Genetic characterization of a locus responsible for low pungency using EMS-induced mutants in Capsicum annuum L.

KEY MESSAGE: The pepper mutants ('221-2-1a' and '1559-1-2h') with very low pungency were genetically characterized. The Pun4 locus, responsible for the reduced pungency of the mutant fruits, was localized to a 208 Mb region on chromosome 6. DEMF06G16460, encoding 3-ketoacyl-CoA synthase, was proposed as a strong candidate gene based on the genetic analyses of bulked segregants, DEG, and expression analyses. Capsaicinoids are unique alkaloids present in pepper (Capsicum spp.), synthesized through the condensation of by-products from the phenylpropanoid and branched-chain fatty acid pathways, and accumulating in the placenta. In this study, we characterized two allelic ethyl methanesulfonate-induced mutant lines with extremely low pungency ('221-2-1a' and '1559-1-2h'). These mutants, derived from the pungent Korean landrace 'Yuwolcho,' exhibited lower capsaicinoid content than Yuwolcho but still contained a small amount of capsaicinoid with functional capsaicinoid biosynthetic genes. Genetic crosses between the mutants and Yuwolcho or pungent lines indicated that a single recessive mutation was responsible for the low-pungency phenotype of mutant 221-2-1a; we named the causal locus Pungency 4 (Pun4). To identify Pun4, we combined genome-wide polymorphism analysis and transcriptome analysis with bulked-segregant analysis. We narrowed down the location of Pun4 to a 208-Mb region on chromosome 6 containing five candidate genes, of which DEMF06G16460, encoding a 3-ketoacyl-CoA synthase associated with branched-chain fatty acid biosynthesis, is the most likely candidate for Pun4. The expression of capsaicinoid biosynthetic genes in placental tissues in Yuwolcho and the mutant was consistent with the branched-chain fatty acid pathway playing a pivotal role in the lower pungency observed in the mutant. We also obtained a list of differentially expressed genes in placental tissues between the mutant and Yuwolcho, from which we selected candidate genes using gene co-expression analysis. In summary, we characterized the capsaicinoid biosynthesis-related locus Pun4 through integrated of genetic, genomic, and transcriptome analyses. These findings will contribute to our understanding of capsaicinoid biosynthesis in pepper.

Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

Development of virus-induced genome editing methods in Solanaceous crops.

Genome editing (GE) using CRISPR/Cas systems has revolutionized plant mutagenesis. However, conventional transgene-mediated GE methods have limitations due to the time-consuming generation of stable transgenic lines expressing the Cas9/single guide RNA (sgRNA) module through tissue cultures. Virus-induced genome editing (VIGE) systems have been successfully employed in model plants, such as Arabidopsis thaliana and Nicotiana spp. In this study, we developed two VIGE methods for Solanaceous plants. First, we used the tobacco rattle virus (TRV) vector to deliver sgRNAs into a transgenic tomato (Solanum lycopersicum) line of cultivar Micro-Tom expressing Cas9. Second, we devised a transgene-free GE method based on a potato virus X (PVX) vector to deliver Cas9 and sgRNAs. We designed and cloned sgRNAs targeting Phytoene desaturase in the VIGE vectors and determined optimal conditions for VIGE. We evaluated VIGE efficiency through deep sequencing of the target gene after viral vector inoculation, detecting 40.3% and 36.5% mutation rates for TRV- and PVX-mediated GE, respectively. To improve editing efficiency, we applied a 37°C heat treatment, which increased the editing efficiency by 33% to 46% and 56% to 76% for TRV- and PVX-mediated VIGE, respectively. To obtain edited plants, we subjected inoculated cotyledons to tissue culture, yielding successful editing events. We also demonstrated that PVX-mediated GE can be applied to other Solanaceous crops, such as potato (Solanum tuberosum) and eggplant (Solanum melongena). These simple and highly efficient VIGE methods have great potential for generating genome-edited plants in Solanaceous crops.

Exploring horticultural traits and disease resistance in Capsicum baccatum through segmental introgression lines.

