A conserved karyotype? Chromosomal rearrangements in Charadrius collaris detected by BAC-FISH.

Charadriidae comprise 142 valid species and the most recent checklist for the occurrence of this family in Brazil describes 11 species. There are few chromosomal studies in Charadriidae, most of them using a conventional approach. In Charadrius, only five species had their karyotypes described by classical cytogenetics, of which four have 2n = 76 (C. hiaticula, C. dubius, C. vociferou and C. collaris) and one 2n = 78 (C. alexandrinus alexandrinus). Among these species, only Charadrius collaris had the karyotype studied by chromosome painting, which allowed the identification of chromosomal homeologies with the karyotypes of Gallus gallus (GGA) and Burhinus oedicnemus (BOE). According to the literature, studies performed with BAC-FISH using probes from Gallus gallus and Taeniopygia guttata (TGU) libraries have shown interactions between macro and microchromosomes and micro inversions in chromosomes previously considered conserved. Other studies have shown the fusion of several microchromosomes, forming new macrochromosomes, leading to a decrease in the 2n of some species. The present study aims to deepen the chromosomal information in Charadrius collaris through the application of BAC-FISH with probes from the GGA and TGU libraries, in order to investigate possible rearrangements within the apparently conserved karyotype of this species, and thus better clarify the evolutionary history of the species. Charadrius collaris presented 2n = 76 and fundamental number (FN) equal to 94. Comparative mapping of BAC probes from GGA and TGU in Charadrius collaris revealed hybridization signals from 26 macrochromosome probes. Probes from microchromosomes 9 to 28 of GGA were also used and revealed 31 hybridization signals. The karyotype is well conserved, but it contains a paracentric and a pericentric inversion on the CCO1 chromosome, a paracentric and a pericentric inversion on the CCO4 and the separation of GGA4 into CCO4 and CCO8, demonstrating that the BAC-FISH approach allows for greater data resolution. More studies are needed to improve the understanding of chromosomal evolution within the order Charadriiformes and thus clarify whether these characteristics demonstrated here are specific traits for Charadrius collaris or if other species share these characteristics.

Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality.

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

Analysis of multiple chromosomal rearrangements in the genome of Willisornis vidua using BAC-FISH and chromosome painting on a supposed conserved karyotype.

BACKGROUND: Thamnophilidae birds are the result of a monophyletic radiation of insectivorous Passeriformes. They are a diverse group of 225 species and 45 genera and occur in lowlands and lower montane forests of Neotropics. Despite the large degree of diversity seen in this family, just four species of Thamnophilidae have been karyotyped with a diploid number ranging from 76 to 82 chromosomes. The karyotypic relationships within and between Thamnophilidae and another Passeriformes therefore remain poorly understood. Recent studies have identified the occurrence of intrachromosomal rearrangements in Passeriformes using in silico data and molecular cytogenetic tools. These results demonstrate that intrachromosomal rearrangements are more common in birds than previously thought and are likely to contribute to speciation events. With this in mind, we investigate the apparently conserved karyotype of Willisornis vidua, the Xingu Scale-backed Antbird, using a combination of molecular cytogenetic techniques including chromosome painting with probes derived from Gallus gallus (chicken) and Burhinus oedicnemus (stone curlew), combined with Bacterial Artificial Chromosome (BAC) probes derived from the same species. The goal was to investigate the occurrence of rearrangements in an apparently conserved karyotype in order to understand the evolutionary history and taxonomy of this species. In total, 78 BAC probes from the Gallus gallus and Taeniopygia guttata (the Zebra Finch) BAC libraries were tested, of which 40 were derived from Gallus gallus macrochromosomes 1-8, and 38 from microchromosomes 9-28. RESULTS: The karyotype is similar to typical Passeriformes karyotypes, with a diploid number of 2n = 80. Our chromosome painting results show that most of the Gallus gallus chromosomes are conserved, except GGA-1, 2 and 4, with some rearrangements identified among macro- and microchromosomes. BAC mapping revealed many intrachromosomal rearrangements, mainly inversions, when comparing Willisornis vidua karyotype with Gallus gallus, and corroborates the fissions revealed by chromosome painting. CONCLUSIONS: Willisornis vidua presents multiple chromosomal rearrangements despite having a supposed conservative karyotype, demonstrating that our approach using a combination of FISH tools provides a higher resolution than previously obtained by chromosome painting alone. We also show that populations of Willisornis vidua appear conserved from a cytogenetic perspective, despite significant phylogeographic structure.

