Molecular sexing of monomorphic endangered Ara birds

Survival of most endangered birds may depend on breeding programs where sex identification plays an important role. Molecular sexing has shown to be a rapid and safe procedure. In this work we established sex identification of monomorphic endangered Ara birds using a chromosome W-linked DNA marker, the Chromo-helicase-DNA-Binding 1 (CHD) gene. Most birds have two CHD sex-linked genes, one W-linked (CHD-W) and one Z-linked (CHD-Z). These markers were characterized from Ara militaris and gender sex was determined by PCR and restriction analyzes. The procedure here reported was successfully applied to five different species of the genus Ara and confirmed the validity of the technique. To our knowledge, this is the first report of molecular sexing of the Ara species. This molecular sexing is currently been used in breeding programs of Ara birds.     

基于CHD基因序列的18种猛禽鸟类系统发育关系

对18个猛禽CHD基因的一段内含子序列进行比较和分析.CHD-W和CHD-Z基因的多态性存在差异,CHD-W基因不适合种间系统发生学的研究.通过对CHD-Z基因扩增序列构建的NJ和ML树显示:隼科与其他猛禽物种关系较远;鹰科鸟类与鸮形目鸟类亲缘关系较近;在白腹鹞的分类地位上与传统形态学分类不一致;长耳鸮、领角鸮、花彩角鸮、西部鸣角鸮的分类地位存在分歧.鸮形目和隼形目鸟类的CHD-W基因大小有明显区别,支持形态学分类结果,与CHD-Z序列分析结果明显不同.     

Biallelic expression of Z-linked genes in male chickens

In birds, females are heterogametic (ZW), while males are homogametic (ZZ). It has been proposed that there is no dosage compensation for the expression of Z-linked genes in birds. In order to examine if the genes are inactivated on one of the two Z chromosomes, we analyzed the allelic expression of the B4GALT1 and CHD-Z genes on Z chromosomes in male chickens. One base substitution was detected among 15 chicken breeds and lines examined for each gene, and cross mating was made between the breeds or lines with polymorphism. cDNAs were synthesized from cultured cell colonies each derived from a single cell of an F1 male embryo. The allelic expression of the B4GALT1 gene was examined by restriction fragment length polymorphism analysis of the PCR products digested with RSAI, and that of the CHD-Z gene by the single nucleotide primer extension (SNuPE) method. Both of the genes displayed biallelic expression, suggesting that these Z-linked genes were not subject to inactivation in male chickens. Comparison between expression levels in males and females by real-time quantitative PCR suggested that expression was compensated for the CHD-Z gene but not for the B4GALT1 gene.     

Improving the reliability of molecular sexing of birds using a W-specific marker

Molecular techniques for identifying sex of birds utilize length differences between CHD-Z and CHD-W introns, but in some cases these methods can lead to sexing errors. Here we show that an additional W-specific primer can be used in conjunction with a pre-existing sexing primer pair to dramatically improve the reliability of molecular sexing methods. We illustrate the approach with American coots (Fulica americana), a species with CHD-Z polymorphism that could not be accurately sexed using traditional methods. We developed a reverse primer GWR2 designed to sit within the intron of the W chromosome and amplify a distinctively small DNA fragment that serves as a W-specific marker. Analysis of known-sex individuals indicates that this W-specific primer provides an efficient and reliable protocol to identify the sex of F. americana. The development of such sex-specific primers will likely increase the reliability of molecular sexing methods in other birds as well. Comparisons between CHD-Z alleles of coots and common moorhens (Gallinula chloropus) revealed that CHD-Z polymorphism evolved separately in these two closely related species. We discuss the implications of repeated evolution of CHD-Z polymorphisms among birds.     

Rooting a phylogeny with homologous genes on opposite sex chromosomes (gametologs): a case study using avian CHD

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.     

