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The tiger genome and comparative analysis with lion and snow leopard genomes

Yun Sung Cho1, Li Hu2, Haolong Hou2, Hang Lee3, Jiaohui Xu2, Soowhan
Kwon4, Sukhun Oh4, Hak-Min Kim1, Sungwoong Jho1, Sangsoo Kim5, Young-Ah
Shin1, Byung Chul Kim1, 6, Hyunmin Kim6, Chang-uk Kim1, Shu-Jin Luo7,
Warren E. Johnson8, Klaus-Peter Koepfli9, Anne Schmidt-K=C3=BCntzel10, Jason
A. Turner11, Laurie Marker12, Cindy Harper13, Susan M. Miller13, 14,
Wilhelm Jacobs15, Laura D. Bertola16, Tae Hyung Kim6, Sunghoon Lee1, 6,
Qian Zhou2, Hyun-Ju Jung6, Xiao Xu7, Priyvrat Gadhvi1, Pengwei Xu2,
Yingqi Xiong2, Yadan Luo2, Shengkai Pan2, Caiyun Gou2, Xiuhui Chu2,
Jilin Zhang2, Sanyang Liu2, Jing He2, Ying Chen2, Linfeng Yang2, Yulan
Yang2, Jiaju He2, Sha Liu2, Junyi Wang2, Chul Hong Kim6, Hwanjong Kwak6,
Jong-Soo Kim1, Seungwoo Hwang17, Junsu Ko6, Chang-Bae Kim18, Sangtae
Kim19, Damdin Bayarlkhagva20, Woon Kee Paek21, Seong-Jin Kim6, 22,
Stephen J. O=E2=80=99Brien9, 23, Jun Wang2, 24, 25 & Jong Bhak1, 6, 26, 27

Nature Communications 4, Article number: 2433 (2013)
Download Citation
Comparative genomicsEvolutionary biology

02 May 2013
13 August 2013
Published online:
17 September 2013


Tigers and their close relatives (Panthera) are some of the world=E2=80=99s=
endangered species. Here we report the de novo assembly of an Amur tiger
whole-genome sequence as well as the genomic sequences of a white Bengal
tiger, African lion, white African lion and snow leopard. Through
comparative genetic analyses of these genomes, we find genetic
signatures that may reflect molecular adaptations consistent with the
big cats=E2=80=99 hypercarnivorous diet and muscle strength. We report a sn=
leopard-specific genetic determinant in EGLN1 (Met39>Lys39), which is
likely to be associated with adaptation to high altitude. We also detect
a TYR260G>A mutation likely responsible for the white lion coat colour.
Tiger and cat genomes show similar repeat composition and an appreciably
conserved synteny. Genomic data from the five big cats provide an
invaluable resource for resolving easily identifiable phenotypes evident
in very close, but distinct, species.

The tiger (Panthera tigris), the largest felid species on Earth and a
widely recognized symbol for wildlife conservation1, is one of the
world=E2=80=99s most endangered species. Tigers are a keystone species and
natural indicators of the health of the ecological communities in which
they are found2. The current estimates of wild tigers range from just
3,050 to 3,950 individuals. It is postulated that without conservation
measures tigers will soon become extinct in the wild, thus turning the
preservation of existing wild tiger populations into a major goal of
conservation efforts3,4. Tigers comprise of nine genetically validated
subspecies1,5,6. Four of these went extinct in the wild during the last
century (Javan, Balinese, South China and Caspian tigers), leaving five
extant subspecies (Amur, Bengal, Indochinese, Malayan and Sumatran
tigers)5. The Amur tiger (Panthera tigris altaica) is the largest in
overall size and the only subspecies inhabiting snow-covered regions.

Previous genetic studies using mitochondrial and nuclear loci have
helped to elucidate the phylogeography and population genetics of
tigers1,5,6,7, and the low coverage genome (1.8 =C3=97 ) of the domestic cat
(Felis catus) has provided insights into felid evolution8,9. However, no
whole-genome reference sequence has been reported for the tiger, or any
of the Panthera species, thus limiting current understanding of genetic
diversity and demography10,11,12.

