Validation of loop‐mediated isothermal amplification for fast and portable sex determination across the phylogeny of birds

Abstract

PCR is a universal tool for the multiplication of specific DNA sequences. For example, PCR-based sex determination is widely used, and a diversity of primer sets is available. However, this protocol requires thermal cycling and electrophoresis, so results are typically obtained in laboratories and several days after sampling. Loop-mediated isothermal amplification (LAMP) is an alternative to PCR that can take molecular ecology outside the laboratory. Although its application has been successfully probed for sex determination in three species of a single avian Family (raptors, Accipitridae), its generality remains untested and suitable primers across taxa are lacking. We designed and tested the first LAMP-based primer set for sex determination across the modern birds (NEO-W) based on a fragment of the gene chromo-helicase-DNA-binding protein located on the female-specific W chromosome. As nucleotide identity is expected to increase among more related taxa, taxonomically targeted primers were also developed for the Order Falconiformes and Families Psittacidae, Ciconiidae, Estrildidae and Icteridae as examples. NEO-W successfully determined sex in a subset of 21 species within 17 Families and 10 Orders and is therefore a candidate primer for all modern birds. Primer sets designed specifically for the selected taxa correctly assigned sex to the evaluated species. A short troubleshooting guide for new LAMP users is provided to identify false negatives and optimize LAMP reactions. This study represents the crucial next step towards the use of LAMP for molecular sex determination in birds and other applications in molecular ecology.

Female-specific SNP markers provide insights into a WZ/ZZ sex determination system for mud crabs Scylla paramamosain, S. tranquebarica and S. serrata with a rapid method for genetic sex identification

“…The ability to correctly identify sex of animals is of critical importance in ecological and evolutionary studies, as well as for artificial breeding and farming purposes [44, 45]. Several approaches have been developed for sex identification in animals, such as behavior observation [46], morphological and external characteristics [47], ultrasonography [48] and hormone levels [49].…”

“…Several approaches have been developed for sex identification in animals, such as behavior observation [46], morphological and external characteristics [47], ultrasonography [48] and hormone levels [49]. However, these methods are prone to high error rates and sometimes technically complex [45]. Genetic sex identification is an essential sex determination tool when the sex of an organism can not be discriminated morphologically [12].…”

“…In the last decade, sex-specific DNA markers have been successfully used to distinguish sex of different species, especially in animals at their early developmental stages without obvious sexual traits [12, 21, 43, 50]. The PCR-based method for genetic sex identification is believed to be expedient, time-saving and cost-effective [12, 45, 51].…”

Rapid sex determination of a wild passerine species using loop‐mediated isothermal amplification (LAMP)

“…Since its development, LAMP has been used widely across clinical research, medicine, microbiology, and parasitology, but less so within ecology and evolution. This method has also been used for sex determination in a number of organisms, including plants (Fujita et al, 2017;Hsu, Gwo, & Lin, 2012;Shiobara et al, 2011), fish (Hsu, Adiputra, Ohta, & Gwo, 2011), mammals (Khamlor et al, 2015;Nogami, Tsutsumi, Komuro, & Mukoyama, 2008), and birds (Centeno-Cuadros et al, 2017;Centeno-Cuadros, Tella, Delibes, Edelaar, & Carrete, 2018;Chan et al, 2012;Kim et al, 2015). Of the avian studies, only one of them has used LAMP as a field application (Centeno-Cuadros et al, 2017;Lee, 2017).…”

“…This method has also been used for sex determination in a number of organisms, including plants (Fujita et al, 2017;Hsu, Gwo, & Lin, 2012;Shiobara et al, 2011), fish (Hsu, Adiputra, Ohta, & Gwo, 2011), mammals (Khamlor et al, 2015;Nogami, Tsutsumi, Komuro, & Mukoyama, 2008), and birds (Centeno-Cuadros et al, 2017;Centeno-Cuadros, Tella, Delibes, Edelaar, & Carrete, 2018;Chan et al, 2012;Kim et al, 2015). This method has also been used for sex determination in a number of organisms, including plants (Fujita et al, 2017;Hsu, Gwo, & Lin, 2012;Shiobara et al, 2011), fish (Hsu, Adiputra, Ohta, & Gwo, 2011), mammals (Khamlor et al, 2015;Nogami, Tsutsumi, Komuro, & Mukoyama, 2008), and birds (Centeno-Cuadros et al, 2017;Centeno-Cuadros, Tella, Delibes, Edelaar, & Carrete, 2018;Chan et al, 2012;Kim et al, 2015).…”