RESEARCH ARTICLE

Genetics of white color and iridophoroma in

“Lemon Frost” leopard geckos
Longhua Guo ID1*, Joshua Bloom ID1, Steve Sykes2, Elaine Huang ID3, Zain Kashif ID1,
Elise Pham ID1, Katarina Ho ID1, Ana Alcaraz4, Xinshu Grace Xiao ID3, Sandra Duarte-
Vogel ID5, Leonid Kruglyak ID1*
1 Department of Human Genetics, Department of Biological Chemistry, Howard Hughes Medical Institute,
University of California, Los Angeles, California, United States of America, 2 Geckos Etc. Herpetoculture,
Rocklin, California, United States of America, 3 Department of Integrative Biology and Physiology, University
a1111111111 of California, Los Angeles, California, United States of America, 4 College of Veterinary Medicine, Western
a1111111111 University of Health Sciences, Pomona, California, United States of America, 5 Division of Laboratory Animal
Medicine, David Geffen School of Medicine, University of California, Los Angeles, California, United States of
a1111111111 America
a1111111111
a1111111111 * longhuaguo@mednet.ucla.edu (LG); lkruglyak@mednet.ucla.edu (LK)

Abstract
OPEN ACCESS
The squamates (lizards and snakes) are close relatives of birds and mammals, with more
Citation: Guo L, Bloom J, Sykes S, Huang E, Kashif than 10,000 described species that display extensive variation in a number of important bio-
Z, Pham E, et al. (2021) Genetics of white color and
iridophoroma in “Lemon Frost” leopard geckos.
logical traits, including coloration, venom production, and regeneration. Due to a lack of
PLoS Genet 17(6): e1009580. https://doi.org/ genomic tools, few genetic studies in squamates have been carried out. The leopard gecko,
10.1371/journal.pgen.1009580 Eublepharis macularius, is a popular companion animal, and displays a variety of coloration
Editor: Hopi E. Hoekstra, Harvard University, patterns. We took advantage of a large breeding colony and used linkage analysis, synteny,
UNITED STATES and homozygosity mapping to investigate a spontaneous semi-dominant mutation, “Lemon
Received: February 1, 2021 Frost”, that produces white coloration and causes skin tumors (iridophoroma). We localized
Accepted: May 4, 2021
the mutation to a single locus which contains a strong candidate gene, SPINT1, a tumor
suppressor implicated in human skin cutaneous melanoma (SKCM) and over-proliferation
Published: June 24, 2021
of epithelial cells in mice and zebrafish. Our work establishes the leopard gecko as a tracta-
Peer Review History: PLOS recognizes the ble genetic system and suggests that a tumor suppressor in melanocytes in humans can
benefits of transparency in the peer review
process; therefore, we enable the publication of
also suppress tumor development in iridophores in lizards.
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pgen.1009580
Author summary
Copyright: © 2021 Guo et al. This is an open
access article distributed under the terms of the The squamates (lizards and snakes) comprise a diverse group of reptiles, with more than
Creative Commons Attribution License, which 10,000 described species that display extensive variation in a number of important biologi-
permits unrestricted use, distribution, and cal traits, including coloration. In this manuscript, we used quantitative genetics and
reproduction in any medium, provided the original
genomics to map the mutation underlying white coloration in the Lemon Frost morph of
author and source are credited.
the common leopard gecko, Eublepharis macularius. Lemon Frost geckos have increased
Data Availability Statement: All sequencing data is white body coloration with brightened yellow and orange areas. This morph also displays
available from the NCBI SRA database (accession
a high incidence of iridophoroma, a tumor of white-colored cells. We obtained phenotype
number PRJNA730084).
information and DNA samples from geckos in a large breeding colony and used genome
Funding: This work is supported by the Howard sequencing and genetic linkage analysis to localize the Lemon Frost mutation to a single
Hughes Medical Institute (to LK) and the Helen Hay

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PLOS GENETICS Genetics in reptiles

Whitney Foundation (to LG). Geckos Etc.

Herpetoculture provided support in the form of locus. This locus contains a strong candidate gene, SPINT1, a tumor suppressor impli-
salary for SS. The funders had no role in study cated in human skin cutaneous melanoma. Together with other recent advances, our
design, data collection and analysis, decision to
work brings reptiles into the modern genetics era.
publish, or preparation of the manuscript.

