Rapid Cis–Trans Coevolution Driven by a Novel Gene Retroposed from a Eukaryotic Conserved CCR4–NOT Component in Drosophila

Young, or newly evolved, genes arise ubiquitously across the tree of life, and they can rapidly acquire novel functions that influence a diverse array of biological processes. Previous work identified a young regulatory duplicate gene in Drosophila, Zeus that unexpectedly diverged rapidly from its parent, Caf40, an extremely conserved component in the CCR4–NOT machinery in post-transcriptional and post-translational regulation of eukaryotic cells, and took on roles in the male reproductive system. This neofunctionalization was accompanied by differential binding of the Zeus protein to loci throughout the Drosophila melanogaster genome. However, the way in which new DNA-binding proteins acquire and coevolve with their targets in the genome is not understood. Here, by comparing Zeus ChIP-Seq data from D. melanogaster and D. simulans to the ancestral Caf40 binding events from D. yakuba, a species that diverged before the duplication event, we found a dynamic pattern in which Zeus binding rapidly coevolved with a previously unknown DNA motif, which we term Caf40 and Zeus-Associated Motif (CAZAM), under the influence of positive selection. Interestingly, while both copies of Zeus acquired targets at male-biased and testis-specific genes, D. melanogaster and D. simulans proteins have specialized binding on different chromosomes, a pattern echoed in the evolution of the associated motif. Using CRISPR-Cas9-mediated gene knockout of Zeus and RNA-Seq, we found that Zeus regulated the expression of 661 differentially expressed genes (DEGs). Our results suggest that the evolution of young regulatory genes can be coupled to substantial rewiring of the transcriptional networks into which they integrate, even over short evolutionary timescales. Our results thus uncover dynamic genome-wide evolutionary processes associated with new genes.

Illegitimate Recombination between Duplicated Genes Generated from Recursive Polyploidizations Accelerated the Divergence of the Genus Arachis

The peanut (Arachis hypogaea L.) is the leading oil and food crop among the legume family. Extensive duplicate gene pairs generated from recursive polyploidizations with high sequence similarity could result from gene conversion, caused by illegitimate DNA recombination. Here, through synteny-based comparisons of two diploid and three tetraploid peanut genomes, we identified the duplicated genes generated from legume common tetraploidy (LCT) and peanut recent allo-tetraploidy (PRT) within genomes. In each peanut genome (or subgenomes), we inferred that 6.8–13.1% of LCT-related and 11.3–16.5% of PRT-related duplicates were affected by gene conversion, in which the LCT-related duplicates were the most affected by partial gene conversion, whereas the PRT-related duplicates were the most affected by whole gene conversion. Notably, we observed the conversion between duplicates as the long-lasting contribution of polyploidizations accelerated the divergence of different Arachis genomes. Moreover, we found that the converted duplicates are unevenly distributed across the chromosomes and are more often near the ends of the chromosomes in each genome. We also confirmed that well-preserved homoeologous chromosome regions may facilitate duplicates’ conversion. In addition, we found that these biological functions contain a higher number of preferentially converted genes, such as catalytic activity-related genes. We identified specific domains that are involved in converted genes, implying that conversions are associated with important traits of peanut growth and development.

Functional Characterization of Aluminum (Al)-Responsive Membrane-Bound NAC Transcription Factors in Soybean Roots

The membrane-bound NAC transcription (NTL) factors have been demonstrated to participate in the regulation of plant development and the responses to multiple environmental stresses. This study is aimed to functionally characterize soybean NTL transcription factors in response to Al-toxicity, which is largely uncharacterized. The qRT-PCR assays in the present study found that thirteen out of fifteen GmNTL genes in the soybean genome were up-regulated by Al toxicity. However, among the Al-up-regulated GmNTLs selected from six duplicate gene pairs, only overexpressing GmNTL1, GmNTL4, and GmNTL10 could confer Arabidopsis Al resistance. Further comprehensive functional characterization of GmNTL4 showed that the expression of this gene in response to Al stress depended on root tissues, as well as the Al concentration and period of Al treatment. Overexpression of GmNTL4 conferred Al tolerance of transgenic Arabidopsis in long-term (48 and 72 h) Al treatments. Moreover, RNA-seq assay identified 517 DEGs regulated by GmNTL4 in Arabidopsis responsive to Al stress, which included MATEs, ALMTs, PMEs, and XTHs. These results suggest that the function of GmNTLs in Al responses is divergent, and GmNTL4 might confer Al resistance partially by regulating the expression of genes involved in organic acid efflux and cell wall modification.

