Intact rDNA arrays of Potentilla-origin detected in Erythronium nucleus suggest recent eudicot-to-monocot horizontal transfer

The occurrence of horizontal gene transfer (HGT) in Eukarya is increasingly gaining recognition. Nuclear-to-nuclear jump of DNA between plant species at high phylogenetic distance and devoid of intimate association (e.g., parasitism) is still scarcely reported. Within eukaryotes, components of ribosomal DNA (rDNA) multigene family have been found to be horizontally transferred in protists, fungi and grasses. However, in neither case HGT occurred between phylogenetic families, nor the transferred rDNA remained tandemly arrayed and transcriptionally active in the recipient organism. This study aimed to characterize an alien eudicot-type of 45S nuclear rDNA, assumingly transferred horizontally to the genome of monocot European Erythronium (Liliaceae). Genome skimming coupled by PacBio HiFi sequencing of a BAC clone were applied to determine DNA sequence of the alien rDNA. A clear phylogenetic signal traced the origin of the alien rDNA of Erythronium back to the Argentea clade of Potentilla (Rosaceae) and deemed the transfer to have occurred in the common ancestor of E. dens-canis and E. caucasicum. Though being discontinuous, transferred rDNA preserved its general tandemly arrayed feature in the host organism. Southern blotting, molecular cytogenetics, and sequencing of a BAC clone derived from flow-sorted nuclei indicated integration of the alien rDNA into the recipient's nuclear genome. Unprecedently, dicot-type alien rDNA was found to be transcribed in the monocot Erythronium albeit much less efficiently than the native counterpart. This study adds a new example to the growing list of naturally transgenic plants while holding the scientific community continually in suspense about the mode of DNA transfer.

The Melon Zym Locus Conferring Resistance to ZYMV: High Resolution Mapping and Candidate Gene Identification

Zucchini yellow mosaic virus (ZYMV; potyviridae) represents a major pathogen of Cucurbitaceae crops. ZYMV resistance in melon PI 414723 is conditioned by a dominant allele at the Zym locus. This resistant accession restricts viral spread and does not develop mosaic symptoms, but necrosis sometimes develops in response to inoculation. In previous studies, Zym has been mapped to linkage group II of the melon genetic map. In the present study, positional cloning of the locus was undertaken, starting from the CM-AG36 SSR marker at approximately 2 cm distance. We utilized five mapping populations that share the same resistant parent, PI 414723, and analyzed a total of 1630 offspring, to construct a high-resolution genetic map of the Zym locus. Two melon BAC libraries were used for chromosome walking and for developing new markers closer to the resistance gene by BAC-end sequencing. A BAC contig was constructed, and we identified a single BAC clone, from the ZYMV susceptible genotype MR-1, that physically encompasses the resistance gene. A second clone was isolated from another susceptible genotype, WMR 29, and the two clones were fully sequenced and annotated. Additional markers derived from the sequenced region delimited the region to 17.6 kb of a sequence that harbors a NAC-like transcription factor and, depending on the genotype, either two or three R-gene homologs with a CC-NBS-LRR structure. Mapping was confirmed by saturating the map with SNP markers using a single mapping population. The same region was amplified and sequenced also in the ZYMV resistant genotype PI 414723. Because numerous polymorphic sites were noted between genotypes, we could not associate resistance with a specific DNA polymorphism; however, this study enables molecular identification of Zym and paves the way to functional studies of this important locus.

Unraveling the Genetic Basis of Fertility Restoration for Cytoplasmic Male Sterile Line WNJ01A Originated From Brassica juncea in Brassica napus

