Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T₀ plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T₁ generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T₀ plants, we could remove foreign DNA easily by genetic segregation in the T₁ generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

     

G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin''s peroxidase activity was discovered. Through our strategy—in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.     

Geographic patterns of genetic variation in nuclear and chloroplast genomes of two related oaks (<Emphasis Type="Italic">Quercus aliena</Emphasis> and <Emphasis Type="Italic">Q. serrata</Emphasis>) in Japan: implications for seed and seedling transfer

In this study, we assessed geographic patterns of genetic variations in nuclear and chloroplast genomes of two related native oaks in Japan, Quercus aliena and Q. serrata, in order to facilitate development of genetic guidelines for transfer of planting stocks for each species. A total of 12 populations of Q. aliena and 44 populations of Q. serrata were analyzed in this study. Genotyping of nuclear microsatellites in Q. aliena was done with only nine populations (n = 212) due to limited numbers of individuals in two populations, while all 12 populations (n = 89) were used in sequencing chloroplast DNA (cpDNA). In Q. serrata, 43 populations (n = 1032) were genotyped by nuclear microsatellite markers, while cpDNA of 44 populations (n = 350) was sequenced. As anticipated, geographic patterns detected in the variations of Q. aliena’s nuclear genome and its chloroplast haplotype distribution clearly distinguished northern and southern groups of populations. However, those of Q. serrata were inconsistent. The geographic distribution of its chloroplast haplotypes tends to show the predicted differentiation between northern and southern lineages, but geographic signals in the genetic structure of its nuclear microsatellites are weak. Therefore, treating northern and southern regions of Japan as genetically distinct transferrable zones for planting stocks is highly warranted for Q. aliena. For Q. serrata, the strong NE-SW geographic structure of cpDNA should be considered.     

Single nucleotide polymorphisms associated with aggressive periodontitis and severe chronic periodontitis in Japanese

Periodontitis is a common inflammatory disease causing destruction of periodontal tissues. It is a multifactor disease involving genetic factors and oral environmental factors. To determine genetic risk factors associated with aggressive periodontitis or severe chronic periodontitis, single nucleotide polymorphisms (SNPs) in multiple candidate genes were investigated in Japanese. We studied 134 patients with aggressive periodontitis, 117 patients with severe chronic periodontitis, and 125 healthy volunteers without periodontitis, under case-control setting, and 310 SNPs in 125 candidate genes were genotyped. Association evaluation by Fisher's exact test (p < 0.01) revealed statistically significant SNPs in multiple genes, not only in inflammatory mediators (IL6ST and PTGDS, associated with aggressive periodontitis; and CTSD, associated with severe chronic periodontitis), but also in structural factors of periodontal tissues (COL4A1, COL1A1, and KRT23, associated with aggressive periodontitis; and HSPG2, COL17A1, and EGF, associated with severe chronic periodontitis). These appear to be good candidates as genetic factors for future study.     

Function of Chemokine (CXC Motif) Ligand 12 in Periodontal Ligament Fibroblasts

The periodontal ligament (PDL) is one of the connective tissues located between the tooth and bone. It is characterized by rapid turnover. Periodontal ligament fibroblasts (PDLFs) play major roles in the rapid turnover of the PDL. Microarray analysis of human PDLFs (HPDLFs) and human dermal fibroblasts (HDFs) demonstrated markedly high expression of chemokine (CXC motif) ligand 12 (CXCL12) in the HPDLFs. CXCL12 plays an important role in the migration of mesenchymal stem cells (MSCs). The function of CXCL12 in the periodontal ligament was investigated in HPDLFs. Expression of CXCL12 in HPDLFs and HDFs was examined by RT-PCR, qRT-PCR and ELISA. Chemotactic ability of CXCL12 was evaluated in both PDLFs and HDFs by migration assay of MSCs. CXCL12 was also immunohistochemically examined in the PDL in vivo. Expression of CXCL12 in the HPDLFs was much higher than that in HDFs in vitro. Migration assay demonstrated that the number of migrated MSCs by HPDLFs was significantly higher than that by HDFs. In addition, the migrated MSCs also expressed CXCL12 and several genes that are familiar to fibroblasts. CXCL12 was immunohistochemically localized in the fibroblasts in the PDL of rat molars. The results suggest that PDLFs synthesize and secrete CXCL12 protein and that CXCL12 induces migration of MSCs in the PDL in order to maintain rapid turnover of the PDL.     