Convergent evolution of polyploid genomes from across the eukaryotic tree of life

By modeling the homoeologous gene losses that occurred in fifty genomes deriving from ten distinct polyploidy events, we show that the evolutionary forces acting on polyploids are remarkably similar, regardless of whether they occur in flowering plants, ciliates, fishes or yeasts. The models suggest these events were nearly all allopolyploidies, with two distinct progenitors contributing to the modern species. We show that many of the events show a relative rate of duplicate gene loss prior to the first post-polyploidy speciation that is significantly higher than in later phases of their evolution. The relatively low selective constraint seen for the single-copy genes these losses produced lead us to suggest that most of the purely selectively neutral duplicate gene losses occur in the immediate post-polyploid period. We also find ongoing and extensive reciprocal gene losses (RGL; alternative losses of duplicated ancestral genes) between these genomes. With the exception of a handful of closely related taxa, all of these polyploid organisms are separated from each other by tens to thousands of reciprocal gene losses. As a result, it is very unlikely that viable diploid hybrid species could form between these taxa, since matings between such hybrids would tend to produce offspring lacking essential genes. It is therefore possible that the relatively high frequency of recurrent polyploidies in some lineages may be due to the ability of new polyploidies to bypass RGL barriers.

Regulation of Survival Motor Neuron Gene Expression by Calcium Signaling

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletion, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, a defect in SMN2 splicing, involving exon 7 skipping, results in a low level of functional SMN protein. Therefore, the upregulation of SMN protein expression from the SMN2 gene is generally considered to be one of the best therapeutic strategies to treat SMA. Most of the SMA drug discovery is based on synthetic compounds, and very few natural compounds have been explored thus far. Here, we performed an unbiased mechanism-independent and image-based screen of a library of microbial metabolites in SMA fibroblasts using an SMN-specific immunoassay. In doing so, we identified brefeldin A (BFA), a well-known inhibitor of ER-Golgi protein trafficking, as a strong inducer of SMN protein. The profound increase in SMN protein was attributed to, in part, the rescue of the SMN2 pre-mRNA splicing defect. Intriguingly, BFA increased the intracellular calcium concentration, and the BFA-induced exon 7 inclusion of SMN2 splicing, was abrogated by the depletion of intracellular calcium and by the pharmacological inhibition of calcium/calmodulin-dependent kinases (CaMKs). Moreover, BFA considerably reduced the expression of Tra2-β and SRSF9 proteins in SMA fibroblasts and enhanced the binding of PSF and hnRNP M to an exonic splicing enhancer (ESE) of exon 7. Together, our results demonstrate a significant role for calcium and its signaling on the regulation of SMN splicing, probably through modulating the expression/activity of splicing factors.

Genome-Wide Characterization and Expression Analysis of CAMTA Gene Family Under Salt Stress in Cucurbita moschata and Cucurbita maxima

Cucurbita Linn. vegetables have a long history of cultivation and have been cultivated all over the world. With the increasing area of saline–alkali soil, Cucurbita Linn. is affected by salt stress, and calmodulin-binding transcription activator (CAMTA) is known for its important biological functions. Although the CAMTA gene family has been identified in several species, there is no comprehensive analysis on Cucurbita species. In this study, we analyzed the genome of Cucurbita maxima and Cucurbita moschata. Five C. moschata calmodulin-binding transcription activators (CmoCAMTAs) and six C. maxima calmodulin-binding transcription activators (CmaCAMTAs) were identified, and they were divided into three subfamilies (Subfamilies I, II, and III) based on the sequence identity of amino acids. CAMTAs from the same subfamily usually have similar exon–intron distribution and conserved domains (CG-1, TIG, IQ, and Ank_2). Chromosome localization analysis showed that CmoCAMTAs and CmaCAMTAs were unevenly distributed across four and five out of 21 chromosomes, respectively. There were a total of three duplicate gene pairs, and all of which had experienced segmental duplication events. The transcriptional profiles of CmoCAMTAs and CmaCAMTAs in roots, stems, leaves, and fruits showed that these CAMTAs have tissue specificity. Cis-acting elements analysis showed that most of CmoCAMTAs and CmaCAMTAs responded to salt stress. By analyzing the transcriptional profiles of CmoCAMTAs and CmaCAMTAs under salt stress, it was shown that both C. moschata and C. maxima shared similarities against salt tolerance and that it is likely to contribute to the development of these species. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) further demonstrated the key role of CmoCAMTAs and CmaCAMTAs under salt stress. This study provided a theoretical basis for studying the function and mechanism of CAMTAs in Cucurbita Linn.

