Abstract
Sex identification provides important information for ecological and evolutionary studies, as well as benefiting snake conservation management. Traditional methods such as cloacal probing or cloacal popping are counterproductive for sex identification concerning very small species, resulting in difficulties in the management of their breeding programs. In this study, the nucleotide sequences of gametologous genes (CTNNB1 and WAC genes) were used for the development of molecular sexing markers in caenophidian snakes. Two candidate markers were developed with the two primer sets, and successfully amplified by a single band on the agarose gel in male (ZZ) and two bands, differing in fragment sizes, in female (ZW) of 16 caenophidian snakes for CTNNB1 and 12 caenophidian snakes for WAC. Another candidate marker was developed with the primer set to amplify the specific sequence for CTNNB1W homolog, and the PCR products were successfully obtained in a female‐specific 250‐bp DNA bands. The three candidate PCR sexing markers provide a simple sex identification method based on the amplification of gametologous genes, and they can be used to facilitate effective caenophidian snake conservation and management programs.
Highlights
Sex identification is important for mating systems and sexual behavior in ecological and conservation research in vertebrates
Sex identification cannot be reliably determined in many squamate reptiles, because males and females have similar morphology, and morphological sexual dimorphism appears in the short period of time before mating (Frýdlová et al, 2011; Garland, 1985; Wangkulangkul, Thirakhupt, & Voris, 2005)
The snakes are considered as excellent model organisms for biomedical research, and snake venom is extracted for developing antivenoms to treat snakebites or for chemotherapeutical development (Blackburn, 2006; Kerkkamp et al, 2016; Ratanabanangkoon et al, 2016)
Summary
Sex identification is important for mating systems and sexual behavior in ecological and conservation research in vertebrates. Improvements in conservation programs are necessary to identify the sex of juveniles before the development of primary and secondary sexual characteristics to reduce the risk of extinction, or when samples are obtained without handling individuals (e.g., noninvasive sampling) This is a very important feature when working with endangered species (Waits & Paetkau, 2005). Size and sequence differences between the Z and W homologs of these genes were found in many caenophidian snakes, leading us to develop molecular sexing markers to identify male and female individuals. We developed novel molecular sexing markers using three primer pairs to amplify fragments from the Z and W homologs of the genes with a clear size difference of PCR products This assay provides a rapid and reliable method to identify genetic sex across different caenophidian snake species
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