Differentiation between wild type and heterozygous albino ball pythons (Python regius) by PCR and qPCR

Diferenciação entre pítons-bola selvagens e albinos heterozigotos(Python regius) por PCR e qPCR

Abstract

Python regius or ball pythons are the famous exotic pets because of their beautiful color and pattern. The albino ball python is one type of ball python, but it is very difficult to determine the difference of phenotype between wildtype and heterozygous genotype of albino (het albino). In this study, PCR and qPCR can distinguish between wildtype and het albino. The PCR product size of wildtype and het albino was 415 bp, but the intensity of PCR product of wildtype was more intense than that of het albinos. No PCR amplicon was found in albinos and the Ct value of wildtype was lower than Ct of het albinos. The molecular detection technique, especially PCR and qPCR, can determine the difference between wildtype and het albinos of ball pythons.

Keywords:
albino; ball python; Python regius; PCR and qPCR

Resumo

Python regius ou pítons-bola são os famosos animais de estimação exóticos por causa de sua bela cor e padrão. A píton-bola albina é um tipo de píton-bola, mas é muito difícil determinar a diferença de fenótipo entre o genótipo selvagem e heterozigoto de albino (albino het). Neste estudo, PCR e qPCR podem distinguir entre albinos selvagens e het. O tamanho do produto de PCR de albinos selvagens e het foi de 415 pb, mas a intensidade do produto de PCR de selvagens foi mais intensa do que a de albinos het. Nenhum amplicon de PCR foi encontrado em albinos e o valor de Ct de selvagens foi menor do que Ct de albinos het. A técnica de detecção molecular, especialmente PCR e qPCR, pode determinar a diferença entre albinos selvagens e het de pítons-bola.

Palavras-chave:
albino; pítons-bola; Python regius; PCR e qPCR

1. Introduction

Python regius or ball pythons, are popular exotic pets traded because they have several beautiful colors and patterns. Wild ball pythons are native to Sub-Saharan Africa with a range extending from Senegal to Uganda and have become popular pets in the United States, Europe and Asia (Amr and Disi, 2011, Bodnar and Bradley, 1996). Colors and patterns are produced through a combination of chemical pigments (Brown et al., 2022). Common melanin pigments are brown to black which are common in birds, reptiles, and lower vertebrates (David et al., 2017). The genetics and development of color patterns in vertebrates have been studied, especially in mammals. However, the knowledge of genetics and development in reptiles is quite limited. A single resource for understanding the genetics of color patterns in reptiles is the ball pythons (David et al., 2017). Wild ball pythons display a mottled color pattern, consisting of brown to black melanin and red to yellow (non-melanin) pigments in the skins. These variants called color morph or morph, include ball pythons with reduced melanin such as albino, increased melanin such as GHI, or complex changes in the replacement of color and pattern on the skin such as clown, spider and enchi (Figure 1).

Figure 1
The variants of color and pattern in ball pythons. (A) Wildtype (B) Albino (C) GHI and (D) Clown.

Many colors and patterns on the skin are heritable and show dominant or recessive patterns of inheritance. In addition, color in reptiles, amphibians, and fish arises from three cell layers (Hofreiter and Schöneberg, 2010). The first layer is the major melanin producing cells, called melanophore. The other two cell layers are xanthophores and iridophores, that contribute to coloration. Xanthophores synthesize yellow pteridine-based pigments and can also contain red carotenoid pigments that are obtained from diet. Iridophores do not contain pigments but instead produce structure coloration by reflecting light on guanine nanocrystals (Teyssier et al., 2015) The three color producing cells interact in 3D space (Grether et al., 2004; Saenko et al., 2013) to generate the remarkable range of color variation observed in reptiles. The morph albino is a good example of a recessive pattern of heredity. The albino ball pythons lack melanin and perform a bright yellow and white skin with pink or red eyes. The albino mutant of several organisms has been studied, and mutation of the tyrosinase gene (TYR) is responsible for this phenotype (Chang et al., 1996; Fukamachi et al., 2001; Iwanishi et al., 2018). As a result of the fact that albino is a recessive pattern of inheritance, the genotype of albino must be homozygous of mutant TYR. However, the ball pythons that have heterozygous mutant TYR (called heterozygous albino or het albino) show the same phenotype as the wildtype (Figure 2). The appearance of wild type and het albino is very similar in color, and pattern. It is very difficult to distinguish their phenotype by naked eye observation. So, this study uses the molecular technique, polymerase chain reaction or PCR and quantitative PCR or qPCR to determine the difference between wildtype and het albino in ball pythons.

