Periodic Reporting for period 1 - birdGRC (Evolution and function of the germline-restricted chromosome in songbirds) Reporting period: 2021-01-01 to 2022-10-31 Summary of the context and overall objectives of the project Metazoans typically keep the same genetic information in all body cells over their life cycle. However, some organisms show exceptions to this rule through programmed DNA elimination from the somatic cell line during embryo development, as is the case of the germline-restricted chromosome (GRC) in songbirds. Recently, it has been demonstrated that the puzzling GRC is likely present in over half of all bird species and that the zebra finch GRC is enriched in developmental genes. However, it remains mysterious why the GRC evolved and how it may affect differential gene expression between tissues, genomic divergence between species, and the determination of cell fate between germline and soma. To address this, we first generated a high-quality reference assembly of the zebra finch GRC comprising over 70% of its expected size. We then found molecular evidence for the presence of the GRC in all the 25 Passeriformes species studied, including two Suboscines. The GRC repeat content has no traceable pattern across the phylogeny but rather seems to have expanded and contracted continuously. The comparison of the gene content of GRCs showed that elavl4 and cpeb1 are the genes more frequently present across Passeriformes GRCs and might have an important role for the GRC. Finally, we started functional analyses to elucidate the role of these genes in the early transmission in embryos with differential expression analyses of mRNA and sRNA, as well as whole-mount in situ hybridization with DNA and RNA probes to spot the GRC location and expression in embryo development. Altogether, we showed that the GRC probably arose once and early during the Passeriformes diversification. Since then, the GRC content has been changing constantly and we detected no genes in common within all the species, so function of the most important genes could be replaced by newly arrived genes. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far First, we generated the first reference-quality multiplatform assembly of the zebra finch GRC (and any large GRC) to date combining different sequencing technologies, which is crucial step towards deciphering the first full genome of a songbird. Second, we compared somatic and testis DNA sequencing libraries from 23 oscine (songbirds) and 2 suboscine species to infer the evolutionary trajectories of the GRC. We found clear evidence for the presence of the GRC in all the 25 species. Repetitive elements in the different GRCs suffered contraction and expansion with little phylogenetic signal. Additionally, we did not find genes present on the GRCs from all the species, but the most frequently present genes were elavl4 and cpeb1 in 19 out of the 25 species. The gene ahnak was present on the GRC of the 2 Suboscines and 9 Oscines. These might be genes relevant for GRC function. Importantly, this is the first time that GRC was detected in Suboscines, locating its origin very early in the Passeriformes phylogeny. We added more evidence of the presence to the GRC in Suboscines by generating a reference-quality multiplatform assembly of the tyrant flycatcher Myiozetetes cayanensis GRC. Third, we analyzed the transmission and putative function of the GRC by sequencing mRNA and sRNA in six different stages to perform differential expression analyses. We also optimized the whole-mount in situ hybridization protocol for DNA probes to trace the location of the GRC during the early stages of the development, and with RNA probes to spot the expression of GRC genes. We expect to have the results from these experiments in the next months. Altogether, we showed that the GRC probably arose once and early during the Passeriformes diversification. Since then, the GRC content has been changing constantly and we detected no genes in common within all the species, so function of the most important genes could be replaced by newly arrived genes. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) How the songbird germline-restricted chromosome evolved and what role it may play during gonad or embryo development is a topic acquiring more relevance over the last five years. Recent technological advances in sequencing, bioinformatics, and developmental biology provided us a unique opportunity to perform the proposed multi-omics analyses. In this project, we generated the first multiplatform assembly of any GRC to date. The technical challenges we solved will help to routinely include the GRC in genome assemblies of Passeriformes and other animals. We also discovered that the GRC originated in a single event, probably very early during the diversification of Passeriformes over 40 million years ago. Thus, around two thirds of all 10,500 bird species likely have a GRC. We found that not a single analyzed gene was shared among all the 25 sampled species, but some of them might be relevant since are present in most of the species. Finally, the optimization of RNA sequencing and cytogenetic analyses in the zebra finch will be useful to understand the role of the GRC in other species. All the progress carried out during this project have significant relevance to diverse areas such as genomics, developmental biology, molecular ecology, and even cancer research. Related documents Novosibirsk_lecture_Dec08.jpg ESEB22_talk.jpg