We report the first tiger genome sequence assembly and annotation as
well as a comparative analysis of the lion (Panthera leo) and snow
leopard (Panthera uncia) genomes. We describe genotypic variation and
genotype association analyses with species-specific phenotypes and
adaptation. Panthera whole-genome sequences provide valuable information
on genome organization, evolutionary divergence and overall endemic
The Amur tiger genome

The DNA of a 9-year-old male Amur tiger from Everland Zoo in Korea was
sequenced by Illumina HiSeq2000 (Supplementary Fig. S1, Supplementary
Tables S1=E2=80=93S3). Sequence reads were assembled using SOAPdenovo13 into
scaffolds (2.4=E2=80=89Gb in length) having an N50 length of 8.84=E2=80=89M=
b (contig N50
length of 29.8=E2=80=89kb; Table 1, Supplementary Figs S2=E2=80=93S4, Suppl=
Tables S4 and S5, Methods). Assembly quality was assessed by aligning
the assembled tiger blood transcripts and cat EST sequences onto the
tiger scaffolds (>96% coverage and 98.9% mapping rate, respectively),
and heterozygous single nucleotide variants (SNVs) were validated by the
Sanger method (Supplementary Tables S6=E2=80=93S9, Supplementary Methods).
Additionally, analysis of the tiger draft genome assembly for core
eukaryotic genes14 revealed homologues for >93.4% of conserved genes in
the assembly (Supplementary Table S10). The tiger genome sequence shows
95.6% similarity to the domestic cat (Supplementary Table S11) from
which it diverged approximately 10.8 million years ago (MYA)15; human
and gorilla have 94.8% similarity and diverged around 8.8 MYA (from
TimeTree). This high similarity allowed us to improve the assembly of
the tiger genome by using the recently completed high coverage (12 =C3=97
coverage) domestic cat genome (Supplementary Fig. S5, Supplementary
Tables S12=E2=80=93S17, Supplementary Methods). For comparative genomic ana=
of big cats, we also sequenced four other Panthera genomes (Table 1,
Supplementary Tables S1 and S18): a white Bengal tiger (Panthera tigris
tigris), an African lion, a white African lion and a snow leopard; their
genome sequences were aligned with the domestic cat and tiger genomes.
Table 1: Global statistics of the Panthera genomes.
Full size table
Adaptation of the big cats

The assembled Amur tiger genome was predicted to contain 20,226
protein-coding genes (Supplementary Tables S19=E2=80=93S23, Supplementary
Methods) and 2,935 non-coding RNAs (Supplementary Table S24,
Supplementary Methods). To create a detailed annotation of the tiger
proteome, gene clusters were constructed using seven mammalian genomes
(tiger, cat, human, dog, mouse, giant panda and opossum). The tiger
proteome contained 14,954 orthologous gene families. Among these, 14,425
orthologous gene families were shared by all seven genomes, whereas 103
orthologous gene families were exclusively shared by the tiger and cat
(Fig. 1a, Supplementary Fig. S6, Supplementary Table S25). The
Felidae-specific gene families contained 287 InterPro domains
(Supplementary Tables S26=E2=80=93S29). Based on the comparison of ortholog=
gene families among seven mammalian species, the Amur tiger genome
displays 381 expanded and 1,790 contracted gene families compared with
the feline common ancestor (Fig. 1b, Supplementary Figs S7 and S8). The
tiger genome is particularly enriched in olfactory receptor activity
(GO:0004984, P=3D5.75 =C3=97 10=E2=88=92185, ChiSquare test followed by a F=
exact test, 289 genes), G-protein coupled receptor signalling pathway
(GO:0007186, P=3D2.98 =C3=97 10=E2=88=92106, 302 genes), signal transducer =
(GO:0004871, P=3D2.25 =C3=97 10=E2=88=9274, 295 genes), amino-acid transport
(GO:0006865, P=3D3.09 =C3=97 10=E2=88=9210, 16 genes) and protein metabolic=
(GO:0019538, P=3D5.72 =C3=97 10=E2=88=9210, 220 genes) (Supplementary Fig. =
Supplementary Table S30). In most cats, smell has an important role in
social behaviour such as territory ownership and mating, while vision
and hearing are important for hunting16.
Figure 1: Relationship of the tiger to other mammalian species.
Figure 1

