New Bird Sexing Strategy Developed in the Order Psittaciformes Involves Multiple Markers to Avoid Sex Misidentification: Debunked Myth of the Universal DNA Marker

Abstract

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.

Polly Wants a Genome: The Lack of Genetic Testing for Pet Parrot Species

“…It was suggested that the three autosomal genes could be used for normalization and autosomal controls in both clades. As found by Kroczak et al, [ 76 ] not all genes amplified in all species.…”

“…Kroczak et al [ 76 ] compared markers and polymorphisms that have been used routinely and commercially as “universal markers” to identify sex in birds and found that no single universal marker can be applied to sex all birds, especially parrots, successfully. These included the two widely used markers P2P8 and CHD1iA as well as four W/Z length polymorphisms ( CHD1iE , CHD1i16 , CHD1i9 and NIPBLi16 ).…”

“…Similarly, the inclusion of polymorphisms linked to sex could simplify the current sexing test available to breeders and SNPs linked to family relationships and identity in more parrot species must be identified. A combination of two to four of the polymorphisms tested by Kroczak et al [ 76 ], could be included on the SNP chip. It should, however, be noted that the Z chromosome (shared between males and females) is euchromatic and gene-rich, while the W chromosome (only found in females) is gene-poor [ 84 ].…”

“…Xu and Zhou [ 84 ] found that despite the independent evolution of recombination suppression, the female W chromosome in birds still consists of highly conserved gene content. Further research on polymorphisms located on the W chromosome that can be combined with the polymorphisms suggested by [ 76 ] is needed before it can be included on a genus-specific SNP chip. Autosomal genes, additional to those used by [ 75 ], linked to male or female trait expression could be identified in addition to Z-linked markers.…”

Short Communication: A new primer set in CHD1 gene for bird sex identification

“…In addition, accurate sex typing is a serious pitfall in many scientific, breeding and conservation programs. Over 50% of the world's bird species, nestlings, and adult individuals show no sexual dimorphism (Kroczak et al 2021). And also, sex determination is the important thing on a population or sex ratio of the population before reintroduction program (Insee et al 2014).…”