An albino mutant of the Japanese rat snake (Elaphe climacophora) carries a nonsense mutation in the tyrosinase gene

The Japanese rat snake (Elaphe climacophora) is a common species in Japan and is widely distributed across the Japanese islands. An albino mutant of the Japanese rat snake (“pet trade” albino) has been bred and traded by hobbyists for around two decades because of its remarkable light-yellowish coloration with red eyes, attributable to a lack of melanin. Another albino Japanese rat snake mutant found in a natural population of the Japanese rat snake at high frequency in Iwakuni City, Yamaguchi Prefecture is known as “Iwakuni no Shirohebi”. It has been conserved by the government as a natural monument. The Iwakuni albino also lacks melanin, having light-yellowish body coloration and red eyes. Albino mutants of several organisms have been studied, and mutation of the tyrosinase gene (TYR) is responsible for this phenotype. By determining the sequence of the TYR coding region of the pet trade albino, we identified a nonsense mutation in the second exon. Furthermore, RT-PCR revealed that TYR transcripts were not detected in this snake. These findings suggest that mutation of TYR is responsible for the albino phenotype of the pet trade line of the Japanese rat snake. However, the Iwakuni albino did not share this TYR mutation; thus, these two albino lines differ in their origins.

Albino mutants lacking dark pigments called melanins have been recorded in many animals, owing to the prominence of their common phenotypes, which comprise white or very pale skin color and red eyes.Albino lines of laboratory animals such as mice (Mus musculus), zebrafish (Danio rerio) and African clawed frogs (Xenopus laevis) have been maintained and analyzed as genetic resources.In humans, this condition is known as nonsyndromic oculocutaneous albinism (OCA), and is classified into seven genetic types (OCA1 to OCA7).The genes responsible for OCA1 to 7 (with the exception of OCA5) are TYR, OCA2, TYRP1, SLC45A2, SLC24A5 and C10ORF11, respectively (Montoliu et al., 2014).Mutations in these genes have also been identified in other animals (Fukamachi et al., 2001;Newton et al., 2001;Lamason et al., 2005;Chang et al., 2006;Miura et al., 2017;Woodcock et al., 2017).TYR encodes tyrosinase, which catalyzes the two reactions, monophenolase and diphenolase activity, at the first step of the synthesis of both the black pigment eumelanin and the brown pigment pheomelanin.The reactions are a single pathway, so there is no bypass pathway.Therefore, the absence of TYR results in entirely white coloration, especially in mammals.Mutations in this gene can thus cause remarkable albino phenotypes (Sánchez-Ferrer et al., 1995).
Reptiles are distinct from other vertebrates in several respects, including skin colorations, reproductive modes (oviparous and viviparous), and body plans (particularly those of snakes, which have no limbs and typically a single lung).Thus, reptiles are intriguing subjects for evolutionary and developmental studies (Di-Poï et al., 2010;Shibata et al., 2017).Notably, reptiles have three types of pigment cell, the melanophore, iridophore and xanthophore, whereas mammals have only a single type, the melanosome, containing melanin (Kuriyama et al., 2006;Kronforst et al., 2012).Body coloration plays various roles in adaptive evolution and speciation for vertebrates, especially for reptiles, including in protection from UV light, sexual selection, mimicry, and other behavioral and physiological traits.Thus, analyses of the mechanisms underlying pigmentation may yield novel biological insights not only for reptiles but also for all vertebrates.
In this study, we attempted to identify the gene responsible for an albino mutation of the Japanese rat snake (Elaphe climacophora) by comparing the TYR sequences of wild-type and albino lines.
