Genome-Wide Characterization and Abiotic Stresses Expression Analysis of Annexin Family Genes in Poplar

Poplar is an illustrious industrial woody plant with rapid growth, providing a range of materials, and having simple post-treatment. Various kinds of environmental stresses limit its output. Plant annexin (ANN) is a calcium-dependent phospholipid-binding protein involved in plant metabolism, growth and development, and cooperatively regulating drought resistance, salt tolerance, and various stress responses. However, the features of the PtANN gene family and different stress responses remain unknown in poplar. This study identified 12 PtANN genes in the P. trichocarpa whole-genome and PtANNs divided into three subfamilies based on the phylogenetic tree. The PtANNs clustered into the same clade shared similar gene structures and conserved motifs. The 12 PtANN genes were located in ten chromosomes, and segmental duplication events were illustrated as the main duplication method. Additionally, the PtANN4 homogenous with AtANN1 was detected localized in the cytoplasm and plasma membrane. In addition, expression levels of PtANNs were induced by multiple abiotic stresses, which indicated that PtANNs could widely participate in response to abiotic stress. These results revealed the molecular evolution of PtANNs and their profiles in response to abiotic stress.

RNA-guided AsCas12a- and SpCas9-catalyzed knockout and homology directed repair of the omega-1 locus of the human blood fluke, Schistosoma mansoni

We compared the efficiency and precision of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. with SpCas9 of Streptococcus pyogenes aiming to advance functional genomics tools for Schistosoma mansoni. Programmed double stranded cleavage catalyzed by AsCas12a results in a staggered strand break whereas SpCas9 produces a blunt ended chromosomal break. The TTTV, the optimal protospacer adjacent motif for AsCas12a is expected frequently within the AT-rich genome of this platyhelminth. We deployed optimized conditions (gRNA:SpCas9:DNA donor ratio and electroporation condition) to edit the ω1 gene. SpCas9 delivered higher efficiency to mutate ω1 target compared to AsCas12a for non-homology end joining (NHEJ)-catalyzed repair (14.04% vs. 10.88%, n = 7 replicates). Most mutations were deletions; SpCas9 induced -3 nt size deletions whereas AsCas12a induced deletions ranging in size from -25 to -2 nt. Although these were less absolute percentage AsCas12a than SpCas9 programmed mutations, the phenotypic outcomes on levels of ω1 transcripts and expressed omega-1 protein were similar. Gene editing efficiency of SpCas9 and AsCas12a markedly increased in the presence of short single stranded donor templates bearing symmetrical homolog arms of 50 nt in length. With AsCas12a, both non-CRISPR target (NT) and target (T) strands of the ω1 gene were tested as homology direct repair (HDR) donor templates. There were 15.67%, 28.71% and 21.43% of NHEJ from 7 pooled genomic DNA from KI_SpCas9, KI_AsCas12a-NT-ssODN and KI_AsCas12a-T-ssODN experiments, respectively. Programmed SpCas9 cleavage led to higher levels than AsCas12a of precise HDR mediated; 17.07%, KI_SpCas9 vs. 14.58%, KI_AsCas12a-NT-ssODN and 12.35%, KI_AsCas12a-T-ssODN (P < 0.0.5), although no significant differences in reduction in ω1 transcripts or of protein levels were apparent. These findings revealed that both AsCas12a and SpCas9 can provide programmed knockout and transgene insertion into genes expressed in the schistosome egg.

Comparative plastomics of Amaryllidaceae: inverted repeat expansion and the degradation of the ndh genes in Strumaria truncata Jacq.

Amaryllidaceae is a widespread and distinctive plant family contributing both food and ornamental plants. Here we present an initial survey of plastomes across the family and report on both structural rearrangements and gene losses. Most plastomes in the family are of similar gene arrangement and content however some taxa have shown gains in plastome length while in several taxa there is evidence of gene loss. Strumaria truncata shows a substantial loss of ndh family genes while three other taxa show loss of cemA, which has been reported only rarely. Our sparse sampling of the family has detected sufficient variation to suggest further sampling across the family could be a rich source of new information on plastome variation and evolution.

