Parallel Nonfunctionalization of CK1δ/ε Kinase Ohnologs Following a Whole-Genome Duplication Event.

Whole-genome duplication (WGD) followed by speciation allows us to examine the parallel evolution of ohnolog pairs. In the yeast family Saccharomycetaceae, HRR25 is a rare case of repeated ohnolog maintenance. This gene has reverted to a single copy in Saccharomyces cerevisiae where it is now essential, but has been maintained as pairs in at least 7 species post-WGD. In S. cerevisiae, HRR25 encodes the casein kinase 1δ/ε and plays a role in a variety of functions through its kinase activity and protein-protein interactions (PPIs). We hypothesized that the maintenance of duplicated HRR25 ohnologs could be a result of repeated subfunctionalization. We tested this hypothesis through a functional complementation assay in S. cerevisiae, testing all pairwise combinations of 25 orthologs (including 7 ohnolog pairs). Contrary to our expectations, we observed no cases of pair-dependent complementation, which would have supported the subfunctionalization hypothesis. Instead, most post-WGD species have one ohnolog that failed to complement, suggesting their nonfunctionalization or neofunctionalization. The ohnologs incapable of complementation have undergone more rapid protein evolution, lost most PPIs that were observed for their functional counterparts and singletons from post-WGD and non-WGD species, and have nonconserved cellular localization, consistent with their ongoing loss of function. The analysis in Naumovozyma castellii shows that the noncomplementing ohnolog is expressed at a lower level and has become nonessential. Taken together, our results indicate that HRR25 orthologs are undergoing gradual nonfunctionalization.

Zebrafish: unraveling genetic complexity through duplicated genes.

The zebrafish is an invaluable model organism for genetic, developmental, and disease research. Although its high conservation with humans is often cited as justification for its use, the zebrafish harbors oft-ignored genetic characteristics that may provide unique insights into gene structure and function. Zebrafish, along with other teleost fish, underwent an additional round of whole genome duplication after their split from tetrapods-resulting in an abundance of duplicated genes when compared to other vertebrates. These duplicated genes have evolved in distinct ways over the ensuing 350 million years. Thus, each gene within a duplicated gene pair has nuanced differences that create a unique identity. By investigating both members of the gene pair together, we can elucidate the mechanisms that underly protein structure and function and drive the complex interplay within biological systems, such as signal transduction cascades, genetic regulatory networks, and evolution of tissue and organ function. It is crucial to leverage such studies to explore these molecular dynamics, which could have far-reaching implications for both basic science and therapeutic development. Here, we will review the role of gene duplications and the existing models for gene divergence and retention following these events. We will also highlight examples within each of these models where studies comparing duplicated genes in the zebrafish have yielded key insights into protein structure, function, and regulation.

A Population-Genetic Lens into the Process of Gene Loss Following Whole-Genome Duplication.

Whole-genome duplications (WGDs) have occurred in many eukaryotic lineages. However, the underlying evolutionary forces and molecular mechanisms responsible for the long-term retention of gene duplicates created by WGDs are not well understood. We employ a population-genomic approach to understand the selective forces acting on paralogs and investigate ongoing duplicate-gene loss in multiple species of Paramecium that share an ancient WGD. We show that mutations that abolish protein function are more likely to be segregating in retained WGD paralogs than in single-copy genes, most likely because of ongoing nonfunctionalization post-WGD. This relaxation of purifying selection occurs in only one WGD paralog, accompanied by the gradual fixation of nonsynonymous mutations and reduction in levels of expression, and occurs over a long period of evolutionary time, "marking" one locus for future loss. Concordantly, the fitness effects of new nonsynonymous mutations and frameshift-causing indels are significantly more deleterious in the highly expressed copy compared with their paralogs with lower expression. Our results provide a novel mechanistic model of gene duplicate loss following WGDs, wherein selection acts on the sum of functional activity of both duplicate genes, allowing the two to wander in expression and functional space, until one duplicate locus eventually degenerates enough in functional efficiency or expression that its contribution to total activity is too insignificant to be retained by purifying selection. Retention of duplicates by such mechanisms predicts long times to duplicate-gene loss, which should not be falsely attributed to retention due to gain/change in function.

Non-functional GoFLA19s are responsible for the male sterility caused by hybrid breakdown in cotton (Gossypium spp.).

