Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

KEY MESSAGE: Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

Sequencing of transcriptomes from two Miscanthus species reveals functional specificity in rhizomes, and clarifies evolutionary relationships.

BACKGROUND: Miscanthus is a promising biomass crop for temperate regions. Despite the increasing interest in this plant, limited sequence information has constrained research into its biology, physiology, and breeding. The whole genome transcriptomes of M. sinensis and M. sacchariflorus presented in this study may provide good resources to understand functional compositions of two important Miscanthus genomes and their evolutionary relationships. RESULTS: For M. sinensis, a total of 457,891 and 512,950 expressed sequence tags (ESTs) were produced from leaf and rhizome tissues, respectively, which were assembled into 12,166 contigs and 89,648 singletons for leaf, and 13,170 contigs and 112,138 singletons for rhizome. For M. sacchariflorus, a total of 288,806 and 267,952 ESTs from leaf and rhizome tissues, respectively, were assembled into 8,732 contigs and 66,881 singletons for leaf, and 8,104 contigs and 63,212 singletons for rhizome. Based on the distributions of synonymous nucleotide substitution (Ks), sorghum and Miscanthus diverged about 6.2 million years ago (MYA), Saccharum and Miscanthus diverged 4.6 MYA, and M. sinensis and M. sacchariflorus diverged 1.5 MYA. The pairwise alignment of predicted protein sequences from sorghum-Miscanthus and two Miscanthus species found a total of 43,770 and 35,818 nsSNPs, respectively. The impacts of striking mutations found by nsSNPs were much lower between sorghum and Miscanthus than those between the two Miscanthus species, perhaps as a consequence of the much higher level of gene duplication in Miscanthus and resulting ability to buffer essential functions against disturbance. CONCLUSIONS: The ESTs generated in the present study represent a significant addition to Miscanthus functional genomics resources, permitting us to discover some candidate genes associated with enhanced biomass production. Ks distributions based on orthologous ESTs may serve as a guideline for future research into the evolution of Miscanthus species as well as its close relatives sorghum and Saccharum.

Decisive indicator for gastrointestinal workup in anemic patients with nondialysis chronic kidney disease.

BACKGROUND: Anemia and iron deficiency are universal problems in patients with chronic kidney disease (CKD). However, decisive indicator to guide the further gastrointestinal (GI) workup has not been determined. METHODS: We included 104 anemic patients with nondialysis-dependent CKD stages 3-5 (38 patients at stage 3, 26 patients at stage 4, and 40 patients at stage 5). Hemoglobin, serum ferritin, transferrin saturation (TSAT), mean corpuscular volume (MCV), and corrected reticulocyte count data were assessed to evaluate diagnostic utility for bleeding-related GI lesions, which were identified by esophagogastroduodenoscopy and colonoscopy. RESULTS: Bleeding-related GI lesions were found in 55 (52.9%) patients, and patients with stage 5 CKD had a higher prevalence of gastric lesions than patients with CKD stage 3 or 4 (all p < 0.05). The areas under the receiver operating characteristic curves used to predict bleeding-related lesions were 0.69 for TSAT (p = 0.002) and 0.61 for serum ferritin (p = 0.085). The sensitivity and specificity of a cutoff value for TSAT < 20% were 0.59 and 0.74, respectively. Hemoglobin, MCV, and corrected reticulocyte levels had no significant diagnostic utility. On multivariable logistic regression, the chance of GI lesions increased by 6% for each 1% reduction in TSAT and increased 4.1-fold for patients with CKD stage 5 (all p < 0.05). CONCLUSIONS: TSAT is a useful indicator for determining the GI workup in anemic patients with nondialysis-dependent CKD stages 3-5. Stage 5 CKD is independently associated with bleeding-related lesions and TSAT should be used cautiously in these patients.

Genetic diversity among Korean bermudagrass (Cynodon spp.) ecotypes characterized by morphological, cytological and molecular approaches.