Figure 2
Difference in phenotype between wild type and heterozygous of albino in ball pythons. (A) Wildtype (B) Heterozygous of albino.

2. Materials and Methods

2.1. Sampling and DNA extraction

Prior to collecting the sample, the Ethic Review Board for Animal Research at Rangsit University granted ethical approval for this study (RSU-AEC 001-2022). We obtained the ball python samples from commercial breeders in Thailand (Reptile Collector by docjavet and Morph Hunter) who supplied us with shed skins. All samples were stored at -20°C to kill any insect larvae infesting the sheds. The total set of animals comprises ten wild types, ten het albinos and three albinos. A 1.5 cm. x 1.5 cm. of each shed sample was immersed into a lysis buffer (1M Tris-HCl pH 7.5 and 10% SDS). The stirring rod was used to crush the shed sample. After that, the solution was used for DNA extraction. The DNA was extracted using GF-1 Tissue DNA extraction kit (Vivantis, Selangor Darul Ehsan, Malaysia). Afterward, these DNA solutions were stored at -20°C until used.

2.2. PCR and qPCR reactions

The PCR primers used in this study were designed base on Tyrosinase gene of Burmese pythons or Python bivittatus (NCBI Reference Sequence: NW_006536432.1). The nucleotide sequences of each primer are shown in Table 1. The PCR primers in this study were designed based on the splicing site of TYR.

Table 1
List of primers for PCR reactions.

Primer name	Sequence 5’-3’
TYR-F	TGG TAG CTT CTG GCC TCT CT
TYR-R	GAG TGT GCA GAA TCA CCC CA

SYBR green I dye was used to enhance the specificity of the qPCR reaction. The PCR amplification contained 1x Taq DNA polymerase buffer, 1.2 mM dNTPs, 0.8 μM TYR-F and TYR-R primers, 8 U Taq DNA polymerase (New England Biolabs), and 10 ng of each DNA extract as a template in a final volume of 25 μl. The cycling conditions comprised of a single initial denaturation at 95°C for 3 min followed by 35 cycles at 90°C for 30 sec (denaturation), 55°C for 30 sec (annealing), 72°C for 30 sec (extension), and a final extension step at 72°C for 5 min. After the PCR amplification, the products of 415 bp were analyzed by electrophoresis using 1.5% agarose gel and Gel Doc^TM XR+ with Image Lab^TM Software (BIO RAD, USA.).

3. Results

3.1. Differentiation between wild type and het albino by PCR

The results of PCR determined the difference between wild type and het albino as shown in Figure 3. All wild types and het albino showed 415 bp. of PCR product. However, the intensity of PCR product band of wild type was higher than that of albino. While no PCR amplicon was observed in albino, it was concluded that this PCR condition could differ the wild type from het albino.

Figure 3
Gel electrophoresis of PCR product. Lane M is a 100 bp DNA ladder, Lanes 1-6 are wild type no.1 to no.6, lanes 7-12 are het albinos no.1 to no.6, lanes 13-16 are wild type no.7 no.10, lanes 17-20 are het albinos no.7 to no.10, and lane 21-23 are albino no.1 to albino no.3.

3.2. Differentiation between wild type and het albino by qPCR

Difference between wild type and het albino by using qPCR was shown in Figure 4. The threshold cycle of Ct of wild type, het albino and albino were shown in Table 2. The average Ct of wild type was 21.15±1.05 while average of Ct of het albinos was 25.94 ±0.90. It meant that qPCR of this condition can distinguish between wild type and het albino.

