Development of an SNP marker set for marker-assisted backcrossing using genotyping-by-sequencing in tetraploid perilla.

High-quality molecular markers are essential for marker-assisted selection to accelerate breeding progress. Compared with diploid species, recently diverged polyploid crop species tend to have highly similar homeologous subgenomes, which is expected to limit the development of broadly applicable locus-specific single-nucleotide polymorphism (SNP) assays. Furthermore, it is particularly challenging to make genome-wide marker sets for species that lack a reference genome. Here, we report the development of a genome-wide set of kompetitive allele specific PCR (KASP) markers for marker-assisted recurrent selection (MARS) in the tetraploid minor crop perilla. To find locus-specific SNP markers across the perilla genome, we used genotyping-by-sequencing (GBS) to construct linkage maps of two F2 populations. The two resulting high-resolution linkage maps comprised 2326 and 2454 SNP markers that spanned a total genetic distance of 2133 cM across 16 linkage groups and 2169 cM across 21 linkage groups, respectively. We then obtained a final genetic map consisting of 22 linkage groups with 1123 common markers from the two genetic maps. We selected 96 genome-wide markers for MARS and confirmed the accuracy of markers in the two F2 populations using a high-throughput Fluidigm system. We confirmed that 91.8% of the SNP genotyping results from the Fluidigm assay were the same as the results obtained through GBS. These results provide a foundation for marker-assisted backcrossing and the development of new varieties of perilla.

Transcriptome Analysis of Differentially Expressed Genes Associated with Salt Stress in Cowpea (Vigna unguiculata L.) during the Early Vegetative Stage.

Cowpea (Vigna unguiculata (L.), 2n = 22) is a tropical crop grown in arid and semiarid regions that is tolerant to abiotic stresses such as heat and drought. However, in these regions, salt in the soil is generally not eluted by rainwater, leading to salt stress for a variety of plant species. This study was conducted to identify genes related to salt stress using the comparative transcriptome analysis of cowpea germplasms with contrasting salt tolerance. Using the Illumina Novaseq 6000 platform, 1.1 billion high-quality short reads, with a total length of over 98.6 billion bp, were obtained from four cowpea germplasms. Of the differentially expressed genes identified for each salt tolerance type following RNA sequencing, 27 were shown to exhibit significant expression levels. These candidate genes were subsequently narrowed down using reference-sequencing analysis, and two salt stress-related genes (Vigun_02G076100 and Vigun_08G125100) with single-nucleotide polymorphism (SNP) variation were selected. Of the five SNPs identified in Vigun_02G076100, one that caused significant amino acid variation was identified, while all nucleotide variations in Vigun_08G125100 was classified as missing in the salt-resistant germplasms. The candidate genes and their variation, identified in this study provide, useful information for the development of molecular markers for cowpea breeding programs.

Identification of quantitative trait loci associated with flowering time in perilla using genotyping-by-sequencing.

Understanding the transition to the reproductive period is important for crop breeding. This information can facilitate the production of novel varieties that are better adapted to local environments or changing climatic conditions. Here, we report the development of a high-density linkage map based on genotyping-by-sequencing (GBS) for the genus perilla. Through GBS library construction and Illumina sequencing of an F2 population, a total of 9607 single-nucleotide polymorphism (SNP) markers were developed. The ten-group linkage map of 1309.39 cM contained 2518 markers, with an average marker density of 0.56 cM per linkage group (LG). Using this map, a total of six QTLs were identified. These quantitative trait loci (QTLs) are associated with three traits related to flowering time: days to visible flower bud, days to flowering, and days to maturity. Ortholog analysis conducted with known genes involved in the regulation of flowering time among different crop species identified GI, CO and ELF4 as putative perilla orthologs that are closely linked to the QTL regions associated with flowering time. These results provide a foundation that will be useful for future studies of flowering time in perilla using fine mapping, and marker-assisted selection for the development of new varieties of perilla.

Molecular characterization of proton beam-induced mutations in soybean using genotyping-by-sequencing.

