Functional Differentiation of the Duplicated Gene BrrCIPK9 in Turnip (Brassica rapa var. rapa).

Gene duplication is a key biological process in the evolutionary history of plants and an important driving force for the diversification of genomic and genetic systems. Interactions between the calcium sensor calcineurin B-like protein (CBL) and its target, CBL-interacting protein kinase (CIPK), play important roles in the plant's response to various environmental stresses. As a food crop with important economic and research value, turnip (Brassica rapa var. rapa) has been well adapted to the environment of the Tibetan Plateau and become a traditional crop in the region. The BrrCIPK9 gene in turnip has not been characterized. In this study, two duplicated genes, BrrCIPK9.1 and BrrCIPK9.2, were screened from the turnip genome. Based on the phylogenetic analysis, BrrCIPK9.1 and BrrCIPK9.2 were found located in different sub-branches on the phylogenetic tree. Real-time fluorescence quantitative PCR analyses revealed their differential expression levels between the leaves and roots and in response to various stress treatments. The differences in their interactions with BrrCBLs were also revealed by yeast two-hybrid analyses. The results indicate that BrrCIPK9.1 and BrrCIPK9.2 have undergone Asparagine-alanine-phenylalanine (NAF) site divergence during turnip evolution, which has resulted in functional differences between them. Furthermore, BrrCIPK9.1 responded to high-pH (pH 8.5) stress, while BrrCIPK9.2 retained its ancestral function (low K+), thus providing further evidence of their functional divergence. These functional divergence genes facilitate turnip's good adaptation to the extreme environment of the Tibetan Plateau. In summary, the results of this study reveal the characteristics of the duplicated BrrCIPK9 genes and provide a basis for further functional studies of BrrCBLs-BrrCIPKs in turnip.

Expectations of duplicate gene retention under the gene duplicability hypothesis.

BACKGROUND: Gene duplication is an important process in evolution. What causes some genes to be retained after duplication and others to be lost is a process not well understood. The most prevalent theory is the gene duplicability hypothesis, that something about the function and number of interacting partners (number of subunits of protein complex, etc.), determines whether copies have more opportunity to be retained for long evolutionary periods. Some genes are also more susceptible to dosage balance effects following WGD events, making them more likely to be retained for longer periods of time. One would expect these processes that affect the retention of duplicate copies to affect the conditional probability ratio after consecutive whole genome duplication events. The probability that a gene will be retained after a second whole genome duplication event (WGD2), given that it was retained after the first whole genome duplication event (WGD1) versus the probability a gene will be retained after WGD2, given it was lost after WGD1 defines the probability ratio that is calculated. RESULTS: Since duplicate gene retention is a time heterogeneous process, the time between the events (t1) and the time since the most recent event (t2) are relevant factors in calculating the expectation for observation in any genome. Here, we use a survival analysis framework to predict the probability ratio for genomes with different values of t1 and t2 under the gene duplicability hypothesis, that some genes are more susceptible to selectable functional shifts, some more susceptible to dosage compensation, and others only drifting. We also predict the probability ratio with different values of t1 and t2 under the mutational opportunity hypothesis, that probability of retention for certain genes changes in subsequent events depending upon how they were previously retained. These models are nested such that the mutational opportunity model encompasses the gene duplicability model with shifting duplicability over time. Here we present a formalization of the gene duplicability and mutational opportunity hypotheses to characterize evolutionary dynamics and explanatory power in a recently developed statistical framework. CONCLUSIONS: This work presents expectations of the gene duplicability and mutational opportunity hypotheses over time under different sets of assumptions. This expectation will enable formal testing of processes leading to duplicate gene retention.

Why old duplicated genes are not thrown away in (paleo)polyploids? Example from the petC gene in Brassica napus.