DNA sexing in Humboldt Penguins (Spheniscus humboldti) from feather samples

Humboldt Penguins (Spheniscus humboldti) show little sexual dimorphism, and although males are usually heavier and larger than females, sexing by direct observation may be difficult, especially in young subjects. In this paper we evaluate the utility of the molecular approach, for sexing impuberal Humboldt Penguins from feathers. Firstly, a PCR test was used employing primers that amplify the homologous region of the CHD-W gene, unique in female, and the CHD-Z gene, occurring in the two sexes. The analysis of the PCR products showed a band of 370 bp in males and two bands of 370 and 380 bp in females. Additionally, to confirm these results, the PCR products were digested with HaeIII and Asp700 for RFLP analysis. Male PCR products showed two bands (310 and 60 bp) after digestion with HaeIII, and a unique band (370 bp) using Asp700, while all fragments obtained from females resolved into three bands using both HaeIII (380, 310 and 60 bp) and Asp700 (370, 270 and 110 bp), confirming the previous PCR sex determination. Results from these two different DNA-based tests were in accordance, in all cases, with sexes checked by preliminary cloacoscopy. Thus, it was found that the PCR method from feather samples alone is sufficient, reliable and without any risks for a rapid sexing in Humboldt Penguin. This non-invasive sexing technique can be useful at any age to verify the sex ratio in field populations and for gender identification in ex situ conservation programs.     

Secrets in the eyes of Black Oystercatchers: a new sexing technique

ABSTRACT.   Sexing oystercatchers in the field is difficult because males and females have identical plumage and are similar in size. Although Black Oystercatchers ( Haematopus bachmani ) are sexually dimorphic, using morphology to determine sex requires either capturing both pair members for comparison or using discriminant analyses to assign sex probabilistically based on morphometric traits. All adult Black Oystercatchers have bright yellow eyes, but some of them have dark specks, or eye flecks, in their irides. We hypothesized that this easily observable trait was sex-linked and could be used as a novel diagnostic tool for identifying sex. To test this, we compared data for oystercatchers from genetic molecular markers (CHD-W/CHD-Z and HINT-W/HINT-Z), morphometric analyses, and eye-fleck category (full eye flecks, slight eye flecks, and no eye flecks). Compared to molecular markers, we found that discriminant analyses based on morphological characteristics yielded variable results that were confounded by geographical differences in morphology. However, we found that eye flecks were sex-linked. Using an eye-fleck model where all females have full eye flecks and males have either slight eye flecks or no eye flecks, we correctly assigned the sex of 117 of 125 (94%) oystercatchers. Using discriminant analysis based on morphological characteristics, we correctly assigned the sex of 105 of 119 (88%) birds. Using the eye-fleck technique for sexing Black Oystercatchers may be preferable for some investigators because it is as accurate as discriminant analysis based on morphology and does not require capturing the birds.     

Sex determination in the Lesser Flamingo (Phoenicopterus minor) using morphological measurements

Morphological measurements and blood samples were taken from 154 Lesser Flamingos Phoenicopterus minor, including adults (>3 years old), immature sub-adults (2–3 years old) and first-year juvenile birds of both sexes, captured at Lake Bogoria, Kenya (0°11'–20' N, 036°06' E) during 2001 and 2002. PCR amplification of the CHD-Z and CHD-W genes using DNA extracted from the blood samples was used to determine the sex of each bird. There were significant differences in mass and tarsus length among the three age groups, indicating that Lesser Flamingos continue to grow in skeletal size and mass between fledging and the attainment of adult plumage at 3–4 years of age. On average, males were significantly larger than females in all age groups, although there was substantial overlap between the sexes in all morphological measurements. The element with the least amount of overlap was head-and-bill length. Discriminant functions utilising head-and-bill length that correctly predict the sex of juvenile and immature birds with approximately 93% accuracy are presented. By adding total tarsus length, the sex of wild adult Lesser Flamingos is correctly predicted with approximately 98% accuracy. The same discriminant function developed for wild adult birds predicted the sex of 19 captive adult Lesser Flamingos of known sex with 100% accuracy.     

