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Sex identification of parrots, toucans, and curassows by PCR: Perspectives for wild and captive population studies
Abstract
Methods for the identification of the sex of bird species without external sexual dimorphism are specially important in field studies and for captive breeding of endangered taxa. We confirmed the accuracy of a polymerase chain reaction (PCR)-based method to identify the sex in three disparate avian orders that included 31 species of parrot, two species of toucan, and eight species of curassow, for which many individuals were previously sexed. In each case, two DNA fragments were amplified in females and one in males with the use of a single set of primers. This method was also tested on unsexed birds of 13 other species of parrot and five species of toucan. The same kind of polymorphism was detected in each. The PCR products of parrots and toucans could be separated in simple agarose gels, while the curassows' products could only be distinguished in acrylamide gels. An advantage of this DNA test is that samples of blood or feathers can be easily collected and stored at room temperature, which is of particular importance for studies of wild birds. Zoo Biol 17:415–423, 1998. © 1998 Wiley-Liss, Inc.
... Initially, the methods were not suitable for large numbers of species (Dos Remedios et al., 2010). This scenario began to change after the publication of the studies by Griffiths et al. (1996), Miyaki et al. (1998) and Ellegren (1996). These studies investigated the existence of a specific gene for molecular sexing of carinatous (non-ratite) birds: CHD-1 (Vieira, 2009;Vucicevic et al., 2012). ...
... Considering the use of set of primers 1, the reaction conditions used in the present work were as follows: Buffer 1X, 3mM MgCl2, 0.2mM dNTP, 0.5uM of each primer (P2/P8), 0.17U/μL Taq DNA Polymerase, 4μL of extracted DNA, for final reaction volume of 16μL. The thermocycling conditions used were adapted from those described by Miyaki et al. (1998), namely: (i) Initial denaturation at 95°C for 5 minutes, (ii) followed by 36 denaturation cycles at 95°C ...
... What is warranted by both sexes has the Z allele, and this is preferably amplified. According to Miyaki et al. (1998), the pair of primers ideal for sexing non-ratites birds is one that, when there is a preference for amplification of a sex-specific allele, has a preference for ringing with the W allele since it differentiates the sexes (occurring exclusively in females). ...
The sexing birds is considered an important tool for behavioral studies and programs for the reintroduction of animals into the wild. Several techniques are used for this purpose, such as laparoscopy, magnetic resonance and molecular sexing. The first are considered more invasive and stressful for the animal, and the last is considered the most accurate. According to it, the aim of this study was to compare the effectiveness of using three sets of primers in the molecular sexing process of true parrots (Amazona aestiva). Blood samples from 10 animals were collected at a Wildlife Screening Center (CETAS) in Bahia, Brazil. The DNA was extracted and the molecular markers amplified by Polymerase Chain Reaction (PCR) using primer pairs P2/P8, 1237L/1272H and 2250F/2718R. The amplified material was visualized with electrophoresis performed at 2% agarose and 12.5% polyacrylamide gels. Among the primer sets used, the 2250F/2718R pair showed the best results for the sexing process, including visualization of the amplified products on an agarose gel. Agarose gel electrophoresis is considered to be faster and cheaper. The results revealed a sample composition of 5 males (0.5) and 5 females (0.5).
... However, over the years, parrots have become real pets due to their beautiful male/female pairs of a phylogenetically representative group of 135 Psittaciformes species. The majority of these species (76) were already sexed [11,13,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33], but 59 taxons were sexed for the first time in our study. Our aims were (1) to find a marker that would combine the advantages of being universal (able to successfully sex as many species as possible) and easy to use and (2) to provide the guidelines for optimizing PCR conditions during the amplification of the markers using DNA templates isolated from fresh blood, feathers and more challenging dried blood samples. ...
... In chicken, the 23rd intron is amplified with P2 and P8 primers, and the products derived from the chromosomes W and Z are 362 bp and 345 bp and differ by 17 bp due to the insertions of 10 bp and 8 bp and a deletion of 1 bp in the CHD1W copy. To separate the PCR products obtained in female individuals, electrophoresis was performed using 3% [16] or 5% [38] agarose gels or even, for some species, polyacrylamide gels [17]. The location of the P2P8, CHD1iA, CHD1i16, CHD1i9, CHD1iE and NIPBLi16 molecular markers within the CHD1 gene gametologous copies in the chromosomes Z and W (a), and the NIPBL gene copy located in the chromosome W (b) of Gallus gallus. ...
... In chicken, the 23rd intron is amplified with P2 and P8 primers, and the products derived from the chromosomes W and Z are 362 bp and 345 bp and differ by 17 bp due to the insertions of 10 bp and 8 bp and a deletion of 1 bp in the CHD1W copy. To separate the PCR products obtained in female individuals, electrophoresis was performed using 3% [16] or 5% [38] agarose gels or even, for some species, polyacrylamide gels [17]. ...
Sexing of birds is indispensable for scientific, breeding and conservation programs but is difficult in many species and is particularly problematic in the case of nestlings showing no sexual dimorphism. Most useful and efficient methods of sex determination are based on unique features of the Z and W sex chromosomes detected via PCR to distinguish males (ZZ) and females (ZW). During the last twenty-five years researchers searched for the universal marker capable of sexing a maximally wide spectrum of species in a single PCR assay. We screened the phylogenetically representative set of 135 Psittaciformes species including 59 species sexed for the first time. Two known (P2P8, CHD1iA) PCR markers and four additional W/Z polymorphisms (CHD1iE, CHD1i16, CHD1i9 and NIPBLi16) located within the Chromo Helicase DNA binding CHD1 or the Nipped-B homolog NIPBL genes were applied. We present the electrophoretic patterns obtained for the PCR products of the analyzed markers including most typical and atypical patterns allowing sex determination, as well as those obtained when the given marker failed in sexing. Technical aspects of molecular sex determination are discussed: the optimization of amplification conditions, direct PCR and potential misinterpretations. A truly universal marker has not been found, and therefore, we propose a sexing strategy based on multiple CHD1i16, NIPBLi16, CHD1i9 and CHD1iE markers. This new strategy confirms the sex of a given bird with at least two markers detecting independent Z/W polymorphisms, reduces the number of necessary PCR reactions and minimizes the risk of sex misidentification.
... Subsequently, the measurement of steroid hormones was used, a noninvasive technique consisting of the determination of the estrogen/testosterone ratio using fecal samples. However, when these samples were not maintained at a constant freezing temperature, their stability was lost, generating false positive results [Swengel et al., 1996;Miyaki et al., 1998;Cerit and Avanus, 2007]. Cytogenetics is another technique of sexing that is based on the analysis of the karyotype of the bird. ...
... The primer set CHD1Wr/NP/CHD1Zr [Ito et al., 2003;Banhos et al., 2008] also shows great capacity to sex males and females in Falconidae. Among the few primers tested in cracids is the P2/P8 set, which has the limitation of producing Z and W fragments with very little difference in size that are only visualized in polyacrylamide gels [Miyaki et al., 1998]. ...
... We therefore believe that an alternative to avoid or minimize incompatibilities and obtain a higher reproductive rate while breeding in captivity is to form potential partners with a much longer adaptation time, i.e., before they reach the reproductive stage, which is possible by performing molecular sexing of white-winged guans between 4 and 6 months of age. Thus, determination of the proportion of sexes, besides being one of the stochastic effects that can drastically affect the size of the next generation [Miyaki et al., 1998], is important for the development of captive breeding programs. ...
The use of accurate and reliable techniques for sex determination of wild birds is of special importance in captive breeding programs, especially in birds with monogamous, aggressive behavior, with absence of copulation, and with a low hatching rate. Using PCR, we evaluated the relative efficacy of primers HPF/HPR and CHD1Wr/NP/CHD1Zr in the amplification of the chromo-helicase-DNA binding 1 (CHD1) gene for sex determination in Penelope albipennis and 8 other species of cracids, 4 species of falconids, 4 species of accipitrids, and 3 species of psittacines. Primer effectiveness was compared using previously sexed bird samples. The HPF/HPR primer set was found to demonstrate a better performance and reliability. Therefore, these primers should be used to determine the sex of juvenile birds to avoid or minimize incompatibilities during the selection of potential breeding pairs.
... Thus, the dichotomy between strongly sexually dimorphic species and apparently monomorphic species, both presumed with similar mating strategies and forms of parental care generates the question of why those species are monomorphic. To answer this question an adequate identification of the sexes and tests for true monomorphism are necessary in monomorphic species (Bertault et al. 1999, Miyaki et al. 1998, Russello and Amato 2001. ...
... Surprisingly, external morphology is similar between the sexes in all five of the species studied here. It is often the case that sexually dichromatic birds have similar morphological measurements (Jehl and Murray-Junior 1989, Miyaki et al. 1998). However, we expected some sexual differences, however subtle in sexually monochromatic species , Winker et al. 1994, since sex differences, even if subtle, could benefit the individuals to recognize themselves sexually, and would show signs of selection . ...
