High‐resolution melting analysis for bird sexing: a successful approach to molecular sex identification using different biological samples

Abstract

High-resolution melting (HRM) analysis is a very attractive and flexible advanced post-PCR method with high sensitivity/specificity for simple, fast and cost-effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real-time PCR systems, along with improved saturating DNA-binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sex-specific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed-tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real-time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing.

Avian gametologs as molecular tags for sex identification in birds of prey of Iran

“…Therefore, for accurate sex identification in these species alternative gametologs such as NIPBL or alternative CHD1 based primers might be used (Suh et al, ). It has recently been shown that high resolution melting curve (HRM) real time PCR and oligonucleotide microarrays may be useful for accurate sex identification in Strigidae (Morinha et al, ; Wang et al, ). P2/P3 is another CHD1 ‐specific primer that has been used for sex identification in birds of prey.…”

Sex‐specific prey partitioning in breeding piscivorous birds examined via a novel, noninvasive approach

“…Regarding the use of consumer DNA, a variety of methods for sex determination ("molecular sexing") and individual identification have been developed (e.g., Kohn & Wayne, 1997;Wultsch, Waits, & Kelly, 2014). For birds, molecular sexing has been commonly applied since the late 1990s to determine the sex of indistinguishable adult birds, juveniles, and embryos (Morinha et al, 2013). Generally, the technique is based on sequence variations of the sex chromosomes, and depending on the species under investigation, different primers have been developed and applied in allele-specific PCRs (Dawson, Dos Remedios, & Horsburgh, 2016;Morinha, Cabral, & Bastos, 2012).…”

“…For European cormorants, Thanou, Giokas, Goutner, Liordos, and Fraguedakis-Tsolis (2013) found the primers 2550F and 2718R (Fridolfsson & Ellegren, 1999), targeting the chromodomain-helicase-DNA-binding protein 1 gene (CHD1) to be best suited for molecular sexing. Traditionally, blood, feathers, muscle tissue, egg shells, and buccal swab samples have been used as a source of bird DNA (Griffiths, Daan, & Dijkstra, 1996;Morinha et al, 2013;Thanou et al, 2013), but recently, molecular sexing has also been successfully carried out with fecal samples (e.g., Faux, McInnes, & Jarman, 2014;Jarman et al, 2013).…”

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

“…This requires the transport of samples and usually implies that sex determination may take several days after collecting the samples. Although high‐resolution melting analysis is an accurate technique for sex determination in birds (Faux, McInnes, & Jarman, ; Morinha et al., ), hand‐held devices for quantitative PCRs required for a portable solution are still expensive and made these protocols unaffordable for most laboratories. Loop‐mediated isothermal amplification (LAMP; Notomi, Mori, Tomita, & Kanda, ; Notomi et al., ) can lift these constraints on time, space and resources.…”