Adaptive benefits of differential post-fledging development patterns in the Lesser Flamingo (Phoenicopterus minor)

Measurements of five morphological components (mass, skull length, culmen, flattened wing and tarsus) and blood samples were taken from 154 fledged wild Lesser Flamingos Phoenicopterus minor captured during 2001 and 2002 at Lake Bogoria, Kenya (0°11'–20'N, 036°06'E). The sample included adults (>3 years old), immature birds (2–3 years old) and first-year juvenile birds of both sexes. The sex of each bird was determined by PCR amplification of the CHD-Z and CHD-W genes, using DNA extracted from blood samples. Within each gender, there were significant differences in mass and tarsus length amongst the three age groups, indicating that the skeletal size and mass of Lesser Flamingos continue to increase between fledging and attainment of adult plumage at three to four years of age. The different morphological components increased in size at different rates, although the same components appeared to increase at similar rates in both males and females. Skull and culmen lengths had reached adult size in juvenile birds, while juvenile wing length, tarsus length and mass were approximately 95%, 85% and 75% of adult size, respectively. The adaptive significance of these findings is discussed.     

New primers for sex identification in the Chinese egret and other ardeid species

Using the universal P2/P8 primers, we were able to obtain the gene segments of chromo-helicase-DNA binding protein (CHD)-Z and CHD-W from ten species of ardeid birds including Chinese egret (Egretta eulophotes), little egret (E. garzetta), eastern reef egret (E. sacra), great egret (Ardea alba), grey heron (A. cinerea), Chinese pond-heron (Ardeola bacchus), cattle egret (Bubulcus ibis), black-crowned night-heron (Nycticorax nycticorax), cinnamon bittern (Ixobrychus cinnamomeus) and yellow bittern (I. sinensis). Based on conserved regions inside the P2/P8-derived sequences, we designed new PCR primers for sex identification in these ardeid species. Using agarose gel electrophoresis, the PCR products showed two bands for females (140 bp derived from CHD-W and the other 250 bp from CHD-ZW), whereas the males showed only the 250 bp band. The results indicated that our new primers could be used for accurate and convenient sex identification in ardeid species.     

Independent Evolution of Transcriptional Inactivation on Sex Chromosomes in Birds and Mammals

X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination). We used RNA-fluorescent in situ hybridization (RNA-FISH) to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains) on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial) silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes.     

Avian sex determination: what, when and where?

Sex is determined genetically in all birds, but the underlying mechanism remains unknown. All species have a ZZ/ZW sex chromosome system characterised by female (ZW) heterogamety, but the chromosomes themselves can be heteromorphic (in most birds) or homomorphic (in the flightless ratites). Sex in birds might be determined by the dosage of a Z-linked gene (two in males, one in females) or by a dominant ovary-determining gene carried on the W sex chromosome, or both. Sex chromosome aneuploidy has not been conclusively documented in birds to differentiate between these possibilities. By definition, the sex chromosomes of birds must carry one or more sex-determining genes. In this review of avian sex determination, we ask what, when and where? What is the nature of the avian sex determinant? When should it be expressed in the developing embryo, and where is it expressed? The last two questions arise due to evidence suggesting that sex-determining genes in birds might be operating prior to overt sexual differentiation of the gonads into testes or ovaries, and in tissues other than the urogenital system.     

All dosage compensation is local: gene-by-gene regulation of sex-biased expression on the chicken Z chromosome

