall
picture of avian genome organization is of a successful and conserved
School of Biosciences, University of Kent, Canterbury, UK
1Current address: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
Correspondence: Professor DK Griffin, School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK.
E-mail: d.k.griffin@kent.ac.uk
Received 1 June 2011; revised 8 September 2011; accepted 12 September 2011; published online 2 November 2011
Breakpoint reuse in avian evolution
BM Skinner and DK Griffin
38
karyotypic pattern, with changes, when they occur, tending to recur
much more than in mammals. Rare, nonrandom patterns in any
evolutionary system, we would assume, usually occur for a reason and
therefore we contend that birds are an appropriate group of animals
on which to study the mechanistic basis of chromosome evolution in
contrast to mammals, in which changes are more commonplace and
apparently more random.
Chromosome fission at centromeric loci has previously been
reported as being a factor of evolutionary change (for example,
Perry et al., 2004). However, other regions of the genome can be
subject to frequent breakage (that is, hotspots). This is commonly
termed ‘breakpoint reuse.’ Where breakpoints are determined to be
between the same base pairs, identity by descent is inferred. Where
breakpoints are not identical but within the same genomic region
then breakpoint reuse is implicated (Mlynarski et al., 2010). Determination of the precise evolutionary breakpoint regions is therefore a
useful tool in distinguishing homoplasy and hemiplasy (Avise and
Robinson, 2008) where apparently convergent changes are in fact
identical by descent.
Recently we (Volker et al., 2010; Warren et al., 2010) generated
comparative maps by aligning whole chromosomal sequences between
chicken (Gallus gallus) and zebra finch (Taeniopygia guttata), and
subsequently verifying rearrangements by fluorescence in situ hybridization (FISH). This showed an apparent combination of multiple
inversions and translocations within chromosomes. Understanding
what rearrangements have occurred to produce the current patterns of
chromosomal synteny requires more species alignments. With the
publication of the turkey (Meleagris gallopavo) genome (Dalloul et al.,
2010), we can identify some lineage-specific rearrangements and
provide evidence for regions of recurrent breakpoints. In this study
therefore we have used whole-chromosomal sequence alignments
among turkey, chicken and zebra finch, combined with our previously
published physical maps (Griffin et al., 2008; Volker et al. 2010)
generated comparative genomic maps among the three avian species.
Using zebra finch as an outgroup, we also tested the hypothesis that
there are genomic regions within which breakpoints recur during
avian evolution.
MATERIALS AND METHODS
To visualize large-scale intrachromosomal rearrangements, we aligned wholechromosome sequences of chicken macrochromosomes 1–10 and their turkey
and zebra finch orthologs using the program GenAlyzer (Choudhuri et al.,
2004) with default settings. The Z-chromosome assembly for turkey was not
complete enough to align properly. The chicken–zebra finch alignments were
already available from our previous study (Volker et al., 2010). Subsequently, to
aid visualization, the GenAlyzer output matches (of 100+ base pairs) were
combined into contiguous blocks using a custom script. This script combined
direct or inverted matches where there was a consecutive run of at least five
matches. If a distance of 40 kb occurred with no matches, a new block was
called. Blocks of at least 250 kb were plotted, to remove spurious matches
caused by repetitive content and to focus on the larger rearrangements. The
chromosomes were manually segmented based on these charts, and the
segments numbered and ordered relative to turkey.
The Multiple Genomes Rearrangement tool on the GRIMM web server
(http://grimm.ucsd.edu/MGR/) (Bourque and Pevzner, 2002) was used to
calculate optimal rearrangement pathways between each species, and to
reconstruct a likely potential chicken–turkey ancestor, in the manner of
Mlynarski et al. (2010) (see also Supplementary Table 3). The series of possible
rearrangements from the chicken–turkey ancestor to each species was considered, and for each rearrangement, the segment ends flanking the breakpoints
were noted. Within each lineage, the number of times a segment end was
involved in a rearrangement was counted. The positions of the segments within
each species genome were noted, to allow correlation with our previously
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published FISH-based physical mapping data (Griffin et al., 2008; Volker et al.,
2010). These data were acquired by cross-species FISH of chicken BACs onto
turkey chromosomes, and same-species FISH of orthologous zebra finch BACs
onto zebra finch chromosomes.
