A nonsense mutation in TFEC is the likely cause of the recessive piebald phenotype in ball
pythons (Python regius)
*Alan Garcia-Elfring, Heather L. Roffey, *Andrew P. Hendry, and *Rowan D. H. Barrett
*Redpath Museum and Department of Biology, McGill University, Montreal, QC, Canada
The corresponding author's email address: firstname.lastname@example.org
Captive-bred ball pythons (Python regius) represent a powerful model system for studying the
genetic basis of colour variation and Mendelian phenotypes in vertebrates. Although hundreds
of Mendelian phenotypes (colour morphs) affecting colouration and patterning have been
described for ball pythons, the genes causing these colour morphs remain unknown. Here, we
used crowdsourcing of samples from commercial ball python breeders to investigate the
genetic basis of a classic phenotype found in the pet trade, the piebald [characterized by
dorsolateral patches of unpigmented (white) skin]. We used whole-genome sequencing of
pooled samples followed by population genetic methods to delineate the genomic region
containing the causal gene. We identified TFEC of the MIT-family of transcription factors as a
candidate gene. Functional annotation of SNPs identified a nonsense mutation in TFEC, which
we conclude is the likely causal variant for the piebald phenotype. Our work shows that ball
python colour morphs have the potential to be an excellent model system for studying the
genetic basis of pigment variation in vertebrates, and highlights how collaborations with
commercial breeders can accelerate discoveries.
The study of colour variation has a long history in evolutionary biology and genetics (Caro 2017;
Cuthill et al. 2017; Harris et al. 2019). Over 150 years ago, crossing yellow and green peas
helped Mendel to discover that traits are inherited as discrete hereditary units or “factors”
(Mendel 1866; reviewed by Ellis et al. 2011). Once Mendel’s work was rediscovered in 1900,
invertebrate colour variation helped establish that Mendel’s “factors”, which we now call
genes, were located on chromosomes (Bridges 1919; Harrison 1920). Further, colour variation
in vertebrates has provided insights in many fields, particularly the biomedical sciences (e.g.
Pingault et al. 1998; Tachibana et al. 2003; Richards et al. 2019), developmental biology (e.g.
Cohen et al. 2016; Haupaix et al. 2018), and evolutionary biology (e.g. Schweizer et al. 2018;
Barrett et al. 2019; Burgon et al. 2020; reviewed by San-Jose and Roulin 2017). However, our
current knowledge of the genetics of vertebrate colouration comes primarily from studies on
model species, particularly the mouse (e.g. Baxter et al. 2004; Hoekstra 2006; San-Jose and
Roulin 2017; but see Ullate-Agote et al. 2020). This bias in the literature provides a limited view
of the ways in which genetic variation can affect vertebrate colouration.
Although mice, as all mammals and birds, have one pigment-producing cell type
(melanocytes) which synthesizes melanin, reptiles, like fish and amphibians, have three colour-
producing cells or chromatophores (reviewed by Bechtel 1978; Mills and Patterson 2009;
Olsson et al. 2013). In addition to melanin-producing cells, called melanophores, reptiles have
xanthophores and iridiophores that contribute to colouration. Xanthphores synthesize yellow
pteridine-based pigments and can also contain red carotenoid pigments that are obtained from
diet (often called erythrophores). Iridiophores do not contain pigments but instead produce
structural colouration by reflecting light on guanine nanocrystals (Teyssier et al. 2015). The
three colour-producing cells interact in 3D space (Grether et al. 2004; Saenko et al. 2013) to
generate the remarkable range of colour variation observed in reptiles.