Integrated synteny- and similarity-based inference on the polyploidization–fractionation cycle

Whole-genome doubling, tripling or replicating to a greater degree, due to fixation of polyploidization events, is attested in almost all lineages of the flowering plants, recurring in the ancestry of some plants two, three or more times in retracing their history to the earliest angiosperm. This major mechanism in plant genome evolution, which generally appears as instantaneous on the evolutionary time scale, sets in operation a compensatory process called fractionation, the loss of duplicate genes, initially rapid, but continuing at a diminishing rate over millions and tens of millions of years. We study this process by statistically comparing the distribution of duplicate gene pairs as a function of their time of creation through polyploidization, as measured by sequence similarity. The stochastic model that accounts for this distribution, though exceedingly simple, still has too many parameters to be estimated based only on the similarity distribution, while the computational procedures for compiling the distribution from annotated genomic data is heavily biased against earlier polyploidization events—syntenic ‘crumble’. Other parameters, such as the size of the initial gene complement and the ploidy of the various events giving rise to duplicate gene pairs, are even more inaccessible to estimation. Here, we show how the frequency of unpaired genes, identified via their embedding in stretches of duplicate pairs, together with previously established constraints among some parameters, adds enormously to the range of successive polyploidization events that can be analysed. This also allows us to estimate the initial gene complement and to correct for the bias due to crumble. We explore the applicability of our methodology to four flowering plant genomes covering a range of different polyploidization histories.

Segmental duplications and their variation in a complete human genome

Despite their importance in disease and evolution, highly identical segmental duplications (SDs) have been among the last regions of the human reference genome (GRCh38) to be finished. Based on a complete telomere-to-telomere human genome (T2T CHM13), we present the first comprehensive view of human SD organization. SDs account for nearly one-third of the additional sequence increasing the genome-wide estimate from 5.4% to 7.0% (218 Mbp). An analysis of 266 human genomes shows that 91% of the new T2T CHM13 SD sequence (68.3 Mbp) better represents human copy number. We find that SDs show increased single-nucleotide variation diversity when compared to unique regions; we characterize methylation signatures that correlate with duplicate gene transcription and predict 182 novel protein-coding gene candidates. We find that 63% (35.11/55.7 Mbp) of acrocentric chromosomes consist of SDs distinct from rDNA and satellite sequences. Acrocentric SDs are 1.75-fold longer (p=0.00034) than other SDs, are frequently shared with autosomal pericentromeric regions, and are heteromorphic among human chromosomes. Comparing long-read assemblies from other human (n=12) and nonhuman primate (n=5) genomes, we use the T2T CHM13 genome to systematically reconstruct the evolution and structural haplotype diversity of biomedically relevant (LPA, SMN) and duplicated genes (TBC1D3, SRGAP2C, ARHGAP11B) important in the expansion of the human frontal cortex. The analysis reveals unprecedented patterns of structural heterozygosity and massive evolutionary differences in SD organization between humans and their closest living relatives.

Assessment of genetics for turcicum leaf blight resistance in maize (Zea mays L.)

Genetics of Turcicum leaf blight (TLB) was to explore the mode of inheritance in maize caused by Exserohilum turcicum is a serious foliar disease. The genetics was estimated with the help of two crosses (CM 212 × V 336 and CM 212 × CM 145) from data of six generations (P1, P2, F1, F2, B1 and B2). The scaling tests as well as joint scaling tests revealed that the inadequacy of simple additive-dominance model and justifying the use of six parameter model for the detection of gene interactions in both the crosses for resistance to Turcicum leaf blight. All the scaling tests (A, B, C and D) were significant for the cross CM 212 × CM 145 and for the cross CM 212 × V 336 only A and C tests were significant. Based on the signs of [h] and [l] gene effects, complementary gene interaction for cross CM 212 × V 336 and duplicate gene interaction for cross CM 212 × CM 145 were evident in the inheritance of Turcicum leaf blight.

Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish

Adult zebrafish are widely used to interrogate mechanisms of disease development and tissue regeneration. Yet, the prospect of large-scale genetics in adult zebrafish has traditionally faced a host of biological and technical challenges, including inaccessibility of adult tissues to high-throughput phenotyping and the spatial and technical demands of adult husbandry. Here, we describe an experimental pipeline that combines high-efficiency CRISPR/Cas9 mutagenesis with functional phenotypic screening to identify genes required for spinal cord repair in adult zebrafish. Using CRISPR/Cas9 dual-guide ribonucleic proteins, we show selective and combinatorial mutagenesis of 17 genes at 28 target sites with efficiencies exceeding 85% in adult F0 “crispants”. We find that capillary electrophoresis is a reliable method to measure indel frequencies. Using a quantifiable behavioral assay, we identify seven single- or duplicate-gene crispants with reduced functional recovery after spinal cord injury. To rule out off-target effects, we generate germline mutations that recapitulate the crispant regeneration phenotypes. This study provides a platform that combines high-efficiency somatic mutagenesis with a functional phenotypic readout to perform medium- to large-scale genetic studies in adult zebrafish.