Hybrid breakdown (HB) functions as a common reproductive barrier and reduces hybrid fitness in many species, including cotton. However, the related genes and the underlying genetic mechanisms of HB in cotton remain unknown. Here, we found that the photosensitive genetic male sterile line CCRI9106 was a hybrid progeny of Gossypium hirsutum and Gossypium barbadense and probably a product of HB. Fine mapping with F2 s (CCRI9106 × G. hirsutum/G. barbadense lines) identified a pair of male sterility genes GoFLA19s (encoding fasciclin-like arabinogalactan family protein) located on chromosomes A12 and D12. Crucial variations occurring in the fasciclin-like domain and the arabinogalactan protein domain were predicted to cause the non-functionalization of GbFLA19-D and GhFLA19-A. CRISPR/Cas9-mediated knockout assay confirmed the effects of GhFLA19s on male sterility. Sequence alignment analyses showed that variations in GbFLA19-D and GhFLA19-A likely occurred after the formation of allotetraploid cotton species. GoFLA19s are specifically expressed in anthers and contribute to tapetal development, exine assembly, intine formation, and pollen grain maturation. RNA-sequencing and quantitative reverse transcriptase-polymerase chain reaction analyses illustrated that genes related to these biological processes were significantly downregulated in the mutant. Our research on male sterility genes, GoFLA19s, improves the understanding of the molecular characteristics and evolutionary significance of HB in interspecific hybrid breeding.

Analysis of a mechanistic Markov model for gene duplicates evolving under subfunctionalization.

BACKGROUND: Gene duplication has been identified as a key process driving functional change in many genomes. Several biological models exist for the evolution of a pair of duplicates after a duplication event, and it is believed that gene duplicates can evolve in different ways, according to one process, or a mix of processes. Subfunctionalization is one such process, under which the two duplicates can be preserved by dividing up the function of the original gene between them. Analysis of genomic data using subfunctionalization and related processes has thus far been relatively coarse-grained, with mathematical treatments usually focusing on the phenomenological features of gene duplicate evolution. RESULTS: Here, we develop and analyze a mathematical model using the mechanics of subfunctionalization and the assumption of Poisson rates of mutation. By making use of the results from the literature on the Phase-Type distribution, we are able to derive exact analytical results for the model. The main advantage of the mechanistic model is that it leads to testable predictions of the phenomenological behavior (instead of building this behavior into the model a priori), and allows for the estimation of biologically meaningful parameters. We fit the survival function implied by this model to real genome data (Homo sapiens, Mus musculus, Rattus norvegicus and Canis familiaris), and compare the fit against commonly used phenomenological survival functions. We estimate the number of regulatory regions, and rates of mutation (relative to silent site mutation) in the coding and regulatory regions. We find that for the four genomes tested the subfunctionalization model predicts that duplicates most-likely have just a few regulatory regions, and the rate of mutation in the coding region is around 5-10 times greater than the rate in the regulatory regions. This is the first model-based estimate of the number of regulatory regions in duplicates. CONCLUSIONS: Strong agreement between empirical results and the predictions of our model suggest that subfunctionalization provides a consistent explanation for the evolution of many gene duplicates.

Subfunctionalization of peroxisome proliferator response elements accounts for retention of duplicated fabp1 genes in zebrafish.

BACKGROUND: In the duplication-degeneration-complementation (DDC) model, a duplicated gene has three possible fates: it may lose functionality through the accumulation of mutations (nonfunctionalization), acquire a new function (neofunctionalization), or each duplicate gene may retain a subset of functions of the ancestral gene (subfunctionalization). The role that promoter evolution plays in retention of duplicated genes in eukaryotic genomes is not well understood. Fatty acid-binding proteins (Fabp) belong to a multigene family that are highly conserved in sequence and function, but differ in their gene regulation, suggesting selective pressure is exerted via regulatory elements in the promoter. RESULTS: In this study, we describe the PPAR regulation of zebrafish fabp1a, fabp1b.1, and fabp1b.2 promoters and compare them to the PPAR regulation of the spotted gar fabp1 promoter, representative of the ancestral fabp1 gene. Evolution of the fabp1 promoter was inferred by sequence analysis, and differential PPAR-agonist activation of fabp1 promoter activity in zebrafish liver and intestine explant cells, and in HEK293A cells transiently transfected with wild-type and mutated fabp1promoter-reporter gene constructs. The promoter activity of spotted gar fabp1, representative of the ancestral fabp1, was induced by both PPARα- and PPARγ-specific agonists, but displayed a biphasic response to PPARα activation. Zebrafish fabp1a was PPARα-selective, fabp1b.1 was PPARγ-selective, and fabp1b.2 was not regulated by PPAR. CONCLUSIONS: The zebrafish fabp1 promoters underwent two successive rounds of subfunctionalization with respect to PPAR regulation leading to retention of three zebrafish fabp1 genes with stimuli-specific regulation. Using a pharmacological approach, we demonstrated here the divergent regulation of the zebrafish fabp1a, fabp1b.1, and fabp1b.2 with regard to subfunctionalization of PPAR regulation following two rounds of gene duplication.