We propose that the ball python (Python regius; Figure 1a) is an excellent model for
studying the genetic basis of vertebrate colouration (Irizarry and Bryden 2016). Ball pythons are
native to western Sub-Saharan Africa, with a range extending from Senegal to Uganda. With a
maximum length of approximately 6 feet long, ball pythons are small relative to their better-
known Asian congener, the Burmese python (Python bivittatus). Owing to their small size,
docility, and simple husbandry, ball pythons rose in popularity in the pet trade, particularly
among reptile enthusiasts. An additional factor that has contributed to the ball python’s
popularity was the discovery of rare phenotypes that affect colouration. In Ghana, claims of
rare wild ball pythons with altered patterning go back as early as 1966. In the early 1990s, one
such ball python was caught, a piebald. This individual had patches of unpigmented (white) skin
(e.g. Figure 1b-d), a pigment deficiency found in many vertebrates (Ahi and Sefc 2017),
including humans (Oiso et al. 2013). However, it would take a few years to rule out
environmental factors as a causal explanation for the piebald phenotype in the ball python
caught from the wild. By 1998, Peter Kahl had reproduced the piebald phenotype in captivity
(called ‘proving’ by commercial breeders) and showed it had a recessive mode of inheritance (A
brief history of Royal Ball Python morphs for beginners, n.d.). This pioneering work of breeders
and hobbyists spurred the discovery and propagation of numerous additional and highly diverse
colour morphs (e.g. Figure 1b-g). Today, 314 Mendelian phenotypes (i.e., phenotypes that
segregate as a single Mendelian factor or major quantitative trait locus) are bred in captivity
(known as ‘basic’ morphs; http://www.worldofballpythons.com/morphs), providing a valuable
resource for studying the genetics of vertebrate colouration in great detail. Indeed, commercial
breeders have taken advantage of epistasis and produced over 6000 unique phenotypes
(‘designer morphs’) with different combinations of the Mendelian colour factors.
Ball pythons therefore provide an outstanding opportunity to study the genetic basis of
Mendelian phenotypes related to pigmentation and patterning. For example, ball python colour
morphs can be characterized by changes to pattern or to pigmentation or both. Phenotypes
with changes to pigmentation but not pattern (Figure 1c) are presumably determined by
changes to genes with roles in a pigment synthesis pathway. On the other hand, aberrations to
pattern, like white-spotting, are thought to occur through mutations in genes with functions in
progenitor cell migration and differentiation from the neural crest (reviewed by Mills and
Patterson 2009). However, most research on colouration, vertebrate and invertebrate, has
focused on pigmentation, particularly melanin-based pigmentation (e.g. San-Jose and Roulin
2018), rather than on patterning (Mills and Patterson 2009). The extensive phenotypic variation
in pigment and pattern found in captive-bred ball pythons can therefore help correct the bias
(e.g. Ullate-Agote et al. 2020) associated with mammalian models and melanin-based
pigmentation in the scientific literature on vertebrate colouration.
In this study, our goal was to discover the candidate mutation(s) that produce the
Mendelian phenotype affecting pattern in ball pythons, the piebald (Figure 1b). We used
crowdsourcing of samples from commercial breeders, thereby bridging a gap between
academia and industry. Genes known to cause white-spotting in other vertebrates, like KIT,
MITF, EDNRB, and SOX10 (Fleischman et al. 1991; Baxter et al. 2004; Ahi and Sefc 2017)
represent a priori candidate genes, but given the differences in pigmentation development
between reptiles and previously studied model systems, we employed a whole-genome
approach to facilitate discovery of unknown candidate genes. We sequenced whole-genomes of
pooled samples and then applied population genetic methods to delineate the genomic region
of interest and identify candidate genes. We then functionally annotated SNPs to identify the
putative causal mutation. Given the character of the piebald phenotype (i.e., dorsolateral
patches of unpigmented skin), we expected the candidate gene to be expressed in the neural
crest and associated with chromatophore migration and differentiation.
Sampling and DNA extraction
We obtained ball python samples by crowd-sourcing from commercial breeders in Canada
(Mutation Creation, T. Dot Exotics, The Ball Room Canada, Designing Morphs), who supplied us
with shed skin. We then used shed skin samples for 47 piebald individuals (i.e., individuals
homozygous for the piebald variant; Table S1) and 52 non-piebald individuals (Table S2).
Although individuals from both sets of samples contained additional basic morphs, the only
consistent difference between the two was the piebald phenotype. We attempted to maximize
the number of individuals that come from different families to minimize the effects of
population structure, although there were some exceptions (Table S2). From each sample we
used approximately 0.1 g of shed skin, cut to small pieces using scissors, for DNA extraction. We
extracted DNA following a standard phenol-chloroform procedure, with a 24-hour proteinase-K
incubation time at 37 °C. Piebald and non-piebald samples were prepared on different working
days to avoid contamination. We quantified all samples using a Picogreen® ds DNA assay
(Thermo Fisher Scientific, Waltham, USA) on an Infinite® 200 Nanoquant (Tecan Group Ltd.
After DNA extraction, we mixed DNA of individuals (according to phenotype) in equimolar
amounts to obtain a single pool for each phenotype, ‘piebald’ and ‘non-piebald.’ We used PCR-
based whole-genome libraries for both pools, which were prepared at the McGill University and
Genome Quebec Innovation Center, Montreal, Canada. We sequenced 150 bp paired-end reads
using two lanes of Illumina HiSeqX.