Competing interests: Steve Sykes is the owner of

Geckos Etc. Herpetoculture. There are no patents, Introduction
products in development or marketed products
associated with this research to declare, and this Color-producing cells [1–5] contribute to animal coloration and patterns. Some cells, such as
does not alter our adherence to PLOS policies on melanocytes, produce pigments chemically. Others, such as iridophores, produce colors struc-
sharing data. turally by making crystal platelets [6–9]. Iridophores are not present in mammals, but are
widespread in insects, fish, birds, amphibians and reptiles. Different types of iridophores can
lead to different colors, including blue [10,11], yellow [12], and white [13]. The size, morphol-
ogy and organization of guanine crystals, which form reflective platelets within the irido-
phores, are considered the mechanisms of different colors [12,14–16]. The form, number and
distribution of the iridophores determine coloration and patterning of the organism [11,17–
20]. We know little of the molecular mechanisms of guanine crystal regulation [21,22]. In con-
trast with well-studied melanocytes, there have been few molecular genetic analyses involving
iridophores. A recent study found that endothelin signaling regulates iridophore development
and proliferation in zebrafish [23]. In mammals, this pathway is required for melanocyte
development [24], suggesting that signaling pathways conserved in evolution can be adapted
to regulate different types of chromatophores.
Many reptile species (e.g., geckos, chameleons, snakes) are bred in captivity as companion
animals, and breeders have established morphs with unique colors and patterns [2]. The inher-
itance of different color morphs is usually carefully documented by breeders. The common
leopard gecko, Eublepharis macularius, is an especially attractive model to study the molecular
regulation of coloration because dozens of color and pattern morphs have been established
over the past 30 years of selective breeding. These morphs either intensify a particular color
(S1A–S1I Fig) or rearrange coloration patterns (S1J–S1L Fig).
Uncontrolled proliferation of iridophores can lead to iridophoroma. White-colored irido-
phoroma is common in many reptile species [25], including green iguanas [26], captive snakes
[27], bearded dragons [28] and veiled chameleons [29]. The genetic causes of iridophoroma in
these species are unknown. Recently, histopathological findings of iridophoroma were
reported in the Lemon Frost morph of leopard geckos [30]. This morph arose as a spontaneous
mutation in a female hatchling from a cross between two wildtype leopard geckos. The muta-
tion leads to increased white body coloration and brightened yellow and orange areas. The
Lemon Frost color morph provides a unique resource for uncovering genetic regulation of iri-
dophores and iridophoroma.
A draft leopard gecko genome assembly has been published, containing 2.02 Gb of
sequence in 22,548 scaffolds, with 24,755 annotated protein-coding genes [31]. Embryonic
development in ovo and blastema-based tail regeneration have also been staged and docu-
mented in great detail [32–34]. Here, we took advantage of these established resources and
used quantitative genetics to gain insight into the molecular regulation of white color and iri-
dophoroma in leopard geckos.

Results
The Lemon Frost allele is a spontaneous semidominant mutation
A male leopard gecko carrying the lemon frost (lf) allele, Mr. Frosty (Fig 1A and 1B), was
crossed to 12 female leopard geckos of different genetic backgrounds. The F1 progeny, which

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Fig 1. The Lemon Frost mutant of the common leopard gecko, Eublepharis macularius. (A) wild type; (B) heterozygous mutant; (C)
homozygous mutant, with red arrow pointing to the eye lid; (D) blizzard mutant with minimal color; (E) Lemon Frost mutation (lf) on the
blizzard background; (F-H) segregation of the lf allele. Lemon Frost (LF) denotes heterozygotes for the mutation; super LF denotes homozygotes
for the mutation. All proportions are consistent with expectations for single-locus Mendelian inheritance (chi-square test p > 0.1).
https://doi.org/10.1371/journal.pgen.1009580.g001

were heterozygous for the lf allele, were backcrossed to the same maternal lines or intercrossed
to establish a colony of more than 900 animals (S2 Fig). Homozygous F2 intercross progeny
were named super Lemon Frost (Fig 1C). These homozygous mutants have an accentuated
color phenotype and thickened skin, which is most apparent in their eyelids (Fig 1C, red
arrow). Heterozygous Lemon Frost animals were also crossed to another mutant, Blizzard,
which is light yellow without other colors or patterns (Fig 1D). The homozygous Blizzard

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progeny carrying the lf allele displayed excessive white color in their heads and trunks, which
brightened Blizzard’s yellow color (Fig 1E). The lf allele also increased white color in the retina
(Fig 1E). The segregation pattern of Lemon Frost in pedigrees is consistent with single-locus
Mendelian inheritance (Fig 1F–1H). The lf allele is semidominant, as homozygous mutants
have more pronounced phenotypes than do heterozygotes (Fig 1B, 1C and 1F–1H).