Crosses that lead to heterosis have been widely used in the rapeseed (Brassica napus L.) industry. Cytoplasmic male sterility (CMS)/restorer-of-fertility (Rf) systems represent one of the most useful tools for rapeseed production. Several CMS types and their restorer lines have been identified in rapeseed, but there are few studies on the mechanisms underlying fertility restoration. Here, we performed morphological observation, map-based cloning, and transcriptomic analysis of the F2 population developed by crossing the CMS line WNJ01A with its restorer line Hui01. Paraffin-embedded sections showed that the sporogenous cell stage was the critical pollen degeneration period, with major sporogenous cells displaying loose and irregular arrangement in sterile anthers. Most mitochondrial electron transport chain (mtETC) complex genes were upregulated in fertile compared to sterile buds. Using bulked segregant analysis (BSA)-seq to analyze mixed DNA pools from sterile and fertile F2 buds, respectively, we identified a 6.25 Mb candidate interval where Rfw is located. Using map-based cloning experiments combined with bacterial artificial chromosome (BAC) clone sequencing, the candidate interval was reduced to 99.75 kb and two pentatricopeptide repeat (PPR) genes were found among 28 predicted genes in this interval. Transcriptome sequencing showed that there were 1679 DEGs (1023 upregulated and 656 downregulated) in fertile compared to sterile F2 buds. The upregulated differentially expressed genes (DEGs) were enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) lysine degradation pathway and phenylalanine metabolism, and the downregulated DEGs were enriched in cutin, suberine, and wax biosynthesis. Furthermore, 44 DEGs were involved in pollen and anther development, such as tapetum, microspores, and pollen wall development. All of them were upregulated except a few such as POE1 genes (which encode Pollen Ole e I allergen and extensin family proteins). There were 261 specifically expressed DEGs (9 and 252 in sterile and fertile buds, respectively). Regarding the fertile bud-specific upregulated DEGs, the ubiquitin–proteasome pathway was enriched. The top four hub genes in the protein–protein interaction network (BnaA09g56400D, BnaA10g18210D, BnaA10g18220D, and BnaC09g41740D) encode RAD23d proteins, which deliver ubiquitinated substrates to the 26S proteasome. These findings provide evidence on the pathways regulated by Rfw and improve our understanding of fertility restoration.

Molecular and FISH analysis of 45S rDNA on BAC molecule of Saccharina Japonica

IGS is abundant in polymorphism, which is widely used in the analysis of intraspecific genetic diversity and phylogenetic relationships among geographical populations. In this study, the 45S rDNA repeat unit of Saccharina japonica was obtained for the first time by BAC clone sequencing. The total length of 45S rDNA repeat unit of S. japonica was 8995 bp, including 5420 bp of 18s-5.8s-25s rDNA and 3575 bp of IGS (Intergenic Spacer), with the GC content of 51.4%. IGS was composed of 465 bp 3’-outer transcribed spacer (ETS), 874 bp 5’-ETS, and 2236 bp non transcribed spacer (NTS), with the GC content of 50.1%.Fiber-FISH (fiber-fluorescence in situ hybridization, fiber-FISH) analysis of 45S rDNA on the BAC molecule of female gametophytes of S. japonica illustrated that each fiber had at least five continuous moniliform hybridization signal points, indicating the distribution of 45S rDNA repeat unit on the bacterial artificial chromosome. This study provided a new candidate molecular marker for detecting intraspecific polymorphisms of S. japonica, and the successful fiber-FISH analysis of 45S rDNA on BAC molecule would contribute to the construction of the physical map and Map-based cloning of this kelp.

A novel high-accuracy genome assembly method utilizing a high-throughput workflow

Across domains of biological research using genome sequence data, high-quality reference genome sequences are essential for characterizing genetic variation and understanding the genetic basis of phenotypes. However, the construction of genome assemblies for various species is often hampered by complexities of genome organization, especially repetitive and complex sequences, leading to mis-assembly and missing regions. Here, we describe a high-throughput gold standard genome assembly workflow using a large-scale bacterial artificial chromosome (BAC) library with a refined two-step pooling strategy and the Lamp assembler algorithm. This strategy minimizes the laborious processes of physical map construction and clone-by-clone sequencing, enabling inexpensive sequencing of several thousand BAC clones. By applying this strategy with a minimum tiling path BAC clone library for the short arm of chromosome 2D (2DS) of bread wheat, 98% of BAC sequences, covering 92.7% of the 2DS chromosome, were assembled correctly for this species with a highly complex and repetitive genome. We also identified 48 large mis-assemblies in the reference wheat genome assembly (IWGSC RefSeq v1.0) and corrected these large mis-assemblies in addition to filling 92.2% of the gaps in RefSeq v1.0. Our 2DS assembly represents a new benchmark for the assembly of complex genomes with both high accuracy and efficiency.

