Chromosome Banding Reveals Hidden Genome Rearrangements in Gamebirds and an Endangered Bustard

Deep inside the cells of quails, partridges, chickens and one of North Africa’s most endangered birds, a team of cytogeneticists has been reading a molecular archive that stretches back to the age of dinosaurs. In a study published in Comparative Cytogenetics, researchers used high-resolution GTG-banding, a classical staining technique that reveals the dark and light stripes of chromosomes under the microscope, to compare the genomes of six bird species: the domestic fowl, the Common and Japanese quail, the Barbary and Chukar partridge, and the Houbara bustard, an endangered Otidiformes. Their findings paint a picture of remarkable genome stability punctuated by a small number of evolutionary events, and they demonstrate that old-fashioned cytogenetics still has plenty to say in the genomic era.

Birds are the most diverse class of tetrapod vertebrates, with roughly 11,032 species worldwide, yet they are also among the least studied genetically. According to the IUCN, about 12 percent of bird populations are threatened with potential extinction, and many scientists argue that we are now living through an irreversible sixth mass extinction that could prove more devastating than all previous ones combined. Birds evolved from theropod dinosaurs between 165 and 150 million years ago and separated from crocodiles about 240 million years ago, which makes their genomes a living record of one of the most successful vertebrate radiations. Understanding how their chromosomes are organized, and how they change, is therefore both an evolutionary question and a conservation imperative.

The domestic chicken occupies a special place in this research. Gallus gallus domesticus is considered a model in phylogeny and comparative genomics and represents the only standardized bird karyotype, established in 1999 under the International System of Standardised Avian Karyotypes. Because the chicken shares several features with other avian species, it is regarded as the closest living representative of the common ancestor of birds, and its chromosomes remain the best studied in the class. The team therefore used the chicken as the reference against which the chromosomes of the five other species were compared, focusing on the first eight autosomal pairs and the ZW sex chromosomes.

The technical work behind the study was meticulous. Embryos were collected during the laying period from fertile eggs of Common quail, Japanese quail, Barbary partridge and Chukar partridge obtained from hunting centres in Algeria, while Houbara bustard embryos came from an Emirati bird breeding centre for conservation. Eggs were incubated under controlled conditions of 55 percent humidity and 39.5 degrees Celsius, and primary fibroblast cultures were established from lung, heart, liver, kidney and muscle fragments of embryos aged 6 to 19 days. The cultures were synchronized using a double thymidine block during S phase to increase the yield of metaphase cells, a standard trick that pushes dividing cells into a narrow window of the cell cycle so that more chromosomes can be harvested at their most condensed and analyzable stage.

GTG-banding itself involves treating aged chromosome slides with a brief trypsin exposure followed by Giemsa staining, producing the characteristic striped pattern that reflects the underlying chromatin structure. Because each chromosome carries a reproducible band signature, researchers can compare chromosomes across species by matching the size, morphology and band patterns of each pair. The team classified macrochromosomes in decreasing size according to centromere position and compared the ratio of long-arm to short-arm length, a measure that reveals even subtle shifts in centromere placement. This combination of criteria allowed the researchers to identify homologous chromosomes and to spot rearrangements that occurred during speciation.

The results revealed a striking degree of conservation. Chromosomes 3, 5 and 6 appeared unchanged across all six species, both morphologically and in their GTG banding patterns, and no rearrangement was detected on them. Where differences did appear, they were mostly intrachromosomal, meaning they occurred within a single chromosome rather than between chromosomes. The Japanese quail chromosome 1 and the Houbara bustard chromosome 1 both showed shorter short arms than the chicken homolog, which the authors attribute to the formation of evolutionary neocentromeres, new centromeres that arise de novo in a euchromatic locus while the old centromere is inactivated. This phenomenon, first demonstrated in birds through studies of the red-legged partridge, has since been reported in pheasants, ducks and guineafowl, and it leaves the order of molecular markers along the chromosome essentially undisturbed.

