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15 pages, 3739 KiB

Open AccessArticle

Identification, Characterization, and Mutational Analysis of a Probable KEAP1 Ortholog in Rice (Oryza sativa L.)

by Yan-Hua Liu, Meng Jiang, Rui-Qing Li, Rasbin Basnet, Jian-Zhong Huang, Shi-Yong Song and Qing-Yao Shu

Plants 2020, 9(11), 1450; https://doi.org/10.3390/plants9111450 - 27 Oct 2020

Cited by 1 | Viewed by 2016

Abstract

The Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor E2-related factor 2 (NRF2) module is a key component in the detoxification and antioxidant system in animals, which plays crucial roles in cell homeostasis and cytoprotection, and consequently in carcinogenesis and disease development. However, this system [...] Read more.

The Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor E2-related factor 2 (NRF2) module is a key component in the detoxification and antioxidant system in animals, which plays crucial roles in cell homeostasis and cytoprotection, and consequently in carcinogenesis and disease development. However, this system seems to have diverged throughout evolution across different organisms, and the question of whether a similar system exists in plants has thus far remained unresolved. In this study, a KEAP1 ortholog was identified in rice (Oryza sativa L., OsKEAP1) and its properties were characterized via in silico and laboratory studies. To reveal OsKEAP1’s function, two knockdown mutants, oskeap1-1 and oskeap1-2, were generated by targeted mutagenesis in the 5′ untranslated region (UTR) using the CRISPR-Cas9 system. In silico analysis showed that OsKEAP1 has a Kelch-repeat domain which is identical to those of animals and a plant-specific development and cell death (DCD) domain in place of the broad-complex, tramtrack, bric-a-brac (BTB) domain found in animals. Orthologs of OsKEAP1 are present across plant species and all have the DCD domain and the Kelch-repeat domain. OsKEAP1 was proven to be localized to both the cytoplasm and nucleus, in contrast to the exclusive cytoplasm localization of animal KEAP1. Single nucleotide insertions in the 5′ UTR significantly reduced the transcription level of OsKEAP1 in the oskeap1-1 and oskeap1-2 mutants. The oskeap1 mutations greatly impaired plant growth and development, resulting in significant declines in a majority of agronomic and yield-related traits, i.e., plant height, panicle length, grain number per plant, and seed-set rate. The downregulation of OsKEAP1 increased the levels of H₂O₂, malondialdehyde, and proline while significantly decreasing the expression of two catalase genes in seedlings grown under normal and salt-stressed conditions. The changes in the above phenotypes are either positively or negatively correlated with the degree of OsKEAP1 downregulation. Altogether, we identified a probable KEAP1 ortholog in rice, revealed its unique subcellular localization, and demonstrated its important functions in vegetative and reproductive growth via regulation of the antioxidant response in plants. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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15 pages, 4141 KiB

Open AccessArticle

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

by Jaihyunk Ryu, Jae Il Lyu, Dong-Gun Kim, Jung-Min Kim, Yeong Deuk Jo, Si-Yong Kang, Jin-Baek Kim, Joon-Woo Ahn and Sang Hoon Kim

Plants 2020, 9(9), 1221; https://doi.org/10.3390/plants9091221 - 17 Sep 2020

Cited by 18 | Viewed by 4129

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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14 pages, 2139 KiB

Open AccessArticle

Discovery of a Novel Induced Polymorphism in SD1 Gene Governing Semi-Dwarfism in Rice and Development of a Functional Marker for Marker-Assisted Selection

by Shivashankar Bhuvaneswari, Subbaiyan Gopala Krishnan, Ranjith Kumar Ellur, Kunnummal Kurungara Vinod, Haritha Bollinedi, Prolay Kumar Bhowmick, Vijay Prakash Bansal, Mariappan Nagarajan and Ashok Kumar Singh

Plants 2020, 9(9), 1198; https://doi.org/10.3390/plants9091198 - 14 Sep 2020

Cited by 14 | Viewed by 3014

Abstract

The semi-dwarfing allele, sd1-d, has been widely utilized in developing high-yielding rice cultivars across the world. Originally identified from the rice cultivar Dee-Geo-Woo-Gen (DGWG), sd1-d, derived from a spontaneous mutation, has a 383-bp deletion in the SD1 gene. To date, as many [...] Read more.