Duplication of genes at different time period, through recurrent and frequent polyploidization events, have played a major role in plant evolution, adaptation and diversification. Interestingly, some of the ancestral duplicated genes (referred as paleologs), have been maintained for millions of years, and there is still a poor knowledge of the reasons of their retention, especially when testing the phenotypic effect of individual copies by using functional genetic approaches. To fill this gap, we performed functional genetic (CRISPR-Cas9), physiological, transcriptomic and evolutionary studies to finely investigate this open question, taking the example of the petC gene (involved in cytochrome b6/f and thus impacting photosynthesis) that is present in four paleologous copies in the oilseed crop Brassica napus. RNA-Seq and selective pressure analyses suggested that all paleologous copies conserved the same function and that they were all highly transcribed. Thereafter, the Knock Out (K.O.) of one, several or all petC copies highlighted that all paleologous copies have to be K.O. to suppress the gene function. In addition, we could determine that phenotypic effects in single and double mutants could only be deciphered in high light conditions. Interestingly, we did not detect any significant differences between single mutants K.O. for either the A03 or A09 copy (despite being differentially transcribed), or even between mutants for a single or two petC copies. Altogether, this work revealed that petC paleologs have retained their ancestral function and that the retention of these copies is explained by their compensatory role, especially in optimal environmental conditions.

Expression profiles in knock-down transgenic plants of high and low diversified duplicate genes in Arabidopsis thaliana.

Duplicated genes show various degrees of functional diversification in plants. We previously identified 1,052 pairs of high diversified duplicates (HDDs) and 600 pairs of low diversified duplicates (LDDs) in Arabidopsis thaliana. Single knock-down of HDDs induced abnormal phenotypic changes because the other gene copy could not compensate for the knock-down effect, while single knock-down of LDDs did not induce abnormal phenotypic changes because of functional compensation by the copy gene. Here, focusing on one pair each of HDDs and LDDs, we performed transcriptome analyses in single-knock-down transgenic plants. The numbers of differentially expressed genes in single-knock-down transgenic plants were not different between HDDs and LDDs. Thus, functional compensation inferred by transcriptomics was similar between HDDs and LDDs. However, the trend of differentially expressed genes was similar in the pair of LDDs, while expression profiles were dissimilar in the pair of HDDs. This result indicates that a pair of LDDs tends to share similar functions but a pair of HDDs tends to have undergone functional divergence. Taking these findings together, as the reason for no phenotypic changes in single knock-down of LDDs but phenotypic changes in double knock-down of LDDs, we concluded that phenotypic changes of LDDs were induced by decreasing gene dosage.

Dynamics of cis-regulatory sequences and transcriptional divergence of duplicated genes in soybean.

Transcriptional divergence of duplicated genes after whole genome duplication (WGD) has been described in many plant lineages and is often associated with subgenome dominance, a genome-wide mechanism. However, it is unknown what underlies the transcriptional divergence of duplicated genes in polyploid species that lack subgenome dominance. Soybean is a paleotetraploid with a WGD that occurred 5 to 13 Mya. Approximately 50% of the duplicated genes retained from this WGD exhibit transcriptional divergence. We developed accessible chromatin region (ACR) datasets from leaf, flower, and seed tissues using MNase-hypersensitivity sequencing. We validated enhancer function of several ACRs associated with known genes using CRISPR/Cas9-mediated genome editing. The ACR datasets were used to examine and correlate the transcriptional patterns of 17,111 pairs of duplicated genes in different tissues. We demonstrate that ACR dynamics are correlated with divergence of both expression level and tissue specificity of individual gene pairs. Gain or loss of flanking ACRs and mutation of cis-regulatory elements (CREs) within the ACRs can change the balance of the expression level and/or tissue specificity of the duplicated genes. Analysis of DNA sequences associated with ACRs revealed that the extensive sequence rearrangement after the WGD reshaped the CRE landscape, which appears to play a key role in the transcriptional divergence of duplicated genes in soybean. This may represent a general mechanism for transcriptional divergence of duplicated genes in polyploids that lack subgenome dominance.

Genomic and Transcriptional Profiles of Kelch-like (klhl) Gene Family in Polyploid Carassius Complex.