(a) Orthologous gene clusters in mammalian species. The Venn diagram
shows the number of unique and shared gene families among seven
mammalian genomes. (b) Gene expansion or contraction in the tiger
genome. Numbers designate the number of gene families that have expanded
(green, +) and contracted (red, =E2=88=92) after the split from the common
ancestor. The most recent common ancestor (MRCA) has 17,841 gene
families. The time lines indicate divergence times among the species.
Full size image

Additionally, we investigated Panthera lineage-specific amino-acid
changes by comparison with the known genes from the human, dog and
mouse. A total of 3,646 genes had amino-acid changes specific to big
cats (Amur tiger, white tiger, snow leopard, African lion and white
lion; Supplementary Tables S31 and S32), and 5,882 genes had amino-acid
changes unique to the felid lineage (big cats plus domestic cat). Among
these, 1,376 genes had big cat-specific amino-acid changes that were
found to be protein functional changes according to computational
predictions (PolyPhen217). Metabolism pathways associated with protein
and fatty acid, which are important sources of energy, were enriched
with genes having Panthera-specific functional changes; histidine
metabolism (P=3D0.00024, Fisher's exact test, six genes), beta-alanine
metabolism (P=3D0.00078, six genes), phenylalanine metabolism (P=3D0.014,
three genes), valine, leucine and isoleucine degradation (P=3D0.035, six
genes), cysteine and methionine metabolism (P=3D0.037, four genes), fatty
acid metabolism (P=3D0.00038, eight genes) and fat digestion and
absorption (P=3D0.025, five genes) (Supplementary Tables S33 and S34).
These signals of amino-acid metabolism have been associated with an
obligatory carnivorous diet18.

In order to detect tiger genes evolving under positive selection, we
used the conserved genome synteny methodology19 (between human and other
mammalian species (cat, dog, mouse and panda)) and a branch-site
likelihood ratio test (Methods). A total of 178 positively selected
genes out of 7,415 high-quality ortholog genes were identified in the
Amur tiger (Supplementary Table S35, Supplementary Data 1). Functional
categories for positively selected genes were over-represented in muscle
filament sliding (GO:0030049, P=3D0.0049, Fisher's exact test, MYH7, TPM4
and TNNC2), filamentous actin (GO:0031941, P=3D0.0062, TPM4 and MYO1A) and
stress fibre (GO:0001725, P=3D0.0039, MYH7, TPM4 and ACTN4) (Supplementary
Table S36, Supplementary Data 2). We also identified GO categories,
which are evolving under significantly high constraints19 (Supplementary
Tables S37=E2=80=93S39, Supplementary Methods). Significantly altered Ka/Ks
ratios of non-synonymous to synonymous substitutions for GO categories
(P<0.01, Binomial test) revealed evidence of rapid evolution in the
tiger for muscle strength (muscle contraction and actin cytoskeleton),
energy metabolism (GTPase activity, ATP binding and energy reserve
metabolic process), and sensory nerves (G-protein coupled receptor
activity, olfactory receptor activity, visual perception and nervous
system development) (Supplementary Tables S40=E2=80=93S43).
Genetic landscape of the snow leopard and white lion