KEY MESSAGE: Segmental introgression and advanced backcross lines were developed and validated as important tools for improving agronomically important traits in pepper, offering improved sensitivity in detecting quantitative trait loci for breeding. Segmental introgression lines (SILs) and advanced backcross lines (ABs) can accelerate genetics and genomics research and breeding in crop plants. This study presents the development of a complete collection of SILs and ABs in pepper using Capsicum annuum cv. 'CM334' as the recipient parent and Capsicum baccatum 'PBC81', which displays various agronomically important traits including powdery mildew and anthracnose resistance, as donor parent. Using embryo rescue to overcome abortion in interspecific crosses, and marker-assisted selection with genotyping-in-thousands by sequencing (GT-seq) to develop SILs and ABs containing different segments of the C. baccatum genome, we obtained 63 SILs and 44 ABs, covering 94.8% of the C. baccatum genome. We characterized them for traits including powdery mildew resistance, anthracnose resistance, anthocyanin accumulation, trichome density, plant architecture, and fruit morphology. We validated previously known loci for these traits and discovered new sources of variation and quantitative trait loci (QTLs). A total of 15 QTLs were identified, including four for anthracnose resistance with three novel loci, seven for plant architecture, and four for fruit morphology. This is the first complete collection of pepper SILs and ABs validated for agronomic traits and will enhance QTL detection and serve as valuable breeding resources. Further, these SILs and ABs will be useful for comparative genomics and to better understand the genetic mechanisms underlying important agronomic traits in pepper, ultimately leading to improved crop productivity and sustainability.

Development of a speed breeding protocol with flowering gene investigation in pepper (Capsicum annuum).

Pepper (Capsicum spp.) is a vegetable and spice crop in the Solanaceae family with many nutritional benefits for human health. During several decades, horticultural traits, including disease resistance, yield, and fruit quality, have been improved through conventional breeding methods. Nevertheless, cultivar development is a time-consuming process because of the long generation time of pepper. Recently, speed breeding has been introduced as a solution for shorting the breeding cycle in long-day or day-neutral field crops, but there have been only a few studies on speed breeding in vegetable crops. In this study, a speed breeding protocol for pepper was developed by controlling the photoperiod and light quality. Under the condition of a low red (R) to far-red (FR) ratio of 0.3 with an extended photoperiod (Epp) of 20 h (95 ± 0 DAT), the time to first harvest was shortened by 75 days after transplant (DAT) compared to that of the control treatment (170 ± 2 DAT), suggesting that Epp with FR light is an essential factor for flowering in pepper. In addition, we established the speed breeding system in a greenhouse with a 20 h photoperiod and a 3.8 R:FR ratio and promoted the breeding cycle of C. annuum for 110 days from seed to seed. To explain the accelerated flowering response to the Epp and supplemented FR light, genome-wide association study (GWAS) and gene expression analysis were performed. As a result of the GWAS, we identified a new flowering gene locus for pepper and suggested four candidate genes for flowering (APETALA2 (AP2), WUSCHEL-RELATED HOMEOBOX4 (WOX4), FLOWERING LOCUS T (FT), and GIGANTEA (GI)). Through expression analysis with the candidate genes, it appeared that Epp and FR induced flowering by up-regulating the flowering-promoting gene GI and down-regulating FT. The results demonstrate the effect of a combination of Epp and FR light by genetic analysis of flowering gene expression. This is the first study that verifies gene expression patterns associated with the flowering responses of pepper in a speed breeding system. Overall, this study demonstrates that speed breeding can shorten the breeding cycle and accelerate genetic research in pepper through reduced generation time.

Genetic mapping revealed that the Pun2 gene in Capsicum chacoense encodes a putative aminotransferase.