To process raw reads, we applied filters based on read quality and length, keeping reads with a
minimum quality of 20 (--quality-threshold 20) and a length of 50 bp (--min-length 50). We then
aligned processed reads to the Burmese python (Python bivittatus) draft assembly Pmo2.0
(Castoe et al. 2013) using the program NextGenMap (Sedlazeck et al. 2013). NextGenMap was
designed for aligning reads to highly polymorphic genomes or genomes of closely related
species. We used SAMtools (Li et al. 2009) to convert SAM files to BAM format and remove
reads with mapping quality below 20 (samtools view -q 20). We filtered for PCR duplicates using
the program MarkDuplicates of Picard Tools (Wysoker et al. 2013). We then created a mpileup
file (samtools mpilep -B) from which the synchronized (sync) file was produced using
Popoolation2 (Kofler et al. 2011b). The sync file contains read counts for all nucleotides
sequenced in the genome and it is used for subsequent downstream analyses (e.g. FST scan).
We also applied the same protocol as above but instead aligned reads to the chromosome-
length Burmese python reference genome, Python_molurus_bivittatus-5.0.2_HiC.assembly
(Dudchenko et al. 2017, 2018).
Identification of fixed SNPs and delineation of the candidate genomic region
We applied an FST scan to identify fixed SNPs across the two pools. For this procedure, we used
the fst-sliding.pl script of Popoolation2 (--min-count 10, --min-coverage 20, --max-coverage 500,
--min-covered-fraction 0, --window-size 1, --step-size 1, --pool-size 47:52, --suppress-
noninformative). We then identified candidate SNPs as those with FST estimates of 1 and
mapped them to genes. We used a custom BASH script to map fixed SNPs to genes in the gene
annotation file using scaffold and SNP position. Because the draft assembly is highly
fragmented (Castoe et al. 2013), we also applied the same FST scan on data aligned to the
chromosome-length genome assembly (Dudchenko et al. 2017, 2018) to get a better
delineation of the genomic region of interest within candidate SNPs. However, this latter
assembly is not annotated with genetic features – hence necessitating the use of both
Functional annotation of SNPs
We next functionally annotated genome-wide SNPs with the software snpEff (Cingolani et al.
2012) to aid in identifying the putative causal mutation for the piebald phenotype. SnpEff was
designed for annotating and predicting the effects of SNPs, such as amino acid changes. This
program provides an assessment of the impact of a SNP, including ‘HIGH’ (e.g. stop codon),
‘MODERATE’ (e.g. non-synonymous change), ‘LOW’ (e.g. synonymous change), or ‘MODIFIER’
(change in an intergenic area).
From the BAM files, we obtained an average depth of coverage of 50.5 and 52.6 for the piebald
and non-piebald pools, respectively. After filtering, our dataset consisted of 3,095,304 SNPs for
the draft assembly and 3,221,285 SNPs in the chromosome-length assembly.
FST scan for fixed SNPs and candidate genomic region
Across all SNPs, we found an average FST of 0.03456 – relatively low, indicating that population
structure was minimized. Using the draft assembly, we identified 129 fixed SNPs (FST = 1.0) and
369 SNPs with FST > 0.9. Plotting the FST values (Figure S1) shows peaks across the reference
genome, demonstrating the fragmented state of the draft assembly. In the chromosome-length
assembly, we found 131 fixed SNPs (FST = 1.0) and 372 SNPs with FST > 0.9. The chromosome-
length assembly also shows that 128/131 fixed SNPs and 365/372 SNPs with FST > 0.9 map to a
single region on scaffold 7, clearly delineating a genomic region of interest (Figure 2). The 128
fixed SNPs map to a region 8 Mb long (scaffold 7: 49526089-57612101). This region becomes
26.5 Mb when considering the 365 SNPs with FST > 0.9 (scaffold 7: 49246858-76754467).
However, most of these 365 SNPs cluster within the 8 Mb region demarcated by fixed SNPs,
with only 11 mapping beyond the bounds of this region (three upstream, eight downstream).