In addition to the Amur tiger data, we used sequence data from the four
other big cats to investigate the genetic basis of several unique
physiological or phenotypic traits. Snow leopards generally live in
alpine areas, 3,350=E2=80=936,700=E2=80=89m above sea level, in Central Asi=
a20. Recent
genome-wide association studies implicated two human loci EGLN1 (Egl
nine homologue 1) and EPAS1 (endothelial PAS domain-containing protein
1) as mediating high-altitude adaptation21. We examined mutational
substitutions in mammalian EGLN1 and EPAS1 genes and found that the snow
leopard had unique amino-acid changes in both genes that were not found
in other mammalian species. Although EGLN1 is highly conserved in
mammals, Met39 (non-polar), instead of Lys39 (positively charged), was
found in the snow leopard (Fig. 2a, Supplementary Fig. S10,
Supplementary Table S44), indicating a significant change in charge that
might alter protein function. This Met39 residue was replicated as a
genetically fixed substitution in 14 additional snow leopards, whereas
the ancestral Lys39 was monomorphic in a sampling of 28 individuals of
Panthera and Neofelis (clouded leopard) (Supplementary Table S45,
Supplementary Methods). Naked mole rats have also adapted to hypoxia22
in a different manner by having unique amino-acid changes in different
positions of EGLN1 (Pro15, Arg17 and Arg36). Ile663 and Arg794 in EPAS1
are two additional snow leopard-specific changes (Supplementary Fig.
S11), and Arg794 was predicted to confer a functional change on the
protein. Taken together, these EGLN1 and possibly EPAS1 variants are
provocative candidates that may have contributed to the snow leopard=E2=80=
acquisition of an alpine, high altitude ecological niche.
Figure 2: EGLN1 and TYR mutations related to hypoxia in snow leopard and
white fur in white lion.
Figure 2

(a) Alignment of mammalian EGLN1 amino-acid sequences. Amino acids
unique to the snow leopard (216th residue in human EGLN1), naked mole
rat and rodents are shown in red, grey and blue, respectively. The
number of individuals genotyped in this study is listed in parentheses.
(b) Alignment of mammalian TYR sequences. Amino-acid sequences unique to
the white lion (87th residue in human TYR) are shown in red, and tawny
lion having heterozygous allele (G/A) are shown in grey; X represents
amino acid of R/Q. The numbers in parentheses are number of individuals.
=E2=80=98w=E2=80=99 denotes white type and =E2=80=98wt=E2=80=99 denotes wil=
d type.
Full size image

Tyrosinase (TYR) mutational variants cause white coat colour in the
domestic cat23, and TYR mutations are related to human oculocutaneous
albinism 1 (refs 24, 25). The genetic basis of the white tiger with
white fur and dark stripes is revealed as an amino-acid change (A477V)
in the transporter protein SLC45A2 (ref. 26). Therefore, we examined the
pigment-associated gene mutation in the white lion and found a unique
nucleotide (TYR260G>A) and corresponding amino-acid change in the white
lion (Fig. 2b, Supplementary Fig. S12), causing the positively charged
Arg87, located in the central domain of TYR, to be changed to uncharged
Gln87. We verified the proposed candidate mutation (TYR260G>A)
underlying the amino-acid change (R87Q) in the TYR gene sequence of 47
lions: 17 of white phenotype and 30 of wild phenotype, of which 11 were
known to be carriers (heterozygotes) based on their pedigree, 14 of
unknown genetic makeup, and 5 of wild phenotype from control populations
where no white lions were found (Supplementary Tables S46=E2=80=93S48,
Supplementary Methods). The concordance between the expected and
observed genotype was 100% for the candidate mutation TYR260G>A. A
second non-synonymous mutation (TYR176C>T), observed in a subset of
animals, did not correlate with the expected genotype, and animals with
this variant did not appear to be phenotypically different.
Genomic comparison between the tiger and other mammals

Although repeat characteristics can vary considerably among closely
related species27, the tiger and cat genomes showed very similar repeat
compositions (39.3% versus 39.2%, respectively), as well as ratios of
repeat components, including tandem repeats and transposable elements
(Supplementary Fig. S13), suggesting a similar genome architecture
between domestic cats and tigers. By contrast, for the great apes, the
ratio of repeat components was considerably different between species,
especially between human and orangutan, which diverged about 12 MYA28.
Additionally, we estimated the evolutionarily conserved sequences
(77=E2=80=89Mb, 3.2%), segmental duplication (11.2=E2=80=89Mb, 0.47%) and
lineage-specific insertions and deletions in the tiger genome
(Supplementary Tables S49=E2=80=93S52, Supplementary Methods).

