Single Nucleotide Polymorphism (SNP) Discovery and Association Study of Flowering Times, Crude Fat and Fatty Acid Composition in Rapeseed (Brassica napus L.) Mutant Lines Using Genotyping-by-Sequencing (GBS)

Rapeseed is the most important oil crop used in the food and biodiesel industries. In this study, based on single nucleotide polymorphism (SNP) identified from genotyping-by-sequencing (GBS), and an association study of flowering time, crude fat and fatty acid contents were investigated in 46 rapeseed mutant lines derived from gamma rays. A total of 623,026,394 clean data reads were generated with 6.6 million reads on average. A set of 37,721 filtered SNPs was used to perform gene ontology and phylogenetic analysis. Hierarchical cluster analysis of the rapeseed mutant lines gave eight groups based on flowering time and fatty acid compositions. Gene ontological analysis of the mutant lines showed that many genes displaying SNPs are involved in cellular processes, cellular anatomy, and binding. A total of 40 SNPs were significantly associated with flowering time (1 SNP), crude fat content (2 SNPs), and fatty acid content (37 SNPs). A total of 21 genes were annotated from fatty acid content SNPs; among them, nine genes were significantly enriched in reproductive processes, such as embryonic development, fruit development, and seed development. This study demonstrated that SNPs are efficient tools for mutant screening and it provides a basis that the improving the oil qualities of rapeseed

Dark/Light Treatments Followed by γ-Irradiation Increase the Frequency of Leaf-Color Mutants in Cymbidium

Radiation randomly induces chromosomal mutations in plants. However, it was recently found that the frequency of flower-color mutants could be specifically increased by upregulating anthocyanin pathway gene expression before radiation treatments. The mechanisms of chlorophyll biosynthesis and degradation are active areas of plant study because chlorophyll metabolism is closely connected to photosynthesis. In this study, we determined the dark/light treatment conditions that resulted in upregulation of the expression levels of six chlorophyll pathway genes, uroporphyrinogen III synthase (HEMD), uroporphyrinogen III decarboxylase (HEME2), NADPH-protochlorophyllide oxidoreductase (POR) A (PORA), chlorophyll synthase (CHLG), chlorophyllase (CLH2), and red chlorophyll catabolite reductase (RCCR), and measured their effects on the γ-irradiation-induced frequencies of leaf-color mutants in two Cymbidium cultivars. To degrade chlorophyll in rhizomes, 60–75 days of dark treatment were required. To upregulate the expressions of chlorophyll pathway genes, 10 days of light treatment appeared to be optimal. Dark/light treatments followed by γ-irradiation increased chlorophyll-related leaf mutants by 1.4- to 2.0-fold compared with γ-ray treatment alone. Dark/light treatments combined with γ-irradiation increased the frequency of leaf-color mutants in Cymbidium, which supports the wider implementation of a plant breeding methodology that increases the mutation frequency of a target trait by controlling the expression of target trait-related genes

Frequency, Spectrum, and Stability of Leaf Mutants Induced by Diverse γ-Ray Treatments in Two Cymbidium Hybrids

Ionizing radiation combined with in vitro tissue culture has been used for development of new cultivars in diverse crops. The effects of ionizing radiation on mutation induction have been analyzed on several orchid species, including Cymbidium. Limited information is available on the comparison of mutation frequency and spectrum based on phenotypes in Cymbidium species. In addition, the stability of induced chimera mutants in Cymbidium is unknown. In this study, we analyzed the radiation sensitivity, mutation frequency, and spectrum of mutants induced by diverse γ-ray treatments, and analyzed the stability of induced chimera mutants in the Cymbidium hybrid cultivars RB003 and RB012. The optimal γ-irradiation conditions of each cultivar differed as follows: RB003, mutation frequency of 4.06% (under 35 Gy/4 h); RB012, 1.51% (20 Gy/1 h). Re-irradiation of γ-rays broadened the mutation spectrum observed in RB012. The stability of leaf-color chimera mutants was higher than that of leaf-shape chimeras, and stability was dependent on the chimera type and location of a mutation in the cell layers of the shoot apical meristem. These results indicated that short-term γ-irradiation was more effective to induce mutations in Cymbidium. Information on the stability of chimera mutants will be useful for mutation breeding of diverse ornamental plants

Comparative Study on Phenolic Compounds and Antioxidant Activities of Hop (Humulus lupulus L.) Strobile Extracts