Several genes regulating capsaicinoid biosynthesis including Pun1 (also known as CS), Pun3, pAMT, and CaKR1 have been studied. However, the gene encoded by Pun2 in the non-pungent Capsicum chacoense is unknown. This study aimed to identify the Pun2 gene by genetic mapping using interspecific (C. chacoense × Capsicum annuum) and intraspecific (C. chacoense × C. chacoense) populations. QTL mapping using the interspecific F2 population revealed two major QTLs on chromosomes 3 and 9. Two bin markers within the QTL regions on two chromosomes were highly correlated with the capsaicinoid content in the interspecific population. The major QTL, Pun2_PJ_Gibbs_3.11 on chromosome 3, contained the pAMT gene, indicating that the non-pungency of C. chacoense may be attributed to a mutation in the pAMT gene. Sequence analysis revealed a 7 bp nucleotide insertion in the 8th exon of pAMT of the non-pungent C. chacoense. This mutation resulted in the generation of an early stop codon, resulting in a truncated mutant lacking the PLP binding site, which is critical for pAMT enzymatic activity. This insertion co-segregated with the pungency phenotype in the intraspecific F2 population. We named this novel pAMT allele pamt11 . Taken together, these data indicate that the non-pungency of C. chacoense is due to the non-functional pAMT allele, and Pun2 encodes the pAMT gene.

Development of selective mercury recovery technology by using iron iodide from waste sludge of non-ferrous metal smelting process.

The waste sludge from non-ferrous metal smelter contains high concentrations of mercury (Hg), arsenic (As) and sulfur (S). The Article 11 of the Minamata Convention on Mercury mandates the recovery of Hg before the disposal stage of Hg waste. However, As compounds have similar boiling points with Hg compounds, and they are considered interfering substances in the recovery of Hg. Moreover, a high concentration of S requires significant energy to volatilize Hg. This study examined the optimal conditions for selective recovery of Hg and energy reduction by introducing FeI2 as an additive during thermal treatment. Thermogravimetric analysis was utilized to evaluate the conversion of HgS to HgI2 under the influence of FeI2. The optimal conditions for thermal treatment such as temperature, treatment time, and molar ratio of [Hg]:[As]:[FeI2] were explored. The simulated waste indicated that the maximum separation efficiency of Hg was ∼95%, thereby allowing a selective separation of 81.5% of Hg from waste sludge with an Hg content of 0.33%, As content of 23.8%, and S content of 30.7%. Sequential extraction procedure was applied to evaluate the stability of Hg and As for residues. As a result, most Hg was vaporized and As was stabilized in sulfide, crystalline, and amorphous forms.

Comparison of the efficiency of metal recovery from wet- and dry-discharged municipal solid waste incineration bottom ash by air table sorting and milling.

Separating high-density fractions from municipal solid waste incineration bottom ash (BA) is a promising approach for collecting metal resources and harmful elements. Herein, the efficiency of the recovery of metals (Cu, Zn, Au, Ag, and Pb) in the high-density fractions of two air-dried BAs discharged from wet-based systems (WBA), was compared with that of two BAs discharged from dry-based systems (DBA). WBAs and DBAs (<8 mm) were sorted using sieving, magnetic separation, milling, and air table sorting techniques. Newton's separation efficiency was used to evaluate three scenarios that combined various sorting processes. WBA contained high-density fractions (D1, 2.73-3.63 g/cm3) with a density similar to that of D1 in DBA (2.68-3.67 g/cm3), but the surface of the particles was covered by more minerals. When D1, and the metal particles were recovered after the milling of the low-density fractions, the overall separation efficiency in WBA (0.30-0.40) was lower than that in DBA (0.49) because of the lower separation efficiency for Zn and Pb compared to that for the other target metals. In contrast, no significant difference was recorded in the separation efficiency for Cu, Au, and Ag between WBA and DBA. D1 (18000-132000 mg-Cu/kg) and metal particles (29900-199800 mg-Cu/kg) in both WBA and DBA recorded abundant Cu contents. Moreover, the Au content in some D1 was similar to that found in natural ores. The elemental contents and separation efficiency results indicated that D1 and metal particles should be collected from both WBA and DBA.

Impact of diatomite addition on lead immobilization in air pollution control residues from a municipal solid waste incinerator.