Mitochondrial Phylogenomics of Fagales Provides Insights Into Plant Mitogenome Mosaic Evolution

Fagales are an order of woody plants and comprise more than 1,100 species, most of which produce economically important timbers, nuts, and fruits. Their nuclear and plastid genomes are well-sequenced and provided valuable resources to study their phylogeny, breeding, resistance, etc. However, little is known about the mitochondrial genomes (mitogenomes), which hinder a full understanding of their genome evolution. In this study, we assembled complete mitogenomes of 23 species, covering five of the seven families of Fagales. These mitogenomes had similar gene sets but varied 2.4 times in size. The mitochondrial genes were highly conserved, and their capacity in phylogeny was challenging. The mitogenomic structure was extremely dynamic, and synteny among species was poor. Further analyses of the Fagales mitogenomes revealed extremely mosaic characteristics, with horizontal transfer (HGT)-like sequences from almost all seed plant taxa and even mitoviruses. The largest mitogenome, Carpinus cordata, did not have large amounts of specific sequences but instead contained a high proportion of sequences homologous to other Fagales. Independent and unequal transfers of third-party DNA, including nuclear genome and other resources, may partially account for the HGT-like fragments and unbalanced size expansions observed in Fagales mitogenomes. Supporting this, a mitochondrial plasmid-like of nuclear origin was found in Carpinus. Overall, we deciphered the last genetic materials of Fagales, and our large-scale analyses provide new insights into plant mitogenome evolution and size variation.

Direct Reprogramming of Non-limb Fibroblasts to Cells with Properties of Limb Progenitors

SUMMARYThe early limb bud consists of mesenchymal progenitors (limb progenitors) derived from the lateral plate mesoderm (LPM) that produce most of the tissues of the mature limb bud. The LPM also gives rise to the mesodermal components of the trunk, flank and neck. However, the mesenchymal cells generated at these other axial levels cannot produce the variety of cell types found in the limb bud, nor can they be directed to form a patterned appendage-like structure, even when placed in the context of the signals responsible for organizing the limb bud. Here, by taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28) normally expressed in the early limb bud, that are capable of imparting limb progenitor-like properties to non-limb fibroblasts. Cells reprogrammed by these factors show similar gene expression profiles, and can differentiate into similar cell types, as endogenous limb progenitors. The further addition of Lin41 potentiates proliferation of the reprogrammed cells while suppressing differentiation. These results suggest that these same four key factors may play pivotal roles in the specification of endogenous limb progenitors.

Vero Cells Gain Renal Tubule Markers in Low-calcium and Magnesium Chemically Defined Media

In this study, a chemically defined, animal component-free media was developed to promote Vero growth in suspension. Key media compounds were screened using Plackett-Burman styled experiments to create a media formulation to support suspension growth. Vero cells remained viable in suspension, but their growth rate was extremely low, conversely, other cell types such as CHO-K1, MDCK and HEK293T were able to grow in single cell suspension in the same media. To investigate the slow growth of Vero cells, RNA-seq analysis was conducted. Vero cells were cultured in three different conditions: adherently in serum-containing medium, adherently in in-house medium, and in suspension in low calcium and magnesium in-house medium. This study illustrates that adherent cells maintain similar gene expression, while the suspension phenotype tends to overexpress genes related to renal tubules.