Population dynamics in hummingbirds in South America is virtually unknown and
unstudied, due to difficulty in their capture in numbers for mark and recapture studies. Yet,
due to species diversity, distributions, the role they play as pollinators of ephemeral
resources and their colorful displays, study of their population dynamics is important for
understanding their ecology and evolution. Here, we begin to fill this lacuna by using
capture-mark-recapture to examine population dynamics, and other aspects of
hummingbird ecology. We examined molt in a variety of species and sexual dimorphism
and molecular identification of the sexes in a group of five apparently monomorphic
species. All hummingbird species in the study area tend to migrate, but details of migration
and comparisons are difficult to determine because marked birds were never recaptured
away from the study area. The most abundant and consistently common species, the Whitethroated
Hummingbird (Leucochloris albicollis) had a population size that varied from
~150–450 individuals (monthly variation) in the study area, with the peak abundance in
March, following after the apparent breeding season in November-December. Survival was
estimated at 33% per year, which is low for a tropical bird. Molt is similar to that of other
hummingbirds, from proximal to distal primary feathers, but in our study species varied
less in the sequence of the most distal feathers as compared to other studies. Secondaries
were quite variable in their sequence of molt, while retrices were more consistent. Molt
took place beginning in December and continued to April, but defining the end of molt was
difficult because species migrate while undergoing molt, and so recaptures to delimit the
end of molt was not possible. Three of the five apparently monomorphic species, once sex
was identified through our new molecular technique for hummingbirds, were sexually
dichromatic (Colibri serrirostris, Eupetomena macroura, L. albicollis) in UV light
reflectance in their feathers. Size and shape, however, were similar in both sexes. With
these results, we find that hummingbird species can be very abundant, but abundance
varies widely over the year, they migrate while molting but the details of their end points
of migration remain unknown, and survival rates are low, suggesting relatively high
reproductive success. We show sexual dichromatism in UV light (which humans do not
perceive) in three of five species, and suggest that the others also are dichromatic, which
will be discovered on further examination of the appropriate feathers. Thus, with this study
we have begun to better understand hummingbird population dynamics.
... Knowing the sex of an individual is also a critical piece of information in captive breeding for conservation programs, for science education, and in terms of contributions to biological diversity. 26 For instance, when birds are exchanged by means of commerce or to prevent inbreeding, knowledge of sex is a prerequisite. 16 Moreover, determining sex in zoos is necessary in order to increase successful reproduction (1993 Meeting of the EEP Penguin TAG, Penguin Conserv 1993;6:10-12, available at: https://goo.gl/J8W6X2). ...
... However, successful results were obtained for those species amplified with the 2550F/2718R and P2/P8 primer sets in both our study and others. 13,21,26,29,34 The sex of Bubo bubo using the 2550F/2718R and P2/P8 primer sets could not be determined because of the lack of length polymorphism. 34,36 However, using the ARMS (amplification refractory mutation system) technique, it was possible to sex the owl species B. bubo and Otus scops. ...
Because many bird species are monomorphic or only sexually dimorphic in adult stages, it is difficult to determine their sexes, which may cause significant problems in population and conservation studies. DNA-based sexing relies on the chromodomain helicase DNA binding (CHD) gene located on the W chromosome and its homolog on the Z chromosome, giving distinct banding patterns on agarose gel as a result of length differences in intronic regions within this gene. We used 3 specific primer sets, CHD1F/CHD1R, 2550F/2718R, and P2/P8, for sex determination of 230 samples from 77 avian species. We report here the records for 70 of those species analyzed using the CHD1F/CHD1R primer set, and 49 species using 2550F/2718R, and 46 species using P2/P8. CHD1F/CHD1R PCR products on agarose gel generally showed an apparent single band in males and 2 bands in females, but the products of 2550F/2718R (61%) and P2/P8 (42%) showed distinct banding patterns for separate bird orders. However, when PCR products of these last 2 primer pairs labeled with fluorescent dye were run in a capillary gel and detected using a DNA analyzer, P2/P8 gave 2 distinguishable peaks in females, whereas 2550F/2718R results remained the same. DNA sexing with any of those 3 primer sets can be used for all sexually monomorphic avian taxa although the primer sets should be compared before choosing the most efficient one for molecular sexing of the studied species.
... Here we report the results of a study on nestling sex-ratio variation in the Yellow-naped Amazon (Amazona auropalliata), a large (550 g), highly social, sex- ually monomorphic Neotropical parrot. These data represent only the second estimate of sex ratios in wild Neotropical parrot populations (Miyaki et al. 1998), and the first attempt to associate sex allocation in parrots with environmental and life history variables. Indirect measures of female dispersal based on mitochondrial control region sequence variation indicate that long-range dispersal of females is common in the Yellow-naped Amazon, but the extent of sex-biased dispersal is unknown (Wright and Wilkinson 2001). ...
... Additionally, the adult sex ratio of the Kakapo is highly skewed toward males in the wild (Trewick 1996). However, the sex ratio of monomorphic Hyacinth Macaw (Anodorhynchus hyacinthinus) chicks in the Pantanal, Brazil was close to 50:50 (Miyaki et al. 1998). ...
Many birds, including some parrots, may adjust the sex ratio of their offspring in relation to the relative fitness benefits of sons and daughters. We investigated nestling sex ratios in Yellow-naped Amazons (Amazona auropalliata) using a molecular sexing technique that amplifies intronic regions of the CHD-W and CHD-Z genes in birds. We examined all nestlings in 37 complete clutches comprising 77 chicks. The overall nestling sex ratio did not differ from unity. Sex allocation was not associated with hatch date, sequence of hatching, or clutch size. We also found no difference in sex ratio between two regional dialects. Female Yellow-naped Amazons may be unable to control their hatchling sex ratio. Alternatively, there may be no fitness benefits to females producing more of one sex in relation to the factors we measured here.
No Existe Evidencia que Indique Modificaciones Adaptativas de la Proporción de Sexos en la Progenie de Amazona auropalliata
Resumen. En muchas aves, incluyendo los loros, la proporción de sexos en la progenie puede ajustarse en relación a los beneficios relativos de adecuación biológica de hembras y machos. Dichas tasas fueron investigadas en Amazona auropalliata por medio de una técnica molecular de determinación sexual por la cual se amplifican regiones intrónicas de los genes CHD-W y CHD-Z de aves. Se examinaron todos los pichones de 37 nidadas completas, constituidas por 77 pichones. La proporción de sexos total no resultó diferente a uno. La asignación sexual no estuvo correlacionada con la fecha de eclosión, la secuencia de eclosión, ni el tamaño de la nidada. Tampoco se encontraron diferencias en las proporciones de sexos entre dos dialectos vocales regionales. Las hembras de A. auropalliata podrían no tener la habilidad de controlar la proporción de sexos de su progenie. Alternativamente, es posible que en términos de adecuación biológica, no haya diferencia en el beneficio de producir una progenie enriquecida en un sexo determinado con respecto a los factores medidos en este estudio.
... Ornithologists have increasingly adopted the PCR analysis technique. Adults of 36 species of Psittaciformes, seven species of rhamphastids, and eight species of cracids were accurately sexed using PCR (Miyaki et al. 1998). Griffiths et al. (1998) used PCR to determine the sex of adults from 11 orders with representatives from 23 families, but it could not sex the Ostrich, Struthio camelus, a ratite. ...
... Adults of 36 species of Psittaciformes, seven species of rhamphastids, and eight species of cracids were accurately sexed using PCR (Miyaki et al. 1998). Griffiths et al. (1998) used PCR to determine the sex of adults from 11 orders with representatives from 23 families, but it could not sex the Ostrich, Struthio camelus, a ratite. In addition to sex determination in adults, PCR provided definitive sexing of immature Greater Flamingos (Phoenicopterus ruber roseus) and Great Bustards (Otis tarda; Bertault et al. 1999;Fridolfsson and Ellegren 1999;Martin et al. 2000). ...
Gender identification is important in demographic studies of bird populations but is difficult with monomorphic bird species. Flow cytometry, a method which estimates differences in DNA content in erythrocyte nuclei, is one potential sexing method. In another sexing method, differences between the autosomal and sex-linked (W) copies of the chromo-helicase-DNA binding (CHD) gene can be determined using the polymerase chain reaction (PCR). Frozen blood samples collected during the breeding season from known-sex adults and juvenile birds were used to test the applicability of flow cytometry and PCR as sexing methods for the Wood Thrush (Hylocichla mustelina). With flow cytometry, 86% of 14 known adults were definitively and correctly sexed. PCR correctly sexed 100% of 11 known adults. Of the 18 juveniles, 15 were sorted definitively by flow cytometry: 14 as male and one as female. PCR sexed the same 18 juveniles as 11 males and seven females, including the three juveniles that were unclassified by flow cytometry. In addition, three juveniles identified as males using flow cytometry were identified as females by PCR, among which were two individuals that consistently fell into the high end of the male genome size range. We conclude that PCR is a reliable sexing method for the Wood Thrush, but flow cytometry has limited reliability due to the lack of disparity in intergender genome size.
... Entre las técnicas más empleadas encontramos el análisis de esteroides fecales, este método mide la proporción entre estrógenos/testosterona en las heces fecales de las aves; donde se espera encontrar proporciones más altas de estrógenos en aquellas muestras provenientes de las hembras. Para lograr un mayor grado de exactitud en los resultados obtenidos es necesario mantener las muestras en congelación desde el momento que se toman con el fin de garantizar la estabilidad de las hormonas, los resultados se ven afectados por la edad del animal y la época de cría (Swengel et al., 1996;Miyaki et al., 1998;Cerit y Avanus, 2007). La citogenética es una técnica que permite identificar el sexo del ave mediante el análisis de los cromosomas. ...
... sangre, pelo, cañón de las plumas) y es conservado durante toda la vida del animal y así mismo este puede ser almacenado durante largos periodos de tiempo in vitro. Se han descrito diversas aproximaciones moleculares para la identificación del sexo en aves; ellas incluyen polimorfismos de DNA amplificados al azar (Lessels y Matemam, 1998), fragmentos amplificados de longitud polimórfica (Griffiths y Orr, 1999), minisatélites (Miyaki et al., 1998), polimorfismo de longitud de fragmentos de restricción (Griffiths y Tiwari, 1995;Griffiths et al., 1996). ...