Air pollution control (APC) residues, which are known to be the byproducts of incineration treatment, exhibit a high leaching potential of toxic metals. Calcium silicate hydrate (C-S-H), which is a major hydration product of hardened cement and immobilizes toxic metal, can be formed by the reaction of Ca with pozzolanic Si in a highly alkaline environment. Toxic metals might be immobilized by the addition of pozzolanic material to APC residues (instead of using cement), which is a Ca source and provides an alkaline condition. In this study, diatomite, which mainly comprises amorphous silica (SiO2·nH2O), was investigated as a pozzolanic material for Pb immobilization in APC residues obtained from a municipal solid waste incinerator. APC residues were cured with and without the addition of diatomite at different temperatures. When diatomite was added to APC residues, pozzolanic phases such as C-S-H gel were formed via the consumption of Ca(OH)2 and CaClOH. Compared to APC residues cured without diatomite, the leaching of Pb decreased by 99% for APC residues cured for 14 days with 10% diatomite at 70 °C. The results of sequential chemical extraction showed that water-soluble Pb in APC residues was reduced from 10.3% to nearly zero by the pozzolanic reaction. Consequently, the leaching amount of Pb dropped below 0.3 mg/L (Japanese criteria for landfill disposal). Overall, these experiments provide promising results regarding the possibility of using diatomite for pretreating APC residues.

Distribution of recoverable metal resources and harmful elements depending on particle size and density in municipal solid waste incineration bottom ash from dry discharge system.

Although municipal solid waste incineration bottom ash (BA) has the potential to be used as a metal resource, it raises concerns about the potential release of harmful elements into the environment. Element distribution in terms of particle size and density should be assessed to determine the fractions for the metal resources' recovery and to remove harmful elements. For this purpose, this study proposed a series of sorting processes based on the distribution of 25 elements in the sorted fractions by sieving, magnetic separation, air table sorting, and milling from dry BA < 8 mm. The Ca, Na, Mg, P, S, Cl, and Ti contents exhibited a decreasing tendency with increasing particle density and could affect the formation of low-density particles. The highest density fraction of non-magnetic components of 0.5-8 mm had abundant metal particles and recorded high Cu, Zn, Cr, Ni, Mo, Fe, Pb, Sb, and Au contents. In particular, the Cu (132000 mg-Cu/kg) and Zn (43000 mg-Zn/kg) contents demonstrated potential as metal resources. The fraction contained considerable proportions of Mo (77%), Cd (46%), Cu (39%), Zn (34%), Pb (26%), Au (40%), and Ag (18%) of the total amount. After milling and sieving of the highest density fraction, a substantial amount of Cd (44%), Cu (18%), Zn (12%), Pb (13%), and Ag (11%) were found in residual minerals; they could become harmful elements when recycled for construction purposes. The results show that air table sorting can separate metal resources and harmful elements before milling of BA.

Determination of metal-abundant high-density particles in municipal solid waste incineration bottom ash by a series of processes: Sieving, magnetic separation, air table sorting, and milling.

Valuable non-ferrous (NFe) metals are contained in municipal solid waste incineration (MSWI) bottom ash (BA). The applicability of an air table for separating NFe metal-abundant particles in MSWI BA (<8 mm) was studied. A stepwise separation procedure was developed based on the performance tests of the air table conducted by changing three variables (air injection, vibration, and end slope). As a result of the stepwise separation, six bulk density ranges (<0.7 to >1.1 g/cm3 at intervals of 0.1 g/cm3) were prepared from non-magnetic fractions with four size ranges (4-8, 2-4, 1-2, and 0.5-1 mm). The effectiveness of air table sorting was evaluated based on dry particle density and proportion of metals. NFe metals were obtained by sieving (>0.5 mm) after a ball mill process, confirming that NFe metals accounted for 3.2% of the < 8 mm MSWI BA. The highest particle density of each size fraction and the proportion of NFe metals in the fractions were 3.19 g/cm3 and 59.9% (4-8 mm), 2.97 g/cm3 and 28.8% (2-4 mm), 2.78 g/cm3 and 10.4% (1-2 mm), and 2.87 g/cm3 and 4.4% (0.5-1 mm). In the 0.5-2 mm particles, the highest density fraction contained 68.7% of the NFe metals, which were expected to be recovered by applying an air table separator to MSWI BA 0.5-2 mm.