To detect genome-wide structural differences, we aligned the tiger
scaffolds to the cat genome using dog genome as a reference after
masking repeats. A total of 571 of the 674 tiger scaffolds (length
>20=E2=80=89kb, 99.6% of the total scaffold length) were aligned with the c=
genome sequence, and as high as 98.8% of gene-coding regions and 98.3%
(2.38=E2=80=89Gb) of conserved synteny blocks were shared by the tiger and =
genomes. We detected a rather high level of genomic synteny, containing
six breaks with large-size chromosomal segmental rearrangement between
the tiger and cat genomes (Fig. 3, Supplementary Fig. S14, Supplementary
Tables S53=E2=80=93S56, Methods). These consisted of two inter- and four
intra-chromosomal rearrangements. Divergence in genomic structure among
closely related species is considered as a major factor underlying
species diversification, as gene flow requires recombination in
collinear chromosomes, and the reduction in recombination associated
with chromosomal rearrangements results in a partial reproductive
barrier29. These structural variations may be one of the important
factors underlying species diversification among the felines.
Figure 3: Synteny blocks between tiger and cat genomes.
Figure 3

Domestic cat chromosomes are shown as grey bars (in Mb scales). The
other six color bars (in Mb scales) are tiger scaffolds with syntenic
break between tiger and cat (2 inter- and 4 intra-chromosomal
rearrangements). The tiger and cat rearrangements were detected using
dog genome as an out-group.
Full size image

The level of within-species genetic diversity, as measured by the rate
of heterozygous SNVs, of the tiger (0.00049=E2=80=930.00073) and lion
(0.00048=E2=80=930.00058) genomes was found to be similar to that of human
(0.00066) (Supplementary Table S57, Supplementary Methods).
Interestingly, diversity of the snow leopard genome was nearly half that
of the other Panthera species and slightly lower than that of the
Tasmanian devil30, which is purported to display a low level of genetic
diversity (Fig. 4a). We also estimated the occurrence of a marked
bottleneck around the last glacial maximum 20 kyr ago (7=E2=80=9370 kyr) us=
ing a
pairwise sequentially Markovian coalescent (PSMC)31 model inference of
tiger demographic history based on SNV distribution (Fig. 4b,
Supplementary Figs S15=E2=80=93S18, Supplementary Tables S58 and S59, Metho=
A similar bottleneck was estimated a bit earlier (72=E2=80=93108 kyr) based=
mitochondrial DNA coalescence1. White lion (0.00048) and domestic cat
(0.00012) have both undergone multiple rounds of close inbreeding during
breed development and therefore would display lower SNV diversity bias
as a consequence. Therefore, we investigated the genetic diversity of
Panthera using the rate of heterozygous SNVs and confirmed that the
genetic diversity within a single individual coincided with that deduced
from mitochondrial sequences of several individuals30.
Figure 4: Genetic diversity and population size history in Panthera species.
Figure 4

(a) Rate of heterozygous SNVs in Panthera species. The heterozygous SNVs
rates (y axis) were calculated by dividing the total number of
heterozygous SNVs by genome size. Individuals that are white colored in
nature (white tiger and white lion) are shown in grey. Tigers, lions,
cat, gorilla, giant panda, chimpanzee and naked mole rat are captive
bred. Snow leopard, orang-utans and Tasmanian devil are wild caught
individuals. (b) Estimated big cat population sizes and climate history
from 2.5 kyr BP to 3 Myr BP. Tsuf, atmospheric surface air temperature;
RSL, relative sea level; 10=E2=80=89m.s.l.e., 10=E2=80=89m sea level equiva=
lent; TG,
Amur tiger; LN, African lion; SL, snow leopard; WTG, white tiger; WLN,
white African lion. =E2=80=98F=E2=80=99 after the species abbreviation mean=
s the data
were generated from comparison with Felis_catus-6.2 as a reference
genome in SNV calling.
Full size image

