bioRxiv preprint doi: https://doi.org/10.1101/2020.02.02.931493; this version posted February 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Evolutionary context can clarify teleosts gene names Gasanov E.V.*, Jędrychowska J.*, Kuźnicki J., Korzh V. International Institute of Molecular and Cell Biology in Warsaw, Poland *equal contribution Corresponding author: Vladimir Korzh Email: [email protected] Phone: +48 22 597 0765 Fax: +48 22 597 0715 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.02.931493; this version posted February 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Nothing in Biology Makes Sense Except in the Light of Evolution Theodosius Dobzhansky, 1973 Summary The initial convention to name genes relied on historical precedent, order in the human genome or mutants in model systems. However, partial duplication of genes in teleosts required naming the duplicated genes, so ohnologs adopted the 'a' or 'b' extension. Rapid advances in deciphering the zebrafish genome in relation to the human genome instituted naming genes in all other fish genomes in the convention of zebrafish. Unfortunately, some ohnologs and their resembling orthologs suffered from incorrect nomenclature, which created confusion in particular instances like establishing disease models. We sought to overcome this barrier by establishing the ex silico evolutionary-based systematic approach to naming the ohnologs in teleosts and other fish. We compared gene synteny using the spotted gar genome as the reference, which represents the unduplicated ancestral state. Using new criteria, we identified several hundreds of potentially misnamed ohnologs and validated manually several ohnologs as a proof pf principle. This may help to establish a standard naming practice resulting in the improved evolutionary-based gene nomenclature. This approach may help to identify and rename ohnologs in all relevant EMBL-EBI and NCBI databases starting from the zebrafish genome to avoid further proliferation of misleading information. Key words: synteny, gene naming, evolutionary-based systematic approach, genome duplication, reference genome, zebrafish, teleosts, spotted gar, ohnologs, orthologs bioRxiv preprint doi: https://doi.org/10.1101/2020.02.02.931493; this version posted February 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Gene naming problem Using scientific terms suggests a certain level of precision in defining a subject. Gene names have quickly become a part of the scientific language in genomics and developmental biology. Their usage then spreads to related areas such as medicine and biotechnology. Correctly naming genes should create a comprehensive tool for communicating across the biological sciences and beyond. Ideally, gene names should utilize a system that reflects the relationship to homologous genes in other species to better define their evolutionary and functional relationship. However, this convention becomes constrained with duplicated genes or ohnologs (Wolfe, 2000) characteristic for teleosts. The existing convention of gene naming relies on historical precedent, an order of genes in the human genome or names of mutants in model systems. So, gene names provide insight into the history of a gene study rather than providing insight into their function or evolutionary history. Our aim is to propose an approach for naming duplicated genes based upon the natural process of genome development, i.e. evolution. Our approach can create a convincing system for gene naming that requires a relatively small effort and minor nomenclature changes. The organized naming of genes is important for proper nomenclature of genes and gene families, synchronizing with mammalian counterparts and working with disease models using zebrafish (Danio rerio) and medaka (Oryzias latipes). Each animal taxon evolved with a continuous process of gene loss and gain, with the latter primarily through gene duplications. Meanwhile, some groups of animals passed through extremely rare but massive gene number changes like whole-genome duplications followed by periods of genome stabilization, i.e. partially duplicated gene inactivation and reduction (Glasauer & Neuhauss, 2014). The significant portion of genes in teleosts, the largest group of vertebrates, are duplicated as a result of whole-genome duplication that occured at the beginning of the taxon evolution (Glasauer & Neuhauss, 2014). Similar gene names in different species should suggest insights into the evolution of the genome. An existent practice is to define ohnologs (two fish orthologs of the mammalian gene) by letters 'a' and 'b' following the gene's name used in the human genome, e.g., kcng4a and kcng4b, the orthologs of human KCNG4 (potassium voltage-gated channel subfamily G member 4 gene). This convention was established at the meeting in Ringberg, Germany, in March 1992 (ZFIN Database). However, this convention did not specify when to use the 'a' or 'b' to distinguish genes. The practice to name the second discovered ohnolog 'b' after adding 'a' to the first discovery reveals complications to follow the precedence of discovery and the independent evolution of ohnologs across teleost species. Some teleost species have both ohnologs, whereas bioRxiv preprint doi: https://doi.org/10.1101/2020.02.02.931493; this version posted February 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. others lost one of them. When a preserved singleton is discovered, corresponding to either 'a' or 'b' in related species, can easily result in incorrect nomenclature. Taken together, these examples reveal an unreliable gene naming system. The insufficiency of the historical approach is clearly described by one example of the tbx-family (T-box family) genes. After cloning, the mammalian Tbx2 zebrafish ohnologs were temporarily named tbx-a and tbx-c (Dheen et al., 1999). When described independently by other authors, tbx-c was named tbx2 (Ruvinsky et al., 2000). tbx-a was later renamed for reasons unknown as tbx2a, whereas tbx-c became tbx2b. In this case, 'a' and 'b' carried no readily apparent information to link to mammalian orthologs. The naming of ohnologs in different teleost species has followed that of the zebrafish. This practice triggered a chain of incorrect naming events like the rather casual approach of naming of zebrafish Tbx2 genes. According to Ensembl GRCz11 (Ensembl Release 98), a bulk of Tbx2 genes in teleosts were named “tbx2b”, whereas “tbx2a” has been defined in addition to “tbx2b” in two species [channel catfish (Ictalurus punctatus) and northern pike (Esox lucius)] to date. Thus, this example represents a problem in gene naming in teleosts, which highlights the problem of establishing a unified criterion for naming ohnolog genes. In the article, we propose using an evolutionary-based systematic approach of naming genes. Sequence similarity criterion We posit that a more reliable criterion should utilize sequence similarity to define 'a' and 'b' orthologs. Sequence similarity relies on evolutionary conservation of a gene function in teleosts compared to other vertebrates. The fish ohnolog gene most resembling the human ortholog (using the traditional gene naming process) or an ancestral gene (using the evolution- based systematic approach proposed here) would hold the name 'a' with the less resembling one holding 'b', respectively. If the fish species possesses only one gene according to this criterion, we propose a second criterion that compares an ortholog to ohnologs of other teleosts to determine a reliable 'a' or 'b' extension. Unfortunately, additional problems arise to systematically assign gene names. One problem arises from uncertainty about which sequence, gene or protein, coding or/and non- coding should be utilized to systematically name genes. Returning to the Tbx2 ohnologs, the genomic DNA similarity of zebrafish tbx2a to human TBX2 is 48.1% and zebrafish tbx2b to human TBX2 is 41.5%, but the coding sequence identity is 36.6% and 47.5%, respectively. So, the protein identity of zebrafish Tbx2a and Tbx2b to human TBX2 is 45.5% and 50.3%, bioRxiv preprint doi: https://doi.org/10.1101/2020.02.02.931493; this version posted February 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. respectively. Using a different sequence, studies defined the non-coding regulatory regions of zebrafish tbx2a and tbx2b (Chi et al., 2008; Kaaij et al., 2016). However, the DNA alignment demonstrated a much closer evolutionary relationship between the enhancers of mammalian Tbx2 and zebrafish tbx2b compared to tbx2a. The