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Genetics of white color and iridophoroma in “Lemon Frost” leopard geckos

['Longhua Guo', 'Department Of Human Genetics', 'Department Of Biological Chemistry', 'Howard Hughes Medical Institute', 'University Of California', 'Los Angeles', 'California', 'United States Of America', 'Joshua Bloom', 'Steve Sykes']

Date: 2021-08

The squamates (lizards and snakes) are close relatives of birds and mammals, with more than 10,000 described species that display extensive variation in a number of important biological traits, including coloration, venom production, and regeneration. Due to a lack of genomic tools, few genetic studies in squamates have been carried out. The leopard gecko, Eublepharis macularius, is a popular companion animal, and displays a variety of coloration patterns. We took advantage of a large breeding colony and used linkage analysis, synteny, and homozygosity mapping to investigate a spontaneous semi-dominant mutation, “Lemon Frost”, that produces white coloration and causes skin tumors (iridophoroma). We localized 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 of epithelial cells in mice and zebrafish. Our work establishes the leopard gecko as a tractable genetic system and suggests that a tumor suppressor in melanocytes in humans can also suppress tumor development in iridophores in lizards.

The squamates (lizards and snakes) comprise a diverse group of reptiles, with more than 10,000 described species that display extensive variation in a number of important biological traits, including coloration. In this manuscript, we used quantitative genetics and genomics to map the mutation underlying white coloration in the Lemon Frost morph of the common leopard gecko, Eublepharis macularius. Lemon Frost geckos have increased white body coloration with brightened yellow and orange areas. This morph also displays a high incidence of iridophoroma, a tumor of white-colored cells. We obtained phenotype information and DNA samples from geckos in a large breeding colony and used genome sequencing and genetic linkage analysis to localize the Lemon Frost mutation to a single locus. This locus contains a strong candidate gene, SPINT1, a tumor suppressor implicated in human skin cutaneous melanoma. Together with other recent advances, our work brings reptiles into the modern genetics era.

Competing interests: Steve Sykes is the owner of Geckos Etc. Herpetoculture. There are no patents, products in development or marketed products associated with this research to declare, and this does not alter our adherence to PLOS policies on sharing data.

Funding: This work is supported by the Howard Hughes Medical Institute (to LK) and the Helen Hay Whitney Foundation (to LG). Geckos Etc. Herpetoculture provided support in the form of salary for SS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Guo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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 documented 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 iridophoroma in leopard geckos.

Uncontrolled proliferation of iridophores can lead to iridophoroma. White-colored iridophoroma 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 mutation 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 iridophores and iridophoroma.

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 inheritance 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 ).

Color-producing cells [ 1 – 5 ] contribute to animal coloration and patterns. Some cells, such as melanocytes, produce pigments chemically. Others, such as iridophores, produce colors structurally 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, morphology and organization of guanine crystals, which form reflective platelets within the iridophores, 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 contrast 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.

Results

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 animals 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. PPT PowerPoint slide

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larger image TIFF original image Download: 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 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

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 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 associated with the Lemon Frost phenotype, as expected for a new mutation with a Mendelian segregation 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 variants 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 statistic was highest for a window on scaffold 996 (S2 Table and Methods), the main candidate scaffold 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 replicated 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. PPT PowerPoint slide

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larger image TIFF original image Download: 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

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