The Amur tiger genome is the first reference genome sequenced from the
Panthera lineage and the second from the Felidae species. For
comparative genomic analyses of big cats, we additionally sequenced four
other Panthera genomes and tried to predict possible big cats=E2=80=99 mole=
adaptations consistent with the obligatory meat eating and muscle
strength of the predatory Panthera lineage. The tiger and cat genomes
showed unexpectedly similar repeat compositions and high genomic
synteny, and these indicated strong genomic conservation in Felidae.
These results could be supported by the recency of the 37
species-Felidae radiation (<11 MYA)15 and well-known hybridizations in
captivity among subspecies in Felidae lineage such as liger and tigon.
By contrast, the ratio of repeat components for the great apes was
considerably different among species, especially between human and
orang-utan28, which diverged about the same time as felines. The breaks
in synteny that we observed are likely occasional rare sporadic
exchanges that accumulated over this short period (<11 MYA) of
evolutionary time. The paucity of exchanges across the mammalian
radiations (by contrast to more reshuffled species such as Canidae,
Gibbons, Ursidae and New World monkeys) is a hallmark of evolutionary

Many whole-reference genome studies used few close species genomes that
can be directly compared with the reference genome constructed. Although
we did not have the resources to construct lion and leopard reference
genomes, and hence were not able to show all the structural variations
on the genomes, our =E2=80=98close species comparative genomics=E2=80=99 ap=
utilizing at least one reference species, heralds a new level of genome
studies. It is because those very close Panthera species have distinct
species-specific and readily identifiable phenotypes that can be
associated quickly to mutations by comparing the homologous genes of
interest as shown in the fur colouring (white lion) and high-altitude
adaptation (snow leopard). If sufficiently distinct phenotypes are
biologically curated, genetic mutations causing species specificity can
be systematically detected using next generation sequencing. Once such
candidate genetic mutations are confirmed in the set of species genomes,
experimental validations can be carried out, as in the additional 47
lion samples here, for targeted genes. This genetic variation comparison
using whole genomes among species and subspecies can thus provide
valuable insight and information for the whole family=E2=80=99s conservatio=
Our data from tigers, lions and snow leopard can provide a rich and
diverse genome resource that could be used in future studies of
conservation and population genomics so that the genetic underpinnings
of local adaptation and potential inbreeding and/or outbreeding10 in
wild and captive populations can be illuminated and thereby help ensure
the future survival of these majestic species.
Genome sequence assembly and annotation

The blood samples used for genome sequencing were acquired from the
Everland Zoo of Korea (Amur tiger, white Bengal tiger, African lion and
white African lion) following the Everland Zoo (Korea) ethical
guidelines and procedures, and a muscle sample was obtained from a
Mongolian snow leopard carcass preserved in the Conservation Genome
Resource Bank for Korean Wildlife, Seoul National University. No animals
were killed or captured as a result of this study. Libraries for the
Amur tiger genome were constructed at BGI, Shenzhen, and the insert
sizes of the libraries were 170=E2=80=89bp, 500=E2=80=89bp, 800=E2=80=89bp,=
2=E2=80=89kb, 5=E2=80=89kb, 10=E2=80=89kb
and 20=E2=80=89kb. The libraries were sequenced using HiSeq2000. Other big =
genomes were sequenced at Theragen BiO Institute (TBI), Korea, using
HiSeq2000 with read and insert lengths of ~90=E2=80=89bp and ~400=E2=80=89b=
p, respectively.