Unmasked chromosome sequences for chicken were downloaded
from Ensembl (ftp://ftp.ensembl.org/pub/release-63/fasta/gallus_gallus/dna/).
RepeatMasker (Smit et al., 1996–2010; http://www.repeatmasker.org) was run
on each chromosome using default settings. For all breakpoints and segments,
the number of repeats wholly located within the breakpoint region or segment
was counted, and expressed as the number of base pairs per megabase of
sequence for each of eight classes of repeat. We used the chicken data for this
analysis due to the higher quality of the sequence assembly compared with
other genomes.
RESULTS
Alignment of orthologous chromosome sequences
Whole-chromosome alignments of draft genome sequences confirmed
previous results demonstrating a high degree of conserved synteny in
the macrochromosomes of all three species, with only two interchromosomal rearrangements distinguishing the chicken and zebra finch
genomes (Itoh and Arnold, 2005), two distinguishing chicken and
turkey (Griffin et al., 2008) and two distinguishing zebra finch and
turkey. Alignment of whole-chromosome sequences of orthologous
chromosomes 1–10 in all three species to visualize large-scale intrachromosomal rearrangements however identified a large number of
differences. The rearrangements predicted agreed with marker order
from existing FISH data where overlapping BACs were available
(Supplementary Table 1).
To better visualize the intrachromosomal differences identified from
the GenAlyzer data, we grouped the short alignments into contiguous
blocks and color-coded as direct (blue) or inverted (red) matches
(Figure 1 and Supplementary Figures S1–S9). Side-by-side alignments
between each of the species of 4250 kb blocks were then plotted to
allow each chromosome to be divided into segments separated by
breakpoints (Figure 2, Supplementary Table 1). The segments were
manually numbered, and an optimal series of rearrangements transforming from one species order to the others was calculated using the
Multiple Genomes Rearrangement tool on the GRIMM web server
(http://grimm.ucsd.edu/MGR/; Bourque and Pevzner, 2002). This
produced potential chicken–turkey ancestral segment orders, and
the pathways from this ancestor to each modern species (see Supplementary Table 3 for details). Figure 3 shows an example pathway of
rearrangements for chromosome 9, with segments ordered from the
perspective of a possible ancestral Neoavian organization. It should be
noted that with the current data, the Neoavian state must be
considered speculative only.
Figure 1 Example alignment between chicken chromosome 9 and
orthologous turkey chromosome 11. (a) Shows the raw Genalyzer output
and (b) shows the same after grouping into synteny blocks. Only synteny
blocks X250 kb are plotted.
Breakpoint reuse in avian evolution
BM Skinner and DK Griffin
39
Evidence for regions prone to breakpoints
In a series of inversions from one genomic arrangement to another,
the breakpoint regions between segments do not remain consistent,
hence, the ends of each segment were used as a proxy for the actual
sequence within the breakpoint. The end point of each segment was
scored based on the potential sequence of rearrangements for the
number of times it was involved in a breakpoint in each lineage
(examples for chromosome 9 in Table 1, full data in Supplementary
Table 2). The distance between segments was calculated for each
species. The median sizes of the regions within which the breakpoints
occurred were: 70 kb for chicken, 102 kb for zebra finch and 125 kb for
turkey, respectively.
In chicken chromosomes 1–10, and their turkey and zebra finch
orthologs, 366 segment ends were identified, of which 318 were
involved in rearrangements. The optimal pathways from the
chicken–turkey ancestor suggested that 32 breakpoint regions
(10.1%) recurred in different lineages, whereas 114 breakpoint regions
(35.8%) recurred in either the same or different lineage.
We have hypothesized that breakpoint regions would be enriched
for repetitive elements. To test this, we used RepeatMasker to identify
classes of repetitive sequences in chromosomes 1–10, and compared
the numbers of repeats in breakpoint regions versus segments
(Table 2). There is an enrichment for all the gross classes of repeat
Table 1 Breakpoints in orthologous chromosomes GGA9, MGA11 and
TGU9. Breakpoints are numbered according to their order in turkey
Segment end;
s, start, e, end
Breakpoints between
chicken–turkey
Total
Breakpoint
Breakpoint
in 41 lineage
recurs
Yes
Yes
ancestor and
Chicken Turkey Zebra finch
Figure 2 Side-by-side alignments between chicken chromosome 9, turkey
chromosome 11 and zebra finch chromosome 9. Colored arrows show the
division of the chromosomes into segments, oriented with respect to turkey.