Proton beam irradiation is a next-generation technique to develop mutant crop varieties. The mutagenic effects and molecular mechanisms of radiation are important multi-disciplinary research subjects. This study was conducted to investigate the types of mutations induced in the soybean genome by proton beam irradiation. In total, 22 plants, including 10 M2 plants treated with proton beam irradiation at 118 and 239 Gy, each, and two wild-type plants (Daepung) were sequenced by genotyping-by-sequencing (GBS). In total, 7453 single nucleotide polymorphisms (SNPs) were detected in the 20 M2 plants, compared with the two wild-type controls. The SNP frequency was 1/36,976 bp with proton beam irradiation at 118 Gy, and 1/32,945 bp at 239 Gy. Of these, 3569 SNPs were detected in genic regions. We observed that proton beam irradiation induced more substitutions than small insertion-deletions (INDELs). Based on the mutagenic effect of proton beam irradiation, the frequency of transition mutations was shown to be higher than that of transversions. The proton beam-induced SNPs were distributed uniformly in most of the chromosomes. Gene ontology (GO) analysis showed that there were many genes involved in protein metabolic process under biological process, intracellular membrane-bounded organelle under cellular component, and nucleic acid binding under molecular function. This study could provide valuable information for investigating the potential mechanisms of mutation, and guidance for developing soybeans cultivars using mutation breeding.

Stomata-Inspired Photomechanical Ion Nanochannels Modified by Azobenzene Composites.

A low-powered and highly selective photomechanical sensor system mimicking stomata in the epidermis of leaves harvested from nature is demonstrated. This device uses a light-responsive composite consisting of 4-amino-1,1'-azobenzene-3,4'-disulfonic acid monosodium salt (AZO) and poly(diallyldimethylammonium chloride) (PDDA) coated on a membrane with tens of nanometer-size pores. The ionic current change through the pore channels as a function of pore size variation is then measured. The tran-cis isomerism of AZO-PDDA during light irradiation and the operation mechanism of photomechanical ion channel sensor are discussed and analyzed using UV-vis spectroscopy and atomic force microscopy analysis. It presents the discriminative current levels to the different light wavelengths. The response time of the photoreceptor is about 0.2 s and it consumes very low operating power (≈15 nW) at 0.1 V bias. In addition, it is found that the change of the pore diameter during the light irradiation is due to the photomechanical effect, which is capable of distinguishing light intensity and wavelength.

Identification of candidate genes for an early-maturing soybean mutant by genome resequencing analysis.

Flowering is indicative of the transition from vegetative to reproductive phase, a critical event in the life cycle of plants. In this study, we performed whole genome resequencing by Illumina HiSeq to identify changes in flowering genes using an early-flowering phenotype of soybean mutant line Josaengserori (JS) derived from Korean landrace, Seoritae (SR), and we obtained mapped reads of 131,769,690 and 167,669,640 bp in JS and SR, respectively. From the whole genome sequencing results between JS and SR, we identified 332,821 polymorphic SNPs and 65,178 indels, respectively. Among these, 30 flowering genes were in SNPs and 25 were in indels. Among 30 flowering genes detected in SNPs, Glyma02g33040, Glyma06g22650, Glyma10g36600, Glyma13g01290, Glyma14g10530, Glyma16g01980, Glyma17g11040, Glyma18g53690, and Glyma20g29300 were non-synonymous substitutions between JS and SR. Changes in Glyma10g36600 (GI), Glya02g33040 (AGL18), Glyma17g11040 (TOC1), and Glyma14g10530 (ELF3) in JS affected the expression of GmFT2a and resulted in early flowering. These results provide insight into the regulatory pathways of flowering in soybean mutants and help to improve our knowledge of soybean mutation breeding.

Strong and Tough Nacre-Inspired Graphene Oxide Composite with Hierarchically Similar Structure.