Specialization for Cell-Free or Cell-to-Cell Spread of BAC-Cloned Human Cytomegalovirus Strains Is Determined by Factors beyond the UL128-131 and RL13 Loci

ABSTRACT It is widely held that clinical isolates of human cytomegalovirus (HCMV) are highly cell associated, and mutations affecting the UL128-131 and RL13 loci that arise in culture lead to the appearance of a cell-free spread phenotype. The bacterial artificial chromosome (BAC) clone Merlin (ME) expresses abundant UL128-131, is RL13 impaired, and produces low infectivity virions in fibroblasts, whereas TB40/e (TB) and TR are low in UL128-131, are RL13 intact, and produce virions of much higher infectivity. Despite these differences, quantification of spread by flow cytometry revealed remarkably similar spread efficiencies in fibroblasts. In epithelial cells, ME spread more efficiently, consistent with robust UL128-131 expression. Strikingly, ME spread far better than did TB or TR in the presence of neutralizing antibodies on both cell types, indicating that ME is not simply deficient at cell-free spread but is particularly efficient at cell-to-cell spread, whereas TB and TR cell-to-cell spread is poor. Sonically disrupted ME-infected cells contained scant infectivity, suggesting that the efficient cell-to-cell spread mechanism of ME depends on features of the intact cells such as junctions or intracellular trafficking processes. Even when UL128-131 was transcriptionally repressed, cell-to-cell spread of ME was still more efficient than that of TB or TR. Moreover, RL13 expression comparably reduced both cell-free and cell-to-cell spread of all three strains, suggesting that it acts at a stage of assembly and/or egress common to both routes of spread. Thus, HCMV strains can be highly specialized for either for cell-free or cell-to-cell spread, and these phenotypes are determined by factors beyond the UL128-131 or RL13 loci. IMPORTANCE Both cell-free and cell-to-cell spread are likely important for the natural biology of HCMV. In culture, strains clearly differ in their capacity for cell-free spread as a result of differences in the quantity and infectivity of extracellular released progeny. However, it has been unclear whether “cell-associated” phenotypes are simply the result of poor cell-free spread or are indicative of particularly efficient cell-to-cell spread mechanisms. By measuring the kinetics of spread at early time points, we were able to show that HCMV strains can be highly specialized to either cell-free or cell-to-cell mechanisms, and this was not strictly linked the efficiency of cell-free spread. Our results provide a conceptual approach to evaluating intervention strategies for their ability to limit cell-free or cell-to-cell spread as independent processes.

Efficient Mutagenesis of Marek’s Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System

The virus-encoded microRNAs (miRNAs) have been demonstrated to have important regulatory roles in herpesvirus biology, including virus replication, latency, pathogenesis and/or tumorigenesis. As an emerging efficient tool for gene editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been successfully applied in manipulating the genomes of large DNA viruses. Herein, utilizing the CRISPR/Cas9 system with a double-guide RNAs transfection/virus infection strategy, we have established a new platform for mutagenesis of viral miRNAs encoded by the Marek’s disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus that can induce rapid-onset T-cell lymphomas in chickens. A series of miRNA-knocked out (miR-KO) mutants with deletions of the Meq- or the mid-clustered miRNAs, namely RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9 and RB-1B∆M11, were generated from vvMDV strain RB-1B virus. Interestingly, mutagenesis of the targeted miRNAs showed changes in the in vitro virus growth kinetics, which is consistent with that of the in vivo proliferation curves of our previously reported GX0101 mutants produced by the bacterial artificial chromosome (BAC) clone and Rec E/T homologous recombination techniques. Our data demonstrate that the CRISPR/Cas9-based gene editing is a simple, efficient and relatively nondisruptive approach for manipulating the small non-coding genes from the genome of herpesvirus and will undoubtedly contribute significantly to the future progress in herpesvirus biology.