The Lemon Frost allele leads to iridophoroma, with potential metastasis in

homozygous animals
Three heterozygous Lemon Frost animals were recently reported to develop iridophoroma
[30], a tumor of iridophores. Histopathological examination of the skin samples from homozy-
gous mutants with accentuated phenotypes showed large solid sheaths of round to polygonal
neoplastic cells that efface and expand the normal tissue architecture (S3 Fig). The cells have
abundant cytoplasm with bright brownish intracytoplasmic pigment. The nuclei are eccentric
and vary from round to fusiform. The white tumor masses stain dark with Hematoxylin and
Eosin (H&E), and remain brightly reflective under dark-field illumination (S4A and S4B Fig),
consistent with their nature as iridophores [10,35–38]. Imaging with Transmission Electron
Microscopy (TEM) showed that the lf allele led to both increased numbers of neoplastic irido-
phores and increased production of reflective platelets within each iridophore [22] (S4C Fig).
In addition to skin, other affected organs in homozygous mutants include liver, eye, and mus-
cle. The interpretation of the widespread neoplastic nodules is that the tumors are malignant
iridophoroma. The increased number of iridophores and increased production of reflective
platelets within the iridophores are the likely mechanisms of increased white color in the
Lemon Frost morph.
More than 80% of both male and female animals carrying the lf allele developed white
tumors 6 months to 5 years after birth. The tumors manifest as patches of white cells in the
skin, which are most evident on the ventral side of the animal (Fig 2A). The tumor skin can be
severely thickened and leathery (Figs 2B and S3). It is resistant to liquid nitrogen freezing, or
to Dounce homogenization, making RNA extraction infeasible. In severe cases in heterozygous
mutants, the tumors develop into skin protrusions (Fig 2C, left), which contain dense white
masses (Fig 2C, right). Tumors cover a greater fraction of the skin of homozygous mutants.
Surprisingly, these tumors rarely develop into skin protrusions as in heterozygous animals.
Instead, they manifest as well-demarcated, white, thickened patches on the ventral skin (Fig
2A), thickened layers of white masses all over the dorsal skin (Fig 2B), white, multifocal, vari-
ably sized, well-demarcated nodules in the liver, and patches of white cells in the oral cavity
(Fig 2D).

Linkage and association analysis in a breeding pedigree

To identify the genetic locus that regulates white color and tumor growth in Lemon Frost
mutants, we used restriction site-associated DNA sequencing (RAD-Seq) to genotype 188 ani-
mals from the breeding pedigree (Figs 3 and S2), including 33 super Lemon Frost (lf/lf), 116
Lemon Frost (lf/+), and 39 wild-type (+/+) individuals. We identified a total of 14,857 variants
covering 2,595 scaffolds of the genome assembly. To map the Lemon Frost locus, we tested the
effect of allelic dosage at each marker on white coloration of the geckos in a standard semi-
dominant association mapping framework, accounting for population structure through the
use of marker-based relatedness. We used a p-value threshold of 7.09e-5 (Methods) to control
the false positive rate at 1%. Forty-eight markers on 31 scaffolds were significantly associated
with white coloration (S1 Table). The top two association signals corresponded to scaffolds
6052 and 996.

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Fig 2. Tumor growth and metastasis in the Lemon Frost mutant. Designations are homozygous mutant (lf/lf); heterozygous
mutant (lf/+); wild type (+/+). (A) tumors in ventral skin; (B) thick layers of white tumor cells (lf/lf) vs. normal white cells (+/+);
(C) outgrowth of white tumor cells (lf/+); (D) metastasis of white tumor cells in the liver and oral cavity. Red arrows: white colored
tumor cells. Arrowhead in B: normal white cells.
https://doi.org/10.1371/journal.pgen.1009580.g002

Synteny analysis and homozygosity mapping

Because the gecko genome assembly is highly fragmented, we used synteny to examine
whether the 31 scaffolds associated with coloration belong to a single genomic interval.
We compared the gecko scaffolds to homologous regions of several vertebrate species with
chromosome-scale genome assemblies: green anole [39], chicken [40] and human [41]. We