Genome duplication supplies raw genetic materials and has been thought to be essential for evolutionary innovation and ecological adaptation. Here, we select Kelch-like (klhl) genes to study the evolution of the duplicated genes in the polyploid Carassius complex, including amphidiploid C. auratus and amphitriploid C. gibelio. Phylogenetic, chromosomal location and read coverage analyses indicate that most of Carassius klhl genes exhibit a 2:1 relationship with zebrafish orthologs and confirm two rounds of polyploidy, an allotetraploidy followed by an autotriploidy, occurred during Carassius evolution. The lineage-specific expansion and biased retention/loss of klhl genes are also found in Carassius. Transcriptome analyses across eight adult tissues and seven embryogenesis stages reveal varied expression dominance and divergence between the two species. The expression of klhls in response to Carassius herpesvirus 2 infection shows different expression changes corresponding to distinct herpesvirus resistances in three C. gibelio gynogenetic clones. Finally, we find that most C. gibelio klhl genes possess three alleles except eight genes that have lost one or two alleles due to genome rearrangement. The allele expression bias is prosperous for Cgklhl genes and varies during embryogenesis owning to the sequential expression manner of the alleles. The current study provides global insights into the genomic and transcriptional evolution of duplicated genes in a given superfamily resulting from multiple rounds of polyploidization.

DNA methylation signatures of duplicate gene evolution in angiosperms.

Gene duplication is a source of evolutionary novelty. DNA methylation may play a role in the evolution of duplicate genes (paralogs) through its association with gene expression. While this relationship has been examined to varying extents in a few individual species, the generalizability of these results at either a broad phylogenetic scale with species of differing duplication histories or across a population remains unknown. We applied a comparative epigenomic approach to 43 angiosperm species across the phylogeny and a population of 928 Arabidopsis (Arabidopsis thaliana) accessions, examining the association of DNA methylation with paralog evolution. Genic DNA methylation was differentially associated with duplication type, the age of duplication, sequence evolution, and gene expression. Whole-genome duplicates were typically enriched for CG-only gene body methylated or unmethylated genes, while single-gene duplications were typically enriched for non-CG methylated or unmethylated genes. Non-CG methylation, in particular, was a characteristic of more recent single-gene duplicates. Core angiosperm gene families were differentiated into those which preferentially retain paralogs and "duplication-resistant" families, which convergently reverted to singletons following duplication. Duplication-resistant families that still have paralogous copies were, uncharacteristically for core angiosperm genes, enriched for non-CG methylation. Non-CG methylated paralogs had higher rates of sequence evolution, higher frequency of presence-absence variation, and more limited expression. This suggests that silencing by non-CG methylation may be important to maintaining dosage following duplication and be a precursor to fractionation. Our results indicate that genic methylation marks differing evolutionary trajectories and fates between paralogous genes and have a role in maintaining dosage following duplication.

Neofunctionalization of tandem duplicate genes encoding putative ß-L-arabinofuranosidases in Arabidopsis.

Tandem duplication, one of the major types of duplication, provides the raw material for the evolution of divergent functions. In this study, we identified 1 pair of tandem duplicate genes (AT5G12950 and AT5G12960) in Arabidopsis (Arabidopsis thaliana) that originated within the last 16 million years after the split of Arabidopsis from the Capsella-Boechera ancestor. We systematically used bioinformatic tools to redefine their putative biochemical function as ß-L-arabinofuranosidases that release L-Arabinose from the ß-L-Araf-containing molecules in Arabidopsis. Comprehensive transcriptomic and proteomic analyses using various datasets showed divergent expression patterns among tissues between the 2 duplicate genes. We further collected phenotypic data from 2 types of measurements to indicate that AT5G12950 and AT5G12960 have different roles resulting in divergent phenotypic effects. Overall, AT5G12950 and AT5G12960 represent putative ß-L-arabinofuranosidase encoding genes in Arabidopsis. After duplication, 1 duplicate copy developed diverged biological functions and contributed to a different phenotypic evolution in Arabidopsis.

HSDecipher: A pipeline for comparative genomic analysis of highly similar duplicate genes in eukaryotic genomes.