Other chromosomes told more dramatic stories. The Japanese quail chromosome 2 carries a large region with an inverted banding pattern, consistent with a double pericentric inversion previously confirmed by comparative mapping with BAC clones and immunodetection. The Houbara bustard, by contrast, showed the most extensive reorganization of any species in the study. Its chromosome 2 appears to have lost the terminal part of its long arm through a terminal fission, and the researchers detected a similarity between that lost region and the bustard’s microchromosome 10, suggesting that the ancestral chromosome split to give rise to both a macrochromosome and a microchromosome. Even more strikingly, the bustard’s chromosome 4 corresponds only to the distal part of the long arm of chromosome 4 in the other species, with the missing regions apparently redistributed onto the bustard’s microchromosomes 11, 14 and 19. These interchromosomal rearrangements involving microchromosomes are rare events in birds, which makes their detection in the bustard particularly significant.

The sex chromosomes added their own chapter to the story. The Z chromosome showed a terminal inversion in the long arm in both the Barbary partridge and the Houbara bustard, and the bustard’s Z also carried a loss of an interstitial segment in the short arm that potentially corresponds to microchromosome 13. The W chromosome, meanwhile, is submetacentric in the partridges and the bustard but subtelocentric in the quails, a difference the authors explain by an evolutionary new centromere. The Z chromosome in birds contains a high number of breakpoints and is particularly subject to structural changes, broadly represented by paracentric and pericentric inversions, and its evolution has been characterized by a complex process of inversions likely related to the suppression of recombination between Z and W during the early stages of sex chromosome differentiation.

Taken together, the findings confirm a paradox that has emerged from decades of avian cytogenetics: the structure of bird karyotypes is more conserved at the interchromosomal scale, where whole chromosomes retain their identity, than at the intrachromosomal scale, where inversions, deletions, fissions and centromere repositioning accumulate over evolutionary time. The appearance of a small number of inter- and intrachromosomal rearrangements across six species suggests a high degree of conservatism of genome organization, and the team proposed a summary diagram tracing the chronology of these events from the presumed ancestral avian karyotype. The Houbara bustard karyotype carries the highest number of rearrangements, including fissions, compared with the ancestral karyotype, which fits its position in a lineage that diverged early from the Galliformes.

The study also carries practical weight for conservation. Classical cytogenetics can detect chromosomal abnormalities responsible for early embryonic mortality, a crucial capability for breeding programs of endangered species like the Houbara bustard, which is classified as vulnerable by the IUCN and has suffered a significant decline over the past thirty years, particularly due to poaching. The storage of chromosomes and nucleotide sequences is a form of biobanking, and sequence analysis can identify genetically important individuals for breeding. As the authors note, avian genomics and stem cell approaches could offer hope not only for saving endangered species such as the green peafowl but also for other birds threatened with extinction, ensuring that the chromosomal record of this extraordinary lineage survives the extinction crisis now unfolding around it.

Subject of Research: Highlighting chromosomal rearrangements of five species of Galliformes (Domestic fowl, Common and Japanese quail, Barbary and Chukar partridge) and the Houbara bustard, an endangered Otidiformes: banding cytogenetic is a powerful tool

Article Title: Highlighting chromosomal rearrangements of five species of Galliformes (Domestic fowl, Common and Japanese quail, Barbary and Chukar partridge) and the Houbara bustard, an endangered Otidiformes : banding cytogenetic is a powerful tool

Article References: Kartout-Benmessaoud, Y., Ouchia-Benissad, S., Mahiddine-Aoudjit, L., & Ladjali-Mohammedi, K. (2024). Highlighting chromosomal rearrangements of five species of Galliformes (Domestic fowl, Common and Japanese quail, Barbary and Chukar partridge) and the Houbara bustard, an endangered Otidiformes: banding cytogenetic is a powerful tool. Comparative Cytogenetics, 18, 213-237. https://doi.org/10.3897/compcytogen.18.135056

Image Credits: AI Generated

DOI: 10.3897/compcytogen.18.135056

Keywords: Highlighting, chromosomal, rearrangements, five, species, Galliformes, Domestic, fowl, Common, Japanese, quail, Barbary