The Japanese rat snake is endemic to Japan and is broadly distributed over the Japanese islands, with its northern and southern limits being Kunashiri Island and Kuchinoshima Island, respectively.This snake is very common and inhabits several environments, including mountainous and peri-urban areas.It has various colorations, from dark green to olive, and patterning comprising a pair of stripes and blotches (Fig. 1).In the northern part of Japan, particularly on Kunashiri Island, Japanese rat snakes have a lighter coloration known as "Kunashiri blue".A number of such individuals have been exported to the United States and bred by hobbyists since the 2000s.In such breeding colonies, albino mutants with red eyes and yellowish body coloration occur, and these mutants, which we refer to as "pet trade" albinos, have been marketed owing to their beautiful coloration (Fig. 1).The juveniles have a pinkish and relatively clear pigmentation pattern, whereas the adults are yellowish with patterning that gradually becomes indistinct as growth continues.In addition to the pet trade line, a natural population of another albino mutant of the Japanese rat snake known as "Iwakuni no Shirohebi" inhabits Iwakuni City (Yamaguchi Prefecture, Japan), and has been conserved by the government as a natural monument (Tokunaga et al., 1991).Iwakuni albinos also lack melanin, having light yellowish or white body coloration and red eyes.
We first attempted to analyze the TYR gene of the pet trade albino Japanese rat snake because of its amelanistic phenotype.TYR coding region sequences are highly conserved in many vertebrates.Moreover, whole-genome sequences are available for several reptile species, including a number of snakes, such as the green anole (Anolis carolinensis), king cobra (Ophiophagus hannah), Burmese python (Python bivittatus), garter snake (Thamnophis sirtalis), corn snake (Pantherophis guttatus) and Taiwan habu, commonly known as the brown spotted pit viper (Protobothrops mucrosquamatus) (Alföldi et al., 2011;Castoe et al., 2013;Vonk et al., 2013;McGlothlin et al., 2014;Ullate-Agote et al., 2014;Aird et al., 2017).When this study began, the only snake genome sequences available were those of the king cobra and Burmese python.Almost all of the PCR primers used to isolate the TYR coding sequence of the Japanese rat snake were designed based on the king cobra sequence, because the former is more closely related to the latter than to the Burmese python.Of the reptiles whose genome sequences have been determined to date, the corn snake is the most closely related to the Japanese rat snake (Ullate-Agote et al., 2014).
Wild-type snakes were captured at the Ito Campus of Kyushu University in Fukuoka City, and total RNA was extracted from the scraped skin tissues of live individuals or frozen samples with an RNeasy Mini Kit (Qiagen, Hilden, Germany).The mRNA was reverse-transcribed Fig. 1.Body colorations and pigment patterns of wild-type and pet trade albino snakes.Scale bars in the whole-animal images and ventral/head images represent 5 cm and 1 cm, respectively.(A) Body, ventral and eye coloration of the wild-type snake.The wild-type snake has olive-green dorsal coloration with four lines and a blotchy pigmentation pattern, and has melanin in the ventral portion of its body.Its eyes have black irises.(B) The albino lacks melanin, manifesting as lighter yellowish coloration, and has no pigmentation pattern in the ventral region.Its irises, lacking melanin, appear red.using a PrimeScript RT-PCR Kit (TaKaRa, Shiga, Japan) and an oligo-dT primer.The complete sequence of the wild-type TYR coding region was then amplified with PrimeSTAR GXL DNA Polymerase (TaKaRa), which is suited to the accurate amplification of long fragments, and the primers TYR_ex1F (5′-AATGAAGTACCAG-GAAGAAATATGC-3′) and TYR_ex5R (5′-TTGGGAC-GTGCCAACACAGATTTTC-3′), which were designed using OLIGO 4.0 (Molecular Biology Insights, Cascade, CO, USA; Fig. 2A).PCR products were loaded on a 1.2% agarose gel and purified with the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA).This DNA was used as a template in a sequencing reaction containing primers designed according to the king cobra sequence (see below) and BigDye Terminator v3.1 reagents (Applied Biosystems, Foster City, CA, USA).Sequencing was carried out on a 3130xl Genetic Analyzer (Applied Biosystems), and assembly was performed with GENETYX-MAC Ver.19 (Genetyx Corporation, Tokyo, Japan).The wild-type TYR coding sequence has been deposited in the DDBJ (accession no.LC365392).
Genomic DNA of wild-type snakes was extracted from blood samples taken from live individuals.That of pet trade and Iwakuni albino mutants was extracted from shed skin homogenized in liquid nitrogen.Cell lysis buffer (10 mM Tris, 10 mM EDTA and 2% SDS; pH 8.0) was added to the blood and shed skin samples.The original DNA extraction protocol for shed skins described by Fetzner (1999) was employed, with the addition of proteinase K digestion at 55 °C for 2 h, and precipitation of protein-SDS complexes with 3 M potassium acetate and centrifugation.