A high-throughput genomic screen identifies a role for the plasmid-borne Type II secretion system of Escherichia coli O157:H7 (Sakai) in plant-microbe interactions

Food-borne illness arising from Shiga-toxigenic Escherichia coli (STEC) is often linked to consumption of fruit and vegetables as the bacteria have the ability to interact with plants and use them as alternative or secondary hosts. The initial stages of the interaction involve chemotaxis, attachment and potentially, responding to the early stages of microbe perception by the plant host. We used a high-throughput positive-selection approach to identify early interaction factors of E. coli O157:H7 isolate Sakai to spinach. A bacterial artificial chromosome (BAC) clone library was quantified by microarray hybridisation, and gene loci enrichment measured using a Bayesian hierarchical model. The screen of four successive rounds of short-term (2 hour) interaction with spinach roots produced in 115 CDS credible candidates, comprising seven contiguous genomic regions. Two candidate regions were selected for functional assessment: a chaperone-usher fimbrial gene cluster (loc6) and the pO157 plasmid-encoded type two secretion system (T2SS). Interaction of bacteria with spinach tissue was reduced in the absence of the pO157 plasmid, which was appeared to involve the T2SS EtpD secretin protein, whereas loss of loc6 did not impact interactions. The T2SS genes, etpD and etpC, were expressed at a plant-relevant temperature of 18 °C, and etpD expressed in planta by E. coli Sakai on spinach plants. Thus, a whole genome screening approach using a combination of computational modelling and functional assays has identified a novel function for STEC T2SS in interactions with plant tissue.

Identification of 1093-bp intron A, SNPS and indels discovered in the porcine pregnancy-associated glycoprotein 2-like (pPAG2-L) gene subfamily in pigs

The objective of this study was to identify the intron A sequence (between exons 1 and 2) of pPAG2-L, novel single nucleotide polymorphisms (SNPs) and mutations (InDels) within intron A in the crossbreed (Landrace x Large White), Hirshmann hybrid and Duroc pigs. Genomic templates were isolated from leukocytes, amplified, and the gel-out were purified and then sequenced. Positive amplification control included CH242-60C13 BAC clone (Duroc) containing pPAG1-L and pPAG2-L. This is the first report that describes the 1093-bp intron A sequence from the pPAG2-L (Acc. No. KF471015; GenBank), which increased general knowledge of the porcine genome. Novel SNPs/InDels were identified within the intron A of the pPAG2-L in the crossbreeds (72), Duroc (45) and Hirshmann hybrids (17). This is a pioneer study describing identification of the intron A and SNPs/InDels in crossbreeds that provides a novel major pattern that represents a large portion of the genetic variation within the porcine genome. This information should be valuable when genotyping (coding and noncoding regions) multiparous sows from many breeds, in which reproductive phenotypes are known.

Lipopolysaccharide-induced expansion of histidine decarboxylase-expressing Ly6G+ myeloid cells identified by exploiting histidine decarboxylase BAC-GFP transgenic mice

Histamine is a biogenic amine that is chiefly produced in mast cells and basophils and elicits an allergic response upon stimulation. Histidine decarboxylase (HDC) is a unique enzyme that catalyzes the synthesis of histamine. Therefore, the spatiotemporally specific Hdc gene expression profile could represent the localization of histamine-producing cells under various pathophysiological conditions. Although the bioactivity of histamine is well defined, the regulatory mechanism of Hdc gene expression and the distribution of histamine-producing cell populations in various disease contexts remains unexplored. To address these issues, we generated a histidine decarboxylase BAC (bacterial artificial chromosome) DNA-directed GFP reporter transgenic mouse employing a 293-kb BAC clone containing the entire Hdc gene locus and extended flanking sequences (Hdc-GFP). We found that the GFP expression pattern in the Hdc-GFP mice faithfully recapitulated that of conventional histamine-producing cells and that the GFP expression level mirrored the increased Hdc expression in lipopolysaccharide (LPS)-induced septic lungs. Notably, a CD11b+Ly6G+Ly6Clow myeloid cell population accumulated in the lung during sepsis, and most of these cells expressed high levels of GFP and indeed contain histamine. This study reveals the accumulation of a histamine-producing myeloid cell population during sepsis, which likely participates in the immune process of sepsis.