The corrected reads were used to complete the genome assembly using
SOAPdenovo13. First, the short insert size library (170=E2=80=89bp, 500=E2=
=80=89bp and
800=E2=80=89bp) data were used to construct a de Bruijn graph. Second, all =
were realigned with the contig sequences. The amount of shared
paired-end relationships between pairs of contigs were calculated and
weighted with the rate of consistent and conflicting paired ends, before
constructing the scaffolds step by step from the short insert size
paired ends to the long distant paired ends. Third, the gaps between the
constructed scaffolds were closed using the paired-end information to
retrieve read pairs where one end mapped to a unique contig while the
other was located in the gap region.

The Amur tiger genes were predicted using three approaches. First, de
novo prediction was performed using the repeat-masked genome using
AUGUSTUS (version 2.5.5)32 and GENSCAN (version 1.0)33. Second,
homologous proteins in other species were mapped to the genome using
tBLASTn (Blast 2.2.23)34 with an E-value cutoff of 1E-5. The aligned
sequence and its query protein were then filtered and passed to GeneWise
(version 2.2.0)35 to search for accurately spliced alignments. Third,
cat EST and full-length cDNA sequences (from UCSC) were aligned to the
genome using BLAT36 to generate spliced alignments. For EST results,
spliced alignments were linked according to overlap using PASA37. Source
evidence generated from the three approaches was integrated with GLEAN38
to produce a consensus gene set. Then, the Amur tiger genome sequence
was aligned to two well-assembled and annotated genomes (human and
domestic cat) using LASTZ (version 1.02). Finally, mapped results
yielding information on homologous proteins were filtered by syntenic
blocks of genome sequences. We also predicted the domestic cat
(Felis_catus-6.2) gene set, because the gene set of the cat genome is
Orthologous gene families

A comparative analysis was used to examine the rate of protein evolution
and the conservation of gene repertoires among orthologs in the genomes
of the Amur tiger, dog, human, mouse, giant panda, domestic cat
(Felis_catus-6.2) and opossum. We used the TreeFam methodology39 to
define a gene family as a group of genes that descended from a single
gene in the last common ancestor of a considered species. We assigned a
connection (edge) between two nodes (genes) if more than 1/3 of the
region was aligned to both genes. An H-score (minimum edge weight) that
ranged from 0 to 100 was used to weigh the similarity (edge). For two
genes, G1 and G2, the H-score was defined as score (G1G2)/max (score
(G1G1), score (G2G2)), where the score shown is the BLAST raw score.
Gene families were extracted by clustering using Hcluster_sg. We used
the average distance for the hierarchical clustering algorithm,
requiring the H-score to be larger than five, and the minimum edge
density (total number of edges/theoretical number of edges) to be larger
than 1/3. The clustering for a gene family would also stop if it already
had one or more of the out-group genes.

We determined the expansion and contraction of the orthologous protein
families among seven mammalian species (tiger, cat (Felis_catus-6.2),
dog, human, mouse, giant panda and opossum) using CAF=C3=89 2.2 (ref. 40)
with 0.001080 of lambda option. GO of all tiger genes was annotated by
InterPro. A =CF=872 test followed by a Fisher=E2=80=99s exact test (P=E2=89=
=A40.01) were used
to test for over-represented functional categories among expanded genes
and =E2=80=98genome background=E2=80=99 genes; Fisher=E2=80=99s exact test =
was used when any
expected value of count was below 5, which would have make the =CF=872 test
Gene evolution

We investigated Panthera lineage-specific amino-acid changes by
comparison with the known genes from the human, dog and mouse (from the
Ensembl 69 release). We used lion and snow leopard gene sets by mapping
reads to the tiger scaffolds and substituting SNVs. Artifacts from the
multiple sequence alignment (ClustalW242) limitations were removed by
filtering option with =E2=89=A51/2 of coverage and =E2=89=A5of well-matched=
amino acids
(consensus string is =E2=80=98*=E2=80=99, =E2=80=98:=E2=80=99 or =E2=80=98.=