In this study, we investigated the phenolic compounds in hop strobile extracts and evaluated their antioxidant property using DPPH and ABTS assay. The total phenolic compound (TPC) and total flavonoid compound (TFC) estimated in two different solvent extracts considerably varied depending on the extraction solvent. The most abundant phenolic compound in hop strobile was humulones (α-acid) with levels ranging from 50.44 to 193.25 µg/g. El Dorado accession revealed higher antioxidant activity in ethanol extracts (DPPH: IC50 124.3 µg/mL; ABTS: IC50 95.4 µg/mL) when compared with that of the other accessions. Correlations between DPPH (IC50) scavenging TFC in ethanol extract (TFC_E, −0.941), and TPC_E (−0.901), and between ABTS (IC50) scavenging TFC_E (−0.853), and TPC_E (−0.826), were statistically significant at p < 0.01 level, whereas no significant correlation was observed between antioxidant activities, TPC and TFC in water extract. This study is the first to report that variations in the level of phenolic contents and antioxidant activity of various hop cultivars depended on the type of extraction solvent used and the cultivation regions. These results could provide valuable information on developing hop products

Comparative Analysis of Volatile Compounds of Gamma-Irradiated Mutants of Rose (Rosa hybrida)

Roses are one of the most important floricultural crops, and their essential oils have long been used for cosmetics and aromatherapy. We investigated the volatile compound compositions of 12 flower-color mutant variants and their original cultivars. Twelve rose mutant genotypes were developed by treatment with 70 Gy of 60Co gamma irradiation of six commercial rose cultivars. Essential oils from the flowers of the 18 genotypes were analyzed by gas chromatography–mass spectrometry. Seventy-seven volatile compounds were detected, which were categorized into six classes: Aliphatic hydrocarbons, aliphatic alcohols, aliphatic ester, aromatic compounds, terpene alcohols, and others. Aliphatic (hydrocarbons, alcohols, and esters) compounds were abundant categories in all rose flowers. The CR-S2 mutant had the highest terpene alcohols and oil content. Three (CR-S1, CR-S3, and CR-S4) mutant genotypes showed higher ester contents than their original cultivar. Nonacosane, 2-methylhexacosane, and 2-methyltricosane were major volatile compounds among all genotypes. Hierarchical cluster analysis (HCA) of the rose genotypes gave four groups according to grouping among the 77 volatile compounds. In addition, the principal component analysis (PCA) model was successfully applied to distinguish most attractive rose lines. These findings will be useful for the selection of rose genotypes with improved volatile compounds

Dramatic Increase in Content of Diverse Flavonoids Accompanied with Down-Regulation of F-Box Genes in a Chrysanthemum (Chrysanthemum × morifolium (Ramat.) Hemsl.) Mutant Cultivar Producing Dark-Purple Ray Florets

Anthocyanins (a subclass of flavonoids) and flavonoids are crucial determinants of flower color and substances of pharmacological efficacy, respectively, in chrysanthemum. However, metabolic and transcriptomic profiling regarding flavonoid accumulation has not been performed simultaneously, thus the understanding of mechanisms gained has been limited. We performed HPLC-DAD-ESI-MS (high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization mass spectrometry) and transcriptome analyses using “ARTI-Dark Chocolate” (AD), which is a chrysanthemum mutant cultivar producing dark-purple ray florets, and the parental cultivar “Noble Wine” for metabolic characterization and elucidation of the genetic mechanism determining flavonoid content. Among 26 phenolic compounds identified, three cyanidins and eight other flavonoids were detected only in AD. The total amounts of diverse flavonoids were 8.0 to 10.3 times higher in AD. Transcriptome analysis showed that genes in the flavonoid biosynthetic pathway were not up-regulated in AD at the early flower stage, implying that the transcriptional regulation of the pathway did not cause flavonoid accumulation. However, genes encoding post-translational regulation-related proteins, especially F-box genes in the mutated gene, were enriched among down-regulated genes in AD. From the combination of metabolic and transcriptomic data, we suggest that the suppression of post-translational regulation is a possible mechanism for flavonoid accumulation in AD. These results will contribute to research on the regulation and manipulation of flavonoid biosynthesis in chrysanthemum

Genotyping-by-sequencing based single nucleotide polymorphisms enabled Kompetitive Allele Specific PCR marker development in mutant Rubus genotypes

