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

Authors

  • Francisco Morinha,

    Corresponding author
    1. Laboratory of Applied Ecology, Centre for the Research and Technology of Agro-Environment and Biological Sciences (CITAB), University of Trás-os-Montes e Alto Douro, Vila Real, Portugal
    • Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes e Alto Douro (IBB/CGB-UTAD), Vila Real, Portugal
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  • Paulo Travassos,

    1. Laboratory of Applied Ecology, Centre for the Research and Technology of Agro-Environment and Biological Sciences (CITAB), University of Trás-os-Montes e Alto Douro, Vila Real, Portugal
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  • Fernanda Seixas,

    1. Histopathology Laboratory, Veterinary Sciences Department, Centre of Animal and Veterinary Sciences (CECAV), University of Trás-os-Montes e Alto Douro, Vila Real, Portugal
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  • Nuno Santos,

    1. Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Campus de Gualtar, Braga, Portugal
    2. ICVS/3B's, PT Government Associate Laboratory, Guimarães, Braga, Portugal
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  • Roberto Sargo,

    1. Veterinary Hospital of the University of Trás-os-Montes e Alto Douro, Vila Real, Portugal
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  • Luís Sousa,

    1. Veterinary Hospital of the University of Trás-os-Montes e Alto Douro, Vila Real, Portugal
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  • Paula Magalhães,

    1. CCGen-IBMC, Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal
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  • João A. Cabral,

    1. Laboratory of Applied Ecology, Centre for the Research and Technology of Agro-Environment and Biological Sciences (CITAB), University of Trás-os-Montes e Alto Douro, Vila Real, Portugal
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  • Estela Bastos

    1. Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes e Alto Douro (IBB/CGB-UTAD), Vila Real, Portugal
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Correspondence: Francisco Morinha, Fax: +351 259 350 266; E-mail: franciscomorinha@hotmail.com

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.

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