Development of Novel High-Resolution Melting (HRM) Assays for Gender Identification of Caribbean Flamingo (Phoenicopterus ruber ruber) and other Birds
Abstract
Unambiguous 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.
Subject
endangered birdsgender of sexually monomorphic birds
sexually monomorphic birds
fledglings
nestlings
DNA test to sex birds
Attwater's Prairie Chicken
Accipiter species
Ría Lagartos Biosphere Reserve
Lesser Flamingo
Flamingo
Chilean Flamingo
Phoenicopterus ruber ruber
Phoenicopterus
Caribbean Flamingo
High-resolution Melting Analysis
CHD1-W
CHD1-Z
polymorphism
W chromosome
Z chromosome
CHD1 gene
CHD gene
gender of birds
sex determination of birds
gender determination of birds
multiple avian species
non-invasive
conservation of birds
sex-ratio of birds
sex determination
molecular avian gender
molecular gender determination
Universal sexing of non-ratite birds
Citation
Chapman, Alexandra (2012). Development of Novel High-Resolution Melting (HRM) Assays for Gender Identification of Caribbean Flamingo (Phoenicopterus ruber ruber) and other Birds. Master's thesis, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /148342.
Related items
Showing items related by title, author, creator and subject.
-
Siegler, Austen MichaelSince the 1980s, wage convergence between men and women has been slow and inconsistent across high-income nations. Moreover, long-established explanations such that of human capital factors more so explain the gender wage ...
-
Flamingo (In Person Press; DIGITAL: Cushing Memorial Library and Archives, 1999)
-
Flamingo (Asbestos Press; DIGITAL: Cushing Memorial Library and Archives, Unknown)