Background: Rubus is an economically important fruit crop across the globe. Recently, several Rubus mutant genotypes with improved agronomic traits have been developed using gamma ray irradiation. This study investigated genetic diversity and variations in Rubus mutant genotypes using single nucleotide polymorphism (SNP) markers generated from genotyping-by-sequencing (GBS) analysis. A GBS library of 14 Rubus genotypes, consisting of seven boysenberry mutant lines, four blackberry mutant lines, and three original varieties, were sequenced on the Illumina Hiseq2000 platform. A set of SNPs were analyzed by Kompetitive Allele Specific PCR (KASP) assay in order to discriminate the Rubus genotypes. Results: A total of 50,831,040 (86.4%) reads of clean data were generated, and the trimmed length ranged from 116,380,840 to 509,806,521 bp, with an average of 228,087,333 bp per line. A total of 19,634 high-quality SNPs were detected, which contained 11,328 homozygous SNPs and 8306 heterozygous SNPs. A set of 1504 SNPs was used to perform a phylogenetic analysis, which showed that there were clear differences among the Rubus genotypes based on their origin. A total of 25 SNPs were used for the KASP assays, of which six KASP primer sets were successfully distinguished among the Rubus genotypes. Conclusions: This study demonstrated that the SNP and KASP method is an economically efficient tool for mutant screening in Rubus breeding programs.How to cite: Ryu J, Kim WJ, Im J, et al. Genotyping-by-sequencing based single nucleotide polymorphisms enabled kompetitive allele specific PCR marker development in mutant Rubus genotypes. Electron J Biotechnol 2018;35. https://doi.org/10.1016/j.ejbt.2018.08.001. Keywords: Blackberry, Boysenberry, Cultivar identification markers, Fruit crop, Gamma irradiation, Genotyping-by-sequencing, KASP methodoloy, Mutation breeding, Phylogenetic tree, Rubus, SN

Phenolic Compounds in Extracts of Hibiscus acetosella (Cranberry Hibiscus) and Their Antioxidant and Antibacterial Properties

Hibiscus species are rich in phenolic compounds and have been traditionally used for improving human health through their bioactive activities. The present study investigated the phenolic compounds of leaf extracts from 18 different H. acetosella accessions and evaluated their biofunctional properties, focusing on antioxidant and antibacterial activity. The most abundant phenolic compound in H. acetosella was caffeic acid, with levels ranging from 14.95 to 42.93 mg/100 g. The antioxidant activity measured by the ABTS assay allowed the accessions to be classified into two groups: a high activity group with red leaf varieties (74.71–84.02%) and a relatively low activity group with green leaf varieties (57.47–65.94%). The antioxidant activity was significantly correlated with TAC (0.933), Dp3-Sam (0.932), Dp3-Glu (0.924), and Cy3-Sam (0.913) contents (p < 0.001). The H. acetosella phenolic extracts exhibited antibacterial activity against two bacteria, with zones of inhibition between 12.00 and 13.67 mm (Staphylococcus aureus), and 10.67 and 13.33 mm (Pseudomonas aeruginosa). All accessions exhibited a basal antibacterial activity level (12 mm) against the Gram-positive S. aureus, with PI500758 and PI500764 exhibiting increased antibacterial activity (13.67 mm), but they exhibited a more dynamic antibacterial activity level against the Gram-negative P. aeruginosa

Genome-Wide Association Study (GWAS) of the Agronomic Traits and Phenolic Content in Sorghum (<i>Sorghum bicolor</i> L.) Genotypes

Sorghum (Sorghum bicolor L.) is a promising biomass crop with high yields of cellulose, hemicellulose, and lignin. Sorghum biomass has emerged as an eco-friendly industrial material useful for producing biofuels and bioplastics. This study conducted genotyping-by-sequencing (GBS)-based genome-wide association studies (GWAS) to establish the genetic basis of traits associated with biomass. Specifically, the researchers evaluated agronomic traits and phenolic compounds using 96 sorghum genotypes. Six phenolic compounds, luteolinidin diglucoside, luteolin glucoside, apigeninidin glucoside, luteolinidin, apigeninidin, and 5-O-Me luteolinidin, were found to be the major phenolic compounds in all genotypes. Out of our six detected phenolic compounds (luteolinidin diglucoside, luteolin glucoside, apigeninidin glucoside, luteolinidin, apigeninidin, and 5-O-Me luteolinidin), luteolinidin was the major phenolic compound in all genotypes. Next, a GWAS analysis was performed to confirm significant associations between 192,040 filtered single-nucleotide polymorphisms (SNPs) and biomass-related traits. The study identified 40 SNPs on 10 chromosomes that were significantly associated with heading date (4 SNPs), plant height (3 SNPs), dry yield (2 SNPs), and phenolic compounds (31 SNPs). The GWAS analysis showed that SbRio.10G099600 (FUT1) was associated with heading date, SbRio.09G149200 with plant height, SbRio.06G211400 (MAFB) with dry yield, SbRio.04G259800 (PDHA1) with total phenolic content and luteolinidin diglucoside, and SbRio.02G343600 (LeETR4) with total phenolic content and luteolinidin, suggesting that these genes could play key roles in sorghum. These findings demonstrate the potential value of sorghum as a biomass resource and the potential for selecting sorghum genotypes with reduced phenolic contents for use in the bioindustry