Recent reports have suggested that birds lack a mechanism of wholesale dosage compensation for the Z sex chromosome. This discovery was rather unexpected, as all other animals investigated with chromosomal mechanisms of sex determination have some method to counteract the effects of gene dosage of the dominant sex chromosome in males and females. Despite the lack of a global mechanism of avian dosage compensation, the pattern of gene expression difference between males and females varies a great deal for individual Z-linked genes. This suggests that some genes may be individually dosage compensated, and that some less-than-global pattern of dosage compensation, such as local or temporal, exists on the avian Z chromosome. We used global gene expression profiling in males and females for both somatic and gonadal tissue at several time points in the life cycle of the chicken to assess the pattern of sex-biased gene expression on the Z chromosome. Average fold-change between males and females varied somewhat among tissue time-point combinations, with embryonic brain samples having the smallest gene dosage effects, and adult gonadal tissue having the largest degree of male bias. Overall, there were no neighborhoods of overall dosage compensation along the Z. Taken together, this suggests that dosage compensation is regulated on the Z chromosome entirely on a gene-by-gene level, and can vary during the life cycle and by tissue type. This regulation may be an indication of how critical a given gene's functionality is, as the expression level for essential genes will be tightly regulated in order to avoid perturbing important pathways and networks with differential expression levels in males and females.     

Sex and death in birds: a model of dosage compensation that predicts lethality of sex chromosome aneuploids

Birds show female heterogamety, with ZZ males and ZW females. It is still not clear whether the W is female-determining, or whether two doses of the Z chromosomes are male-determining, or both. This question could easily be settled by the sexual phenotypes of ZZW and ZO birds, in the same way that the sexual phenotypes of XXY and XO showed that the Y is male determining in humans, but that the dosage of an X-borne gene determines sex in Drosophila. However, despite extensive searches, no ZZW or ZO diploid birds have been satisfactorily documented, so we must assume that these genotypes are embryonic lethals. Given that ZW and ZZ are viable and the W contains few genes it is not clear why this should be so. Here I propose that sex chromosome aneuploids are lethal in chicken because, to achieve dosage compensation, a locus on the W chromosome controls the upregulation of genes on the Z in ZW females. ZO birds would therefore have only half the normal dose of Z-linked gene product and ZZW would have twice the amount, both of which would undoubtedly be incompatible with life. Reports of other aneuploids and triploids are also consistent with this hypothesis.     

Mammalian sex--Origin and evolution of the Y chromosome and SRY

Sex determination in vertebrates is accomplished through a highly conserved genetic pathway. But surprisingly, the downstream events may be activated by a variety of triggers, including sex determining genes and environmental cues. Amongst species with genetic sex determination, the sex determining gene is anything but conserved, and the chromosomes that bear this master switch subscribe to special rules of evolution and function. In mammals, with a few notable exceptions, female are homogametic (XX) and males have a single X and a small, heterochromatic and gene poor Y that bears a male dominant sex determining gene SRY. The bird sex chromosome system is the converse in that females are the heterogametic sex (ZW) and males the homogametic sex (ZZ). There is no SRY in birds, and the dosage-sensitive Z-borne DMRT1 gene is a credible candidate sex determining gene. Different sex determining switches seem therefore to have evolved independently in different lineages, although the complex sex chromosomes of the platypus offer us tantalizing clues that the mammal XY system may have evolved directly from an ancient reptile ZW system. In this review we will discuss the organization and evolution of the sex chromosomes across a broad range of mammals, and speculate on how the Y chromosome, and SRY, evolved.     

Molecular sexing of unusually large numbers of Spheniscus magellanicus (Spheniscidae) washed ashore along the Brazilian coast in 2008

There have been few studies on Magellanic penguins (Spheniscus magellanicus). In 2008, these penguins washed ashore along the Brazilian coast in unusually high numbers, some reaching as far as northeast Brazil. As Magellanic penguins show little sexual dimorphism, sex determination by morphological features is not accurate. Here, we tested a molecular procedure for sexing specimens of S. magellanicus washed ashore along the coasts of Sergipe, Rio de Janeiro and Rio Grande do Sul in 2008, comparing the sex ratio between these localities. Tissue samples were collected from 135 dead, beached specimens. We carried out total genomic DNA extraction and CHD-Z/CHD-W gene amplification by PCR using P2 and P8 primers. Amplicons were separated by 12% acrylamide gel electrophoresis. We found a greater proportion of females (70%). Sex could be determined because females have two intronic regions of CHD gene of different size in the sex chromosomes, visualized as two bands on the gel (380 and 400 bp approximately), while males have only one (400 bp). Therefore, this method proved to be effective and sensitive for sex determination of S. magellanicus individuals. Data on sex ratios are useful for understanding the dynamics and ecology of Magellanic penguin populations.     