The semi-dwarfing allele, sd1-d, has been widely utilized in developing high-yielding rice cultivars across the world. Originally identified from the rice cultivar Dee-Geo-Woo-Gen (DGWG), sd1-d, derived from a spontaneous mutation, has a 383-bp deletion in the SD1 gene. To date, as many as seven alleles of the SD1 gene have been identified and used in rice improvement, either with a functional single-nucleotide polymorphism (SNP), with insertion–deletions (InDels), or both. Here, we report discovery of a novel SNP in the SD1 gene from the rice genotype, Pusa 1652. Genetic analysis revealed that the inheritance of the semi-dwarfism in Pusa 1652 is monogenic and recessive, but it did not carry the sd1-d allele. However, response to exogenous gibberellic acid (GA₃) application and the subsequent bulked segregant and linkage analyses confirmed that the SD1 gene is involved in the plant height reduction in Pusa 1652. Sequencing of the SD1 gene from Pusa 1652 revealed a novel transition in exon 3 (T/A) causing a nonsense mutation at the 300th codon. The stop codon leads to premature termination, resulting in a truncated protein of OsGA20ox2 obstructing the GA₃ biosynthesis pathway. This novel recessive allele, named sd1-bm, is derived from Bindli Mutant 34 (BM34), a γ-ray induced mutant of a short-grain aromatic landrace, Bindli. BM34 is the parent of an aromatic semi-dwarf cultivar, Pusa 1176, from which Pusa 1652 is derived. The semi-dwarfing allele, sd1-bm, was further validated by developing a derived cleaved amplified polymorphic sequence (dCAPS) marker, AKS-sd1. This allele provides an alternative to the most widely used sd1-d in rice improvement programs and the functional dCAPS marker will facilitate marker-assisted introgression of the semi-dwarf trait into tall genotypes. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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10 pages, 1859 KiB

Open AccessArticle

Fast and Inexpensive Phenotyping and Genotyping Methods for Evaluation of Barley Mutant Population

by Yudai Kawamoto, Hirotaka Toda, Hiroshi Inoue, Kappei Kobayashi, Naoto Yamaoka, Takuya Araki and Takashi Yaeno

Plants 2020, 9(9), 1153; https://doi.org/10.3390/plants9091153 - 06 Sep 2020

Cited by 3 | Viewed by 2643

Abstract

To further develop barley breeding and genetics, more information on gene functions based on the analysis of the mutants of each gene is needed. However, the mutant resources are not as well developed as the model plants, such as Arabidopsis and rice. Although [...] Read more.

To further develop barley breeding and genetics, more information on gene functions based on the analysis of the mutants of each gene is needed. However, the mutant resources are not as well developed as the model plants, such as Arabidopsis and rice. Although genome editing techniques have been able to generate mutants, it is not yet an effective method as it can only be used to transform a limited number of cultivars. Here, we developed a mutant population using ‘Mannenboshi’, which produces good quality grains with high yields but is susceptible to disease, to establish a Targeting Induced Local Lesions IN Genomes (TILLING) system that can isolate mutants in a high-throughput manner. To evaluate the availability of the prepared 8043 M₃ lines, we investigated the frequency of mutant occurrence using a rapid, visually detectable waxy phenotype as an indicator. Four mutants were isolated and single nucleotide polymorphisms (SNPs) were identified in the Waxy gene as novel alleles. It was confirmed that the mutations could be easily detected using the mismatch endonuclease CELI, revealing that a sufficient number of mutants could be rapidly isolated from our TILLING population. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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11 pages, 1794 KiB

Open AccessArticle

Epigenetic Variation Induced by Gamma Rays, DNA Methyltransferase Inhibitors, and Their Combination in Rice

by Sung-Il Lee, Jae Wan Park, Soon-Jae Kwon, Yeong Deuk Jo, Min Jeong Hong, Jin-Baek Kim and Hong-Il Choi

Plants 2020, 9(9), 1088; https://doi.org/10.3390/plants9091088 - 24 Aug 2020

Cited by 2 | Viewed by 2296

Abstract

DNA methylation plays important roles in the regulation of gene expression and maintenance of genome stability in many organisms, including plants. In this study, we treated rice with gamma rays (GRs) and DNA methyltransferase inhibitors (DNMTis) to induce variations in DNA methylation and [...] Read more.