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Fig 3. Localization of the Lemon Frost mutation. (A) p-value for association with white color and (B) linkage disequilibrium for 28 markers syntenic to chicken
chromosome 5 (red, ordered by synteny), 4 markers syntenic to chromosome 7 (cyan), and 16 markers without synteny information (green). Randomly selected markers
that are not associated with white color (purple). (C) A schematic of the region showing synteny and gene annotation. (D) Fraction of markers showing expected allele
frequency pattern in pools, plotted for 10kb windows along scaffold 996. The four windows with the highest fraction are marked by asterisks and span the location of the

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gene SPINT1. Windows with fewer than 5 variants were not plotted (dashed red lines). (E) Genome-wide distribution of the fraction of markers showing expected allele
frequency pattern in pools for all 10 kb windows. The 4 highest windows on scaffold 996 (red arrows) marked in D are among the 6 highest windows in the entire genome.
https://doi.org/10.1371/journal.pgen.1009580.g003

found that 17 of the 22 scaffolds that have synteny information (including scaffolds 6052 and
996) correspond to one region on chicken chromosome 5, human chromosome 15, and green
anole chromosome 1 (Fig 3A–3C and S1 Table). Three additional scaffolds also have synteny
to the green anole chromosome 1. The remaining two scaffolds without synteny to the green
anole chromosome 1 were more weakly associated with coloration. The 28 markers on these
17 scaffolds are in linkage disequilibrium (Fig 3B), which decays with distance when markers
are ordered by synteny (Fig 3B). These results indicate that a single genomic region is associ-
ated with the Lemon Frost phenotype, as expected for a new mutation with a Mendelian segre-
gation pattern.
To narrow down the location of the causal gene within this genomic region, we used whole
genome sequencing and homozygosity mapping. We pooled DNA from 25 super Lemon Frost
genomes (lf/lf), 63 Lemon Frost genomes (lf/+), and 71 wildtype geckos (+/+) and sequenced
each pool to 30x coverage. We reasoned that the lf mutation in Mr. Frosty and its flanking vari-
ants should form a haplotype that would be found in the super Lemon Frost pool with 100%
frequency, in the Lemon Frost pool with 50% frequency, and would not be seen in the wildtype
pool. We scanned the genome in 10 kb windows and measured the fraction of heterozygous
variants from Mr. Frosty that followed this expected pattern in the pools (S2 Table). This statis-
tic was highest for a window on scaffold 996 (S2 Table and Methods), the main candidate scaf-
fold from statistical mapping (S1 Table). The expected frequency pattern was observed for 20
of 22 variants in this window (630-640kb on scaffold 996). Four of the top six intervals fall in
the region from 570kb to 640kb on scaffold 996, with the signal decaying with distance away
from this region (Fig 3D and 3E). The linkage between this region and Lemon Frost was repli-
cated in an independent 3-generation backcross between Mr. Frosty and a Sunburst Tangerine
morph (Fig 4 and S3 Table). These results indicate that scaffold 996 contains the Lemon Frost
mutation.

SPINT1 is a strong candidate gene for the Lemon Frost phenotype

The genomic interval spanning positions 570kb-640kb on scaffold 996 contains a single gene,
SPINT1. SPINT1 (serine peptidase inhibitor, Kunitz type 1), also known as hepatocyte growth
factor activator inhibitor type 1 (HAI-1), is a transmembrane serine protease inhibitor
expressed mainly in epithelial cells [42–44]. It is the only gene in the larger associated region
reported to be a suppressor of epithelial cell tumors in model organisms and in humans
[42,45–56]. Because the breeding and transmission data indicate that the lf allele arose from a
single spontaneous mutation, we reasoned that a mutation disrupting SPINT1 causes the over-
proliferation of white-colored skin cells in Lemon Frost geckos.
The Lemon Frost SPINT1 allele differs from the reference genome assembly at two posi-
tions in the exons, as well as at 147 positions in the introns and the 3’UTR (S4 Table). This
large number of variants is a consequence of differences in genetic background between Mr.
Frosty’s parents and the non-Lemon Frost individual used to generate the reference, and
makes it challenging to identify the causal mutation. Both differences in the coding sequence
of SPINT1 are synonymous. Notable differences in non-coding regions include 7 large
insertion/deletions (indels) in the introns and a 13-nucleotide insertion in the 3’UTR
(CAAGTGTATGTAT). Indels in introns and promoters of SPINT1 have been reported to
lead to loss of SPINT1 function in fish and mice [51,52,56].