1s
1e
1
0
0
0
1
0
2
0
2s
2e
0
1
0
0
3
0
3
1
3s
3e
1
0
0
0
0
0
1
0
4s
4e
0
0
0
0
0
1
0
1
5s
5e
0
0
0
0
2
1
2
1
6s
6e
0
0
0
1
1
0
1
1
7s
7e
0
0
1
1
0
0
1
1
8s
0
1
0
1
Yes
Yes
Indication is given as to whether breakpoints occur in more than one lineage, and simply
whether they recur. The full list is given in Supplementary Table 2.
Figure 3 Example of a potential series of inversions from a Neoaves common ancestor leading to the organization seen in chicken chromosome 9, turkey
chromosome 11 and zebra finch chromosome 9. Inverted segments are indicated with dotted arrows. Note that in this figure, segment order is presented
with respect to the hypothetical Neoaves common ancestor. Note also that rearrangements shown in the path to zebra finch could have occurred in either the
path from the avian common ancestor to zebra finch or from the avian common ancestor to the chicken–turkey ancestor, depending on the true ancestral
organization.
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Breakpoint reuse in avian evolution
BM Skinner and DK Griffin
40
Table 2 Repeat density in breakpoint regions versus segments for
various repeat classes as measured by base pairs of repeat per
megabase of sequence
DNA transposon
Breakpoint regions
Segments
Ratio
Avg bp/Mb
Avg bp/Mb
EBR/segment
2518.71
433.75
5.81
39864.73
13605.16
13545.98
759.30
2.94
17.92
Low complexity
Pseudogene
5089.52
2600.95
4857.61
67.81
1.05
38.36
SINE
Simple repeat
2283.39
5548.28
1180.83
4511.47
1.93
1.23
864.00
510.81
1.69
LINE
LTR
Unknown
Abbreviations: Avg, average; EBR, evolutionary breakpoint region; LTR, long terminal repeat.
The difference is significant (Wilcoxon-signed rank, P¼0.01). For a more detailed breakdown of
repeat content, see Supplementary Table 4.
(see Supplementary Table 4 for detailed breakdown), and this enrichment is significant (Wilcoxon signed-rank test, W¼36, n¼8, P¼0.01).
DISCUSSION
This paper is the first to describe the comparative genomics among
three bird species and thereby provide the basis through which we
might define the chromosomal rearrangements and syntenies that
have occurred during avian evolution. Recently, in carrying out the
same with only two species, we suggested that a series of inversions
and intrachromosomal translocations were commonplace. The advantage of having more than two species however allows us to make initial
inferences about the direction of change. Contrary to the suppositions
in our previous analysis of chicken and zebra finch alone (Volker et al.,
2010), we therefore now believe that all of the observed rearrangements could be explained by a series of inversions and there is no need
to invoke intrachromosomal translocations as a mechanism. This
agrees with recent findings on the nature of complex rearrangements
in other species (Schubert and Lysak, 2011). The reason for the
conservation of interchromosomal but not intrachromosomal synteny
(and apparent paucity of translocations of any kind) is not clear. It
may however imply that there is a selective advantage to birds in
keeping certain blocks of synteny together in the interphase nuclei.
Our studies on interphase nuclei in birds (Skinner et al., 2009b) and
pigs (Quilter et al., 2002; Foster et al., 2005) provide proof of principle
about how future studies in this area might proceed.
Our data show that the median breakpoint sizes are lowest in the
chicken (70 kb) and highest in the turkey (125 kb). This may reflect
the different methods used in the sequencing of the three genomes.
The chicken and zebra finch genomes were both sequenced from a
combination of plasmid, fosmid and BAC-end read pairs, and
assembled with PCAP (Hillier et al., 2004; Warren et al., 2010). The
turkey genome was generated by Roche 454 (Roche, Branford, CI,
USA) and Illumina (San Diego, CA, USA) sequencing from 3 kb and
20 kb libraries and assembled with Celera Assembler 5.3 (Dalloul et al.,
2010). Poorer alignments between the turkey assembly and the others
could manifest as a higher apparent breakpoint size.