Identification of Putative Biosynthetic Gene Clusters for Tolyporphins in Multiple Filamentous Cyanobacteria

Tolyporphins A–R are unusual tetrapyrrole macrocycles produced by the non-axenic filamentous cyanobacterium HT-58-2. A putative biosynthetic gene cluster for biosynthesis of tolyporphins (here termed BGC-1) was previously identified in the genome of HT-58-2. Here, homology searching of BGC-1 in HT-58-2 led to identification of similar BGCs in seven other filamentous cyanobacteria, including strains Nostoc sp. 106C, Nostoc sp. RF31YmG, Nostoc sp. FACHB-892, Brasilonema octagenarum UFV-OR1, Brasilonema octagenarum UFV-E1, Brasilonema sennae CENA114 and Oculatella sp. LEGE 06141, suggesting their potential for tolyporphins production. A similar gene cluster (BGC-2) also was identified unexpectedly in HT-58-2. Tolyporphins BGCs were not identified in unicellular cyanobacteria. Phylogenetic analysis based on 16S rRNA and a common component of the BGCs, TolD, points to a close evolutionary history between each strain and their respective tolyporphins BGC. Though identified with putative tolyporphins BGCs, examination of pigments extracted from three cyanobacteria has not revealed the presence of tolyporphins. Overall, the identification of BGCs and potential producers of tolyporphins presents a collection of candidate cyanobacteria for genetic and biochemical analysis pertaining to these unusual tetrapyrrole macrocycles.

Comparative plastomics of Amaryllidaceae: Inverted repeat expansion and the degradation of the ndh genes in Strumaria truncata Jacq.

Amaryllidaceae is a widespread and distinctive plant family contributing both food and ornamental plants. Here we present an initial survey of plastomes across the family and report on both structural rearrangements and gene losses. Most plastomes in the family are of similar gene arrangement and content however some taxa have shown gains in plastome length while in several taxa there is evidence of gene loss. Strumaria truncata shows a substantial loss of ndh family genes while three other taxa show loss of cemA, which has been reported only rarely. Our sparse sampling of the family has detected sufficient variation to suggest further sampling across the family could be a rich source of new information on plastome variation and evolution.

Vero cells gain renal tubule markers in low-calcium and magnesium chemically defined media

In this study, a chemically defined, animal component-free media was developed to promote Vero growth in suspension. Key media compounds were screened using Plackett-Burman styled experiments to create a media formulation to support suspension growth. Vero cells remained viable in suspension, but their growth rate was extremely low, conversely, other cell types such as CHO-K1, MDCK and HEK293T were able to grow in single cell suspension in the same media. To investigate the slow growth of Vero cells, RNA-Seq analysis was conducted. Vero cells were cultured in three different conditions: adherently in serum-containing medium, adherently in in-house medium, and in suspension in low calcium and magnesium in-house medium. This study illustrates that adherent cells maintain similar gene expression, while the suspension phenotype tends to overexpress genes related to renal tubules.

Density-based binning of gene clusters to infer function or evolutionary history using GeneGrouper

Motivation: Identifying gene clusters of interest in phylogenetically proximate and distant taxa can help to infer phenotypes of interest. Conserved gene clusters may differ by only a few genes, which can be biologically meaningful, such as the formation of pseudogenes or insertions interrupting regulation. These qualities may allow for unsupervised clustering of similar gene clusters into bins that provide a population-level understanding of the genetic variation in similar gene clusters. Results: We developed GeneGrouper, a command-line tool that uses a density-based clustering method to group gene clusters into bins. GeneGrouper demonstrated high recall and precision in benchmarks for the detection of the 23-gene Salmonella enterica LT2 Pdu gene cluster and four-gene Pseudomonas aeruginosa PAO1 Mex gene cluster in 435 genomes containing mixed taxa. In a subsequent application investigating the diversity and impact of gene complete and incomplete LT2 Pdu gene clusters in 1130 S. enterica genomes, GeneGrouper identified a novel, frequently occurring pduN pseudogene. When replicated in vivo, disruption of pduN with a frameshift mutation negatively impacted microcompartment formation. We next demonstrated the versatility of GeneGrouper by clustering both distant homologous gene clusters and variable gene clusters found in integrative and conjugative elements.