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

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.

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

Objectives1. 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.

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

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)

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

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.

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

Genome Enrichment Areas

▪ Olfactory receptor activity▪ G-protein coupled receptor

signaling pathway▪ Signal transducer activity▪ Amino-acid transport▪ Protein metabolic process

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

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.

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.

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.

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.

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 as TYR260G>A.

▪ The concordance between the expected and observed genotype was 100% for the mutation in 47 examined lions.

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.

Synteny and Collinear Rearrangement

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.

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

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.

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.

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 of White 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.

Questions?