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Fig 4. The lemon frost allele in a backcross. (A) We genotyped 7 progeny with the Lemon Frost phenotype and 6 wild type progeny from the third generation of
a backcross of Mr. Frosty to the Sunburst line for markers in the SPINT1 region and observed a consistent inheritance pattern. (B) Sequencing chromatogram of a
heterozygous animal (lf/+) at an insertion marker. (C) Sequencing chromatogram of a homozygous animal (+/+) at the same insertion marker.
https://doi.org/10.1371/journal.pgen.1009580.g004

We used BLASTn to examine whether some of the non-coding differences between the ref-
erence and the Lemon Frost SPINT1 alleles are located in well-conserved vertebrate sequences.
We found that intron 1 has >90% sequence identity to Gekko japonicus and >80% sequence
identity to Zootoca vivipara. Intron 8 has >80% sequence identity to Saurodactylus brosseti.
The 3’ UTR has 79% identity to Gekko japonicus. We also found that human SPINT1 introns
1/2 and the 3’ UTR are the most conserved non-coding regions of the gene among 100

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vertebrates by PhyloP analysis [57–59], with conservation scores that are comparable to those
for exons 8 and 10. The ENCODE candidate cis-regulatory elements (cCRE) analysis showed
that introns 1/2 and the 3’ UTR are the major regions with multiple candidate enhancers
[57,60]. Similar results were obtained for the mouse SPINT1 gene. These observations suggest
that some of the 22 sequence variants in intron 1 or 20 variants in the 3’ UTR of the leopard
gecko SPINT1 may be functionally important.
Sequencing of RNA extracted from normal gecko skin and from skin peripheral to tumors
in homozygous mutants confirmed that SPINT1 is expressed in this tissue (S5 Fig). However,
we did not observe a significant difference between homozygous mutants and wildtype geckos
in SPINT1 mRNA levels or splicing patterns. This result suggests that the putative causal muta-
tion in SPINT1 may alter translation or protein activity, rather than transcription. Alterna-
tively, the mutation might reduce SPINT1 expression only in tumors, which are refractory to
RNA extraction as noted above.

Discussion
Several lines of evidence support our hypothesis that a defect in SPINT1 causes iridophoroma
in Lemon Frost geckos. First, SPINT1 function is dosage-dependent, consistent with our
observation that Lemon Frost is a semi-dominant phenotype. In humans, carcinoma tissues
in vivo and carcinoma-derived cell lines in vitro have reduced SPINT1 on the cell membrane
[61,62] through enhanced shedding of the extracellular domain or decreased mRNA or protein
expression. Reduced expression of SPINT1 has been associated with a negative prognosis of
human Skin Cutaneous Melanoma (SKCM) [45] and pancreatic ductal adenocarcinoma [46].
Knockdown of SPINT1 expression by siRNA in cancer cell lines led to increased invasion or
metastasis [47,62,63]. Second, loss of SPINT1 function in fish and mice leads to tumor forma-
tion in epithelial cells. In mice, homozygous deletion of SPINT1 leads to disrupted placental
basement membranes and embryonic lethality [52,54]. Rescued mosaic animals developed
scaly skin with hyperkeratinization [55]. Intestine-specific deletion of SPINT1 leads to
increased tumor growth of intestine epithelium [48]. Increased expression of SPINT1 in the
skin abrogated matriptase-induced spontaneous skin squamous cell carcinoma [64]. In zebra-
fish, reduced expression led to hyperproliferation of basal keratinocytes [51] and enhanced
proliferation of epithelial cells [56]. Furthermore, SPINT1 deficiency was used to establish a
disease model for Skin Cutaneous Melanoma (SKCM) in zebrafish [45]. In all three studies in
zebrafish, skin inflammation was observed. Third, insertions in introns [51,52] and promoters
[56] have caused loss of SPINT1 function. Together with our genetic localization of the lf locus
to SPINT1, these lines of evidence make this gene a very strong candidate for the Lemon Frost
phenotype.
Molecular genetics in reptiles is not well established due to long reproductive cycles and
challenges in laboratory breeding. Early work focused on careful documentation of patterns of
inheritance [2,65]. Molecular studies have examined sequence variants in a candidate pigmen-
tation gene, melanocortin-1 receptor, and their association with melanic or blanched pheno-
types in different species and ecological niches [66–73]. Recent work in the wall lizard [74] and
the corn snake [75] used a similar sequencing-based approach to our study to identify the
molecular basis of coloration polymorphisms in these species. Successful use of CRISPR-
Cas9-mediated gene editing to mutate the tyrosinase gene has been reported in the lizard
Anolis sagrei [76]. Although this species is only distantly related to the leopard gecko, this
advance offers promise that targeted studies of the role of SPINT1 mutations in the Lemon
Frost phenotype will become possible.

