qPCR‐based molecular sexing by copy number variation in rRNA genes and its utility for sex identification in soft‐shell turtles

Abstract

Summary1 Sex diagnosis is important in ecology, evolution, conservation biology, medicine, and food production. However, sex diagnosis is difficult in species without conspicuous sexual dimorphism or at life stages before such differences develop. This problem is exacerbated when the diagnostic trait is a continuous (non-discrete) variable to which general analytical methods are not commonly applied. 2 Here we demonstrate the use of copy-number variation between males and females of the nucleolar organizing region (NOR) in the genome of Apalone spinifera softshell turtles, which we quantify by real-time PCR. We analyze these continuous data using mixture models that can be applied either in discriminant analysis when a subset of individuals of known sex is used as a training set, or in clustering procedures when all individuals are of unknown sex. 3 Using individuals of known sex, the discriminant analysis exhibited 100% accurate classification rate for both the training set and the test set. Classification rates were also 100% when using the clustering procedure to identify groups and classify individuals in the absence of sex information. Standard curves using only male DNA provided better discrimination than using mixed-sex DNA during qPCR. NOR copy number is an effective sex diagnostic for A. spinifera turtles. Our sexing approach using qPCR of 18S genes should prove useful for other taxa that also possess dimorphic NORs, as is known in some vertebrates and insects. While the 18S copy numbers in our dataset exhibited a non-overlapping binomial distribution, this may not always be the case in future studies of A. spinifera or for other taxa. 4 Importantly however, our sex-typing approach using mixture models provides an attractive alternative under overlapping distributions of these and of other continuous data such as hormone levels, gene expression levels, shape or behavior. We present an example using overlapping distributions of hormone levels in Chelydra serpentina turtles, to demonstrate the broader utility of mixture models for sex-typing, and obtain a high correct classification of 90%.

Cytogenetic Insights into the Evolution of Chromosomes and Sex Determination Reveal Striking Homology of Turtle Sex Chromosomes to Amphibian Autosomes

“…4 ; Apalone [Badenhorst et al, 2013], this study, fig. 4 ), as it also occurs in diverse taxa [reviewed in Literman et al, 2014] such as some reptiles, mammals, amphibians, and fish [Goodpasture and Bloom, 1975;Hsu et al, 1975;Schmid et al, 1983Schmid et al, , 1993Porter et al, 1994;Born and Bertollo, 2000;Wiley, 2003;Abramyan et al, 2009;Takehana et al, 2012], as well as some invertebrates [e.g., Monti et al, 2011;Montiel et al, 2012]. The NOR in ASPW and PSIW is much larger than the Z-NOR in these turtles, a dimorphic trait that can be leveraged to diagnose the genotypic sex in these turtles using molecular techniques .…”

Mammalian X homolog acts as sex chromosome in lacertid lizards

“…This difference in copy number between sexes can be determined by qPCR, allowing the reliable identification of sex-specific genes (Rovatsos et al, 2014a(Rovatsos et al, -c, 2015; for similar application of qPCR see also Nguyen et al, 2013;Gamble et al, 2014;Literman et al, 2014).…”

Effects of Incubation Temperature on Sex Determination in the Endangered Magdalena River Turtle,Podocnemis lewyana

“…-Diagnosing the sex of individuals is important to study the ecology and evolution of species, and nonintrusive methods are particularly crucial to monitor the sex ratios of endangered species. Molecular methods have been developed for some taxa (reviewed in Literman et al 2014), including Podocnemis (Lance et al 1992), but they can be cumbersome and expensive and may require highly specialized equipment. Geometric-morphometric techniques are extremely powerful to compare the shape of individuals based on photographic records alone and can distinguish very subtle differences that are undetectable to the naked eye.…”

“…In species that lack a strong enough sexual dimorphism that can be observed by the naked eye at early life stages, sex information must be obtained destructively (e.g., by gonadal histology) or via indirect methods (e.g., morphometric or molecular approaches; reviewed in Literman et al 2014). Thus, conservation efforts for endangered species benefit from indirect methods that are nonintrusive and inexpensive.…”