With our current resolution of 250 kb minimum size for a segment,
we cannot state unequivocally that exact breakpoints have been
reused; however, our data provide evidence that there are regions of
avian genomes that seem prone to breakage. Of the segment ends
participating in breakpoint regions, about one-third of them appear to
occur in more than one rearrangement. This broadly agrees with our
previous observations (Volker et al., 2010); whereas there are few
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rearrangements at the interchromosomal level, rearrangements within
chromosomes are far more frequent. Especially notable is the finding
that more than a third of the breakpoint regions found in this study
appears to have been reused in the same or in a different lineage.
Furthermore, 10% of all the breakpoint regions seem to be reused in
different lineages. We must note that the order of rearrangements is a
supposition, based on the most parsimonious way to transform the
three genomes; the actual sequence of rearrangements may have been
slightly different. However, even if a longer sequence of rearrangements had occurred than we suggest here, breakpoint region reuse
would still be implicated.
It has been suggested that the small number of interchromosomal
rearrangements is a consequence of the small numbers of interspersed
repeats, segmental duplications and pseudogenes in avian genomes,
which provide few substrates for non-allelic homologous recombination (Burt et al., 1999; Burt, 2002). Our analysis of the sequence
composition of these breakpoint regions reflects this, showing especial
enrichment for pseudogenes, long terminal repeats, DNA transposon
and LINEs. We would expect that breakpoint regions also harbor other
small inversions beyond the detection of this study. A recent study
(Braun et al., 2011) examining microinversions (inversions o50 kb)
in non-coding DNA from a range of bird species suggested that these
are more common than previously suspected, most appeared lineagespecific and that there was evidence for hotspots of microinversion.
This is consistent with our hypothesis; regions prone to breakage cause
apparent breakpoint reuse at large-scale (4250 kb) resolution while
harboring many smaller independent microinversions. We predict
that, as with repetitive content, these breakpoint regions will be
found to overlap regions of non-allelic homologous recombination,
segmental duplications and copy number variation. Our previous
finding that regions of copy number variation and chromosomal
rearrangement between chicken and zebra finch are associated with
elevated recombination rates already support this (Volker et al. 2010);
such studies extended to further avian genomes will be of interest.
We expect there to be a significant overlap between the chromosomal
segments described here and the homologous synteny blocks described
by Larkin et al. (2009). Larkin et al (2009) suggested that homologous
synteny blocks and evolutionary breakpoint regions are subject to
different evolutionary pressures. As further avian genomes are published, especially those with atypical karyotypes (for example, Falconiformes and Psittaciformes (de Oliveira et al., 2005, 2010; Nanda et al.,
2007; Nishida et al., 2008; Nie et al., 2009), we will be able to test the
hypothesis that blocks of ordered genes have been preserved through
evolution, and that these are reflected in the syntenic blocks seen here.
Further species that have been of interest in zoo-FISH studies and
would thus be likely candidates for studies in addition to the Falconiformes and Psittaciformes mentioned above include other Galliformes
(Shibusawa et al., 2004a, b) and a range of species from orders such as
Anseriformes and Passeriformes (Guttenbach et al., 2003). Comparisons with Paleognathous birds, whose chromosomes are thought to
resemble closely the ancestral avian karyotype (Shetty et al., 1999;
Nishida-Umehara et al., 2007) should provide a useful outgroup for
understanding the chromosomal evolution of the Neoaves.
This analysis of the macrochromosomes of chicken, turkey and
zebra finch has provided evidence in support of our hypothesis that
there are regions of bird genomes that are prone to breakage, and thus
facilitate chromosomal rearrangements. As more bird genomes are
sequenced, the relationship between karyotypic rearrangements,
evolutionarily conserved synteny blocks and their mechanisms will
become clearer. It is becoming clearer that much of the structural
variation in bird genomes is only visible with high-resolution (that is,
Breakpoint reuse in avian evolution
BM Skinner and DK Griffin
41
sequence level) comparisons. Thus, it remains to be seen whether the
36% breakpoint reuse and 10% cross-lineage breakpoint reuse values
found here are comparable across other bird orders.
DATA ARCHIVING
Synteny block data and RepeatMasker data have been deposited in the
Dryad repository: doi:10.5061/dryad.8h7k64hh.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Supplementary Information accompanies the paper on Heredity website (http://www.nature.com/hdy)
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