We report a graphene oxide (GO)-based composite, featuring GO/cross-linking agent (CA) nanoparticles, inspired by a nacre-like hierarchical structure present in nature. The as-prepared GO/CA composite was powdered to nanoscale particles and then mixed with pure GO to be GO/CA/GO (GCG) composite forming hierarchical GO/CA nanoasperities on the GO surface. The strength and toughness of the nacre-inspired GCG composite films were simultaneously improved by adjusting the nanoparticle concentration and hierarchical level of the GO-based films. Compared to pristine GO films and GO/CA composites, which exhibit a low level of hierarchy in their structures, the tensile strength and toughness of the GCG composites with higher hierarchy were enhanced 3.1 and 1.6 times and 47.6 and 10.9 times, respectively. Furthermore, a plausible mechanism of increasing mechanical properties based on nanoscale asperities and homogeneous interactions between GO and CA has been discussed.

Asymmetric Dichroic Colors in Stretchable Film with Embedded Au/Ag Alloy Nanoparticles.

We present a new type of stretchable dichroic film in which Au and Ag alloy nanoparticles (NPs) are dispersed in polydimethylsiloxane (PDMS). The alloy NPs are synthesized with different atomic compositions and sizes to modulate their plasmonic resonance frequencies and absorption and scattering cross sections. The PDMS dichroic film in which 100 nm alloy NPs with a Au/Ag ratio of 7:3 are dispersed shows exotic optical properties under tensile strain. When 40% tensile strain is applied, the film exhibits a strain-sensitive transmission and strain-insensitive reflection behavior in which the transmittance is increased up to 2.6 times, whereas the reflectance remains unchanged. Moreover, we demonstrate a stretchable anticounterfeiting film and a flexible dichroic sculpture fabricated with the PDMS composite. This work demonstrates a new type of plasmonic application that has great potential in various applications, such as special-purpose optical films, security patterns, and smart windows.

Comparative analysis of pepper and tomato reveals euchromatin expansion of pepper genome caused by differential accumulation of Ty3/Gypsy-like elements.

BACKGROUND: Among the Solanaceae plants, the pepper genome is three times larger than that of tomato. Although the gene repertoire and gene order of both species are well conserved, the cause of the genome-size difference is not known. To determine the causes for the expansion of pepper euchromatic regions, we compared the pepper genome to that of tomato. RESULTS: For sequence-level analysis, we generated 35.6 Mb of pepper genomic sequences from euchromatin enriched 1,245 pepper BAC clones. The comparative analysis of orthologous gene-rich regions between both species revealed insertion of transposons exclusively in the pepper sequences, maintaining the gene order and content. The most common type of the transposon found was the LTR retrotransposon. Phylogenetic comparison of the LTR retrotransposons revealed that two groups of Ty3/Gypsy-like elements (Tat and Athila) were overly accumulated in the pepper genome. The FISH analysis of the pepper Tat elements showed a random distribution in heterochromatic and euchromatic regions, whereas the tomato Tat elements showed heterochromatin-preferential accumulation. CONCLUSIONS: Compared to tomato pepper euchromatin doubled its size by differential accumulation of a specific group of Ty3/Gypsy-like elements. Our results could provide an insight on the mechanism of genome evolution in the Solanaceae family.

Molecular mapping and characterization of a single dominant gene controlling CMV resistance in peppers (Capsicum annuum L.).

Cucumber mosaic virus (CMV) is one of the most destructive viruses in the Solanaceae family. Simple inheritance of CMV resistance in peppers has not previously been documented; all previous studies have reported that resistance to this virus is mediated by several partially dominant and recessive genes. In this study, we showed that the Capsicum annuum cultivar 'Bukang' contains a single dominant resistance gene against CMV(Korean) and CMV(FNY) strains. We named this resistance gene Cmr1 (Cucumber mosaic resistance 1). Analysis of the cellular localization of CMV using a CMV green fluorescent protein construct showed that in 'Bukang,' systemic movement of the virus from the epidermal cell layer to mesophyll cells is inhibited. Genetic mapping and FISH analysis revealed that the Cmr1 gene is located at the centromeric region of LG2, a position syntenic to the ToMV resistance locus (Tm-1) in tomatoes. Three SNP markers were developed by comparative genetic mapping: one intron-based marker using a pepper homolog of Tm-1, and two SNP markers using tomato and pepper BAC sequences mapped near Cmr1. We expect that the SNP markers developed in this study will be useful for developing CMV-resistant cultivars and for fine mapping the Cmr1 gene.