Sequences of exons and adjacent introns of wild-type and albino mutants were determined, and an alignment was generated using CLUSTAL W (Thompson et al., 1994).All splicing junction sequences were conserved in wild-type and albino mutants; however, the nonsense mutation p.R299X (c.895C > T) was present in exon 2 of the pet trade albino (Fig. 2B; the accession number of the coding sequence of the pet trade albino is LC367643).Moreover, this mutation was not observed in Iwakuni albinos (Fig. 2B).
Levels of the TYR transcript were tested by RT-PCR.The central region of the sequence was amplified using TYR_RT-F (5′-TCTCAGGTCCAGGGTTCAGCCAATG-3′) and TYR_RT-R (5′-CACAGCAGCTCCGAGTAACCAT-GGC-3′), which were designed based on the Japanese rat snake coding sequence (Fig. 2A).TYR mRNA was not detected in tissue from pet trade albinos by RT-PCR (Fig. 2C).As the mutation noted in these snakes resulted in a premature stop codon, it is likely that the mutant TYR transcripts were degraded by nonsense-mediated mRNA decay (Maquat, 2002).Thus, we expect that the pet trade albino lacks functional tyrosinase and associated melanin synthesis activity.
In conclusion, we detected a nonsense mutation in the TYR coding region of the pet trade albino and a lack of the corresponding transcript in this snake.Thus, mutation of the TYR gene contributes to this albino phenotype of the Japanese rat snake.Following the discovery of an albino OCA2 mutation in the corn snake that is caused by the insertion of an LTR retrotransposon into OCA2 (Saenko et al., 2015), our work represents the second study to have indicated a gene whose mutation is responsible for albinism in a reptile.Furthermore, albino phenotypes caused by mutations in TYR have not been reported to date in other reptiles.This study also revealed that the Iwakuni albino carried no mutations in the coding region of TYR; therefore, the origin of this mutation differs from that of the pet trade albino.

Fig. 2 .
Fig. 2. Structure of the TYR gene and the mutation site in the albino mutant.(A) Structure of the TYR gene, which consists of five exons (indicated by filled boxes).Filled arrowheads (a, b, c, d, e, f, g, h, i and j) indicate the positions of the primers used to amplify each exon and adjacent introns (TYR_ex1F, TYR_ex1R, TYR_ex2F, TYR_ex2R, TYR_ex3F, TYR_ex3R, TYR_ex4F, TYR_ ex4R, TYR_ex5F and TYR_ex5R, respectively).Gray arrowheads (k, l, m, n and o) indicate the positions of the primers used for Vectorette PCR (TYR_ex3V1, TYR_ex3V2, TYR_ex5V-F, TYR_ ex5V1 and TYR_ex5V2, respectively).Open arrowheads (p and q) indicate the positions of the RT-PCR primers (RT-F and RT-R, respectively).TYR_ex1F (a) and TYR_ex5R (j) were also used for amplification of the coding region by RT-PCR.The direction of TYR transcription is from left to right.This diagram was created with reference to the Taiwan habu (brown spotted pit viper) sequence, and the uncertain lengths of introns 1 and 3 are indicated by wavy lines.(B) Comparison of the TYR sequences of the wild-type (WT) and albino mutants.The region around the nonsense mutation p.R299X (c.895C > T) in exon 2 is shown by ABI chromatograms.(C) Levels of the TYR transcript in wildtype and pet trade albino snakes.TYR fragments were amplified using the primer pair RT-F and RT-R.As a loading control, β-actin fragments were amplified with the specific primers actin-F (5′-TGACATTGCTGCGCTCGTGGTCGAC-3′) and actin-R (5′-AGACGGAGGATGGCATGGGGCAAGG-3′), which were designed based on the Burmese python sequence.β-actin is a single-copy gene and is expressed in almost all tissues(Zhong and Simons, 1999).The values to the right of these images are the number of cycles in the PCR amplifications.There is essentially no TYR transcript in the albino samples.