The lack of sexual dimorphism in nestling, juvenile or adult birds of large number of avian species, makes it difficult or impossible sex determination based on phenotipic characteristics. To use molecular markers for bird sex determination is a rapid and safe procedure; moreover this methodology minimizes the stress during sampling, compared to other invasive techniques that could affect the health or biological stability of the animal. The early sex determination in birds is of particular importance when considering ex situ conservation programs, production, exploitation or population ecology studies. This review presents the methodologies used to sex determination, making emphasize on molecular tools, showing its advantages and limitations.
... It is necessary to correctly identify sex for wildlife conservation as well as evolutionary and ecological studies (Miyaki et al., 1998; Birkhead et al., 2001; Dubiec et al., 2006). In avian species, females have heterogametic sex chromosomes Z and W, whereas male birds are homogametic ZZ (Ellegren, 2001). ...
... However , a correct identification of this EE0.6 fragment from crested ibis required a duplex PCR involving species specific primers (Park et al., 2011; Kasuga et al., 2012) as well as an internal control, e.g., the conserved exon of common W and Z chromosome-linked spindlin gene (Itoh et al., 2001) or the mitochondrial 12S rRNA gene (Park et al., 2011), to exclude possible false negatives due to technical failure (Itoh et al., 2001). The P2/P8 has been a commonly used primer set for sex identification in avian species (e.g., Griffiths, et al., 1998; Jensen et al., 2003; Cerit and Avanus, 2007); however, length differences between the CHD1-Z and CHD1-W introns were found to be varied among species and it may be necessary to use a polyacrylamide gel if such differences are to be limited (e.g., Miyaki et al., 1998; Dawson et al., 2001; Jensen et al., 2003). ...
Sex Identification of monomorphic birds, especially endangered avian species, is essential for ecological study and biodiversity conservation. In this study, two popular primer sets of 2550F/2718R and P2/P8, which were designed to amplify different fragments of chromodomain-helicase-DNA binding protein 1 (CHD1) genes mapped on both Z and W chromosomes in birds, were used to identify for the first time the sex of individuals of the endangered species crested ibis (Nipponia nippon) in a large number of samples. An improved primer set of 2467F/2530R was re-designed to be specific to crested ibis following their conserved sequences derived from the 2550F/2718R primers. PCR products of the new primers were conveniently visualized with two bands of 552 base pairs (bp) and 358 bp for females, but a single band of 552 bp for males in routine 1.8% agarose gel. Similarly, the P2/P8 primer set amplified two fragments of 398 bp and 381 bp from females but one fragment of 398 bp from males; however, a high resolution involving 10% Polyacrylamide gel had to be employed to resolve the 17 bp insertions/deletions (in/dels) present between the two amplicons in females. In addition, a microsatellite locus NnNF05 was validated to be sex-linked and shown to be effective in the sexing of crested ibis, supporting its utility in non-invasive sampling. This study provides a rapid, convenient, and reliable molecular assay for improving sex identification in the monomorphic and monogamous crested ibis, and thus facilitates the selection of breeding pairs in captive programs and reintroduction initiatives.
... Entre las técnicas más empleadas encontramos el análisis de esteroides fecales, este método mide la proporción entre estrógenos/testosterona en las heces fecales de las aves; donde se espera encontrar proporciones más altas de estrógenos en aquellas muestras provenientes de las hembras. Para lograr un mayor grado de exactitud en los resultados obtenidos es necesario mantener las muestras en congelación desde el momento que se toman con el fin de garantizar la estabilidad de las hormonas, los resultados se ven afectados por la edad del animal y la época de cría (Swengel et al., 1996; Miyaki et al., 1998; Cerit y Avanus, 2007). La citogenética es una técnica que permite identificar el sexo del ave mediante el análisis de los cromosomas. ...
... sangre, pelo, cañón de las plumas) y es conservado durante toda la vida del animal y así mismo este puede ser almacenado durante largos periodos de tiempo in vitro. Se han descrito diversas aproximaciones moleculares para la identificación del sexo en aves; ellas incluyen polimorfismos de DNA amplificados al azar (Lessels y Matemam, 1998), fragmentos amplificados de longitud polimórfica (Griffiths y Orr, 1999), minisatélites (Miyaki et al., 1998 ), polimorfismo de longitud de fragmentos de restricción (Griffiths y Tiwari, 1995; Griffiths et al., 1996)., 1993). Los microsatélites son vulnerables a errores en la replicación, a causa de su estructura repetitiva como consecuencia de una deficiencia postreplicativa durante la reparación de apareamientos erróneos (Toledo y Cruz, 2005). ...
La ausencia de dimorfismo sexual en los estadios juveniles y durante la edad adulta
de gran cantidad de especies de aves, dificulta o imposibilita la determinación del
sexo basados en el fenotipo. El empleo de marcadores moleculares para determinar
el sexo de las aves es una herramienta útil debido a la exactitud y rapidez de los resultados
y a su vez se constituye en un método que minimiza el estrés durante la toma
de muestra, comparado con otras técnicas invasivas que pudieran afectar la salud o
estabilidad biológica del animal. La determinación temprana del sexo en aves resulta
de especial relevancia cuando se consideran programas de conservación ex situ, producción,
explotación y estudios de ecología de poblaciones. Esta revisión presenta las
metodologías usadas para determinar el sexo, haciendo especial énfasis en herramientas
moleculares, presentando sus ventajas y limitaciones.
... The sexual identification of birds is very important for population and conservation studies, particularly for reproduction programs of threatened species (Ellegren & Sheldon 1997, Miyaki et al. 1998), identification of morphological aspects (King & Griffiths 1994, Burns 1998), behavioural ecology and evolutionary biology (Ellegren 1996, Lessells & Mateman 1998). Sex is one of the most important variables to distinguish individuals in a population. ...
... Techniques for sex determination in birds include laparoscopy, karyotyping (Basrur et al. 1998), analysis of the faecal steroid, DNA fingerprinting and molecular techniques based on the chromo-helicase-DNA binding (CHD) gene (Ellegren 1996, Griffiths et al. 1998). The CHD gene has been used successfully in many bird species (Griffiths et al. 1998, Miyaki et al. 1998, Ito et al. 2003, Sacchi et al. 2004, Lee et al. 2007, 2010), since the gene is preserved in most bird species (Griffiths & Tiwari 1995). ...
Sex determination is important for conservation and population studies, particularly for reproduction programs of threatened species and behavioural ecology. Turdus amaurochalinus, Creamy-bellied Thrush, only exhibits sexual dimorphism during the breeding season, when males are considered to show intense yellow bills, and females and immature males show dark brown bills. The objectives of this study were: 1) to determine the sex of individuals using genetic techniques, and 2) to test the hypothesis that sex dimorphism can be detected by morphometry. This study was carried out at Parque Nacional da Restinga de Jurubatiba, a preserved area located on the North coast of Rio de Janeiro State. The birds were captured using ornithological nets, singly marked with metal rings, weighed, measured and had blood samples collected before being released. The sex of 42 T. amaurochalinus individuals was determined using the CHD gene marker. A total of 20 males and 22 females were identified from June to August, with peak capture frequency in June. Turdus amaurochalinus females and males differed significantly in morphometrical measures. The most important traits to distinguish males from females were wing length (Student t-test = 4.34, df = 40, p = 0.0001) and weight (Student t-test = 2.08, d f = 40, p = 0.044): females were heavier and had significantly shorter wing length than males. Females and males were correctly classified in 86% and 75% of cases, respectively, using Discriminant Analysis. The molecular analysis was the most secure method for sex determination in the studied species.
... Although both random Mendelian sex allocation and the adaptive sex determination model suggested by Fisher [23] predicts a stabilized number of males and females in a population, a review made by Donald [24] supported that skewed adult sex ratios are actually common in wild birds. An asymmetric sex ratio (significantly different than 1:1) is a type of stochastic phenomenon that has the potential to erode the genetic variability of populations, by limiting the number of couples in monogamous birds and consequently reducing the number of individuals generated in the population [25]. In this way, estimating sex ratio is very important to inform conservation actions. ...
The Brown-backed Parrotlet, Touit melanonotus, is a rare endemic bird to the Brazilian Atlantic Forest, currently considered as "Vulnerable" in the Brazilian Red List of Threatened Species. We estimated the sex ratio of a wild flock of T. melanonotus using molecular markers, examined morphological variation in 34 museum specimens to test for sexual dimorphism, and conducted a literature review about sex ratio in Psittacidae for comparative purposes. We found a sex ratio of 0.8:1 (male/female; n = 29) in T. melanonotus, and a χ2 Goodness-of-fit test showed no significant difference from equality (p > 0.05). We describe three main categories in plumage: the first (and most common) comprises uniformly lime green birds, slightly darker on the head. The second is composed of individuals who are overall lighter, with the breast feathers washed with light greenish gray, and feathers of the head being dark lime green, presenting a sharp contrast with the breast feathers. The third and the rarest one is composed of birds with light greenish gray underparts with emerald green and darker upper parts. T. melanonotus has no apparent sexual dimorphism. We found no evidence of geographic variation. Sex ratio deviation may not be a parameter increasing the vulnerability of the species. Data like these represent a big leap in the knowledge of the species and has the potential to help and inform conservation efforts.