Angle-Insensitive Transmission and Reflection of Nanopatterned Dielectric Multilayer Films for Colorful Solar Cells.

Aesthetically appealing photovoltaic (PV) panels with colorful layers are used in numerous applications involving color matching with the surroundings. To develop a colored film for a PV system, appropriate optical properties such as high transparency and low angle sensitivity are necessary because the colored layers can reduce the efficiency of the PV system by causing variations in the transmittance and angle of incidence. Herein, we propose a facile fabrication method for bioinspired three-dimensional (3D) photonic crystal (PC) films that exhibit broad angle-insensitive transmission and reflection, for application in colorful PV. This structure, patterned on a sequentially stacked 11-layer film of SiO2 and TiO2, is fabricated via nanoimprint lithography and a one-step dry-etching process, without using a metal mask. The changes in transmission and reflection are observed via ultraviolet-visible spectroscopy and from the reflected images obtained under various angles. The transmittance dips of the 3D PC film shift by less than 10 nm in wavelength, for angles from 0 to 45°, indicating low angle dependency. In addition, the change in the observed color, with respect to the viewing position, is less in the fabricated film. Once the 3D PC film was added to a commercial PV cell, it exhibited a higher efficiency (approximately 6% upper) when compared to a cell with a one-dimensional PC film, during the duration of the experiment, from 0 to 30°. Thus, the proposed method demonstrates excellent potential for developing structural color films for achieving aesthetically appealing PV cells.

Evolution of ribosomal DNA-derived satellite repeat in tomato genome.

BACKGROUND: Tandemly repeated DNA, also called as satellite DNA, is a common feature of eukaryotic genomes. Satellite repeats can expand and contract dramatically, which may cause genome size variation among genetically-related species. However, the origin and expansion mechanism are not clear yet and needed to be elucidated. RESULTS: FISH analysis revealed that the satellite repeat showing homology with intergenic spacer (IGS) of rDNA present in the tomato genome. By comparing the sequences representing distinct stages in the divergence of rDNA repeat with those of canonical rDNA arrays, the molecular mechanism of the evolution of satellite repeat is described. Comprehensive sequence analysis and phylogenetic analysis demonstrated that a long terminal repeat retrotransposon was interrupted into each copy of the 18S rDNA and polymerized by recombination rather than transposition via an RNA intermediate. The repeat was expanded through doubling the number of IGS into the 25S rRNA gene, and also greatly increasing the copy number of type I subrepeat in the IGS of 25-18S rDNA by segmental duplication. Homogenization to a single type of subrepeat in the satellite repeat was achieved as the result of amplifying copy number of the type I subrepeat but eliminating neighboring sequences including the type II subrepeat and rRNA coding sequence from the array. FISH analysis revealed that the satellite repeats are commonly present in closely-related Solanum species, but vary in their distribution and abundance among species. CONCLUSION: These results represent that the dynamic satellite repeats were originated from intergenic spacer of rDNA unit in the tomato genome. This result could serve as an example towards understanding the initiation and the expansion of the satellite repeats in complex eukaryotic genome.

Integration of cytogenetic and genetic linkage maps unveils the physical architecture of tomato chromosome 2.

We report the integration of the linkage map of tomato chromosome 2 with a high-density bacterial artificial chromosome fluorescence in situ hybridization (BAC-FISH)-based cytogenetic map. The euchromatic block of chromosome 2 resides between 13 and 142 cM and has a physical length of 48.12 microm, with 1 microm equivalent to 540 kb. BAC-FISH resolved a pair of loci that were 3.7-3.9 Mb apart and were not resolved on the linkage map. Most of the regions had crossover densities close to the mean of approximately 200 kb/cM. Relatively hot and cold spots of recombination were unevenly distributed along the chromosome. The distribution of centimorgan/micrometer values was similar to the previously reported recombination nodule distribution along the pachytene chromosome. FISH-based physical maps will play an important role in advanced genomics research for tomato, including map-based cloning of agronomically important traits and whole-genome sequencing.