Rapid Multi-Hybridisation FISH Screening for Balanced Porcine Reciprocal Translocations Suggests a Much Higher Abnormality Rate Than Previously Appreciated.

With demand rising, pigs are the world's leading source of meat protein; however significant economic loss and environmental damage can be incurred if boars used for artificial insemination (AI) are hypoprolific (sub-fertile). Growing evidence suggests that semen analysis is an unreliable tool for diagnosing hypoprolificacy, with litter size and farrowing rate being more applicable. Once such data are available, however, any affected boar will have been in service for some time, with significant financial and environmental losses incurred. Reciprocal translocations (RTs) are the leading cause of porcine hypoprolificacy, reportedly present in 0.47% of AI boars. Traditional standard karyotyping, however, relies on animal specific expertise and does not detect more subtle (cryptic) translocations. Previously, we reported development of a multiple hybridisation fluorescence in situ hybridisation (FISH) strategy; here, we report on its use in 1641 AI boars. A total of 15 different RTs were identified in 69 boars, with four further animals XX/XY chimeric. Therefore, 4.5% had a chromosome abnormality (4.2% with an RT), a 0.88% incidence. Revisiting cases with both karyotype and FISH information, we reanalysed captured images, asking whether the translocation was detectable by karyotyping alone. The results suggest that chromosome translocations in boars may be significantly under-reported, thereby highlighting the need for pre-emptive screening by this method before a boar enters a breeding programme.

Comparative Mapping of the Macrochromosomes of Eight Avian Species Provides Further Insight into Their Phylogenetic Relationships and Avian Karyotype Evolution.

Avian genomes typically consist of ~10 pairs of macro- and ~30 pairs of microchromosomes. While inter-chromosomally, a pattern emerges of very little change (with notable exceptions) throughout evolution, intrachromosomal changes remain relatively poorly studied. To rectify this, here we use a pan-avian universally hybridising set of 74 chicken bacterial artificial chromosome (BAC) probes on the macrochromosomes of eight bird species: common blackbird, Atlantic canary, Eurasian woodcock, helmeted guinea fowl, houbara bustard, mallard duck, and rock dove. A combination of molecular cytogenetic, bioinformatics, and mathematical analyses allowed the building of comparative cytogenetic maps, reconstruction of a putative Neognathae ancestor, and assessment of chromosome rearrangement patterns and phylogenetic relationships in the studied neognath lineages. We observe that, as with our previous studies, chicken appears to have the karyotype most similar to the ancestor; however, previous reports of an increased rate of intrachromosomal change in Passeriformes (songbirds) appear not to be the case in our dataset. The use of this universally hybridizing probe set is applicable not only for the re-tracing of avian karyotype evolution but, potentially, for reconstructing genome assemblies.

A Comprehensive Cytogenetic Analysis of Several Members of the Family Columbidae (Aves, Columbiformes).

The Columbidae species (Aves, Columbiformes) show considerable variation in their diploid numbers (2n = 68-86), but there is limited understanding of the events that shaped the extant karyotypes. Hence, we performed whole chromosome painting (wcp) for paints GGA1-10 and bacterial artificial chromosome (BAC) probes for chromosomes GGA11-28 for Columbina passerina, Columbina talpacoti, Patagioenas cayennensis, Geotrygon violacea and Geotrygon montana. Streptopelia decaocto was only investigated with paints because BACs for GGA10-28 had been previously analyzed. We also performed phylogenetic analyses in order to trace the evolutionary history of this family in light of chromosomal changes using our wcp data with chicken probes and from Zenaida auriculata, Columbina picui, Columba livia and Leptotila verreauxi, previously published. G-banding was performed on all these species. Comparative chromosome paint and G-banding results suggested that at least one interchromosomal and many intrachromosomal rearrangements had occurred in the diversification of Columbidae species. On the other hand, a high degree of conservation of microchromosome organization was observed in these species. Our cladistic analysis, considering all the chromosome rearrangements detected, provided strong support for L. verreauxi and P. cayennensis, G. montana and G. violacea, C. passerina and C. talpacoti having sister taxa relationships, as well as for all Columbidae species analyzed herein. Additionally, the chromosome characters were mapped in a consensus phylogenetic topology previously proposed, revealing a pericentric inversion in the chromosome homologous to GGA4 in a chromosomal signature unique to small New World ground doves.