... and Molecular Evolution Laboratory at the Biology Institute of the University of São Paulo, Brazil. In the laboratory, DNA was extracted and PCR was run for sex identification following Miyaki et al. 35,36 . Due to logistical challenges in sending the samples, as well as to the remoteness of the nests and unfavorable weather conditions, not all the chicks were sexed (sex identification method details see Miyaki et al. 36 ). ...
Studies on the breeding of vulnerable and endangered bird species are hindered by low numbers of individuals, inaccessible location of nests, unfavourable environmental conditions, and complex behavioural patterns. In addition, intraspecific variation may emerge only following long-term, systematic observations of little-known patterns and processes. Here, data collected over 30 years were used to determine growth model of hyacinth macaw ( Anodorhynchus hyacinthinus ) chicks in the Pantanal biome of Brazil. During this period, the speed of growth and body mass of chicks varied widely. Four growth models were tested: logistic, Gompertz, Richards, and cubic polynomial. They were fitted using three biometric measurements: body mass, total length, and tail length. The best-fitting growth curves were identified using Akaike’s information criterion. The best models were the cubic polynomial for body mass, Richards for total length, and Gompertz for tail length. We confirmed the occurrence of dwarf individuals, whose body mass, total length, and tail length were 20%, 22%, and 70% smaller, respectively, than in the overall population. The dwarfs remain small in size after having fledged and are easily identified as adults. We discuss the importance of long-term studies to identify windows of opportunity for further research that will help in the conservation of endangered macaw species.
... It would be interesting to perform combined analyses with cranial shape and attributes of the feet and hypotarsal structures to measure the differences between the arboreal species and those parrots which are conservatively feeding on grasslands. However numerous of species are sexually monomorphic (Miyaki et al. 1998), it is possible that there are species -e.g. the Great-billed Parrot (Tanygnathus megalorynchos) -where the sexual dimorphism might have role in the cranial shape, as it was observed previously in the case of the Kea . Similarities in the skull shape can reflect also the phylogenetic relation. ...
Parrots (Psittaciformes) are a unique and diverse avian group and vary tremendously in size, shape, and colour. Mainly distributed throughout the tropics and subtropics, most species of parrots are largely or exclusively arboreal with several exceptions. The species also differ in diet and habitat, which led to different musculoskeletal adaptations of the skull. However, parrots have conspicuous generalized external features; in this recent study, we tried to increase our knowledge of the cranial shape and foraging habits. A geometric morphometric approach was used to analyse two-dimensional cranial landmarks. We used principal component (PC) analyses on measurements that may be related to diet. The PCs described the relative height of the cranium , the relative length and curvature of the beak, differences in the orientation and curvature of the lacrimal bone and the upper margin of orbits, variation in the size and position of the palatine bone and the relative width of the cranium, and variation in the relative size of the neurocranium compared to the viscerocranium . The dietary categories overlap in the morphospace but the analysis in lateral and ventral view resulted in significant differences.
... Individuals were banded with metal bands provided by CEMAVE (Centro Nacional de Pesquisa para Conservação das Aves Silvestres) as part of the Brazil National Banding System, as well as with 2 colored bands for individual identification. We weighed each individual and collected blood samples (SISBIO permit 30336-3) to sex the birds (Myiaki et al. 1998). ...
We present the first detailed account of the breeding biology and egg description of the Restinga Tyrannulet (Phylloscartes kronei). Restinga Tyrannulets build their nests (n = 10) 0.7-3.0 m above ground level. The nests were closed and ovoid, with an interior of dry grass leaves and an exterior covered with moss, all adhered with spider webs to fix the nest material. Nest chambers were lined with plumose seeds. Nest construction lasted ∼19 d and was apparently accomplished exclusively by females. We recorded a clutch size of 2 to 3 white eggs (15.9-16.8 × 12.7-13.1 mm). Incubation was performed only by females and lasted ∼12 d (constancy of incubation = 0.4), but both adults fed the nestlings and fledglings and removed fecal sacs from the nests. Nests and eggs were within the general pattern found for genus Phylloscartes. Female P. kronei had a more important role than males during the breeding period and seemed to be solely responsible for nest construction, which differs from reports for other Phylloscartes species.
... Yet several species are sexually monomorphic and monochromatic (Ruschi 1982, Sick 1997, Schuchmann 1999, Fogden et al. 2014, Winkler et al. 2015, which is an enigma because female-only parental care typically goes hand-in-hand with sexual selection of males that result in bright colors or energetic displays (Hurly et al. 2001, Rico-Guevara & Araya-Salas 2015. To understand the lack of sexual dimorphism among hummingbirds we must first identify sex and then test for possible cryptic dimorphism (Miyaki et al. 1998, Bertault et al. 1999, Russello & Amato 2001. ...
Many species of hummingbirds are sexually monochromatic. This is surprising because in all hummingbird species parental care is carried out by females, while males are promiscuous, and this mating system is usually associated with some form of sexual dimorphism. To study sexual selection in monochromatic species, the identification of sex is a prerequisite. Using blood and feathers from live individuals we apply a simple method to identify sex. We amplified fragments of the CHD-1 gene through PCR, using P2 and P8 primers with fluorescence, followed by genotyping in an automatic sequencer. We sexed 110 individuals of five species: Versicolored Emerald (Amazilia versicolor), White-throated Hummingbird (Leucochloris albicollis), White-vented Violetear (Colibri serrirostris), Black Jacobin (Florisuga fusca), and Swallow-tailed Hummingbird (Eupetomena macroura). We found gene fragments of two sizes: 374 bp in both sexes (Z chromosome) and either 379 or 383 bp in females (W chromosome). Using this method for monochromatic species permits sexing in species with similar fragment sizes between sexes (i.e., hummingbirds). Sexing monochromatic birds will be very useful for field studies that require continuous monitoring of individuals in the wild.
... 21 (1) enero -junio 2017. 112-121 por machos y hembras en edad reproductiva o si se liberan parejas ya establecidas (WHITINGHAMM et al., 2000;JONES et al., 2003;KOCIJAN et al., 2011;MIYAKI et al., 1998). ...
resumen La familia Psittacidae es uno de los grupos con mayor declive poblacional a causa de la cacería, fragmentación y degradación de hábitat. Los programas de conservación y de reintroducción requieren la identificación del sexo en todos los individuos para realizar análisis de parentesco, estimar parámetros demográficos y lograr que las liberaciones o reintroducciones de individuos tengan mayor probabilidad de éxito al liberar grupos conformados por machos y hembras en edad reproductiva o de parejas ya establecidas. En los psitácidos la discriminación sexual presenta dificultades debido a la ausencia de dimorfismo sexual y a que los métodos tradicionales de sexaje son traumáticos o requieren protocolos extensos que no funcionan en individuos juveniles. En este sentido, las herramientas moleculares y algunas citogenéticas resultan efectivas por ser poco invasivas y tener una mayor sensibilidad. Tanto las herramientas moleculares como las citogenéticas empleadas en este estudio permitieron la diferenciación entre machos y hembras de ocho especies de psitácidos (Amazona amazónica, Amazona festiva, Amazona ochrocephala, Ara ararauna, Ara macao, Ara severa, Aratinga wagleri y Pionus menstruus). En términos generales, la eficiencia de la reacción de PCR para amplificar los genes CHD-Z y CHD-W para los individuos muestreados fue del 83 % (34 de 41 individuos amplificados). En un total de 32 individuos de seis especies de psitácidos (A. ararauna, A. macao, A. festiva, A. ochrocephala, P. menstruus) se hizo posible la diferenciación por cariotipo en 25 de ellas, lo que representa una sensibilidad del 78 % para la prueba citogenética de sexaje. No fue posible obtener un número suficiente de metafases con el fin de establecer el patrón de cariotipo de A. wagleri y A. severa por el bajo número de individuos. El sexaje de psitácidos puede realizarse a través de cualquiera de estos dos métodos, aunque el sexaje citogenético resulta inconveniente para las especies que no tienen cariotipo definido tal como es el caso de A. severa y A. wagleri. El sexaje molecular proporciona una técnica poco invasiva, eficaz y rápida para obtener el sexo de individuos mediante la utilización de los iniciadores P2 y P8.
... Hence, P2/P8 primers successfully discriminated sex in chicken. Similar findings were also reported by Miyaki et al. (1998) and in non-ratite birds, however, in ratite birds, only single fragment of 380 bp was observed in either sex (Fig. 1). Similar results were also reported by Constantini et al. (2008) in juvenile emu and by Cerit and Avanus (2006) in ostrich and emu. ...
In spite of number of methods for sex determination in birds, it is very difficult to distinguish sex especially in ratite birds due to lack of sexual dimorphism. Chromodomain helicase DNA binding 1 gene (CHD 1) is the choice of gene for gender differentiation using PCR based molecular method. In present study, non ratite CHD gene specific primers viz. 1237L/1272H, 2550F/2718R, P2/P8, P2/P3 and ratite bird specific primers viz.W5/ W7 and
W1/ K7 were used for gender differentiation in ratite birds. The ratite bird specific primer W5/W7 was the only primer, which determined the sex in emu as well as ostrich successfully, while 1237L/1272H, 2550F/2718R, P2/P8, P2/P3 primers were unable to discriminate sex in emu and ostrich but ratite and non ratite primers can be used to discriminate the sex in non-ratite bird, primarily in chicken. In an alternative approach of PCR-RFLP, the high resolution melting curve (HRM) analysis showed conflicting pattern in both sexes of ratite birds but in chicken HRM analysis showed clear cut differential melting temperature in both sexes, hence HRM can be used for gender differentiation successfully.