Nacre-Mimetic Graphene Oxide/Cross-Linking Agent Composite Films with Superior Mechanical Properties.

We report a graphene oxide/cross-linking agent (GO/CA) composite inspired by the nacre structure. Based on the "brick-and-mortar" concept of nacre, graphene oxide and a cross-linking agent are covalently conjugated in the form of nacre. The mechanical characteristics of the nacre-mimetic GO/CA composite film can be controlled by adjusting the preparation method, degree of cross-linking, and cross-linking times. As a result, the cross-linking strategy can drastically enhance the tensile strength [142.9 ± 6.4 MPa (∼2.3-fold)], modulus [4.7 ± 0.36 GPa (∼15.7-fold)], and hardness [917.4 ± 85.7 MPa (∼9.0-fold)], which are superior to those of pristine materials. The cross-linking agent-based chemical bonding method for mechanically improved integration is mainly attributed to the formation of strong cross-linked networks between the GO-based 2D interfaces and CA. The facile fabrication process provides many opportunities to design advanced, robust, and integrated nacre-like GO/CA composites, which can be applied to future aerospace utilizations, electronic protectors, robotic elements, and permeable membranes.

Iridescent and Glossy Effect on Polymer Surface Using Micro-/Nanohierarchical Structure: Artificial Queen of the Night Tulip Petals.

Many petals in nature have a hierarchical structure that imparts various optical properties. Among these, the petals of the Queen of the Night tulip exhibit an iridescent and glossy color due to the diffraction and scattering of light. Herein, we report a bioinspired micro-/nanohierarchical structure that mimics Queen of the Night tulip petal surfaces. Using a method that combined soft lithography and UV-ozone treatment, we fabricated nanoscale line patterns with a linewidth of 600 nm on microwrinkles of 15 µm width and 3 µm height. Using optical microscopy in the dark-field mode and monochromatic light diffraction measurements, we found that these hierarchical structures on a poly(dimethylsiloxane) (PDMS) substrate synergistically improved the scattering and diffraction effects, unlike the pristine, nano-, and microstructures. In addition, using a dye-colored PDMS material, we fabricated artificial Queen of the Night petals with iridescent and glossy effects. They show great potential for a range of applications, such as coloring, smart displays, dynamic gratings, and light-control devices.

Surface-diffusion-limited growth of atomically thin WS2 crystals from core-shell nuclei.

Atomically thin transition metal dichalcogenides (TMDs) have recently attracted great attention since the unique and fascinating physical properties have been found in various TMDs, implying potential applications in next-generation devices. The progress towards developing new functional and high-performance devices based on TMDs, however, is limited by the difficulty in producing large-area monolayer TMDs due to a lack of knowledge of the growth processes of monolayer TMDs. In this work, we have investigated the growth processes of monolayer WS2 crystals using a thermal chemical vapor deposition method, in which the growth conditions were adjusted in a systematic manner. It was found that, after forming WO3-WS2 core-shell nanoparticles as nucleation sites on a substrate, the growth of three-dimensional WS2 islands proceeds by ripening and crystallization processes. Lateral growth of monolayer WS2 crystals subsequently occurs by the surface diffusion process of adatoms toward the step edge of the three-dimensional WS2 islands. Our results provide understanding of the growth processes of monolayer WS2 by using chemical vapor deposition methods.

A comparative synteny analysis tool for target-gene SNP marker discovery: connecting genomics data to breeding in Solanaceae.