Genetic inhomogeneous Poisson processes describing the roles of an isolated mature tree in forest regeneration

The spatial distribution of genotyped adult plants and offspring can be modeled by genetic inhomogeneous Poisson processes. This paper reports the development of a previously proposed point process model to cover safe site conditions for sapling survival, unknown seed sources and wider ranges of dispersal kernels. Suppose that a species has limited seed dispersal and shade-tolerance, and that young trees are clustered around highly isolated adults. The clustering might be formed solely by dispersed seeds from adults. However, the survival of the offspring might be influenced by shading by the adults crowns. The new genetic processes are applicable to such cases, as demonstrated for a young population of Fagus crenata, a shade-tolerant canopy tree species, in a 55-year-old stand regenerated after shelterwood logging. Isozyme analysis revealed that the regenerating trees were genetically related to the nearest adults, but some were not their respective daughters. The maximum likelihood method has led us to the following regeneration: seeds were dispersed mostly within 20 m of their mothers; two residual adults in the plot had equal female reproductive success; about 10% of offspring even close to the adult were not their daughters; the adults crowns reduced the survival of offspring up to 20–25 m away. However, the degree of model fitting was unsatisfactory. Hence, our models, in principle, can quantify two roles of highly isolated adults: providing seed sources and safe sites, but the case study suggests that other, unknown factors influence F. crenata regeneration after shelterwood harvesting.     

The response of forest floor vegetation and tree regeneration to deer exclusion and disturbance in a riparian deciduous forest,central Japan

The response of forest floor vegetation and regeneration of major treespecies to deer exclusion in a riparian forest were studied for 3 years withtheinteractive effects of natural disturbances. At the start of this study, deerdensity had quickly increased to a fairly high level (29–31 individualskm^–2) during the last decade and had severely reduced theamount of forest floor vegetation in the study area. Dwarf bamboos, which weredominant before, had almost diminished from the forest floor. During the studyperiod, aboveground biomass increased steadily but species diversity did notchange much in the exclosures. Outside the exclosures, the seedlings of alltreespecies were damaged greatly by deer browsing, especially the taller ones. Deerbrowsing had little effect on the emergence of tree seedlings, but deertrampling might have accelerated emergence indirectly by disturbing the soilsurface for some species. Differences in plant responses were also observed fordeer browsing and the presence of dwarf bamboo that strongly inhibits therecruitment of tree seedlings. The nine major species were classified intothreegroups according to the response of seedlings to deer browsing and disturbance.Group 1 consists of the species whose seedling survival is affected bybrowsing,but seldom by disturbances (Phellodendron amurense,Kalopanax pictus, Quercus crispulaandMalus toringo). Groups 2 and 3 consist of species adaptedto tree-fall disturbance (Betula spp.) and ripariandisturbance (Alnus hirsuta var.sibirica, Ulmus davidiana var.japonica, Populus maximowiczii andSalix sachalinensis), respectively, and seedling survivalof these two groups is principally affected by light conditions. The effect ofdeer browsing on seedling survival and growth was greater for Group 2 than forGroup 3.     

Riparian disturbance and community structure of a Quercus-Ulmus forest in central Japan

Population structure and ecological characteristics of component species of a riparian Ulmus-Quercus forest in central Japan were analyzed with special reference to riparian disturbance regime. Though the dbh distribution of the whole community was L-shaped, those of several component tree populations had several modes, suggesting intermittent regeneration periods in the past. Correlation of spatial distributions among tree populations and subpopulations showed 6 major groups reflecting riparian disturbances in the past and different establishment patterns among species. A cluster of small-sized tree populations (Salix sachalinensis, Alnus hirsuta and Populus maximowiczii <30 cm dbh) were distributed on the lower terrace along the active river channel, while large-sized subpopulations (dbh 60 cm) of Quercus crispula and Ulumus davidiana var. japonica occurred on the higher terrace. The Phellodendron amurense population also occurred on the higher terrace in small clumps though the trees were small (less than 55 cm dbh). Subpopulations of intermediate-sized individuals (30 dbh < 60 cm) of Q. crispula and U. davidiana var. japonica, together with Betula and Acer spp. populations occurred on the intermediate terrace. Dendrochronological analyses indicated that the large and intermediate-sized tree groups were established about 330 and 90 years ago, respectively, while the small-sized tree group established about 35 years ago. A survey of historical disturbances showed that these periods of establishment of the former two groups almost coincided with the historically major floods occurring in 1662 and 1902. However, the disturbance that resulted in the establishment of the youngest group could not be precisely identified. Thus, the forest is a mosaic of three differently-aged patches, which is closely related to the frequency and scale of riparian disturbances. Longevity of trees and the preferred conditions for seed germination and/or seedling establishment were particularly important for the guild structure in this forest community.