Graduate presentation in Advanced Genetics.

1. What can a tiger’s genome tell us about
mammalian evolution?
A review of Cho, et al. 2013. The tiger genome and comparative analysis with lion and snow
leopard genomes. Nature. 4:2433. by Courtney Dunn

3. Introduction
▪ Panthera tigris exists as the largest
felid species on Earth as well as one
of the most endangered species
known, functioning as a keystone
species.
▪ Population estimates range from
3,050 to 3,950 individuals
▪ Nine genetically identifiable
subspecies have been named of
which four have went extinct in the
last century
▪ The Amur subspecies is the largest
and the only one which does not live
in a tropical, warm climate.

4. A New Genetic Realm
▪ Previous studies have elucidated the
phylogeography and population genetics of tigers
▪ Relied primarily on mitochondrial and nuclear loci
▪ Domestic cat (Felis catus), although a low
coverage genome, has provided further insights.
▪ No whole-genome reference sequences have
been reported for any Panthera species

5. Objectives
1. Sequence the first tiger genome through
assembly and annotation.
2. Compare sequences to Panthera uncia to
determine genetic adaptations specific to
high-altitude habitats.
3. Determine mutations responsible for white
coat coloration in Panthera leo and
Panthera tigris.

6. Methods
▪ Genome sequence assembly and annotation
▪ Orthologous gene families
▪ Gene evolution
▪ Chromosomal rearrangement
▪ Demographic history

7. Genome Sequence Assembly and Annotation
▪ Blood samples for Amur tiger, white Bengal tiger, African
lion, and African lion acquired from the Everland Zoo of
Korea.
▪ Muscle sample for a Mongolian snow leopard was obtained
from the Conservation Genome Resource Bank at Seoul
National University
▪ Sequenced using HiSeq2000 with read and insert lengths of
approx. 90 bp and 400 bp.
▪ Assembled by SOAPdenovo
▪ De novo prediction using AUGUSTUS (version 2.5.5) and
GENSCAN (version 1.0)

8. Orthologous gene families
▪ The expansion and contraction of the orthologous
protein families using seven mammalian species
(tiger, cat, dog, human, mouse, giant panda and
opossum) via CAFE´ 2.2 and Fisher’s exact test.
▪ Multiple sequence alignment (CLUSTALW2)
▪ Chromosomal rearrangement from SyMap and
LASTZ software.
▪ Markovian coalescent model for population size
history analysis

9. The Amur tiger genome
▪ Core eukaryotic genes revealed
homologues for >93.4% of
conserved genes.
▪ Tiger genome has 95.6%
similarity to the domestic cat –
evolutionary divergence 10.8
million years ago (MYA)
▪ Such a similarity was used to
improve the tiger genome
assembly via a recently completed
high coverage domestic cat
genome.

10. Adaptation of the big cats
▪ Assembled Amur genome predicted to
contain 2,935 non-coding RNAs and
20,226 protein-coding genes.
▪ Gene clusters constructed using seven
mammalian genomes
▪ Tiger proteome contained 14,954
orthologous gene families – of which:
▪ 14,425 shared by all seven comparison
genomes
▪ 103 exclusively shared by the cat and tiger
▪ Amur tiger genome displays 381
expanded and 1,70 contracted gene
families compared with the feline
common ancestor

12. Genome Enrichment Areas
▪ Olfactory receptor activity
▪ G-protein coupled receptor signaling
pathway
▪ Signal transducer activity
▪ Amino-acid transport
▪ Protein metabolic process

13. Lineage-specific amino acid changes
▪ Compared to human, dog, and mouse
▪ 3,646 gene changes specific to big cats
▪ 5,882 gene changes unique to the felid
lineage
▪ 1,376 related to protein function changes

14. Metabolism Pathway Alteration
▪ Panthera specific changes associated with proteins
and fatty acids a.k.a energy acquisition.
▪ Histidine, beta-alanine, phenylalanine valine,
leucine and isoleucine degradation, cysteine and
methoionine , fatty acid, and fat digestion and
absorption.
▪ Reflective of an obligatory carnivorous diet.