Candidate genes and SNP annotation
We found that 59 fixed SNPs map to 12 different protein-coding genes (BMT2, CAPZA2, CNTN1,
DOCK4, FOXP2, GPR85, KCND2, LOC103067393, LSMEM1, ST7, TES, and TFEC). We annotated
SNPs for predicted impact (see methods) and found that all fixed SNPs have the lowest impact
classification (MODIFIER), often assigned to mutations in non-functional regions, and most map
to introns. However, we found other impact classifications among SNPs with FST greater than
0.9. One synonymous (i.e., silent) SNP (LOW impact, NW_006539084.1 26867, FST = 0.92) is
located within the gene LSMEM1 and one nonsense SNP (HIGH impact, NW_006534020.1
160458, FST = 0.96) is located within the gene TFEC. This nonsense variant consisted of a C>T
mutation on Arg165, resulting in a premature Opal stop codon (Table S3).
We regard the ball python as a powerful new model for understanding the genetic basis of
pigmentation in vertebrates. As proof-of-principle, we set out to discover candidate genes and
causal mutations for the classic colour morph found in the pet trade, the piebald. Over the last
thirty years, ball python breeders have discovered and successfully propagated in captivity
more than three hundred Mendelian phenotypes. Yet, researchers working on the genetics of
vertebrate colouration have been slow to appreciate the ball python as a powerful new model
(but see Irizarry and Bryden 2016). We set out to bridge this gap. To do so, we appealed to
commercial ball python breeders to join our efforts in discovery, starting with uncovering the
genetic basis of the piebald phenotype. Commercial breeders have a vested interest in
acquiring the ability to identify heterozygotes for recessive phenotypes (i.e., carriers of
recessive alleles affecting colouration), and piebald is one such phenotype, although subtle
phenotypic differences on heterozygotes are not uncommon (e.g. ring of unpigmented skin,
often near the vent).
To elucidate the genetic basis of the piebald phenotype, we combined a case-control
experimental design with whole-genome sequencing, population genetic methods, and
functional annotation of the candidate SNPs. We identified a premature stop codon in the
protein-coding region of the gene TFEC as the putative genetic basis for this Mendelian
phenotype. TFEC belongs to a family of transcription factors (MITF) known to be expressed in
the neural crest and pigment cells. However, functional validation through gene editing
techniques, like CRISPR cas9 (Rasys et al. 2019), is required to establish that the candidate
nonsense mutation is the causal variant.
With knowledge of the specific genetic variants underlying pigmentation variation in ball
pythons, gene editing could pave the way to produce colour morphs free of secondary
phenotypes that are undesired from a commercial or ethical perspective. Genes that contribute
to colouration are also expressed in many other tissues derived from neural crest cells
(Donoghue et al. 2008). Thus, mutations that alter colour or patterning often alter other traits,
such as behaviour (Ducrest et al. 2008; McKinnon and Pierotti 2010). Not surprisingly, there are
ball python colour morphs linked to morphological deformities and neurological dysfunction
(Rose and Williams 2014). For example, the incomplete dominant ‘spider’ morph is associated
with a neurological syndrome called the ‘head wobble’ (Fox and Hogan 2020). The propagation
of colour morphs with correlated traits have implications for the health and welfare of ball
pythons in captivity (Rose and Williams 2014) and are a source of current controversy among
hobbyists and commercial breeders (Riera 2015). In 2017, the International Herpetological
Society in the U. K. banned ball pythons with the spider mutation, which is thought to be
homozygous lethal, from their reptile shows (International Herpetological Society 2020).
However, it is not known if the correlated trait(s) for any particular colour morph is due to
pleiotropic effects or linked variation. If due to linked variation, targeted mutation on a
different genetic background (i.e., without the linked variant) could allow the reproduction of
colour phenotypes free of behavioural and morphological abnormalities. If due to pleiotropy,
targeted mutation at different nearby loci could achieve the same effect.
The wide variety of ball python phenotypes found in the pet trade offers the
opportunity to study how genes control vertebrate pigmentation and pattern formation.
Changes to pigmentation are often caused by mutations in genes involved in the pigment
synthesis pathway (Mills and Patterson 2009; Hubbard et al. 2010). For example, albinism
occurs when an individual inherits two copies of a gene involved in melanin synthesis (e.g.
OCA2) that is non-functional. Snakes with two non-functional OCA2 genes lack dark melanin-
based pigment though contain yellow pigments produced in the xanthophores (Saenko et al.