Many tools have been developed to measure the degree of similarity between gene duplicates within and between species. Here, we present HSDecipher, a bioinformatics pipeline to assist users in the analysis and visualization of highly similar duplicate genes (HSDs). We describe the steps for analysis of HSDs statistics, expanding HSD gene sets, and visualizing the results of comparative genomic analyses. HSDecipher represents a useful tool for researchers exploring the evolution of duplicate genes in select eukaryotic species. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021)1 and Zhang et al. (2022).2.

Models for the retention of duplicate genes and their biological underpinnings.

Gene content in genomes changes through several different processes, with gene duplication being an important contributor to such changes. Gene duplication occurs over a range of scales from individual genes to whole genomes, and the dynamics of this process can be context dependent. Still, there are rules by which genes are retained or lost from genomes after duplication, and probabilistic modeling has enabled characterization of these rules, including their context-dependence. Here, we describe the biology and corresponding mathematical models that are used to understand duplicate gene retention and its contribution to the set of biochemical functions encoded in a genome.

Conversion between duplicated genes generated by polyploidization contributes to the divergence of poplar and willow.

BACKGROUND: Gene conversion has an important effect on duplicate genes produced by polyploidization. Poplar (Populus trichocarpa) and willow (Salix brachista) are leading models and excellent green plants in the Salicaceae. Although much attention has been paid to the evolution of duplicated genes in poplar and willow, the role of conversion between duplicates generated from polyploidization remains poorly understood. RESULTS: Here, through genomic synteny analyses, we identified duplicate genes generated by the Salicaceae common tetraploidization (SCT) in the poplar and willow genomes. We estimated that at least 0.58% and 0.25% of poplar and willow duplicates were affected by whole-gene conversion after the poplar-willow divergence, with more (5.73% and 2.66%) affected by partial-gene conversion. Moreover, we found that the converted duplicated genes were unevenly distributed on each chromosome in the two genomes, and the well-preserved homoeologous chromosome regions may facilitate the conversion of duplicates. Notably, we found that conversion maintained the similarity of duplicates, likely contributing to the conservation of certain sequences, but is essentially accelerated the rate of evolution and increased species divergence. In addition, we found that converted duplicates tended to have more similar expression patterns than nonconverted duplicates. We found that genes associated with multigene families were preferentially converted. We also found that the genes encoding conserved structural domains associated with specific traits exhibited a high frequency of conversion. CONCLUSIONS: Extensive conversion between duplicate genes generated from the SCT contributes to the diversification of the family Salicaceae and has had long-lasting effects on those genes with important biological functions.

The multiple fates of gene duplications: Deletion, hypofunctionalization, subfunctionalization, neofunctionalization, dosage balance constraints, and neutral variation.

Gene duplications have long been recognized as a contributor to the evolution of genes with new functions. Multiple copies of genes can result from tandem duplication, from transposition to new chromosomes, or from whole-genome duplication (polyploidy). The most common fate is that one member of the pair is deleted to return the gene to the singleton state. Other paths involve the reduced expression of both copies (hypofunctionalization) that are held in duplicate to maintain sufficient quantity of function. The two copies can split functions (subfunctionalization) or can diverge to generate a new function (neofunctionalization). Retention of duplicates resulting from doubling of the whole genome occurs for genes involved with multicomponent interactions such as transcription factors and signal transduction components. In contrast, these classes of genes are underrepresented in small segmental duplications. This complementary pattern suggests that the balance of interactors affects the fate of the duplicate pair. We discuss the different mechanisms that maintain duplicated genes, which may change over time and intersect.

Chromosome-level genome assembly of Bactrocera dorsalis reveals its adaptation and invasion mechanisms.

Bactrocera dorsalis is an invasive polyphagous pest causing considerable ecological and economic damage worldwide. We report a high-quality chromosome-level genome assembly and combine various transcriptome data to explore the molecular mechanisms of its rapid adaptation to new environments. The expansions of the DDE transposase superfamily and key gene families related to environmental adaptation and enrichment of the expanded and unique gene families in metabolism and defence response pathways explain its environmental adaptability. The relatively high but not significantly different expression of heat-shock proteins, regardless of the environmental conditions, suggests an intrinsic mechanism underlying its adaptation to high temperatures. The mitogen-activated protein kinase pathway plays a key role in adaptation to new environments. The prevalence of duplicated genes in its genome explains the diversity in the B. dorsalis complex. These findings provide insights into the genetic basis of the invasiveness and diversity of B. dorsalis, explaining its rapid adaptation and expansion.