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Most of our knowledge about molecular and cellular regulation of iridophores derives from
work in zebrafish [11,13,17,19,23,77–85]. Interestingly, few cases of iridophoroma have been
reported in zebrafish [86]. Our data suggest that an evolutionarily conserved gene, SPINT1,
regulates the proliferation of white iridophores in the leopard gecko. Increased production of
reflective platelets in Lemon Frost iridophores is a novel finding that warrants further investi-
gation, for instance by manipulating SPINT1 expression. The tumor suppressor function of
SPINT1 establishes a potential link between iridophoroma and regulation of white coloration
in reptiles. Our work suggests that cancer genes can play as important a role in iridophores as
they do in melanocytes and melanoma [87], and that Lemon Frost leopard geckos may serve
as a disease model to study Skin Cutaneous Melanoma.

Methods
Ethics statement
All activities involving animals included in this manuscript were approved by the University of
California, Los Angeles (UCLA) Institutional Animal Care and Use Committee, approval
number: ARC #2018–035 (6/19/2018~6/18/2021).

Gecko maintenance and experimental procedures

Leopard geckos were acquired from a commercial breeder. Housing conditions at UCLA
included: room temperature of 70–80 F, cage temperature of 72–95 F, room relative humidity
between 30–60%, and a 12:12 hours light cycle. A heating pad was provided at one side of the
cage to establish a temperature gradient. Animals were singly housed in polycarbonate cages
with cardboard lines (Techboard) at the bottom, water was provided in bowls inside the cage,
and PVC pipe pieces and plastic plants were offered as environmental enrichment. Geckos
were fed 2–6 fresh crickets and 2–4 mealworms three times per week.
Geckos were euthanized with an intracoelomic injection of sodium pentobarbital (Eutha-
sol) at a dose of 100–200 mg/Kg. Immediately after euthanasia, a necropsy was performed,
including external examination, body and organ weighing, gross assessment of normal and
abnormal tissues, and tissue collection for histopathology processing and assessment. Normal
and abnormal tissues were fixed in 10% formalin, embedded in paraffin, sectioned, and stained
with H&E for pathologic evaluation.

Phenotyping
Lemon Frost and super Lemon Frost phenotypes were scored by experienced breeders who
used visual inspection to determine an increase in white color of the body, eye, and belly com-
pared to normal wildtype animals. The Lemon Frost mutation increases the white base color
of the gecko over its entire body. This results in an overall brightening of white, yellow, and
orange colors of the gecko. The effects of the mutation can also be observed in a whitening of
the eyes and in white color spreading down the sides onto the belly. Pictures were taken for
each animal to document the phenotypes.

Genotyping
Genomic DNA was extracted from fresh tail tips with Easy-DNA gDNA purification kit
(K180001, ThermoFisher), or from the saliva with PERFORMAgene (PG-100, DNAgenotek).
Genomic DNA extracted from saliva was further purified with ethanol precipitation before
genotyping assays. DNA libraries for whole genome sequencing were prepared with Nextera
DNA Library Prep Kit (FC-121-1031, Illumina). Libraries for RADseq were prepared

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according to the procedures of Adapterama III [88] with few modifications. Libraries were
sequenced on a HiSeq 3000 (Illumina).
Only scaffolds larger than 5kb in the draft genome assembly were used as a reference. RAD-
seq reads and Whole Genome Sequencing (WGS) reads were aligned to the leopard gecko
draft genome [31] with bwa mem (version 0.7.17) [89]. Variants for WGS were identified with
GATK (version 4.1.4.1) [90]. Variants for RADseq were identified with Stacks (version 2.41)
[91,92]. All variants were filtered with VCFtools (version 0.1.14) [93]. First, variants had to be
are bi-allelic and heterozygous in Mr. Frosty. The ratio of the reads for the two alleles was
required to be in the range 0.4–0.6. Second, only high-quality variants were used in homozy-
gosity mapping or statistical mapping (DP> = 30, GQ> = 30).