Parallel nonfunctionalization of CK1δ/ε kinase ohnologs following a whole-genome duplication event.

Whole genome duplication (WGD) followed by speciation allows us to examine the parallel evolution of ohnolog pairs. In the yeast family Saccharomycetaceae, HRR25 is a rare case of repeated ohnolog maintenance. This gene has reverted to a single copy in S. cerevisiae where it is now essential, but has been maintained as pairs in at least 7 species post WGD. In S. cerevisiae, HRR25 encodes the casein kinase (CK) 1δ/ε and plays a role in a variety of functions through its kinase activity and protein-protein interactions (PPIs). We hypothesized that the maintenance of duplicated HRR25 ohnologs could be a result of repeated subfunctionalization. We tested this hypothesis through a functional complementation assay in S. cerevisiae, testing all pairwise combinations of 25 orthologs (including 7 ohnolog pairs). Contrary to our expectations, we observed no cases of pair-dependent complementation, which would have supported the subfunctionalization hypothesis. Instead, most post-WGD species have one ohnolog that failed to complement, suggesting their nonfunctionalization or neofunctionalization. The ohnologs incapable of complementation have undergone more rapid protein evolution, lost most PPIs that were observed for their functional counterparts and singletons from post and non-WGD species, and have non-conserved cellular localization, consistent with their ongoing loss of function. The analysis in N. castelli shows that the non-complementing ohnolog is expressed at a lower level and has become non-essential. Taken together, our results indicate that HRR25 orthologs are undergoing gradual nonfunctionalization.

Duplication and Loss of Function of Genes Encoding RNA Polymerase III Subunit C4 Causes Hybrid Incompatibility in Rice.

Reproductive barriers are commonly observed in both animals and plants, in which they maintain species integrity and contribute to speciation. This report shows that a combination of loss-of-function alleles at two duplicated loci, DUPLICATED GAMETOPHYTIC STERILITY 1 (DGS1) on chromosome 4 and DGS2 on chromosome 7, causes pollen sterility in hybrid progeny derived from an interspecific cross between cultivated rice, Oryza sativa, and an Asian annual wild rice, O. nivara Male gametes carrying the DGS1 allele from O. nivara (DGS1-nivaras ) and the DGS2 allele from O. sativa (DGS2-T65s ) were sterile, but female gametes carrying the same genotype were fertile. We isolated the causal gene, which encodes a protein homologous to DNA-dependent RNA polymerase (RNAP) III subunit C4 (RPC4). RPC4 facilitates the transcription of 5S rRNAs and tRNAs. The loss-of-function alleles at DGS1-nivaras and DGS2-T65s were caused by weak or nonexpression of RPC4 and an absence of RPC4, respectively. Phylogenetic analysis demonstrated that gene duplication of RPC4 at DGS1 and DGS2 was a recent event that occurred after divergence of the ancestral population of Oryza from other Poaceae or during diversification of AA-genome species.

What Fraction of Duplicates Observed in Recently Sequenced Genomes Is Segregating and Destined to Fail to Fix?

Most sequenced eukaryotic genomes show a large excess of recent duplicates. As duplicates age, both the population genetic process of failed fixation and the mutation-driven process of nonfunctionalization act to reduce the observed number of duplicates. Understanding the processes generating the age distributions of recent duplicates is important to also understand the role of duplicate genes in the functional divergence of genomes. To date, mechanistic models for duplicate gene retention only account for the mutation-driven nonfunctionalization process. Here, a neutral model for the distribution of synonymous substitutions in duplicated genes which are segregating and expected to never fix in a population is introduced. This model enables differentiation of neutral loss due to failed fixation from loss due to mutation-driven nonfunctionalization. The model has been validated on simulated data and subsequent analysis with the model on genomic data from human and mouse shows that conclusions about the underlying mechanisms for duplicate gene retention can be sensitive to consideration of population genetic processes.