2015). Changes to pattern, on the other hand, are often attributed to genes with functions in
cell migration from the neural crest and differentiation into colour-producing cells (Mills and
Patterson 2009). However, Mendelian phenotypes characterized by changes to either
pigmentation or patterning are likely to map to the protein-coding regions of genes. Genomic
research on the most extensively studied vertebrate, humans, shows that Mendelian
phenotypes are generally caused by mutations arising in protein-coding regions and affecting
protein function (Cooper et al. 2010; Chong et al. 2015). The progenitor cells of
chromatophores originate in the neural crest and mutations to these genes result in partial or
total loss of pigment and structural colouration. Mutations to genes such as MITF, KIT, EDNRB,
and SOX10, which are expressed in the neural crest, results in white spotting or a complete lack
of pigment across a wide range of vertebrates, including fish, amphibians, birds, and mammals
(Baxter et al. 2004; Kelsh 2006; George et al. 2016; Ahi and Sefc 2017; Woodcock et al. 2017;
Camargo-Sosa et al. 2019; Goding and Arnheiter 2019). These genes represented a priori
candidates for the piebald phenotype in ball pythons, but did not show differentiation between
our phenotypic pools.
We identified a putative causal mutation for the piebald phenotype in ball pythons
located in a new candidate gene for vertebrate pigmentation: the gene TFEC (Figure 3, Table
S3). This variant, which produces a premature stop codon, was nearly fixed (FST = 0.96).
Complete differentiation between the piebald and non-piebald pools was prevented by a single
read for the reference allele that was sequenced in the piebald samples. It is possible this is the
result of a sequencing error or the inclusion of an unknown heterozygote. TFEC was not one of
the candidate genes expected a priori. This particular gene has not been implicated in
mammalian colouration. However, TFEC is part of the MIT-family of transcription factors known
to be involved in animal patterning. For example, this family consists of the gene MITF, which
forms heterodimers with the other three members, TFEB, TFE3, and TFEC (Goding and
Arnheiter 2019). In mouse models, double knockout mutations to MITF and TFE3 cause all-
white phenotypes (Steingrímsson et al. 2002). Interestingly, the same study showed that
knocking out TFEC did not result in a phenotype different from the wild-type mouse. There is
evidence that shows TFEC is expressed in the retinal pigment epithelium of mammals (Rowan et
al. 2004). No evidence suggests, however, that TFEC is expressed in cells that descend from the
mammalian neural crest, which includes pigment-producing cells. In contrast, recent evidence
from the zebrafish model shows that TFEC is highly expressed in neural crest cells, specifically
progenitor pigment cells and iridophores (Petratou et al. 2019; Saunders et al. 2019). In fact,
TFEC is expressed in two dorsolateral patches on the torso during embryogenesis (Lister et al.
2011; Petratou et al. 2018; Petratou et al. 2019), reminiscent of where the white-spotting
occurs in the piebald phenotype in ball pythons. The expression patterns of TFEC in zebrafish,
together with our results, makes a strong case that TFEC is the piebald gene in ball pythons and
that a premature stop codon is the likely causal mutation.
In this study, we aimed to uncover, for the first time, the genetic basis of a ball python colour
morph. We discovered that a premature stop codon in the gene TFEC is the likely cause for the
Mendelian piebald phenotype found in ball pythons from the pet trade. The lack of evidence for
TFEC affecting mammalian pigmentation highlights the need to study the genetic basis of
colouration in non-mammalian vertebrate models. Future work will be aimed at demonstrating
causality through direct functional manipulation. These efforts would produce the first
genetically engineered ball python, a step that would open the door to safe reproduction of
other colour morphs through targeted mutation.
RDHB was supported by an NSERC Discovery Grant and Canada Research Chair. We thank
Mutation Creation, T Dot Exotics, The Ball Room Canada, and Designing Morphs for supplying
The data that support the findings of this manuscript will be uploaded to Dryad.
AGE, HR, APH, and RDHB conceived and designed the study. HR collected samples. AGE
performed the molecular work. AGE performed the bioinformatics and analyzed the genomic
data. AGE wrote the manuscript with input from all authors.
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Figure 1. A small sample of the phenotypic variation found in captive-bred ball pythons (Python regius).
(a) wild-type, (b) piebald, (c) banana piebald, (d) pastel piebald, (e) pastel HRA enhancer, (f) ultramel
clown, (g) banana champagne. Photo credit (b-g): Designing Morphs.
Figure 2. Fst plot between piebald and non-piebald samples on the chromosome-length assembly
Figure 3. A premature stop codon on Arg165 is the
putative causal variant for the piebald phenotype.
Figure S1. Fst plot of SNPs mapped to the Burmese python draft assembly (Pmo2.0). The 129 fixed SNPs
in this plot were used to map to candidate genes.