Retention of duplicated genes in evolution.

Gene duplication is a prevalent phenomenon across the tree of life. The processes that lead to the retention of duplicated genes are not well understood. Functional genomics approaches in model organisms, such as yeast, provide useful tools to test the mechanisms underlying retention with functional redundancy and divergence of duplicated genes, including fates associated with neofunctionalization, subfunctionalization, back-up compensation, and dosage amplification. Duplicated genes may also be retained as a consequence of structural and functional entanglement. Advances in human gene editing have enabled the interrogation of duplicated genes in the human genome, providing new tools to evaluate the relative contributions of each of these factors to duplicate gene retention and the evolution of genome structure.

Genome-wide identification and characterization of 14-3-3 genes in fishes.

The 14-3-3 family genes are highly conserved regulatory factors in eukaryotes with involvement in multiple important cellular processes. However, detailed investigations of this family in fishes are very limited. Here, a comparative genomic and transcriptomic survey were performed to investigate the 14-3-3 family in fishes. We confirmed that the numbers of 14-3-3 genes ranged from 5 to 7 in non-teleost fishes, as well as additional 14-3-3 genes (9 to 11) in teleost fishes. In addition, some special teleost fishes possess 17 to 25 14-3-3s, which undergone the fourth whole-genome duplication (WGD). We also found that six pairs of fish 14-3-3 genes were clustered with mammalian ε, γ, ς, η, τand ß isotypes, respectively, while σ was absent with a potential specificity within mammals, on the basis of their phylogenetic and synteny analyses. According to our results, we inferred that the diversity of 14-3-3 genes in fishes seems to be generated from a combination of WGD and gene loss. Comparative transcriptomic analysis revealed that there are differences in tissue distribution, and we speculated that 14-3-3 genes may contribute to terrestrial adaptations in mudskippers. In addition, protein sequence alignments of 14-3-3s supported their differential roles in fishes. In summary, our present comparative genomic and transcriptomic survey will benefit for further functional investigations of these fish genes.

Divergent DNA methylation contributes to duplicated gene evolution and chilling response in tea plants.

The tea plant (Camellia sinensis) is a thermophilic cash crop and contains a highly duplicated and repeat-rich genome. It is still unclear how DNA methylation regulates the evolution of duplicated genes and chilling stress in tea plants. We therefore generated a single-base-resolution DNA methylation map of tea plants under chilling stress. We found that, compared with other plants, the tea plant genome is highly methylated in all three sequence contexts, including CG, CHG and CHH (where H = A, T, or C), which is further proven to be correlated with its repeat content and genome size. We show that DNA methylation in the gene body negatively regulates the gene expression of tea plants, whereas non-CG methylation in the flanking region enables a positive regulation of gene expression. We demonstrate that transposable element-mediated methylation dynamics significantly drives the expression divergence of duplicated genes in tea plants. The DNA methylation and expression divergence of duplicated genes in the tea plant increases with evolutionary age and selective pressure. Moreover, we detect thousands of differentially methylated genes, some of which are functionally associated with chilling stress. We also experimentally reveal that DNA methyltransferase genes of tea plants are significantly downregulated, whereas demethylase genes are upregulated at the initial stage of chilling stress, which is in line with the significant loss of DNA methylation of three well-known cold-responsive genes at their promoter and gene body regions. Overall, our findings underscore the importance of DNA methylation regulation and offer new insights into duplicated gene evolution and chilling tolerance in tea plants.

Copy number variations and young duplicate genes have high methylation levels in sticklebacks.