Figure 4
The graph of qPCR to determine wild type and het albino.

Table 2
Ct of wild type, het albino and albino.

Sample	Ct	Sample	Ct	Sample	Ct
Wild type 1	20.09	Het albino 1	27.81	Albino 1	NA
Wild type 2	20.17	Het albino 2	25.19	Albino 2	NA
Wild type 3	21.48	Het albino 3	25.25	Albino 3	NA
Wild type 4	22.76	Het albino 4	26.27
Wild type 5	21.08	Het albino 5	25.33
Wild type 6	20.22	Het albino 6	27.13
Wild type 7	20.51	Het albino 7	25.45
Wild type 8	22.81	Het albino 8	26.01
Wild type 9	20.44	Het albino 9	25.64
Wild type 10	21.89	Het albino 10	25.27
average	21.15	average	25.94
SD	1.05	SD	0.90

4. Discussion

The results (Figure 3) showed that the intensity of PCR product in the wild type was about two times greater than that of PCR product in het albino because the wild type contained two alleles of normal TYR which the PCR primer can amplify, while het albino had only one normal allele of TYR. Based on the DNA region in which the primers were designed, our study implies that the DNA region of TYR splicing site is missing in albino ball python. The albino ball python lacks this region in both alleles while het albino presents only one allele consistent with the study of Brown et al. (2022). Our study was similar to Wrona et al. (2019) who determined the heterozygosity of neutrophil cytosolic factor 1 (NCF1) gene in chronic granulomatous disease (CGD) by PCR. They found that the PCR product of heterozygous NCF1 gene in CGD patients had less intensity than the healthy people (Wrona et al., 2019). In our studies, we can determine the albino genotype by PCR, the same as Iwanishi et al. (2018) which used PCR to detect the albino mutation in Japanese rat snakes (Elaphe climaccophora), therefore, this is the first report identify the heterozygous of albino in ball python by PCR method.

Furthermore, this study determined the different genotypes between normal and het albino in ball python by qPCR. Our findings (Figure 4 and Table 2) indicated that, the average Ct of wild type was 21.15 ±1.05 whereas the average Ct of het albino was 25.94 ± 0.90. There was 22.65% difference. Our results were similar to Mizugaki et al. (2000) which determined CYP2C18 genotypes by qPCR. Mizugaki et al. (2000) showed the difference between Ct of wild type CYP2C18 and mutant CYP2C18. They found that the Ct of wild type was 21.90 and 23.20 in mutant. It was 5.94% difference. This is the first report using qPCR to distinguish between wild type and het albino in ball python. Our findings will be useful for the ball python breeders for planning the breeding program of albino ball pythons. By using this method, the ball python breeders can select only the het albino to produce albino ball pythons more efficiently.

The genes that regulate melanin synthesis in reptiles have been described in Brown et al. (2022). These genes encode enzymes that synthesize melanin (TYR) (Ikuo et al., 2017), a chloride channel required for maintaining the pH of melanosome (OCA2), and transporters thought to import solutes into the cell or into the organisms (SLC7A11, SLC23A5, and SLC45A2) (Chintala et al., 2005; Ginger et al., 2008; Nicholas et al., 2014; Vitavska and Wieczorek, 2013). Brown et al. (2022) hypothesized that the major cause of albino ball pythons was loss of function of TYR, which encodes the enzyme catalyzing the rate-limiting step of melanin production. They also found that three haplotypes of TYR among albinos might carry a distinct loss of function variant in the gene. The first one, a missense variant in the third coding region of TYR which leads to an aspartic acid to glycine exchange (D394G) (Brown et al., 2022). The second was a missense variant in the third coding region of TYR which leads to a proline to leucine exchange (P384L), which is a similar site as a variant (P384A) associated with oculocutaneous albinism in humans (Simeonov et al., 2013), and the last one, lacked coding or splicing site variants (Brown et al.,2022). Based on Brown's hypothesis, which lacks coding or splicing sites, the PCR primers of this study were designed on splicing regions of TYR and they showed the ability to distinguish between wild type and het albino in ball pythons. This provides further evidence to support the hypothesis of Brown et al. (2022). Meanwhile, there is only one qPCR application in ball pythons. Blanak et al. (2020) used RT-qPCR for detecting nidovirus in ball python. However, this study is the first time using qPCR to distinguish the genetic between wildtype and het albino in ball pythons.