15. Positive Selection Genes
▪ Over-represented in muscle
filament sliding (MYH7,TPM4,
MYO1A), stress fiber (MYH7,
TPM4, and ACTN4)
▪ Significantly altered Ka/Ks ratios
of non-synonymous to
synonymous substitutions
revealed evidence of rapid
evolution for muscle strength,
energy metabolism, and sensory
nerves.

16. Genetic Landscape of the Snow Leopard
▪ The study investigated the
genetic basis of several unique
physiological and phenotypic
traits.
▪ Snow leopards are adapted to life
in extreme alpine areas in Central
Asia
▪ Previous genome-wide
association studies have revealed
two human loci responsible for
high-altitude adaptation –
EGLN1 and EPAS1 - as well as in
Naked mole rats.

17. Genetic Landscape of the Snow Leopard
▪ The study revealed Snow Leopards have unique
amino-acid changes in both which were not found
in any other species including Lys39 (Polar) ->
Met39 (Non-Polar)
▪ Lys39 has been shown to occur monomorphically
in Panthera and Neofelis individuals.
▪ Variants may have contributed to this species
acquisition of a unique ecological niche.

18. White Tigers and Lions – Mystery solved?
▪ Tyrosinase (TYR) variants are responsible for
albinism in humans and white coats in
domestic cats.
▪ However, an amino-acid change in the
transporter protein SLC45A2 was found
responsible for White tigers.
▪ Examination of pigment-associated gene for
White lion revealed a change from +Arg87 to
Gln87, a mutation known asTYR260G>A.
▪ The concordance between the expected and
observed genotype was 100% for the
mutation in 47 examined lions.

20. Genomic Comparison between Tiger and
other Mammals
▪ Tiger and cat genomes showed very
similar repeat compositions – 39.3% vs
39.2% respectively – as well as
transposable elements and repeat
components suggesting a similar
genome architecture.
▪ Alignment of tiger scaffolds to cat
genomes revealed 571 out of 674 tiger
scaffolds were alignment – 98.8% gene-
coding regions and 98.3% of conserved
synteny blocks.
▪ High level of genomic synteny – six
breaks with large chromosomal
segment rearrangement.

21. Synteny and Collinear Rearrangement

22. Genomic Comparison between Tiger and
other Mammals
▪ Divergence among closely-related species is
an important factor underlying species
diversification.
▪ Gene flow requires recombination in collinear
chromosomes.
▪ Such recombination results in a partial
reproductive barrier.

23. Within-Species Diversity
▪ Measured by the rate of heterozygous
SNVS – single nucleotide variants
▪ Tiger (0.00049 – 0.00073), Lion (0.00048 –
0.00058), Human (0.00066).
▪ Diversity of Snow leopard genomes was
nearly half that of other Panthera species
and slightly lower than that of the
Tasmanian devil.
▪ Based on mitochondrial DNA coalescence,
a marked bottleneck occurred around the
last glacial maximum 20,000 years ago and
72-108,000 years ago

24. Within-Species Diversity
▪ White lion (0.00048)
▪ Domestic cat (0.00012)
▪ Multiple rounds of close in-
breeding may have resulted
in such low SNV diversity.

26. Discussion
▪ The Amur tiger genome was the first reference genome for
the Panthera lineage and only the second for Felidae
species.
▪ Predicted possible molecular adaptations consistent with
big cats obligatory meat diet, muscle strength, and
predatory behavior.
▪ Similarity between cat and tiger genomes could be
supported by their recent species divergence – approx. 11
million years ago.
▪ Breaks in synteny could be due to rare, sporadic
accumulated exchanges over evolutionary time.
▪ Close species comparative genomics approach with one
reference species heralds a new level of genomic studies.

27. Discussion
▪ If sufficiently distinct phenotypes are biologically
curated, genetic mutations causing species
specificity can be systematically detected using
next generation sequencing.
▪ e.g. phenotypic analysis ofWhite lions using 47
individuals
▪ Utilizing whole genomes for variation comparison
has and can provide valuable insight for a whole
family’s conservation – especially related to local
adaptation and potential inbreeding/outbreeding.

28. Questions?