... Although molecular sex typing of birds initially required a blood sample (Fridolfsson and Ellegren 1999;Griffiths et al. 1998) primers are now available for freshly plucked or collected feathers (Bosnjak et al. 2013;Ong and Vellayan 2008;Presti et al. 2013). Typically the molecular sexing of birds targets sex specific DNA fragments that are visualized on agarose gel electrophoresis, providing a low cost and simple laboratory assay (Miyaki et al. 1998;Ong and Vellayan 2008). For this technique the DNA rich avian blood with nucleated erythrocytes is usually used (Fridolfsson and Ellegren 1999;Griffiths et al. 1998). ...
Genetic tagging is the unique identification of individuals by their DNA profile. This technique is well established in mammals, but it has not yet been widely adopted for birds. Extraction methods for minute amounts of DNA even enable the use of genetic tagging from non-invasive samples, like hair, scat, or feather. In this study, we evaluate the potential for non-invasive genetic tagging by using molted feathers of two sympatric macaw species in the Peruvian Amazon. Correct species identification is critical when relying on feathers for genetic analysis, so we describe multilocus methods for species identification. We evaluate the quality of naturally shed macaw feathers in tropical environmental conditions and present new primers for molecular sexing on the feather samples. We successfully validated 11 microsatellite markers for use in genetic tagging studies on large macaws and confirmed that DNA from blood and feather samples yields equivalent population genetic patterns. The techniques described here can be implemented for other birds with higher conservation concern.
... The thermal protocol consisted of an initial denaturation step at 95°C for 4 min, followed by 35 cycles of denaturation (95°C, 30 sec), annealing (55°C, 30 sec) and DNA extension (72°C, 45 sec), and a final extension step at 72°C for 4 min. Wang et al., 2007 Miyaki et al., 1998 Not available Wang et al., 2007 Wang et al., 2007 Cuculiformes ...
... A razão entre estrógeno e testosterona, utilizada na análise de esteróides fecais, não é altamente confiável para determinação do sexo. Por último, embora a sexagem por meio de análises cariotípicas seja adequada em aves, já que cromossomos sexuais ZW e ZZ são encontrados em fêmeas e machos, respectivamente, esta demanda grande tempo e é difícil em espécies que possuem cromossomos sexuais praticamente idênticos 28 . ...
Entre os animais silvestres envolvidos em tráfico/comércio ilegal no Brasil, as aves compreendem um dos grupos mais atingidos, especialmente devido a características como canto e colorido das penas. Atualmente, análises genéticas compreendem uma das formas mais eficazes de gerar dados para solucionar e minimizar os resultados de crimes ambientais e comércio ilegal de animais silvestres. Uma das análises genéticas que podem ser utilizadas com o intuito de subsidiar programas conservacionistas associados ao tráfico ilegal de aves refere-se à sexagem molecular, dado que neste grupo de vertebrados nem sempre é possível identificar o gênero por meio de caracteres morfológicos. A sexagem molecular pode ser feita com base em amostras de DNA obtidas de diferentes fontes, como penas e sangue, e posterior amplificação de regiões dos genes CHD-Z e CHD-W (chromo helicase-DNA binding). Os dados de perfis genéticos sexoespecíficos servem de subsídio a programas conservacionistas de manutenção e/ou reprodução em cativeiro e posterior soltura ou reintrodução dos animais em ambiente natural.
... Desert bighorn sheep (Epps et al. 2005) Sex determination Eurasian otter (Dallas et al. 2000); red deer (Huber et al. 2002); canid species (Ortega et al. 2004;Seddon 2005); felid species (Pilgrim et al. 2005); wolverine ); Asian elephant ; birds (Miyaki et al. 1998) Molecular tracking or genetic tagging, genetic monitoring Brown bear (Taberlet et al. 1997;Lorenzini et al. 2004;Tallmon et al. 2004); mountain lion (Ernest et al. 2000); gray wolf ; chimpanzees ; humpback whales (Palsbøll et al. 1997); black and brown bears (Woods et al. 1999), wolverine ) Disease status ...
INTRODUCTION A key component of the emergence of conservation genetics as a recognisable subdiscipline of conservation biology over the last ten years has been the development of methods to genetically assess and monitor populations of endangered species non-invasively. The rapid development of methodologies for ensuring the accurate capture of molecular data from elusive, easily stressed or potentially dangerous (!) organisms and concerns over the accuracy of the data produced have prompted a number of excellent reviews on the subject in recent times (e.g. Taberlet et al. 1999; Taberlet and Luikart 1999; Piggott and Taylor 2003; Woodruff 2003; Wayne and Morin 2004). Here, we will review the issues and wide-ranging applications of non-invasive genetic analysis without focusing on the molecular technicalities in great detail. Why non-invasive genetics? Before the advent of non-invasive genetics and the use of animal by-products such as faeces, shed hair, feathers, bones, fish scales, teeth, etc., obtaining genetic material from wild populations was often ethically (in particular for species listed as endangered and critically endangered under CITES regulations) and logistically extremely difficult. Now such analysis is increasingly possible and the sampling of large populations without visual/physical contact is particularly beneficial for endangered species or if the species studied can transmit or are susceptible to disease (e.g. great ape species whose pathogens are often extremely similar to those of the researchers studying them). In a century where linking behaviour, social structure, dispersal and population genetic structure has become a new challenge for conservation geneticists, the development of non-invasive sampling and genotyping has provided the opportunity to explore these links and has dramatically opened new areas for research. The characterisation of non-invasive material using molecular markers allows biologists to identify and count individuals in wild populations,identify the sex of those individuals and determine their movement patterns, infer parentage, kinship and relatedness, and assess pathogens and diet (see Kohn and Wayne 1997 for a review).
... The thermal protocol consisted of an initial denaturation step at 95°C for 4 min, followed by 35 cycles of denaturation (95°C, 30 sec), annealing (55°C, 30 sec) and DNA extension (72°C, 45 sec), and a final extension step at 72°C for 4 min. Wang et al., 2007 Miyaki et al., 1998 Not available Wang et al., 2007 Wang et al., 2007 Cuculiformes Tauraco persa ...
The aim of this research was to test the CHD gene (Chromo Helicase DNA-binding gene) as a universal molecular
marker for sexing birds of relatively distant species. The CHD gene corresponds to the aim because of its high degree of conservation and different lengths in Z and W chromosomes due to different intron sizes. DNA was isolated from feathers and the amplification of the CHD gene was performed with the following sets of polymerase chain reaction (PCR) primers: 2550F/2718R and P2/P8. Sex determination was attempted in 284 samples of 58 bird species. It was successful in 50 bird species; in 16 of those (Alopochen aegyptiacus, Ara severus, Aratinga acuticaudata, Bucorvus leadbeateri, Cereopsis novaehollandiae, Columba arquatrix, Corvus corax, C. frugilegus, Cyanoliseus patagonus, Guttera plumifera, Lamprotornis superbus, Milvus milvus, Neophron percnopterus, Ocyphaps lophotes, Podiceps cristatus, and Poicephalus senegalus), it was carried out for the first time using molecular markers and PCR. It is reasonable to assume that extensive research is necessary to define the CHD gene as a universal molecular marker for successful sex determination in all bird species (with exception of ratites). The results of this study may largely contribute to the aim.
... It is known that sex identification of birds is essential part of ex-situ conservation breeding programmes. Although the CHD gene has been used successfully in many bird species Miyaki et al. 1998;Ito et al. 2003;Sacchi et al. 2004;Lee et al. 2007Lee et al. , 2010, but we mainly discussed the merits of two such methods for the molecular sexing of captived birds in this study, the Griffith et al. (1998; P8/P2) and Fridolfsson and Ellegren (1999;2550F/2718R). The aim of this work was to test the 2-molecular sexing method on bird species, particularly for birds kept in captivity in Indonesia. ...
Visually identifying the sex of a bird can be difficult. It cannot be done in half the world’s species when they are adults, and virtually none can be sexed as chicks. Despite this, the sex of a bird is vital for captive breeding. An increased number of birds are being sexed using DNA amplification techniques. In this approach, the CHD-W and CHD-Z are distinguished by the amplification of an intron present in both genes. PCR products on the gel electrophoresis vary in size revealing one band in males at the CHD-Z, and two bands in females corresponding to both the CHD-W and CHD-Z. Two independent sets of primer (P8/P2 and 2550F/2718R) were used to amplify the CHD gene region from both the Z and W chromosome. One hundred and ten (110) birds were sexed using first pair of primers: (P8/P2). Sexing results indicated that 81.8% were successfully determined, 12.7% failed to be amplified and 5.5% were not perfectly determined because the PCR products showed thick band. The thick band caused misidentified female to male birds. An alternative primer (2550F/2718R) was applied to solve the problem. Two hundreds and twenty-nine birds were sexed and the results showed 100% successfully determined. From this study, it is suggested to use a pair of 2550F and 2718R primers for distinguishing a male from a female bird.
... In that regard, the aim of this study was to assess the effectiveness of a molecular method for sex determination in parrots and samples were taken from species with a relatively wide distribution throughout the Psittaciformes order. In previous studies (Miyaki et al., 1998; Taylor and Parkin, 2008), sexing various species of parrots was mostly conducted with P2/P8 primer set described by Griffits et al. (1998). Ong and Vellayan (2008) indicated that 2550F/2718R primer set, which was used in this research, provides a higher confidence level of establishing the sex of birds even without the use of polyacrylamide gels as is required in some bird species with the use of primers P2/P8 and 1237L/1272H (Kahn et al., 1998). ...
