Avian cell culture is widely applied for cytogenetic studies, the improvement of which increasingly allows for the production of high-quality chromosomes, essential to perform both classical and molecular cytogenetic studies. Among these approaches, there are two main types: fibroblast and bone marrow culture. Despite its high cost and complexity, fibroblast culture is considered the superior approach due to the quality of the metaphases produced. Short-term bone marrow cultivation provides more condensed chromosomes but nonetheless is quicker and easier. In the search for a quicker, cheaper way to prepare metaphases without losing quality, the present work developed a novel, widely applicable protocol for avian chromosome preparation. Twenty-one bird embryos from distinct families were sampled: Icteridae, Columbidae, Furnariidae, Estrildidae, Thraupidae, Troglodytidae and Ardeidae. The protocol was based on a combination of modified fibroblast culture and bone marrow cultivation, taking the advantages of both. The results show that all species consistently presented good mitotic indexes and high-quality chromosomes. Overall, the application of this protocol for bird cytogenetics can optimize the time, considering that most fibroblast cultures take at least 3 days and often much longer. However, our protocol can be performed in 3 h with a much-reduced cost of reagents and equipment.

Furnariidae (Ovenbirds) is one of the most diversified families in the Passeriformes order and Suboscines suborder. Despite their great diversity of species, cytogenetic research is still in its early stages, restricting our knowledge of their karyotype evolution. We combined traditional and molecular cytogenetic analyses in three representative species, Synallaxis frontalis, Syndactyla rufosuperciliata, and Cranioleuca obsoleta, to examine the chromosomal structure and evolution of Ovenbirds. Our findings reveal that all the species studied had the same diploid number (2n= 82). Differences in chromosomal morphology of some macrochromosomes indicate the presence of intrachromosomal rearrangements. Although the three species only had the 18S rDNA on one microchromosome pair, chromosomal mapping of six simple short repeats revealed a varied pattern of chromosome distribution among them, suggesting that each species underwent different repetitive DNA accumulation upon their divergence. The interspecific comparative genomic hybridization (CGH) experiment revealed that the Furnariidae species investigated carry centromeric regions enriched in similar repetitive sequences, bolstering the Furnariidae family's karyotype conservation. Nonetheless, the outgroup species Turdus rufiventris (Turdidae) demonstrated an advanced stage of sequence divergence with hybridization signals that were almost entirely limited to a few microchromosomes. Overall, the findings imply that Furnariidae species have a high degree of chromosomal conservation, and also we could observe a differentiation of repetitive sequences in both Passeriformes suborders (Suboscines and Oscines).

Passeriformes birds are widely recognized for their remarkable diversity, with over 5700 species described so far. Like most bird species, they possess a karyotype characteristic of modern birds, which includes a bimodal karyotype consisting of a few pairs of macrochromosomes and many pairs of microchromosomes. Although the karyotype is typically 2n = 80, the diploid number can atypically vary greatly, ranging from 56 to approximately 100 chromosomes. In this study, we aimed to understand the extent of conservation of the karyotype’s organizational structure within four species of this group using Bacterial Artificial Chromosomes via Fluorescence In Situ Hybridization (BAC-FISH) with microchromosome probes from Chicken (Gallus gallus) or Zebra Finch (Taeniopygia guttata) per microchromosomes (GGA10-28, except GGA16). By examining the chromosome complement of four passerine species—the Streaked Flycatcher (Myiodynastes maculatus), Shiny Cowbird (Molothrus bonariensis), Southern House Wren (Troglodytes aedon), and Double-collared Seedeater (Sporophila caerulescens)—we discovered a new chromosome number for Southern House Wren. Through FISH experiments, we were able to observe the same pattern of microchromosome organization as in the common ancestor of birds. As a result, we propose a new diploid number for Southern House Wren and confirm the conservation status of microchromosome organization, which may confer evolutionary advantages to this group.