Questions to Address  What Are the Genetic Mechanisms Underlying the Coat Color Variations in Tiger?

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The Genetic Basis of Tiger Pelage Variation Three most commonly asked and Conservation Implications questions about tigers

1. If a tiger and a lion live in the same forest, who is the king of the jungle? 2. Is China where tiger came from? 3. Is white tiger a defective animal? Shu-Jin Luo Peking University, China PAG XXVI, 2018.1.15

The most obvious phenotypes in wildlife are Individuals of the same species often share an overall COAT COLOR and PATTERN, uniform coat colour and pattern. which play important roles in camouflage and survival However, there are variations…… Jaguar Jaguarundi

Lion Asiatic golden cat

Questions to Address  What are the genetic mechanisms underlying the coat color variations in tiger?

 Are these tiger color variants “genetic defects” or normal polymorphisms?

(Akbar Nama 1561)  White tiger: found in the wild of India as early as the 1500s.  A variant of Bengal tiger subspecies, characterized by a white fur with dark brown (to black) strips, ice blue eyes, pink nose and paw pads  A white tiger is NOT an albino!  The white phenotype is a recessive trait at an autosomal locus.  White tigers went extinct from the wild since the 1950s, and hundreds are now kept in captivity, many suffering health problems. The tiger is known to have at least four color phases…

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Tiger Agouti Hair in Mammals Hair Morphology

The Agouti gene is responsible for determining whether a Tiger hairs from non-striped area are are typically agouti patterned. mammal's coat is banded (agouti) or of a solid color (non-agouti) Tiger hairs from stripes are solid-colored.

Basic Mammalian Pigmentation Pathways Hypothesis of Tiger Coat Color Variations Established by Genetic Studies of Mice

Inheritance model: autosomal recessive white locus; Putative genotype: ww Cysteine Pheomelanin

Inheritance model: ASIP SLC7A11 autosomal recessive wideband locus; ? Putative genotype: bb MC1R TYR cAMP Tyrosine DOPA

TYRP2 Inheritance model: double recessive at both loci; Putative genotype: ww/bb TYRP1 w: ?? a-MSH Eumelanin b: ?? Melanocyte

Strategies of Gene Mapping

Candidate gene Sequencing NO of SNPs Phenotype  Classical linkage mapping region identified associated SNPs  Multi-generation pedigree with phenotype segregation MC1R Exon1, UTRs 2 none  Existing genetic map ASIP Exon1-3, UTRs 0 none TYR Exon1-5, UTRs 2 none  Genome-wide association studies (GWAS) TYRP1 Exon1-8, UTRs 1 none  Large case/control groups of unrelated individuals Exon1-12, UTRs SLC7A11 4 none  Only associated genetic region identified SLC18A2 Exon1-15, UTRs 0 none  Whole-genome sequencing GWAS in a pedigree - None of the tested genes are responsible for the tiger coat color variations. ✔  - There are 378 coat color-related genes reported from human, mouse and Only a pedigree of small sample size required zebra fish, 171 cloned and 207 uncloned, http://www.espcr.org/micemut/  Power to identify the causative mutation - Where to begin with?

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Tiger Pedigree Segregating at the wideband Locus (N=19)

BB bb wwbb Wwbb wwbb wwbb wwbb

GZXJ34 GZXJ39 GZXJ35 GZXJ37 GZXJ36

Bb Bb Bb Bb Bb Bb Bb 30x WGS

GZXJ008 GZXJ007 GZXJ043 GZXJ044 N=14 from pedigree 4 Snow (wwbb) 10 White (wwBb)

N=5 unrelated individuals Bb Bb Bb bb bb bb bb Bb 1 Golden (Wwbb) All individuals in this Bb bb 4 Snow (wwbb)

pedigree are homozygous GZXJ041 GZXJ042 GZXJ045 GZXJ046 GZXJ048 GZXJ049 GZXJ050 GZXJ051 GZXJ040 GZXJ047 bb at the wideband locus All individuals in this pedigree are homozygous mutant (ww) at the white locus Tiger Pedigree Segregating at the white Locus (N=16) 19 individuals were use for linkage mapping, 9bb + 10Bb; 3 WGS+16 RAD-seq