The aim of this study was to test the feasibility and efficacy of a non-invasive molecular method for
gender identification of parrots, using different types of samples and the Chromo Helicase DNA-binding (CHD) gene as a molecular marker. DNA was isolated primarily from feathers and the amplification of the CHD gene was performed using 2550F/2718R primers. In order to compare the reliability of different sources of DNA, we used buccal swab, blood and feces. All sample types exerted successful sexing results with the exception of feces samples where the success rate was 25%. Sexing was successfully determined in 239 birds belonging to 32 species of parrots. In 6 species (Amazona finschi, A. leucocephala, Aratinga aurea, Barnardius zonarius, Coracopsis nigra and Nymphicus hollandicus), 2550F/2718R primers proved to work well for the first time. Species used in this study are on the IUCN red list of threatened species. Furthermore, Amazona finschi, A. leucocephala, Cacatua moluccensis and C. sulphurea are on the list of CITES Apendix I. Since the failure in reproduction is one of the main causes of illegal trafficking of parrots, the non-invasive and universal molecular sexing method we tested may be a very useful tool in the preservation of endangered parrot species.
... Cloacal examination is applicable only to certain adults, specifically females that have recently laid an egg, and their respective mates (Boersma and Davis, 1987). Surgical procedures should be avoided in live endangered birds (Miyaki et al., 1998). Morphometry requires multivariate analysis (Cruz and Cruz, 1990) and vocalization records can take some years to generate sufficient resolution (Genevois and Bretagnolle, 1995). ...
Pterodroma phaeopygia is a critically endangered avian species of the Galápagos Islands. This bird is sexually monomorphic, making it difficult to identify the sex. This information, however, is relevant to studies of behavior, ecology, and management of wild or captive populations. Here, we aimed to implement a molecular approach for determining sex in this petrel. DNA was extracted from the blood and the feathers of 24 adult P. phaeopygia, with samples from a female and a male Gallus gallus for comparison. We amplified the cromo-helicase DNA binding protein 1 (CHD-1) gene by PCR, using primers P2 and P8. Allele CHD-1W is unique to females and CHD-1Z occurs in both sexes. We then digested these PCR products using the restriction enzyme HaeIII. The PCR amplified a 400-bp product for both alleles. The digestion of the G. gallus, amplicons split the CHD-1Z allele into two fragments (of 320 and 80 bp), while CHD-1W remained intact. Thus, the male exhibited two bands (digested CHD- 1Z) and the female three bands (undigested CHD-1W and digested CHD-1Z). Applying this RFLP method on DNA derived from blood, 9 of the 24 petrels were found to be male, while 15 were females. The same results were obtained using feathers as the source of DNA. To our knowledge, this is the first report of molecular method for sexing this species. The potential of sexing this petrel from feathers is remarkable as it minimizes blood sampling induced stress. This method could be used to reinforce the conservation efforts for this bird, to investigate population sex ratios and to develop new conservation strategies.
... formación de dos bandas para las hembras (WZ) y solo una banda para los machos (ZZ), el tamaño de las bandas fue de aproximadamente 350 bp, lo cual concuerda con lo publicado por Griffiths et al., 1998. La prueba para sexado por PCR funcionó correctamente en especies de la familia Psittacidae, de acuerdo con Miyaki et al., 1998 la prueba podría utilizarse en toda esta familia. ...
... Several authors have reported the use of genetics-based techniques to establish sex ratios for fledgling and adult birds in both field and captive breeding projects. Projects involving the sexing of flamingos [Bertault et al., 1999]; whooping cranes [Duan and Fuerst, 2001]; black-eared miners [Ewen et al., 2001]; Tengmalm's owl [Hornfeldt et al., 2000]; Hawaiian honeycreepers [Jarvi and Banko, 2000]; macaws, parrots, toucans, and curassows [Miyaki et al., 1992[Miyaki et al., , 1998Griffiths and Tiwari, 1995;Russello and Amato, 2001]; and Old World vultures [Wink et al., 1998] have emphasized the importance of this technique in conservation projects. ...
The ability to rapidly and reliably determine the sex of birds is very important for successful captive-bird breeding programs, as well as for field research. Visual inspection of adult birds is sufficient for sexually dimorphic species, but nestlings and monomorphic species are difficult, if not impossible, to sex by sight only. A method for rapid extraction of gDNA from blood, shell-membrane blood vessels, and fully grown feathers, using Chelex, and the PCR conditions for determination of sex-specific bands in 47 species (39 genera, 21 families, and 10 orders) are described. The PCR primers used amplify a length of DNA spanning an intron in the CHD-1 gene, which is present on both the W and Z chromosomes. The intron differs in size between the two sex chromosomes, resulting in PCR products that separate into two bands for females and a single band for males in most avian species (except ratites). Because this simple technique uses Chelex, a rapid gDNA isolation protocol, and sets of PCR primers independent of restriction enzyme digestion, birds can be accurately sexed within 5 hr of sample collection. Zoo Biol 22:561–571, 2003. © 2003 Wiley-Liss, Inc.
... However, the polymorphic nature of the intron length, along with the variability in electrophoresis resolution power, does not allow for accurate sex identification in some bird species [26,43]. The use of denaturing acrylamide gels instead of agarose gels increases the resolution of electrophoresis [26,46,53]. Nevertheless, difficulties in CHD1-based molecular sexing caused by the intraspecific variations at the CHD1Z homologues have been reported [54 -56]. ...
Accurate identification of sex in birds is important for the management and conservation of avian wildlife in several ways, namely in the development of population, behavioral and ecological studies, as well as in the improvement of ex situ captive breeding programs. In general, nestlings, juveniles and adult birds of a wide number of sexually monomorphic species cannot be sexed based on phenotypic traits. The development of molecular methodologies for avian sexing overcame these difficulties, allowing a reliable gender differentiation for these species. The polymerase chain reaction (PCR)-based methods have been widely applied in molecular sexing of birds, using a large diversity of sex-linked markers. During the last 15 yrs, there was a continuous improvement in the PCR-based protocols for bird sexing, increasing the accuracy, speed and high-throughput applicability of these techniques. The recent advances in real-time PCR platforms and whole genome analysis methods provided new resources for the detection and analysis of novel specific markers and protocols. This review presents a comparative guide of classical and recent advances in PCR-based methods for avian molecular sexing, highlighting its strengths and limitations. Future research opportunities in this field are also addressed.
... The thermal protocol consisted of an initial denaturation step at 95°C for 4 min, followed by 35 cycles of denaturation (95°C, 30 sec), annealing (55°C, 30 sec) and DNA extension (72°C, 45 sec), and a final extension step at 72°C for 4 min. Wang et al., 2007 Miyaki et al., 1998 Not available Wang et al., 2007 Wang et al., 2007 Cuculiformes ...
The aim of this research was to test the CHD gene (Chromo Helicase DNA-binding gene) as a universal molecular marker for sexing birds of relatively distant species. The CHD gene corresponds to the aim because of its high degree of conservation and different lengths in Z and W chromosomes due to different intron sizes. DNA was isolated from feathers and the amplification of the CHD gene was performed with the following sets of polymerase chain reaction (PCR) primers: 2550F/2718R and P2/P8. Sex determination was attempted in 284 samples of 58 bird species. It was successful in 50 bird species; in 16 of those (Alopochen aegyptiacus, Ara severus, Aratinga acuticaudata, Bucorvus leadbeateri, Cereopsis novaehollandiae, Columba arquatrix, Corvus corax, C. frugilegus, Cyanoliseus patagonus, Guttera plumifera, Lamprotornis superbus, Milvus milvus, Neophron percnopterus, Ocyphaps lophotes, Podiceps cristatus, and Poicephalus senegalus), it was carried out for the first time using molecular markers and PCR. It is reasonable to assume that extensive research is necessary to define the CHD gene as a universal molecular marker for successful sex determination in all bird species (with exception of ratites). The results of this study may largely contribute to the aim. Zoo Biol. 00:1-13, 2012. © 2012 Wiley Periodicals, Inc.
... Sex was determined through chromohelicase-DNA-binding gene amplification, by using blood samples (Miyaki et al., 1998). Positive male and female samples as well as negative controls (water) were used in each amplification reaction. ...
This study evaluated the health status and established hematologic and serum biochemistry parameters for free-living nestlings of the Hyacinth Macaw (Anodorhynchus hyacinthinus) from the Brazilian Pantanal (19 degrees 51'-19 degrees 58'S; 56 degrees 17'-56 degrees 24'W), for four consecutive years (from December 2003 through December 2006). Physical examinations indicated that all the birds were in good health. Endoparasites and blood parasites were not detected in any of the nestlings, and ectoparasites seemed to be limited to Philornis sp. (Diptera: Muscidae). Significantly higher levels of total white blood cells and heterophils, glucose, total protein, triglycerides, and phosphorus were observed in females. In females, higher cholesterol levels and packed cell volumes were observed in older birds, and total white blood cell and heterophil counts were higher in young animals. In males, uric acid levels were higher in older individuals. Wild Pantanal Hyacinth Macaws feed on only two species of palm nuts (Acrocomia totai and Scheelea phalerta). This limited food habit has a strong impact on population size and may alter the clinical pathology parameters of these birds. Therefore, knowledge of blood levels in normal individuals is essential to assess the physiologic and pathologic condition of wild macaws, to assess the effects of environmental changes on their health, and to contribute to conservation strategies of this endangered species.