Sexually dimorphic expression of the sex chromosome-linked genes cntfa and pdlim3a in the medaka brain

In vertebrates, sex differences in the brain have been attributed to differences in gonadal hormone secretion; however, recent evidence in mammals and birds shows that sex chromosome-linked genes, independent of gonadal hormones, also mediate sex differences in the brain. In this study, we searched for genes that were differentially expressed between the sexes in the brain of a teleost fish, medaka (Oryzias latipes), and identified two sex chromosome genes with male-biased expression, cntfa (encoding ciliary neurotrophic factor a) and pdlim3a (encoding PDZ and LIM domain 3 a). These genes were found to be located 3–4 Mb from and on opposite sides of the Y chromosome-specific region containing the sex-determining gene (the medaka X and Y chromosomes are genetically identical, differing only in this region). The male-biased expression of both genes was evident prior to the onset of sexual maturity. Sex-reversed XY females, as well as wild-type XY males, had more pronounced expression of these genes than XX males and XX females, indicating that the Y allele confers higher expression than the X allele for both genes. In addition, their expression was affected to some extent by sex steroid hormones, thereby possibly serving as focal points of the crosstalk between the genetic and hormonal pathways underlying brain sex differences. Given that sex chromosomes of lower vertebrates, including teleost fish, have evolved independently in different genera or species, sex chromosome genes with sexually dimorphic expression in the brain may contribute to genus- or species-specific sex differences in a variety of traits.     

Variation in the X-Linked EFHC2 Gene Is Associated with Social Cognitive Abilities in Males

Females outperform males on many social cognitive tasks. X-linked genes may contribute to this sex difference. Males possess one X chromosome, while females possess two X chromosomes. Functional variations in X-linked genes are therefore likely to impact more on males than females. Previous studies of X-monosomic women with Turner syndrome suggest a genetic association with facial fear recognition abilities at Xp11.3, specifically at a single nucleotide polymorphism (SNP rs7055196) within the EFHC2 gene. Based on a strong hypothesis, we investigated an association between variation at SNP rs7055196 and facial fear recognition and theory of mind abilities in males. As predicted, males possessing the G allele had significantly poorer facial fear detection accuracy and theory of mind abilities than males possessing the A allele (with SNP variant accounting for up to 4.6% of variance). Variation in the X-linked EFHC2 gene at SNP rs7055196 is therefore associated with social cognitive abilities in males.     

No evidence that Y‐chromosome differentiation affects male fitness in a Swiss population of common frogs

The canonical model of sex‐chromosome evolution assigns a key role to sexually antagonistic (SA) genes on the arrest of recombination and ensuing degeneration of Y chromosomes. This assumption cannot be tested in organisms with highly differentiated sex chromosomes, such as mammals or birds, owing to the lack of polymorphism. Fixation of SA alleles, furthermore, might be the consequence rather than the cause of recombination arrest. Here we focus on a population of common frogs (Rana temporaria) where XY males with genetically differentiated Y chromosomes (nonrecombinant Y haplotypes) coexist with both XY° males with proto‐Y chromosomes (only differentiated from X chromosomes in the immediate vicinity of the candidate sex‐determining locus Dmrt1) and XX males with undifferentiated sex chromosomes (genetically identical to XX females). Our study finds no effect of sex‐chromosome differentiation on male phenotype, mating success or fathering success. Our conclusions rejoin genomic studies that found no differences in gene expression between XY, XY° and XX males. Sexual dimorphism in common frogs might result more from the differential expression of autosomal genes than from sex‐linked SA genes. Among‐male variance in sex‐chromosome differentiation seems better explained by a polymorphism in the penetrance of alleles at the sex locus, resulting in variable levels of sex reversal (and thus of X‐Y recombination in XY females), independent of sex‐linked SA genes.