Effect of substitution reaction with tin chloride in thermal treatment of mercury contaminated tailings.

Sites contaminated by mercury (Hg) from artisanal small-scale gold mine tailings have been found near agricultural land. For the active implementation of the Minamata Convention on Mercury, development of technology for the remediation of Hg contaminated sites is required. This study examined the conditions for the thermal treatment of Hg contaminated tailings at reduced temperature by introducing SnCl2 as an additive. Thermogravimetric analysis (TGA) was used to identify the possibility of converting typical Hg compounds (HgO, HgS) in the environment to HgCl2. The operation conditions for thermal treatment such as temperature, retention time, and ratio of [Cl2]/[Hg] were derived from lab scale experiments using commercial Hg compounds (HgO, HgS), additive (SnCl2), and tailings. The tailings with Hg content of 26.39 mg-Hg/kg were reduced to 3.87 mg-Hg/kg and 4.57 µg-g/L of leaching concentration through the application of the Korea standard leaching test. Both concentrations were below the standard limit of soil pollution and hazardous waste classification criteria. The sequential extraction procedure was applied to evaluate the Hg stability of residual tailings. The results show that this method will be effective for remediation of small scale Hg contaminated areas.

Development and Characterization of an Ethyl Methane Sulfonate (EMS) Induced Mutant Population in Capsicum annuum L.

Plant breeding explores genetic diversity in useful traits to develop new, high-yielding, and improved cultivars. Ethyl methane sulfonate (EMS) is a chemical widely used to induce mutations at loci that regulate economically essential traits. Additionally, it can knock out genes, facilitating efforts to elucidate gene functions through the analysis of mutant phenotypes. Here, we developed a mutant population using the small and pungent ornamental Capsicum annuum pepper "Micro-Pep". This accession is particularly suitable for mutation studies and molecular research due to its compact growth habit and small size. We treated 9500 seeds with 1.3% EMS and harvested 3996 M2 lines. We then selected 1300 (32.5%) independent M2 families and evaluated their phenotypes over four years. The mutants displayed phenotypic variations in plant growth, habit, leaf color and shape, and flower and fruit morphology. An experiment to optimize Targeting Induced Local Lesions IN Genomes (TILLING) in pepper detected nine EMS-induced mutations in the eIF4E gene. The M2 families developed here exhibited broad phenotypic variation and should be valuable genetic resources for functional gene analysis in pepper molecular breeding programs using reverse genetics tools, including TILLING.

The effect of NaOH for the recovery of elemental mercury from simulated mixture wastes and waste sludge from an industrial process using a thermal desorption process.

The need for appropriate management of mercury (Hg) wastes is increasing for active implementation of the Minamata Convention on Mercury. Though Hg can be a contaminant if it is not dealt with properly, recovered Hg can become a resource. Besides, a recovered Hg with reduced volume can be managed efficiently. This study examined the effect of NaOH for the recovery of elemental Hg from a waste sludge from an industrial process using the thermal desorption and condensation. For this purpose, the operating conditions, including temperature and pressure of the apparatus, were derived based on the experiments using Hg compounds (HgS and HgO), simulated waste (mixtures of HgCl2/As2O3 and HgS/As2O3). The reduced chamber pressure promoted to the recovery of elemental Hg via the thermal desorption. NaOH was introduced to increase the recovery efficiency of Hg in the presence of interfering substances such as S and As compounds. The Hg recovery efficiency increased, and 62.5% of Hg was recovered as elemental form by adding NaOH via thermal desorption and gas condensation with a lab-scale apparatus. Interfering substances such as Cl, S, and As compounds were captured in the bottom ash when bound with Na.

Correlation between mercury content and leaching characteristics in waste phosphor powder from spent UV curing lamp after thermal treatment.