... Two different sized PCR products result in females, Z and W, but just one in males, ZZ. This is possible because the primers span differential intronic sequences (Miyaki et al. 1998). As the CHD-gene is located on the avian sex chromosomes of all species (except ratites) this technique has been widely used. ...
The Brazilian tanager, Ramphocelus bresilius is an endemic species from Brazil that is sexually dimorphic in adult plumage. Young males are similar to adult and young females until their second year. Adults and young females are not distinguishable in plumage. We tested whether iris colour can be used to separate adult females from immature females. We used for the first time the molecular sexing technique based on CHD-genes to confirm the sex of the individuals classified as "female plumage with red iris", and to identify the sex of individuals classified as "female plumage and brown iris". The adult males were used as a positive control. DNA samples from 190 individuals were analysed. The sizes of the PCR products were identified as 350 base pairs (bp) for CHD-Z and 388 bp for CHD-W. We confirmed that adult females have a red iris and the young females a brown iris. We could also separate young males and females which present the same iris colour and plumage. Although there are indications that the iris colour can be used by birds to identify the adults in co-operative breeding species such as the Brazilian tanager, more behavioural data are required to understand the role of iris coloration in this species.
... Single products for homogametic male neognaths and for paleognaths of both sexes were cleaned by column purification using QIAquick columns (QIAgen Inc.) following the supplier's protocol. Two products for heterogametic female neognaths were resolvable in 3% LMP agarose gels, as the amplified fragment spanned an intron that varied in length between CHD-W and CHD-Z Miyaki et al. 1998). The two resulting bands were each excised and cleaned using the gel extraction version of the QIAquick kit. ...
We describe a previously unrecognized form of gene homology using the term "gametology," which we define as homology arising through lack of recombination and subsequent differentiation of sex chromosomes. We demonstrate use of gametologous genes to root each other in phylogenetic analyses of sex-specific avian Chromo-helicase-DNA binding gene (CHD) sequences. Phylogenetic analyses of a set of neognath bird sequences yield monophyletic groups for CHD-W and CHD-Z gametologs, as well as congruent relationships between these two clades and between them and current views of avian taxonomy. Phylogenetic analyses including paleognath bird CHD sequences and rooting with crocodilian CHD sequences, suggest an early divergence for paleognath CHD within the avian CHD clade. Based on our CHD analyses calibrated with avian fossil dates, we estimate the divergence between CHD-W and CHD-Z at 123 MYA, suggesting an early differentiation of sex chromosomes that predates most extant avian orders. In agreement with the notion of male-driven evolution, we find a faster rate of change in male-linked CHD-Z sequences.
The one remaining wild male Spix's macaw (Cyanopsitta spixii) chose a female Illiger's macaw (Propyrrhura maracana) as a mate. Eggs were found in the nest used by the couple and one of them contained an embryo. Sequence variation in CHD1-Z and CHD1-W genes showed that it was a natural hybrid.
I monitored Blue fronted Parrot nests during five consecutive breeding seasons (2002-2003 to 2006-2007) at Loro Hablador Provincial Park and neighboring areas (Chaco Province, Argentina). Parrots used cavities wide and deep in live and mature trees with high entrance holes. Clutch size was 3.7 eggs and it decreased along the breeding season. Number of eggs at the end of incubation was 3.6 and number of chicks hatched was 2.7. I observed brood reduction in 20% of the nests and it was more frequent in broods of three and four chicks. The length of the chick’s period was positively associated with brood size. Male chicks reached a higher body mass and size than females. On average 2.2 chicks fledged per nest and young’s survival during the first month was 94%. Adult males differ from adult females in seven morphometric variables and in the percentage of yellow plumage of the head, indicating that this species is sexually dimorphic and dichromatic. Nest survival varied between 27% and 67% depending of the year and method of estimation used. Seventy-four percent of the nests found during construction and early laying completed the incubation and 49% produced at least one fledgling. Most nests failed during the incubation and the first 10 days of the chick’s period. Daily survival rates varied between 98.5% and 99.5%. The best models to explain survival included as covariables age of the nest, laying date, high and orientation of entrance hole. Nest site fidelity was 68% and cavity reoccupation was 62%. Cavity characteristics that best predicted reoccupation were deep and thick of the cavity wall. The probability of reoccupation was lower if the nest had failed the previous year than if it was successful. The number of cavities destroyed per year by deforestation and selective logging at Almirante Brown and General Güemes departments was 20,000 while chick’s harvest affected 486 cavities per year and did not affect cavity reoccupation if the hole was correctly repaired. Harvested nests had a low survival than non-harvested nests (73% vs. 93%). The information presented in this thesis represents a contribution to the general knowledge of the reproductive strategies of parrots. In addition, this information could be relevant to develop management and conservation strategies for Blue-fronted Parrots.
Arising from a zoo survey carried out in the 1990s, this set of husbandry guidelines for the four species of white cockatoo commonly kept in zoos in Europe and bred as part of European breeding programmes (EEPs) provides information on diet, enrichment, reproduction, veterinary issues and enclosure needs, amongst others.
We used DNA fingerprinting to examine genetic variation in wild populations of two species of Macaw: the Blue and Yellow Macaw (Ara ararauna, in the Ema National Park, state of Goiás, Brazil) and the Green-winged Macaw (A. chloroptera, Pantanal, state of Mato Grosso do Sul/Brazil). Mean heterozygosity and genetic relationship between chicks from the same nest were estimated with the human multilocus minisatellite probes 33.6 and 33.15. The Green-winged Macaw has greater heterozygosity than the Blue-and-Yellow Macaw. The latter species showed a mean genetic similarity index similar to those in species considered vulnerable or endangered. Chicks from the same nest had genetic similarity indices close to those expected for first degree relatives in a monogamous species. In only one nest of the Green-winged Macaw did the index of similarity suggest that the chicks were from different parents. The sex ratio of both populations was close to 1:1. In the Green-winged Macaw population a sex- specific polymorphism was observed. Use of DNA fingerprinting can provide a tool to identify animal populations with low genetic variability,
An extraordinary abundance of aquatic birds, a highly diversified avifauna and phenomena as the formation of ninhais (nesting colonies) are outstanding peculiarities that contributed to the recognition of the Pantanal wetland as one of the most attractive places in the world to watch or study birds. Its peculiarities, however, have been attracting researchers not only recently, but since the beginning of the nineteenth century, when Johann Natterer coordinated expressive collections of about 150 bird species in the northern portion of Pantanal (Pelzeln 1870, Paynter and Traylor 1991a, b, Vanzolini 1993). In the end of the same century, expeditions coordinated by Borelli (Salvadori 1895, 1900) and Smith (Allen 1891, 1892, 1893) at the surroundings of Corumbá collected a less extensive number of bird species. These and additional records of one or few species published by several authors were joined in a remarkable publication on bird species collected in the current States of Mato Grosso and Mato Grosso do Sul (Naumburg 1930). In the begining of the twentieth century, the most outstanding collections were those coordinated by Roosevelt-Rondon in the former State of Mato Grosso (Naumburg 1930), by J. A. G. Rehn at Descalvados (Stone and Roberts 1934), and by members of the Museu de Zoologia da Universidade de São Paulo at several localities of Pantanal (Pinto 1932, 1938, 1940, 1944, 1945). The mid twentieth century was characterised by greater participation of national institutions which considerably increased the number and distribution of inventories in the Pantanal through several expeditions involving bird collection (Moojen et al.. According to the literature, collections of birds in the wetland occurred until the early 1980s, when Dubs collected specimens in the Rio Negro region for the Zoologisches Museum der Universität Zürich (Dubs 1983a). On the other Bird species of the Pantanal wetland, Brazil
Many birds, including some parrots, may adjust the sex ratio of their offspring in relation to the relative fitness benefits of sons and daughters. We investigated nestling sex ratios in Yellow-naped Amazons (Amazona auropalliata) using a molecular sexing technique that amplifies intronic regions of the CHD-W and CHD-Z genes in birds. We examined all nestlings in 37 complete clutches comprising 77 chicks. The overall nestling sex ratio did not differ from unity. Sex allocation was not associated with hatch date, sequence of hatching, or clutch size. We also found no difference in sex ratio between two regional dialects. Female Yellow-naped Amazons may be unable to control their hatchling sex ratio. Alternatively, there may be no fitness benefits to females producing more of one sex in relation to the factors we measured here. No Existe Evidencia que Indique Modificaciones Adaptativas de la Proporción de Sexos en la Progenie de Amazona auropalliata Resumen. En muchas aves, incluyendo los loros, la proporción de sexos en la progenie puede ajustarse en relación a los beneficios relativos de adecuación biológica de hembras y machos. Dichas tasas fueron investigadas en Amazona auropalliata por medio de una técnica molecular de determinación sexual por la cual se amplifican regiones intrónicas de los genes CHD-W y CHD-Z de aves. Se examinaron todos los pichones de 37 nidadas completas, constituidas por 77 pichones. La proporción de sexos total no resultó diferente a uno. La asignación sexual no estuvo correlacionada con la fecha de eclosión, la secuencia de eclosión, ni el tamaño de la nidada. Tampoco se encontraron diferencias en las proporciones de sexos entre dos dialectos vocales regionales. Las hembras de A. auropalliata podrían no tener la habilidad de controlar la proporción de sexos de su progenie. Alternativamente, es posible que en términos de adecuación biológica, no haya diferencia en el beneficio de producir una progenie enriquecida en un sexo determinado con respecto a los factores medidos en este estudio.