DNA methylation plays important roles in the regulation of gene expression and maintenance of genome stability in many organisms, including plants. In this study, we treated rice with gamma rays (GRs) and DNA methyltransferase inhibitors (DNMTis) to induce variations in DNA methylation and evaluated epigenetic diversity using methylation-sensitive amplified polymorphism (MSAP) and transposon methylation display (TMD) marker systems. Comparative and integrated analyses of the data revealed that both GRs and DNMTis alone have epimutagenic effects and that combined treatment enhanced these effects. Calculation of methylation rates based on band scoring suggested that both GRs and DNMTis induce epigenetic diversity by demethylation in a dose-dependent manner, and combined treatment can induce variations more synergistically. The difference in the changes in full and hemi-methylation rates between MSAP and TMD is presumed to be caused by the different genomic contexts of the loci amplified in the two marker systems. Principal coordinate, phylogenic, and population structure analyses commonly yielded two clusters of individuals divided by DNMTi treatment. The clustering pattern was more apparent in TMD, indicating that DNMTis have a stronger effect on hypermethylated repetitive regions. These findings provide a foundation for understanding epigenetic variations induced by GRs and DNMTis and for epigenetic mutation breeding. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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16 pages, 1170 KiB

Open AccessArticle

The Selection of Gamma-Ray Irradiated Higher Yield Rice Mutants by Directed Evolution Method

by Hiroshi Kato, Feng Li and Akemi Shimizu

Plants 2020, 9(8), 1004; https://doi.org/10.3390/plants9081004 - 07 Aug 2020

Cited by 8 | Viewed by 3233

Abstract

We have succeeded in selecting four higher yield mutants from five gamma-ray irradiated high-yielding Japanese rice varieties using a novel approach. A total of 464 M₂ plants which had heavier total panicle weights per plant were first selected from 9801 irradiated M [...] Read more.

We have succeeded in selecting four higher yield mutants from five gamma-ray irradiated high-yielding Japanese rice varieties using a novel approach. A total of 464 M₂ plants which had heavier total panicle weights per plant were first selected from 9801 irradiated M₂ plants. Their higher yields were confirmed by yield trials conducted for three years with a six to ten-pairwise replicated plot design. FukuhibikiH6 and FukuhibikiH8 were selected from an irradiated high-yielding variety Fukuhibiki and showed 1.2% to 22.5% higher yield than their original significantly. YamadawaraH3 was selected from an irradiated high-yielding variety Yamadawara and its yield advantages were 2.7% to 3.9%. However, there was no difference in the genotypes of the 96 SNP (single nucleotide polymorphism) markers between the higher yield mutants and their respective original varieties. The differences in the measured phenotypical traits between each mutant and its original variety were not constant and the actual differences were marginal. Therefore, the higher yields of the selected mutants were likely to have been caused by physiological traits rather than phenotypical traits. The selection method used in this study is an application of the directed evolution method which has long been commonly used in the substantial improvements of microorganisms and their proteins. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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11 pages, 2230 KiB

Open AccessCommunication

Digital Absolute Gene Expression Analysis of Essential Starch-Related Genes in a Radiation Developed Amaranthus cruentus L. Variety in Comparison with Real-Time PCR

by Veronika Lancíková and Andrea Hricová

Plants 2020, 9(8), 966; https://doi.org/10.3390/plants9080966 - 30 Jul 2020

Cited by 7 | Viewed by 3221

Abstract

We investigated the expression pattern of four major starch genes at different seed developmental stages in the radiation-bred amaranth variety “Pribina” (Amaranthus cruentus L.) and corresponding control genotype “Ficha” (Amaranthus cruentus L.). Two platforms were used and compared for the gene [...] Read more.

We investigated the expression pattern of four major starch genes at different seed developmental stages in the radiation-bred amaranth variety “Pribina” (Amaranthus cruentus L.) and corresponding control genotype “Ficha” (Amaranthus cruentus L.). Two platforms were used and compared for the gene expression analysis of GBSSI, SSSI, SBE, and DBE amaranth genes, including a standard quantitative real-time PCR (qPCR) technique and relatively novel droplet digital PCR (ddPCR) assay. In our conditions, both methods showed great accuracy and revealed higher expression of the investigated genes in the mutant variety than in the control genotype. Here we report for the first time, a ddPCR gene expression assay for the cultivated grain amaranth, as the most important group of the species in the genus Amaranthus. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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15 pages, 802 KiB

Open AccessArticle

The Effect of Single and Combined Use of Gamma Radiation and Ethylmethane Sulfonate on Early Growth Parameters in Sorghum

by Maliata Athon Wanga, Hussein Shimelis, Lydia N. Horn and Fatma Sarsu

Plants 2020, 9(7), 827; https://doi.org/10.3390/plants9070827 - 30 Jun 2020

Cited by 20 | Viewed by 3171

Abstract

Success in inducing genetic variation through mutagenic agents is dependent on the source and dose of application. The objective of this study was to determine the optimum doses of a single and combined use of gamma radiation and ethylmethane sulfonate (EMS) for effective [...] Read more.