It is necessary for molecular breeders to overcome the difficulties in applying abundant genomic information to crop breeding. Candidate orthologs would be discovered more efficiently in less-studied crops if the information gained from studies of related crops were used. We developed a comparative analysis tool and web-based genome viewer to identify orthologous genes based synteny as well as sequence similarity between tomato, pepper and potato. The tool has a step-by-step interface with multiple viewing levels to support the easy and accurate exploration of functional orthologs. Furthermore, it provides access to single nucleotide-polymorphism markers from the massive genetic resource pool in order to accelerate the development of molecular markers for candidate orthologs in the Solanaceae. This tool provides a bridge between genome data and breeding by supporting effective marker development, data utilization and communication.Database URL: http://tgsol.seeders.co.kr/scomp/.

Effect of interlayer interactions on exciton luminescence in atomic-layered MoS2 crystals.

The atomic-layered semiconducting materials of transition metal dichalcogenides are considered effective light sources with both potential applications in thin and flexible optoelectronics and novel functionalities. In spite of the great interest in optoelectronic properties of two-dimensional transition metal dichalcogenides, the excitonic properties still need to be addressed, specifically in terms of the interlayer interactions. Here, we report the distinct behavior of the A and B excitons in the presence of interlayer interactions of layered MoS2 crystals. Micro-photoluminescence spectroscopic studies reveal that on the interlayer interactions in double layer MoS2 crystals, the emission quantum yield of the A exciton is drastically changed, whereas that of the B exciton remains nearly constant for both single and double layer MoS2 crystals. First-principles density functional theory calculations confirm that a significant charge redistribution occurs in the double layer MoS2 due to the interlayer interactions producing a local electric field at the interfacial region. Analogous to the quantum-confined Stark effect, we suggest that the distinct behavior of the A and B excitons can be explained by a simplified band-bending model.

Whole-transcriptome analyses of the Sapsaree, a Korean natural monument, before and after exercise-induced stress.

BACKGROUND: The Sapsaree (Canis familiaris) is a Korean native dog that is very friendly, protective, and loyal to its owner, and is registered as a natural monument in Korea (number: 368). To investigate large-scale gene expression profiles and identify the genes related to exercise-induced stress in the Sapsaree, we performed whole-transcriptome RNA sequencing and analyzed gene expression patterns before and after exercise performance. RESULTS: We identified 525 differentially expressed genes in ten dogs before and after exercise. Gene Ontology classification and KEGG pathway analysis revealed that the genes were mainly involved in metabolic processes, such as programmed cell death, protein metabolic process, phosphatidylinositol signaling system, and cation binding in cytoplasm. The ten Sapsarees could be divided into two groups based on the gene expression patterns before and after exercise. The two groups were significantly different in terms of their basic body type (p ≤ 0.05). Seven representative genes with significantly different expression patterns before and after exercise between the two groups were chosen and characterized. CONCLUSIONS: Body type had a significant effect on the patterns of differential gene expression induced by exercise. Whole-transcriptome sequencing is a useful method for investigating the biological characteristics of the Sapsaree and the large-scale genomic differences of canines in general.

De Novo Transcriptome Analysis of Cucumis melo L. var. makuwa.

Oriental melon (Cucumis melo L. var. makuwa) is one of six subspecies of melon and is cultivated widely in East Asia, including China, Japan, and Korea. Although oriental melon is economically valuable in Asia and is genetically distinct from other subspecies, few reports of genome-scale research on oriental melon have been published. We generated 30.5 and 36.8 Gb of raw RNA sequence data from the female and male flowers, leaves, roots, and fruit of two oriental melon varieties, Korean landrace (KM) and Breeding line of NongWoo Bio Co. (NW), respectively. From the raw reads, 64,998 transcripts from KM and 100,234 transcripts from NW were de novo assembled. The assembled transcripts were used to identify molecular markers (e.g., single-nucleotide polymorphisms and simple sequence repeats), detect tissue-specific expressed genes, and construct a genetic linkage map. In total, 234 single-nucleotide polymorphisms and 25 simple sequence repeats were screened from 7,871 and 8,052 candidates, respectively, between the KM and NW varieties and used for construction of a genetic map with 94 F2 population specimens. The genetic linkage map consisted of 12 linkage groups, and 248 markers were assigned. These transcriptome and molecular marker data provide information useful for molecular breeding of oriental melon and further comparative studies of the Cucurbitaceae family.