ALBERT

Description: Volvariella volvacea is an important crop in Southeast Asia, but erratic fruiting presents a serious challenge for its production and breeding. Efforts to explain inconsistent fruiting have been complicated by the multinucleate nature, typical lack of clamp connections, and an incompletely identified sexual reproductive system. In this study, we addressed the life cycle of V. volvacea using whole genome sequencing, cloning of MAT loci, karyotyping of spores, and fruiting assays. Microscopy analysis of spores had previously indicated the possible coexistence of heterothallic and homothallic life cycles. Our analysis of the MAT loci showed that only MAT-A , and not MAT-B , controlled heterokaryotization. Thus, the heterothallic life cycle was bipolar. Karyotyping of single spore isolates (SSIs) using molecular markers supported the existence of heterokaryotic spores. However, most SSIs were clearly not heterokaryotic, yet contained structural variation (SV) markers relating to both alleles of both parents. Heterokaryons from crossed, self-sterile homokaryons could produce fruiting bodies, agreeing with bipolar heterothallism. Meanwhile, some SSIs with two different MAT-A loci also produced fruiting bodies, which supported secondary homothallism. Next, SSIs that clearly contained only one MAT-A locus (homothallism) were also able to fruit, demonstrating that self-fertile SSIs were not, per definition, secondary homothallic, and that a third life cycle or genetic mechanism must exist. Finally, recombination between SV markers was normal, yet 10 out of 24 SV markers showed 1:2 or 1:3 distributions in the spores, and large numbers of SSIs contained doubled SV markers. This indicated selfish genes, and possibly partial aneuploidy.

Description: Foxtail millet ( Setaria italica ) is an important crop possessing C4 photosynthesis capability. The S. italica genome was de novo sequenced in 2012, but the sequence lacked high-density genetic maps with agronomic and yield trait linkages. In the present study, we resequenced a foxtail millet population of 439 recombinant inbred lines (RILs) and developed high-resolution bin map and high-density SNP markers, which could provide an effective approach for gene identification. A total of 59 QTL for 14 agronomic traits in plants grown under long- and short-day photoperiods were identified. The phenotypic variation explained ranged from 4.9 to 43.94%. In addition, we suggested that there may be segregation distortion on chromosome 6 that is significantly distorted toward Zhang gu. The newly identified QTL will provide a platform for sequence-based research on the S. italica genome, and for molecular marker-assisted breeding.

Description: Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stoichiometry of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa , we performed micrococcal nuclease digestion in the wild-type and the $$\Delta $$ hH1 strain followed by sequencing (MNase-seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq) revealed a modest but global increase in DNA methylation in the $$\Delta $$ hH1 mutant. Together, these data suggest that H1 acts as a nonspecific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa .

Description: The development of clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technologies promises a quantum leap in genome engineering of model organisms. However, CRISPR-mediated gene targeting reports in Drosophila melanogaster are still restricted to a few genes, use variable experimental conditions, and vary in efficiency, questioning the universal applicability of the method. Here, we developed an efficient two-step strategy to flexibly engineer the fly genome by combining CRISPR with recombinase-mediated cassette exchange (RMCE). In the first step, two sgRNAs, whose activity had been tested in cell culture, were co-injected together with a donor plasmid into transgenic Act5C-Cas9 , Ligase4 mutant embryos and the homologous integration events were identified by eye fluorescence. In the second step, the eye marker was replaced with DNA sequences of choice using RMCE enabling flexible gene modification. We applied this strategy to engineer four different locations in the genome, including a gene on the fourth chromosome, at comparably high efficiencies. Our data suggest that any fly laboratory can engineer their favorite gene for a broad range of applications within approximately 3 months.