This study evaluated the correlation between the amount of mercury (Hg) compounds in waste phosphor powder from spent UV curing lamps and their leaching characteristics. The appropriate thermal treatment conditions and Hg content in the residue necessary to satisfy the leaching criteria for classification as non-hazardous waste were identified. The decomposition of Hg compounds by thermal treatment was also evaluated by comparing sequential extraction results based on thermal stability and leaching potential of Hg compounds. Before the thermal treatment, the Hg content in waste phosphor powder and concentration in the leaching extract were 108.7 mg-Hg/kg and 0.56 mg-Hg/L, respectively. Hg compounds with low thermal stability were removed rapidly during the initial stage of thermal treatment at temperatures between 400 °C and 600 °C. After thermal treatment, Hg in the form of an intermetallic compound, such as Sr-Hg, was expected to be remained mainly, and the Hg content was reduced to 13 mg-Hg/kg in the waste phosphor powder, at that point the residue satisfied the leaching standard limit (5 µg-Hg/L) for non-hazardous waste stipulated in the legislation of Republic of Korea.

Application of powdered activated carbon coating to fabrics in a hybrid filter to enhance mercury removal.

Elemental mercury (Hg0) is predominant constituent of flue gas emitted from coal-fired power plants. Adsorption has been considered the best available technology for removal of Hg0 from flue gas. However, adsorbent injection increases the amount of ash generated. In the present study, powdered activated carbon (PAC) was coated on polytetrafluoroethylene/glass fiber filters to increase Hg0 removal while concurrently reducing the amount of ash generated. The optimal PAC coating rate was determined in laboratory experiments to ensure better Hg0 removal with low pressure drop. When PAC of particle size less than 45 µm was used, and the areal density was 50 g/m2, the pressure drop remained under 30 Pa while the Hg0 removal efficiency increased to 15.8% from 4.3%. The Hg0 removal efficiency also increased with decrease in filtration velocity. The optimal PAC coating rate was applied on a hybrid filter (HF), which was combined with a bag filter and an electrostatic precipitator in a single chamber. Originally designed to remove fine particulates matter, it was retrofitted to the flue gas control device for simultaneous Hg0 removal. By employing the PAC coating, the Hg removal efficiency of the HF increased to 79.79% from 66.35%. Also, a temporary reduction in Hg removal was seen but this was resolved following a cleaning cycle in which the dust layer was removed.

The simultaneous capture of mercury and fine particles by hybrid filter with powder activated carbon injection.

The hybrid filter (HF) was newly designed and operated with powder activated carbon (PAC) injection to capture mercury and fine particulate matter in the coal power plant. With PAC injection in HF operation, the capture efficiency of elemental mercury was clearly enhanced. When the injection rate of PAC increased from 0 to 20 mg/m3, the speciation fraction of elemental mercury significantly decreased from 85.19% to 3.76% at the inlet of the hybrid filter. The speciation fraction of oxidized mercury did not vary greatly, whereas the particulate mercury increased from 1.31% to 94.04%. It was clearly observed that the HF played a role in the capture of mercury and fine PM by leading the conversion of elemental mercury as particulate mercury and the growth of PM via electrode discharge in the HF operation with PAC injection.

Mercury distribution characteristics in primary manganese smelting plants.

The mercury (Hg) distribution characteristics were investigated in three primary manganese smelting plants in Korea for the assessment of anthropogenic Hg released. Input and output materials were sampled from each process, and Hg concentrations in the samples were analyzed. Among the input materials, the most mercury was found in the manganese ore (83.1-99.7%) and mercury was mainly released through fly ash or off gas, depending on the condition of off gas cleaning system. As off gas temperature decreases, proportion and concentration of emitted gaseous elemental mercury (Hg0) in off gas decreases. Based on mass balance study from these three plants and national manganese production data, the total amount of mercury released from those Korean plants was estimated to 644 kg/yr. About half of it was emitted into the air while the rest was released to waste as fly ash. With the results of this investigation, national inventory for Hg emission and release could be updated for the response to Minamata Convention on Mercury.