Early attempts to identify sex in sexually monomorphic birds were based on morphological or chromosomal characters but since avian W-specific DNA sequences were identified, their PCR amplification has become commonly used molecular sexing method. We report here a DNA technique that amplifies an intrinsic CHD region without a DNA extraction. This test was applied to twelve species belonging to three waterbird orders, Ciconiiformes, Pelecaniformes and Phoenicopteriformes. All birds were sexed successfully with high reproducibility. There was complete agreement between our results and sex diagnosis by parallel methods. Because the proposed method eliminates the DNA extraction step, this protocol is more efficient and simple. It demands only minute amounts of DNA and reduces both time and the quantities of reagents consumed.
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Capítulo 1. Origem da vida: um tempo curto para uma experiência bem-sucedida
Carlos Frederico Martins Menck, Eduardo Gorab e Mariana Cabral de Oliveira
Capítulo 2. O mundo de RNA e a origem da complexidade da vida
Mariana Cabral de Oliveira e Carlos Frederico Martins Menck
Capítulo 3. Genoma não codificante - Uma breve introdução
Alysson Renato Muotri e Cassiano Carromeu
Capítulo 4. O papel do RNA de interferência na célula eucariótica
Stephano Spanó Mello, Luciana Nogueira de Sousa Andrade e Carlos Frederico Martins Menck
Capítulo 5. Estabilidade do material genético: mutagênese e reparo
Luis Eduardo Soares Netto e Carlos Frederico Martins Menck
Capítulo 6. Sexo, por quê?
Sergio Russo Matioli e Anita Wajntal
Capítulo 7. Taxas de evolução e o relógio molecular
Daniela Calcagnotto
Capítulo 8. Evolução dos genes nucleares de RNA ribossômico
Eduardo Gorab
Capítulo 9. O genoma instável, sequências genéticas móveis
Marie-Anne Van Sluys, Nathalia de Setta, Katia C. Scortecci e Ana Paula Pimentel Costa
Capítulo 10. Evolução dos genomas
Laila Alves Nahum
Capítulo 11. Biologia evolutiva do desenvolvimento
Luis Paulo de Moura Andrioli
Capítulo 12. Reconstrução filogenética. Introdução e o método da máxima parcimônia
Cristina Yumi Miyaki, Cláudia A. de Moraes Russo e Sergio Luiz Pereira
Capítulo 13. Reconstrução filogenética: Métodos geométricos
Cláudia A. de Moraes Russo, Cristina Yumi Miyaki, Sergio Luiz Pereira
Capítulo 14. Reconstrução filogenética: Métodos probabilísticos
Sergio Luiz Pereira, Cristina Yumi Miyaki, Cláudia A. de Moraes Russo
Capítulo 15. Reconstrução filogenética: Inferência bayesiana
Sergio Luiz Pereira
Capítulo 16. Como escolher genes para problemas filogenéticos específicos
Claudia A. M. Russo
Capítulo 17. Polimorfismos de isozimas
Vera Nisaka Solferini e Denise Selivon Scheepmaker
Capítulo 18. RFLP: O emprego de enzimas de restrição para a detecção de polimorfismos no DNA
Maria Cristina Arias e Maria Elena Infante-Malachias
Capítulo 19. Métodos baseados em PCR para análise de polimorfismos de ácidos nucléicos
Sergio Russo Matioli e Maria Rita dos Santos e Passos-Bueno
Capítulo 20. Genealogias e o processo de coalescência
Flora Maria de Campos Fernandes
Capítulo 21. Análise filogeográfica
Haydée A. Cunha e Antonio M. Solé-Cava
Capítulo 22. Biodiversidade molecular e genética da conservação
Antonio M. Solé-Cava e Haydée A. Cunha
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The St. Vincent Amazon Parrot Consortium was established during the 1980s in an effort to cooperatively manage the captive population of the St. Vincent parrot (Amazona guildingii) and support conservation of the wild population. Ex situ management of A. guildingii has been hindered by the sexual monomorphism of the group, mediated in the past through the use of time-consuming, expert-driven, and sometimes hazardous sexing procedures. In this article, we apply a noninvasive, molecular sexing technique, using the polymerase chain reaction (PCR) and DNA from a single feather tip to the captive populations of A. guildingii residing on St. Vincent (66 individuals) and Barbados (13 individuals). This study allowed for the rapid assessment of gender, while posing no threat to individual health, and will facilitate the efforts of the consortium breeding programs in the United States, Europe and on the islands of St. Vincent and Barbados. Zoo Biol 20:41-45, 2001. Copyright 2001 Wiley-Liss, Inc.
In theory, birds should control the sex ratio of the offspring they produce. In practice, we have very limited evidence to support this idea because of our difficulty in sexing nestling birds. In addition, extinction is facing an increasing number of birds. Our ability to help includes captive breeding which, again, is difficult if male and female adults cannot be recognized. Here we describe the discovery of a W-linked gene in the Great tit (Parus major). It is named CHD-W (chromodomain-helicase-DNA-binding protein W-linked), it is highly conserved and it is W-chromosome linked in a range of bird species. These birds also possess a second, non-W-linked CHD gene (CHD-NW). A single, simple polymerase chain reaction technique based on both genes can be used to identify the sex in a wide variety of birds.
Birds are difficult to sex. Nestlings rarely show sex-linked morphology and we estimate that adult females appear identical to males in over 50% of the world's bird species. This problem can hinder both evolutionary studies and human-assisted breeding of birds. DNA-based sex identification provides a solution. We describe a test based on two conserved CHD (chromo-helicase-DNA-binding) genes that are located on the avian sex chromosomes of all birds, with the possible exception of the ratites (ostriches, etc.; Struthioniformes). The CHD-W gene is located on the W chromosome; therefore it is unique to females. The other gene, CHD-Z, is found on the Z chromosome and therefore occurs in both sexes (female, ZW; male, ZZ). The test employs PCR with a single set of primers. It amplifies homologous sections of both genes and incorporates introns whose lengths usually differ. When examined on a gel there is a single CHD-Z band in males but females have a second, distinctive CHD-W band.
The destruction of natural habitats and illegal trading are endangering many species of Brazilian parrots. Survival of some species may depend on breeding programmes in captivity. It is therefore important to be able to confirm the identity and parentage of the birds, and to maintain the genetic variability of captive populations. We studied here DNA fingerprints of the endangered Aratinga guarouba and five other species of Aratinga using two human minisatellite multilocus probes (33.6, 33.15) and the restriction enzyme HaeIII. Hybridization with 33.6 produces individual-specific patterns with 18 to 33 bands, depending on the species. The index of similarity obtained between unrelated birds was of the same order in the endangered A. guarouba (0.16) as in other Aratinga species (0.31 to 0.12) and in wild populations of birds reported in the literature. It was possible to perform segregation analysis of the bands only in A. aurea, for which we studied a pedigree including five chicks. There were at least 14 unlinked loci. Also, we assigned the parentage of three A. guarouba from a group of seven potential parents. A novel fragment was present in two siblings and absent from the third, this was interpreted as a gonadal 'mutation' in one of the parents. Hybridization with 33.15 revealed a low number of bands in all Aratinga species but A. jandaya where 33.2 ± 2.5 bands were scored. However, in all species the probe 33.15 hybridizes strongly to one or several possibly W-chromosome specific bands. Multilocus fingerprints should allow individual identification, parentage assignment and probably sex determination in the genus Aratinga. Moreover, band sharing indices can be used to prevent consanguineous matings and to maintain the genetic diversity of these species in captivity.
This is the first attempt to study wild Brazilian endangered macaws with human multilocus minisatellite probes 33.6 and 33.15. Twenty individuals belonging to four species (Ara ararauna, A. chloroptera, A. macao, A. nobilis) were studied. The band sharing values observed between unrelated individuals vary from 0.16 to 0.25 using the minisatellite probe 33.6. The number of bands detected with 33.15 is low, with possibly one or more intense W chromosome-specific fragments detected in all species. The application of multilocus fingerprints in the genetic management of captive and wild populations of macaw is discussed in the light of these results.
Chromosomes were studied in female specimens of the ostrich, Struthio camelus L., cassowary, Casuarius casuarius (L.), emu, Dromiceius novaehollandiae (Lath.) and rhea, Rhea americana L. by means of blood and feather pulp culture techniques. Male karyotypes were also studied in the emu and rhea. The diploid chromosome number was most likely 80 in the ostrich and rhea and 82 in the emu, while the exact number could not be determined in the cassowary. Karyotypes of the 4 species were strikingly similar and apparently interchangeable with one another with slight modifications of the centromeric position in one or two pairs of macrochromosomes. No heteromorphic macrochromosomal pair was found either in female specimens or in male ones of the ratite species so far examined, except for a female rhea. This specimen was found to possess an acrocentric chromosome which was evidently a member of nos. 4–6, but considerably smaller than any other chromosome of the group. 3H-thymidine autoradiography provided no more information than the straightforward morphological analysis with regard to the differentiation of the sex-chromosomes.
Chromo-helicase-DNA binding 1 (CHD1) is a conserved protein with a putative role in chromatin architecture. Single homologues have been found in mouse, Drosophila and yeast. In birds the situation is different as they possess two homologues. One is known to be W-linked, we show the second, closely related gene is linked to the Z sex chromosome. The basic structure of the Z-linked gene is similar to the homologous genes, however, it does possess an additional, internal 88 amino acid hydrophilic domain, rich in glutamic acid and lysine. Studies on pairs of genes sex-linked in mammals suggests rapid divergence of DNA sequence and function. We suggest the DNA sequences of CHD-W and CHD-Z do not follow this pattern.
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