GWAS in a tiger pedigree segregating at w locus (N=16) Mutation Validation (n=130)

The genome-wide P value of association mapping based on A

Fisher’s exact test by allele counts (Human, OCA4) under recessive model (172,554 P58A P58S W202C T302S R348C L374F A486V SNPs, 1 marker/14kb) T500P M42I G64S D157N G188V delF221 E272K Y317C L361P A477T V507L Linkage analysis in the two candidate

scaffolds revealed one haplotype TM TM TM TM TM TM TM TM TM TM TM TM block of 3.3 Mb at complete linkage disequilibrium (LD) with white trait. D153N S435P (horse, cream; mouse, Uwdbr ) (mouse, uwb)

B white tiger A477V mutation SNPs causing non -synonymous substitution were validated in unrelated tiger R-QQAPGGSLDGSERGQGLDCAVLTCMVQLAQILVGGGLGFLA477VVNKAGS-VIVVIT white20 tiger white and.-...... 110 orange tigers. VMutation...... -...... slc45a2 correlates cat .-...... -...... perfectly with the white tiger phenotype. dog(Number represent .-...R.....SG...... -...... unrelated individuals used in the extended study. Numbers in parenthesis panda represent.-H.....GG..DG...... -...... related individuals from the pedigree used in GWAS) cattle K-.R.L..GP...S...... A..S...... L...T...V.V.... pig .-...Q..G..S.R...... A...... VV..V.V.... horse .-R..Q..DV.S.G...... A...... I...V.V.... elephant .-.ALSE.-P.S....K.V...A..S...... L...T...VIV.... human .-...... DP.N.V..K.M...T...... T..TV.V.... chimpanzee .-...... DP.NNV..K.M...T...... T..TV.V.... mouse KG.E....-P.NQG..K.V...A...... M...V.V.... chicken L-..KEQ.TEH.--..K.I...A...... IL.V...L..SV...-AVT..S clawed frog M-.PNGKTPTPSDN..K.I...A...... I...... SL...V.V...S zebrafish .-KLG-.DGVAPEG..T.M...A...... VI..A...A...L...VIV..LS Polymorphisms in SLC45A2 have been reported in human, medaka fish, chimpanzee, house mouse, C D Norway rats, chicken, Japanese quail, dog, and cattle Periplasm TM1 TM8

TM6 TM5 TM4 TM10 TM9 TM3

A477V A477 TM12 TM11 TM2 TM7 C N

Cytoplasm

A 3D homology model of SLC45A2 proposed that the A477V substitution in the white tiger might partially block the cavity of the transporter channel and hinder the substrate translocation, therefore affecting the function of SLC45A2 and inhibiting melanogenesis.

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Coat Patterns in Mammals – the Cat’s Contribution How the white tiger lost its color, but kept its stripes? A proposed model for tiger coat color formation through interaction of Mc1r and Ednrb signaling pathways under the effect of Slc45a2. A, In an orange tiger, Slc45a2 maintains proper pH in melanosomes, the Mc1r pathway engages in downstream eumelanin and pheomelanin syntheses, and Edn3 stimulates eumelanin synthesis through the Ednrb pathway; the high level of Edn3 expression increases eumelanin production and suppresses pheomelanin in striped fur. B, In a white tiger, melanin synthesis is abolished by Slc45a2 deficiency in background fur leading to white coloration, Region-specific high expression of Edn3 contributes to but eumelanin synthesis is sustained by high eumelanin production in stripes and spots (Kaelin et al. 2012) expression of Edn3 in striped fur.

GWAS of tigers segregating at the wideband locus (N=19) Mutation Mining in CORIN

6 H587Y scaffold1457

5 scaffold97 scaffold91 TM Fz1 LDLR Fz2 LDLR SR Protease 4

)