The genus Cynodon comprises ten species. The objective of this study was to evaluate the genetic diversity of Korean bermudagrasses at the morphological, cytological and molecular levels. Morphological parameters, the nuclear DNA content and ploidy levels were observed in 43 bermudagrass ecotypes. AFLP markers were evaluated to define the genetic diversity, and chromosome counts were made to confirm the inferred cytotypes. Nuclear DNA contents were in the ranges 1.42-1.56, 1.94-2.19, 2.54, and 2.77-2.85 pg/2C for the triploid, tetraploid, pentaploid, and hexaploid accessions, respectively. The inferred cytotypes were triploid (2n = 3x = 27), tetraploid (2n = 4x = 36), pentaploid (2n = 5x = 45), and hexaploid (2n = 6x = 54), but the majority of the collections were tetraploid (81%). Mitotic chromosome counts verified the corresponding ploidy levels. The fast growing fine-textured ecotypes had lower ploidy levels, while the pentaploids and hexaploids were coarse types. The genetic similarity ranged from 0.42 to 0.94 with an average of 0.64. UPGMA cluster analysis and principle coordinate analysis separated the ecotypes into 6 distinct groups. The genetic similarity suggests natural hybridization between the different cytotypes, which could be useful resources for future breeding and genetic studies.

A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae.

BACKGROUND: All previously reported eukaryotic nuclear genome sequences have been incomplete, especially in highly repeated units and chromosomal ends. Because repetitive DNA is important for many aspects of biology, complete chromosomal structures are fundamental for understanding eukaryotic cells. Our earlier, nearly complete genome sequence of the hot-spring red alga Cyanidioschyzon merolae revealed several unique features, including just three ribosomal DNA copies, very few introns, and a small total number of genes. However, because the exact structures of certain functionally important repeated elements remained ambiguous, that sequence was not complete. Obviously, those ambiguities needed to be resolved before the unique features of the C. merolae genome could be summarized, and the ambiguities could only be resolved by completing the sequence. Therefore, we aimed to complete all previous gaps and sequence all remaining chromosomal ends, and now report the first nuclear-genome sequence for any eukaryote that is 100% complete. RESULTS: Our present complete sequence consists of 16546747 nucleotides covering 100% of the 20 linear chromosomes from telomere to telomere, representing the simple and unique chromosomal structures of the eukaryotic cell. We have unambiguously established that the C. merolae genome contains the smallest known histone-gene cluster, a unique telomeric repeat for all chromosomal ends, and an extremely low number of transposons. CONCLUSION: By virtue of these attributes and others that we had discovered previously, C. merolae appears to have the simplest nuclear genome of the non-symbiotic eukaryotes. These unusually simple genomic features in the 100% complete genome sequence of C. merolae are extremely useful for further studies of eukaryotic cells.

Genes for the peptidoglycan synthesis pathway are essential for chloroplast division in moss.

The general consensus is that a cyanobacterium phagocytosed by a host cell evolved into the plastids of red and green algae, land plants, and glaucophytes. In contrast to the plastids of glaucophytes, which retain a cyanobacterial-type peptidoglycan layer, no wall-like structures have been detected in plastids from other sources. Although the genome of Arabidopsis thaliana contains five genes that are essential for peptidoglycan synthesis, MurE, MurG, two genes for D-Ala-D-Ala ligase (Ddl), and the gene for translocase I (MraY), their functions have not been determined. We report that the moss Physcomitrella patens has nine homologous genes related to peptidoglycan biosynthesis: MurA, B, C, D, E, and F, Ddl, genes for the penicillin-binding protein Pbp, and dd-carboxypeptidase (Dac). Corroborating a computer prediction, analysis of the GFP fusion proteins with the N terminus of PpMurE or of PpPbp suggests that these proteins are located in the chloroplasts. Gene disruption of the PpMurE gene in P. patens resulted in the appearance of macrochloroplasts both in protonema and in leaf cells. Moreover, gene knockout of the P. patens Pbp gene showed inhibition of chloroplast division in this moss; however, no Pbp gene was found in A. thaliana.