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

Hanna R Koch,Elke Blohm‐Sievers,Miriam Liedvogel

doi:10.1002/ece3.5168

Abstract

Many bird species are sexually monomorphic and cannot be sexed based on phenotypic traits. Rapid sex determination is often a necessary component of avian studies focusing on behavior, ecology, evolution, and conservation. While PCR‐based methods are the most common technique for molecularly sexing birds in the laboratory, a simpler, faster, and cheaper method has emerged, which can be used in the laboratory, but importantly also in the field. Herein, we used loop‐mediated isothermal amplification (LAMP) for rapid sex determination of blood samples from juvenile European blackcaps, Sylvia atricapilla, sampled in the wild. We designed LAMP primers unique to S. atricapilla based on the sex chromosome‐specific gene, chromo‐helicase‐DNA‐binding protein (CHD), optimized the primers for laboratory and field application, and then used them to test a subset of wild‐caught juvenile blackcaps of unknown gender at the time of capture. Sex determination results were fast and accurate. The advantages of this technique are that it allows researchers to identify the sex of individual birds within hours of sampling and eliminates the need for direct access to a laboratory if implemented at a remote field site. This work adds to the increasing list of available LAMP primers for different bird species and is a new addition within the Passeriformes order.

Highlights

With more than half of all avian species being monomorphic, deter‐ mining the sex of a bird based on external morphology or obvious phenotypic differences, such as plumage color or patterns, becomes impossible
After testing different combinations of incubation temperature and time, we found that males and females were clearly and consistently distinguished when loop‐mediated isothermal am‐ plification (LAMP) reactions were run at 65°C for 60 min (CHD‐W primers) and 80 min (CHD‐Z primers; Table 1)
Visualization of amplicons under ambient light conditions was immediate after staining with SYBR Green I and seeing the sample color change from orange to yellow for all 18 reactions targeting CHD‐Z (Figure 3a) but only for eight of the 18 reactions targeting CHD‐W (Figure 3b), indicating the presence of eight females and ten males

Summary

| INTRODUCTION

With more than half of all avian species being monomorphic, deter‐ mining the sex of a bird based on external morphology or obvious phenotypic differences, such as plumage color or patterns, becomes impossible. Relatively recently a new amplification technique has been developed, called loop‐me‐ diated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and speed under isothermal conditions (Notomi et al, 2000), and without the need for direct access to a laboratory (Lee, 2017) This new method offers a simpler protocol with the LAMP reaction requiring a single enzyme (DNA polymerase with strand displacement activity that can greatly amplify from a minimal number of DNA copies) and a single temperature, which removes the need for an expensive, high‐precision thermal cycler. Product detection can be achieved directly within the reaction tube by a diagnostic color change and fluorescence, eliminating the need for electrophoretic techniques and related equipment (Centeno‐ Cuadros, Abbasi, & Nathan, 2017; Lee, 2017; Mori, Nagamine, Tomita, & Notomi, 2001) The principle of this method, based on autocycling strand displacement, is more complex than traditional PCR and requires four specially designed primers that recognize six distinct regions on the target DNA (Figure 1). Our optimized LAMP‐based protocol proved to be just as accurate, but much more time efficient compared to the PCR‐based protocol

| MATERIALS AND METHODS

| DISCUSSION

CONFLICT OF INTEREST