Transcriptome sequencing
Skin tissue samples around 6mm in diameter were taken from the ventral side of the geckos
after anesthetization with 1–5% isoflurane. As tumor tissues are refractory to RNA extraction,
flanking tumor-free tissue samples were taken for homozygous Lemon Frost animals. All sam-
ples were homogenized with TissueRuptor in buffer RLT immediately after collection. Lysates
were immediately frozen on dry ice until all tissues were collected from animals. Then all
lysates were centrifuged for 5 minutes at 13,000 rpm to remove debris. Supernatants were
taken to fresh tubes, and mRNA was extracted according to the procedures of RNeasy Fibrous
Tissue Mini Kit (74704, QIAGEN).
Libraries of extracted mRNA were prepared with RNA HyperPrep kit (KAPA) and
sequenced on a HiSeq 3000 (Illumina). RNA-seq reads were mapped to the leopard gecko
draft genome [31] using HISAT2 with default parameters. Identification of alternative and dif-
ferential splicing events was performed using JuncBase [94]. Gene expression was compared
using Sleuth [95] after RNA transcript abundance was quantified using Kallisto [96].

Pathology
Complete postmortem examination was performed, and representative tissue samples were
obtained. All tissues obtained at necropsy were preserved in 10% neutral-buffered formalin
solution for up to 5 days before being processed and embedded in paraffin. All tissues were
sectioned at 5 μm, and routinely stained with Hematoxylin and Eosin.

Statistical mapping
Biallelic markers with minor allele frequency of less than 5% and with fewer than 10 individu-
als called as homozygous for both the reference and alternative alleles were excluded from
mapping and kinship matrix construction. A kinship matrix was calculated using the function
A.mat with default parameters from the rrBLUP [97] R package. Phenotype was encoded as 0
for wild type, 1 for Lemon Frost, and 2 for super Lemon Frost. Association statistics between
this phenotype vector and marker genotypes were computed using the function gwas2 in the
NAM [98] R package using a linear mixed model with a random effect of kinship to control
for population structure. The effective number of tests was computed to be 141.1 based on the
procedure of Galwey et al [99]. A family-wise error rate significance threshold was calculated
as 0.01/141.1 or p<7.09e-5.

Homozygosity mapping
Pooled animals and Mr. Frosty were sequenced to ~30x coverage on a HiSeq 3000 (Illumina).
Variants were identified with GATK and filtered with VCFtools. Biallelic heterozygous

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variants from Mr. Frosty, including indels, were used as markers to localize the Lemon Frost
mutation. Allele ratios (AF) were calculated by dividing the read count of alternative alleles by
the sum of the counts of reference alleles and alternative alleles. Variants closely linked to the
Lemon Frost mutation are expected to have AF between 0.4 and 0.6 in the Lemon Frost pool
and in Mr. Frosty, AF > 0.85 in the super Lemon Frost pool, and AF < 0.15 in the wildtype
pool. The number of variants meeting these criteria was counted for every 10kb genome inter-
val. The fraction of such variants among all variants heterozygous in Mr. Frosty within the
interval was then calculated. Intervals with fewer than 5 variants were excluded because they
could not provide statistically meaningful results.

Transmission electron microscopy

Dissected skin tissues were fixed in 2.5% glutaraldehyde and 4% formaldehyde in 0.1 M
sodium cacodylate buffer overnight at 4˚C. After being washed in PBS, samples were post-
fixed in 1% osmium tetroxide in 0.1M sodium cacodylate, and dehydrated through a graded
series of ethanol concentrations. After infiltration with Eponate 12 resin, the samples were
embedded in fresh Eponate 12 resin and polymerized at 60˚C for 48 hours. Ultrathin sections
of 70 nm thickness were prepared, placed on formvar-coated copper grids, and stained with
uranyl acetate and Reynolds’ lead citrate. The grids were examined using a JEOL 100CX trans-
mission electron microscope at 60 kV, and images were captured by an AMT digital camera
(Advanced Microscopy Techniques Corporation, model XR611).