(

1 3 H587

o l tiger(orange) ENSDNQTCLMPNEDVEECSPSHFKCGSGRCVLASRRCDGQADCDDDSDEENCGCKERDLW

- tiger(golden) ...... Y...... 2 cat ...... D...... ferret ...... D.A...... R...M. 1 dog ...E...... D.G...... panda ...... D.A...... R...... cattle ..A...... DDE...... R...... 0 sheep ..A...... DDE...... S...... 0 A1 A2 A3 B1 B2 B3 B4 C1 C2 D1 D2 D3 D4 E1 E2 E3 F1 F2 X UN pig ...... DA...... S...... Chromosome whale ....S...... DD...... S...... horse ...... D...... S...... E..E.. rabbit ...... DQ...... R...... Tiger scaffolds were arranged and grouped into putative chromosomes (x axis) based mice .S...... L...... R...... G...... A.. human ...... D.Y...... R..Q...... on homology to cat genome (Felis_catus 6.2). SNPs within every 500 Kb window were gorilla .....R.....D.Y...... R...... plotted in a single column. A total of 112 SNPs from tiger genome scaffolds 91, 97 and bat ...... D...... D...... EP...... A...... G.. elephant ...... D...... R....I...... E..N.. 1457 were associated with golden trait (p < 0.0001), among which 47 SNPs from chicken ...... T.D...... R...... E.....DS...... G.. scaffold 97 and 1457 showed the strongest association (p = 1.08E-5, 9.3Mb LD region). LDL-receptor class A 6 domain

CORIN: a transmembrane serine protease

Mutation Validation (n=197) Secreted extracellular ASIP was Corin-knockout agouti mouse degraded in the presence of wild- typed CORIN, but not the CORIN p.H587Y variant Correlation between tiger coat color variations and genotype of slc45a2A477V(w) and corinH587Y(b)

CORIN is a transmembrane serine protease involved in regulations of blood pressure in mammals and participates in pigmentation process by suppressing the agouti pathway. (Enshell-Seijffers et al. 2008; Yan et al. 1999; Yan et al. 2000; Xu et al. 2017)

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Conclusions of Tiger Coat Color Variations Summary of Tiger Coat Color Genetics

Inheritance model: autosomal recessive;  Genetic basis of white, golden tabby and snow Putative genotype: ww tigers was identified through pedigree-based whole genome sequencing, contributing to our understanding of adaptive pigmentation.  Slc45a2p.A477V substitution results in the white Inheritance model: autosomal recessive; Putative genotype: bb tiger coat color and is a natural polymorphism in wild tiger populations (not genetic defect), highlighting its conservation value.  Corinp.H587Y substitution results in the golden Inheritance model: double recessive at both loci; tiger coat color whose physiological effect Putative genotype: ww × bb remains to be examined. w: slc45a2p.A477V  Double recessive mutations result in a snow tiger coat color phenotype. b: corinp.H587Y Xu X et al. (2013). The genetic basis of white tigers. Current Biology 23: 1031-1035. Xu X et al. (2017). The genetics of tiger pelage color variations. Cell Research 27: 954-957 Xu et al. (2013) Current Biology; Xu et al. Cell Research (2017)

Acknowledgements  Lab Members: Xiao Xu, Yan Zhuang, Xuesong Hu, Lin Miao, Zhengting Zou, Tingting Zhang, Haoran Xue, Yuechen Liu  Sampling and Veterinary Assistance: Chimelong Wildlife Safari: Dong Guixin, Zhang Xueli, Zhang Tianyou,Yang Handong, Zhang Delu, Zhong Yongshan, Qiu Renan, Li Baohong, Lei Zhenqiu, Yang Wenhui, Dong jian Cheetah Conservation Fund: Anne Schmidt-Küntzel, Lusia Mhuulu Dr. Xiao Xu Learning from Exotic Species Laurie Marker NIH-LGD: Stephen O’Brien, Marilyn Raymond Cell - 18 July 2013, 154(2) 257-259 Zoos, Museums and Private owners (hundreds….) Although much of what we know about biology has come from the  Genome Sequencing: Novogene (Li Ruiqiang), PKU-BIOPIC study of a handful of organisms,  Computational Analysis: Jiang Taijiao (CAS-IBP) these existing models only provide  Functional Analysis: Xie Can, Jiang Zhengfan, Fang Run (PKU-SLS) a sampling of the interesting traits and adaptations of life. Techno- logical advances have enabled biologists to take a much broader look, with this Select highlighting recent insights into fundamental Zhuang Yan Sun Xin Meng Hao Yu He Liu Yuechen Xing Yueting Si Si processes revealed from the study of non-model organisms.