To detect tiger genes evolving under positive selection, we used
conserved genome synteny methodology19 to establish a high-confidence
orthologous gene set. Briefly, whole-genome multiple alignments were
performed between human (hg19) and other species (cat (Felis_catus-6.2),
dog (CanFam2.0), mouse (mm9) and panda (ailMel1) genomes) by the LASTZ
alignment pipeline. We collected all the human protein-coding genes from
RefSeq43, KnownGene44 and VEGA45, and mapped them to the other species
via the syntenic regions. We then filtered the resulting blocks with
rigorous conditions to get large-scale synteny of high-alignment
quality, and a conservation of exon=E2=80=93intron structure. Finally, we f=
7,415 1:1 high-quality ortholog genes to analyse, most of which also
correspond to genes in the panda, dog and mouse genomes. Then, we
aligned ortholog genes by PRANK46 and used the optimized branch-site
model of PAML (version 4.5) and likelihood ratio tests (LRTs) (P=E2=89=A40.=
A GO annotation download from Ensembl was used to assign GO categories
to 7,415 orthologs. A =CF=872 test followed by a Fisher=E2=80=99s exact tes=
t (P=E2=89=A40.01)
were used to test for over-represented functional categories among
positively selected genes; a Fisher=E2=80=99s exact test was used when any
expected value of count was below 5, which would have made the =CF=872 test

We also used an approach based on Ka/Ks47,48 to identify GO categories
significantly above or below average in the tiger genome. The Ka and Ks
rates are estimated by PAML from all aligned bases with a quality score
>20 in orthologs, using the F3 =C3=97 4 codon frequency model and the REV
substitution matrix. To determine whether the GO categories are evolving
under significantly high constraints, we repeated this procedure 10,000
times on the same data set after randomly permuting the GO annotations.
Then, we acquired the GO categories if the P-value was less than 0.05.
Chromosomal rearrangement

Among the alignment data generated from SyMAP49, when one scaffold
happened to be mapped to several physically distant cat
(Felis_catus-6.2) chromosomal locations, they were considered to be
inter- or intra-chromosomal rearrangement events of the Amur tiger
genome relative to the cat genome. The species (tiger and domestic
cat)-specific genomic rearrangements were also analysed. We performed
the dog versus tiger and cat versus tiger whole-genome pair-wise
alignments using LASTZ software on the repeat-masked genomes. Using
these methods, we identified clusters of unique alignments with
well-defined order and orientation. There was a total of 18 chromosomal
rearrangement (12 inter- and 6 intra-chromosomal rearrangements)
overlaps when the results from SyMAP and LASTZ were integrated by
comparing syntenic break positions. As the tiger assembly was generally
fragmented, we carefully validated the 18 syntenic breaks to examine the
assembly integrity by aligning long insert mate-pair libraries (2=E2=80=89k=
5=E2=80=89kb, 10=E2=80=89kb and 20=E2=80=89kb) to the tiger scaffolds. Fina=
lly, we reported six
putative chromosomal rearrangements (two inter- and four
intra-chromosomal rearrangements) between the tiger and cat. All six
rearrangements were validated by long-range PCR experiments followed by
the Sanger sequencing method.
Demographic history

The history of population size helps to develop insights into evolution.
Based on the pairwise sequentially Markovian coalescent model (PSMC)31,
we inferred detailed population size histories of Amur tiger (TG),
African lion (LN), snow leopard (SL), white tiger (WTG) and white lion
(WLN). Using SNV data sets scanned with all the big cat sequencing reads
mapped to Felis_catus-6.2, the consensus sequences of each big cat were
constructed and then divided into non-overlapping 100-bp bins marked as
homozygous or heterozygous. The resultant bin sequences for their sex
chromosomal parts were removed, and then they were taken as the input of
the PSMC estimation. To test the estimation accuracy, bootstrapping was
performed by randomly resampling 100 sequences from the original
sequences. Using the neutral mutation rates, the raw PSMC outputs were
scaled to time and population sizes. We obtained atmospheric surface air
temperature and global relative sea level data of the past 3 million

-- =

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DRM is THEFT - We are the STAKEHOLDERS - RI Safir 2002 - Leadership Development in Free Software - Unpublished Archive - coins!

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