While, Iwanishi et al. (2018) identified the nonsense mutation in tyrosinase gene in an albino mutant of the Japanese rat snake (Elaphe climacophora). Furthermore, Garcia-Elfring et al. (2023) explored the nonsense mutation in the piebald phenotype of ball pythons and discovered that a premature stop codon in the TFEC gene (transcription factor EC) of the MIT-family of transcription factors is the likely cause of this Mendelian trait.

In addition to albino (severe loss of melanin), the other two phenotypes that show loss of melanin in the skin and eyes are lavender (moderate loss of melanin) and ultramel (mild loss of melanin) that shown in Figure 5. The lavender morph shows lavender instead of brown or black patches on the skin. Reports by Mizugaki et al. (2000) and Gardner et al. (1992) determined that this lavender phenotype is caused by a loss function of OCA2 gene. When the OCA2 protein is absent or non-functional, the enzymes that synthesize melanin are less active, and only small amounts of melanin are produced (Ni-Komatsu and Orlow, 2006; Puri et al., 2000). The ultramel color morph is performed by skin patches that are tan or light brown, rather than dark brown or black, shown in Figure 5. This phenotype is caused by loss of function in five genes: TYR1, TYR2, SLC7A11, SLC24A5 and SLC45A2, (Puri et al., 2000). TYR1 and TYR2 encode enzymes involved in melanin synthesis (Lai et al., 2018). SLC7A11 encodes a transporter for importing cystine into the cell (Sato et al., 1999). SLC24A5 encodes K⁺-dependent Na⁺-Ca²⁺ exchanger (Nicholas et al., 2014). SLC45A2 encodes a putative sugar transporter (Chintala et al., 2005). Loss of their encoded proteins decreases melanin through mechanisms that may involve defects in the regulation of melanosome pH (Nicholas et al., 2014; Newton et al., 2001; Bin et al., 2015).

Figure 5
The variants of loss melanin phenotype. (A) Albino (B) Lavender and (C) Ultramel.

However, the limitation of this study is the small number of the samples. Even though the samples size is small, the results clearly distinguish between wild type and het albino.

Although, PCR is the standard molecular technique for detecting the albino genotype, but there are many non-convenient factors such as being time-consuming and requiring complicated equipment such as thermal cycler. To reduce these hindrances, the loop-mediated isothermal amplification or LAMP technique may be used. There are many LAMP applications to determine human male by using blood stains (Kanchanaphum, 2018; Kanchanaphum et al., 2013; Kumsiri and Kanchanaphum, 2021), detecting Salmonella spp. (Vichaibun and Kanchanaphum, 2020), contamination to detect identifying Aspergillus flavus contamination (Kumsiri and Kanchanaphum, 2020), and monitoring environmental biosecurity (Deliveyne et al., 2023). Therefore, further studies may focus on developing the LAMP technique instead of PCR. Furthermore, the PCR conditions may be developed to differentiate between wildtype and piebald ball pythons.

5. Conclusion

The results showed that the molecular detection techniques, especially, PCR and qPCR could distinguish between wildtype and het albino of ball pythons. This study is the first to apply the qPCR technique to molecular genetics in ball pythons.

Acknowledgements

We would like to sincerely thank Mr. Stewart Miller for critical correcting English grammar. We would like to thank Biochemistry unit, faculty of Science, Rangsit University for funding in this study.

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Publication Dates

• Publication in this collection
  04 Oct 2024
• Date of issue
  2024

History

• Received
  17 May 2024
• Accepted
  05 Aug 2024