Description: To identify genes that regulate the dynamics of lipid droplet (LD) size, we have used the genetically tractable model organism Caenorhabditis elegans , whose wild-type LD population displays a steady state of size with an upper limit of 3 μm in diameter. From a saturated forward genetic screen of 6.7 x 10 5 mutagenized haploid genomes, we isolated 118 mutants with supersized intestinal LDs often reaching 10 μm. These mutants define nine novel complementation groups, in addition to four known genes ( maoc-1 , dhs-28 , daf-22 , and prx-10 ). The nine groups are named drop (lipid droplet abnormal) and categorized into four classes. Class I mutants drop-5 and drop-9 , similar to prx-10 , are up-regulated in ACS-22 - DGAT-2 -dependent LD growth, resistant to LD hydrolysis, and defective in peroxisome import. Class II mutants drop-2 , drop-3 , drop-6 , and drop-7 are up-regulated in LD growth, are resistant to LD hydrolysis, but are not defective in peroxisome import. Class III mutants drop-1 and drop-8 are neither up-regulated in LD growth nor resistant to LD hydrolysis, but seemingly up-regulated in LD fusion. Class IV mutant drop-4 is cloned as sams-1 and, different to the other three classes, is ACS-22 -independent and hydrolysis-resistant. These four classes of supersized LD mutants should be valuable for mechanistic studies of LD cellular processes including growth, hydrolysis, and fusion.

Description: We aimed to elucidate the cell types, tissues, and pathways influenced by common variants in systemic lupus erythematosus (SLE). We applied a nonparameter enrichment statistical approach, termed SNPsea, in 181 single nucleotide polymorphisms (SNPs) that have been identified to be associated with the risk of SLE through genome-wide association studies (GWAS) in Eastern Asian and Caucasian populations, to manipulate the critical cell types, tissues, and pathways. In the two most significant cells’ findings (B lymphocytes and CD14+ monocytes), we subjected the GWAS association evidence in the Han Chinese population to an enrichment test of expression quantitative trait locus (QTL) sites and DNase I hypersensitivity, respectively. In both Eastern Asian and Caucasian populations, we observed that the expression level of SLE GWAS implicated genes was significantly elevated in xeroderma pigentosum B cells ( P  ≤ 1.00 10 –6 ), CD14+ monocytes ( P  ≤ 2.74 10 –4 ) and CD19+ B cells ( P  ≤ 2.00 10 –6 ), and plasmacytoid dendritic cells (pDCs) ( P  ≤ 9.00 10 –6 ). We revealed that the SLE GWAS-associated variants were more likely to reside in expression QTL in B lymphocytes (q 1 /q 0  = 2.15, P  = 1.23 10 –44 ) and DNase I hypersensitivity sites (DHSs) in CD14+ monocytes (q 1 /q 0  = 1.41, P  = 0.08). We observed the common variants affected the risk of SLE mostly through by regulating multiple immune system processes and immune response signaling. This study sheds light on several immune cells and responses, as well as the regulatory effect of common variants in the pathogenesis of SLE.

Description: We describe a suite of predictive models, coined FAST m C , for nonreference, cost-effective exploration and comparative analysis of context-specific DNA methylation levels. Accurate estimations of true DNA methylation levels can be obtained from as few as several thousand short-reads generated from whole-genome bisulfite sequencing. These models make high-resolution time course or developmental and large diversity studies practical regardless of species, genome size, and availability of a reference genome.

Description: Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila . Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach.

Description: The cucumber ( Cucumis sativus ) is characterized by its diversity and seasonal plasticity in sexual type. A long day length condition significantly decreased the cucumber female flower ratio by 17.7–52.9%, and the effect of photoperiod treatment is more significant under low temperature than under high temperature. Transcriptome analysis indicates that the photoperiod treatment preferentially significantly influenced flower development processes, particularly MADS-box genes in shoot apices. The long-day treatment resulted in predominantly transposable element (TE)- and gene-associated CHH-types of DNA methylation changes. Nevertheless, there was significant enrichment of CG- and CHG-types of DNA methylation changes nearing transcription start sites (TSSs)/transcription end sites (TESs) and gene bodies, respectively. Predominantly negative association between differentially methylated regions (DMRs) and differentially expressed genes (DEGs) were observed which implied epiregulation of DEGs. Two MADS-box genes that were significantly downregulated by long photoperiod showed significant hypermethylation in promoter regions that is essentially TE-rich. This study indicates MADS-box genes which are partially regulated by promoter methylation state may mediate photoperiod-dependent regulation of cucumber sex expression.

Description: The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast Schizosaccharomyces pombe by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid. Both fragments contain only a portion of the ura4 or bsdMX marker so that only the correctly assembled plasmid can confer uracil prototrophy or blasticidin resistance. We show that this gap-repair-based and cloning-free CRISPR/Cas9 procedure permits rapid and efficient point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion in fission yeast.