Supporting information
S1 Fig. Coloration and pattern diversity of the common leopard gecko, Eublepharis macu-
larius. (A) wild type; (B) black night; (C) variant of black night; (D) granite snow; (E) gem
snow; (F) white knight; (G) sunburst tangerine; (H-I) variants of sunburst tangerine; (J) red
stripes; (K) bold stripes; (L) rainbow.
(TIF)
S2 Fig. Breeding pedigree of the Lemon Frost mutation. Mr. Frosty, the original carrier of
the spontaneous Lemon Frost mutation, was bred to 12 female geckos from different genetic
backgrounds. F1s carrying the lf allele were bred among themselves or back to their female
parent, producing the second generation of animals heterozygous or homozygous for the lf
allele. Blue: lf/lf; green: lf/+; red: +/+. Dashed line: same individual/line.
(TIF)
S3 Fig. Histopathology of skin tumors. (A) Thick layers of white tumor tissue (star) infiltrat-
ing white skin (arrow). (B) Skin biopsies organized and fixed in a paper roll for sectioning. (C)
H&E staining of the skin sections. Arrow: skin; star: infiltrated tumor mass. (D) H&E staining
of the skin sections showing normal skin cells and neoplastic cells (star). Neoplastic cells have
eccentric and condensed nuclei.
(TIF)
S4 Fig. Potential metastasis of iridophoroma. (A) In normal skin, cell nuclei are oval and
perpendicular to the skin surface. In Lemon Frost skin, cell nuclei are flat, elongated and paral-
lel to the skin, reminiscent of epithelial-to-mesenchymal transition. (B) Iridophoroma in the
liver, stained dark in H&E sections. In dark field imaging, iridophores are bright white. Such
iridophores invade blood vessels in the tissue (red arrows). (C) In TEM imaging, white tumor
skins in super LF are filled with abundant iridophores with excessive brightly reflective crystals
(Tumor). In normal skin, iridophores are much fewer and have less crystals (Normal).
(TIF)

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PLOS GENETICS Genetics in reptiles

S5 Fig. SPINT1 expression in gecko skin. SPINT1 mRNA reads from transcriptome sequenc-
ing were aligned to the genome and visualized in IGV. Top 3 rows show samples from homo-
zygous mutants. Bottom 3 rows show samples from wild type geckos. Skin tissue adjacent to
the tumors was used in the mutants. Peaks mark SPINT1 exons. The last exon on the right is
transcribed together with the 3’UTR.
(TIF)
S1 Table. Scaffolds associated with Lemon Frost phenotype.
(XLSX)
S2 Table. Candidate Lemon Frost mutations on SPINT1.
(XLSX)
S3 Table. Genotyping results of the backcross.
(XLSX)
S4 Table. Candidate and background mutations in homozygosity mapping.
(XLSX)

Acknowledgments
We thank Aaron Miller, Jasmine Gonzalez, Kendall Placido and James Walter for their assis-
tance in gecko DNA collection and phenotyping. We thank members of the Kruglyak lab for
helpful feedbacks on the project, and Giancarlo Bruni, Stefan Zdraljevic, Eyal Ben-David and
Olga Schubert for helpful comments on the manuscript. We thank Chunni Zhu (Electron
Microscopy Core Facility, UCLA Brain Research Institute) for her assistance in TEM sample
processing and imaging. We thank Jonathan Eggenschwiler and Douglas Menke for helpful
discussions.

Author Contributions
Conceptualization: Longhua Guo, Leonid Kruglyak.
Data curation: Longhua Guo, Zain Kashif, Elise Pham, Katarina Ho.
Formal analysis: Longhua Guo, Joshua Bloom, Elaine Huang, Xinshu Grace Xiao.
Funding acquisition: Longhua Guo, Leonid Kruglyak.
Investigation: Longhua Guo, Joshua Bloom, Steve Sykes, Ana Alcaraz, Sandra Duarte-Vogel,
Leonid Kruglyak.
Methodology: Longhua Guo, Joshua Bloom, Ana Alcaraz, Sandra Duarte-Vogel, Leonid
Kruglyak.
Project administration: Longhua Guo, Leonid Kruglyak.
Resources: Steve Sykes.
Software: Joshua Bloom, Elaine Huang.
Supervision: Xinshu Grace Xiao, Leonid Kruglyak.
Validation: Longhua Guo, Leonid Kruglyak.
Visualization: Longhua Guo, Leonid Kruglyak.
Writing – original draft: Longhua Guo, Leonid Kruglyak.

PLOS Genetics | https://doi.org/10.1371/journal.pgen.1009580 June 24, 2021 13 / 19

PLOS GENETICS Genetics in reptiles

Writing – review & editing: Longhua Guo, Zain Kashif, Katarina Ho, Ana Alcaraz, Sandra
Duarte-Vogel, Leonid Kruglyak.

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