Gene duplication is an important driver of genomic diversity that can promote adaptive evolution. However, like most mutations, a newly duplicated gene is often deleterious and removed from the genome by drift or natural selection. The early molecular changes that occur soon after duplication therefore may influence the long-term survival of gene duplicates, but relatively little empirical data exist on the events near the onset of duplication before mutations have time to accumulate. In this study, we contrast gene expression and DNA methylation levels of duplicate genes in the threespine stickleback, Gasterosteus aculeatus, including recently emerged duplications that segregate as copy number variations (CNVs). We find that younger duplicate genes have higher levels of promoter methylation than older genes, and that gene CNVs have higher promoter methylation than non-CNVs. These results suggest preferential duplication of highly methylated genes or rapid methylation changes soon after duplication. We also find a negative association between methylation and expression, providing a putative role for methylation in suppressing transcription that compensates for increases in gene copy numbers and promoting paralog retention. We propose that methylation contributes to the longevity of young duplicate genes, extending the window of opportunity for functional divergence via mutation.

Resistance to pirimiphos-methyl in West African Anopheles is spreading via duplication and introgression of the Ace1 locus.

Vector population control using insecticides is a key element of current strategies to prevent malaria transmission in Africa. The introduction of effective insecticides, such as the organophosphate pirimiphos-methyl, is essential to overcome the recurrent emergence of resistance driven by the highly diverse Anopheles genomes. Here, we use a population genomic approach to investigate the basis of pirimiphos-methyl resistance in the major malaria vectors Anopheles gambiae and A. coluzzii. A combination of copy number variation and a single non-synonymous substitution in the acetylcholinesterase gene, Ace1, provides the key resistance diagnostic in an A. coluzzii population from Côte d'Ivoire that we used for sequence-based association mapping, with replication in other West African populations. The Ace1 substitution and duplications occur on a unique resistance haplotype that evolved in A. gambiae and introgressed into A. coluzzii, and is now common in West Africa primarily due to selection imposed by other organophosphate or carbamate insecticides. Our findings highlight the predictive value of this complex resistance haplotype for phenotypic resistance and clarify its evolutionary history, providing tools to for molecular surveillance of the current and future effectiveness of pirimiphos-methyl based interventions.

A comprehensive analysis of chimpanzee (Pan Troglodytes)-specific AluYb8 element.

BACKGROUND: Alu elements are most abundant retrotransposons with > 1.2 million copies in the primate genome. AluYb8 subfamily was diverged from AluY lineage, and has accumulated eight diagnostic mutations and 7-bp duplication during primate evolution. A total of 1851 AluYb copies are present in the human genome, and most of them are human-specific. On the other hand, only a few AluYb8 copies were identified in the chimpanzee genome by previous studies on AluYb8. The significantly different number of species-specific AluYb8 elements between human and chimpanzee might result from the incompletion of chimpanzee reference genome sequences at the time of the previous study. OBJECTIVE: AluYb8 elements could generate genomic structural variations in the chimpanzee genome. This study aimed to identify and characterize chimpanzee-specific AluYb elements using the most updated chimpanzee reference genome sequences (Jan. 2018, panTro6). METHODS: To identify chimpanzee-specific AluYb8, we carried out genomic comparison with non-chimpanzee primate genome using the UCSC table browser. In addition, chimpanzee-specific AluYb8 candidates were manually inspected and experimentally verified using PCR and Sanger sequencing. RESULTS: Among a total of 231 chimpanzee-specific AluYb8 candidates, 11 of the candidates are chimpanzee-specific AluYb8, and 29 elements are shared between the chimpanzee and non-chimpanzee primate genomes. Through the sequence analysis of AluYb8 and other Alu subfamilies, we were able to observe various diagnostic mutations and variable length duplications in 7-bp duplication region of AluYb8 element. In addition, we further validated two of the chimpanzee-specific AluYb8 elements (CS8 and CS20) that were not previously discovered by display PCR and Sanger sequencing. Interestingly, we identified a AluYb8 insertion-mediated deletion (CS8 locus) in the chimpanzee genome. CONCLUSION: Our study found that AluYb8 elements are much more abundant in the human genome than chimpanzee genome, and that it could be due to the absence of hyperactive "master" AluYb8 elements in the chimpanzee genome.

The Evolution of Duplicated Genes of the Cpi-17/Phi-1 (ppp1r14) Family of Protein Phosphatase 1 Inhibitors in Teleosts.

The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.