Development of Novel High-Resolution Melting (HRM) Assays for Gender Identification of Caribbean Flamingo (Phoenicopterus ruber ruber) and other Birds

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dc.contributorAlvarado-Bremer, Jaime R
dc.creatorChapman, Alexandra
dc.date.accessioned2013-03-14T16:21:51Z
dc.date.accessioned2017-04-07T20:03:23Z
dc.date.available2013-03-14T16:21:51Z
dc.date.available2017-04-07T20:03:23Z
dc.date.created2012-12
dc.date.issued2012-08-27
dc.description.abstractUnambiguous gender identification (ID) is needed to assess parameters in studies of population dynamics, behavior, and evolutionary biology of Caribbean Flamingo (Phoenicopterus ruber ruber) and other birds. Due to its importance for management and conservation, molecular (DNA-based) avian gender ID assays targeting intron-size differences of the Chromosome Helicase ATPase DNA Binding (CHD) gene of males (CHD-Z) and females (CHD-W) have been developed. Male (ZZ) and female (WZ) genotypes are usually scored as size polymorphisms through agarose or acrylamide gels. For certain species, W-specific restriction sites or multiplex polymerase chain-reaction (PCR) involving CHD-W specific primers are needed. These approaches involve a minimum of three steps following DNA isolation: PCR, gel electrophoresis, and photo-documentation, which limit high throughput scoring and automation potential. In here, a short amplicon (SA) High-resolution Melting Analysis (HRMA) assay for avian gender ID is developed. SA-HRMA of an 81-Base Pair (bp) segment differentiates heteroduplex female (WZ) from homoduplex male (ZZ) genotypes by targeting Single-nucleotide Polymorphisms (SNPs) instead of intron-size differences between CHD-Z and CHD-W genes. To demonstrate the utility of the approach, the gender of Caribbean Flamingo (P. ruber ruber) (17 captive from the Dallas Zoo and 359 wild from Ria Lagartos, Yucatan, Mexico) was determined. The assay was also tested on specimens of Lesser Flamingo (P. minor), Chilean Flamingo (P. chilensis), Saddle-billed Stork (Ephippiorhynchus senegalensis), Scarlet Ibis (Eudocimus ruber), White-bellied Stork (Ciconia abdimii), Roseate Spoonbill (Platalea ajaja), Marabou Stork (Leptoptilos crumeniferus), Greater Roadrunner (Geococcyx californianus), and Attwater's Prairie Chicken (Tympanuchus cupido attwateri). Although the orthologous 81 bp segments of Z and W are highly conserved, sequence alignments with 50 avian species across 15 families revealed mismatches affecting one or more nucleotides within the SA-HRMA forward or reverse primers. Most mismatches were located along the CHD-Z gene that may generate heteroduplex curves and thus gender ID errors. For such cases, taxon and species-specific primer sets were designed. The SA-HRMA gender ID assay can be used in studies of avian ecology and behavior, to assess sex-associated demographics and migratory patterns, and as a proxy to determine the health of the flock and the degree by which conservation and captive breeding programs are functioning.
dc.identifier.urihttp://hdl.handle.net/1969.1/148342
dc.subjectendangered birds
dc.subjectgender of sexually monomorphic birds
dc.subjectsexually monomorphic birds
dc.subjectfledglings
dc.subjectnestlings
dc.subjectDNA test to sex birds
dc.subjectAttwater's Prairie Chicken
dc.subjectAccipiter species
dc.subjectR?a Lagartos Biosphere Reserve
dc.subjectLesser Flamingo
dc.subjectFlamingo
dc.subjectChilean Flamingo
dc.subjectPhoenicopterus ruber ruber
dc.subjectPhoenicopterus
dc.subjectCaribbean Flamingo
dc.subjectHigh-resolution Melting Analysis
dc.subjectCHD1-W
dc.subjectCHD1-Z
dc.subjectpolymorphism
dc.subjectW chromosome
dc.subjectZ chromosome
dc.subjectCHD1 gene
dc.subjectCHD gene
dc.subjectgender of birds
dc.subjectsex determination of birds
dc.subjectgender determination of birds
dc.subjectmultiple avian species
dc.subjectnon-invasive
dc.subjectconservation of birds
dc.subjectsex-ratio of birds
dc.subjectsex determination
dc.subjectmolecular avian gender
dc.subjectmolecular gender determination
dc.subjectUniversal sexing of non-ratite birds
dc.titleDevelopment of Novel High-Resolution Melting (HRM) Assays for Gender Identification of Caribbean Flamingo (Phoenicopterus ruber ruber) and other Birds
dc.typeThesis

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