Patterns of microchromosome organization remain highly conserved throughout avian evolution.

The structure and organization of a species genome at a karyotypic level, and in interphase nuclei, have broad functional significance. Although regular sized chromosomes are studied extensively in this regard, microchromosomes, which are present in many terrestrial vertebrates, remain poorly explored. Birds have more cytologically indistinguishable microchromosomes (~ 30 pairs) than other vertebrates; however, the degree to which genome organization patterns at a karyotypic and interphase level differ between species is unknown. In species where microchromosomes have fused to other chromosomes, they retain genomic features such as gene density and GC content; however, the extent to which they retain a central nuclear position has not been investigated. In studying 22 avian species from 10 orders, we established that, other than in species where microchromosomal fusion is obvious (Falconiformes and Psittaciformes), there was no evidence of microchromosomal rearrangement, suggesting an evolutionarily stable avian genome (karyotypic) organization. Moreover, in species where microchromosomal fusion has occurred, they retain a central nuclear location, suggesting that the nuclear position of microchromosomes is a function of their genomic features rather than their physical size.

Chromosome-level assembly reveals extensive rearrangement in saker falcon and budgerigar, but not ostrich, genomes.

BACKGROUND: The number of de novo genome sequence assemblies is increasing exponentially; however, relatively few contain one scaffold/contig per chromosome. Such assemblies are essential for studies of genotype-to-phenotype association, gross genomic evolution, and speciation. Inter-species differences can arise from chromosomal changes fixed during evolution, and we previously hypothesized that a higher fraction of elements under negative selection contributed to avian-specific phenotypes and avian genome organization stability. The objective of this study is to generate chromosome-level assemblies of three avian species (saker falcon, budgerigar, and ostrich) previously reported as karyotypically rearranged compared to most birds. We also test the hypothesis that the density of conserved non-coding elements is associated with the positions of evolutionary breakpoint regions. RESULTS: We used reference-assisted chromosome assembly, PCR, and lab-based molecular approaches, to generate chromosome-level assemblies of the three species. We mapped inter- and intrachromosomal changes from the avian ancestor, finding no interchromosomal rearrangements in the ostrich genome, despite it being previously described as chromosomally rearranged. We found that the average density of conserved non-coding elements in evolutionary breakpoint regions is significantly reduced. Fission evolutionary breakpoint regions have the lowest conserved non-coding element density, and intrachromomosomal evolutionary breakpoint regions have the highest. CONCLUSIONS: The tools used here can generate inexpensive, efficient chromosome-level assemblies, with > 80% assigned to chromosomes, which is comparable to genomes assembled using high-density physical or genetic mapping. Moreover, conserved non-coding elements are important factors in defining where rearrangements, especially interchromosomal, are fixed during evolution without deleterious effects.

Reconstruction of the diapsid ancestral genome permits chromosome evolution tracing in avian and non-avian dinosaurs.

Genomic organisation of extinct lineages can be inferred from extant chromosome-level genome assemblies. Here, we apply bioinformatic and molecular cytogenetic approaches to determine the genomic structure of the diapsid common ancestor. We then infer the events that likely occurred along this lineage from theropod dinosaurs through to modern birds. Our results suggest that most elements of a typical 'avian-like' karyotype (40 chromosome pairs, including 30 microchromosomes) were in place before the divergence of turtles from birds ~255 mya. This genome organisation therefore predates the emergence of early dinosaurs and pterosaurs and the evolution of flight. Remaining largely unchanged interchromosomally through the dinosaur-theropod route that led to modern birds, intrachromosomal changes nonetheless reveal evolutionary breakpoint regions enriched for genes with ontology terms related to chromatin organisation and transcription. This genomic structure therefore appears highly stable yet contributes to a large degree of phenotypic diversity, as well as underpinning adaptive responses to major environmental disruptions via intrachromosomal repatterning.