Success in inducing genetic variation through mutagenic agents is dependent on the source and dose of application. The objective of this study was to determine the optimum doses of a single and combined use of gamma radiation and ethylmethane sulfonate (EMS) for effective mutation breeding in sorghum. The study involved two concurrent experiments as follows: in experiment I, the seeds of four sorghum genotypes (‘Parbhani Moti’, ‘Parbhani Shakti’, ‘ICSV 15013′, and ‘Macia’) were treated using gamma radiation (0, 300, 400, 500 and 600 Gy), EMS (0, 0.5 and 1.0%), and gamma radiation followed by EMS (0 and 300 Gy and 0.1% EMS; 400 Gy and 0.05% EMS). In experiment II, the seeds of two genotypes (‘Macia’ and ‘Red sorghum’) were treated with seven doses of gamma radiation only (0, 100, 200, 300, 400, 500 and 600 Gy). Overall, the combined applied doses of gamma radiation and EMS are not recommended due to poor seedling emergence and seedling survival rate below LD₅₀. The best dosage of gamma radiation for genotypes Red sorghum, Parbhani Moti, Macia, ICSV 15013 and Parbhani Shakti ranged between 392 and 419 Gy, 311 and 354 Gy, 256 and 355 Gy, 273 and 304 Gy, and 266 and 297 Gy, respectively. The EMS optimum dosage ranges for genotypes Parbhani Shakti, ICSV 15013, Parbhani Moti and Macia were between 0.41% and 0.60%, 0.48% and 0.58%, 0.46% and 0.51%, and 0.36% and 0.45%, respectively. The above dose rates are useful to induce genetic variation in the tested sorghum genotypes for greater mutation events in sorghum breeding programs. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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9 pages, 1022 KiB

Open AccessEditor’s ChoiceArticle

Composition and Antioxidant Activities of Volatile Organic Compounds in Radiation-Bred Coreopsis Cultivars

by Bo-Ram Kim, Hyun Mi Kim, Chang Hyun Jin, Si-Yong Kang, Jin-Baek Kim, Yeo Gyeong Jeon, Kong Young Park, Ik-Soo Lee and Ah-Reum Han

Plants 2020, 9(6), 717; https://doi.org/10.3390/plants9060717 - 04 Jun 2020

Cited by 45 | Viewed by 2917

Abstract

Coreopsis is a flowering plant belonging to the Asteraceae family. It is an ornamental plant native to the Americas, Asia and Oceania and its flower is used as a raw material for tea and food manufacture in China. In this study, new cultivars [...] Read more.

Coreopsis is a flowering plant belonging to the Asteraceae family. It is an ornamental plant native to the Americas, Asia and Oceania and its flower is used as a raw material for tea and food manufacture in China. In this study, new cultivars of C. rosea (“golden ring”) were developed via radiation-induced mutation of the original cultivar, “pumpkin pie”. The chemical composition and antioxidant activities of flowers belonging to three different Coreopsis cultivars were evaluated: “golden ring”, “pumpkin pie” and “snow chrysanthemum” (coreopsis tea; C. tinctoria). The volatile compounds were characterized via gas chromatography-mass spectrometry (GC-MS) and 50–59 oils representing 95.3–96.8% of the total volatile compounds in these flower materials were identified. ”Golden ring” contained a high amount of fatty acids (38.13%), while “pumpkin pie” and “snow chrysanthemum” teas were rich in aliphatic amides (43.01%) and esters (67.22%), respectively. The antioxidant activities of the volatile oils of these cultivars were evaluated using 1,1-diphenyl-2-picrylhydraxyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assays. The volatile extract of “golden ring” showed higher antioxidant activities compared with the extracts of the other cultivars. Therefore, “golden ring” can be used for further development as a raw material for tea manufacture or as a dietary supplement. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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13 pages, 1427 KiB

Open AccessArticle

High-Resolution Mapping of the Novel Early Leaf Senescence Gene Els2 in Common Wheat

by Na Wang, Yanzhou Xie, Yingzhuang Li, Shengnan Wu, Shuxian Li, Yu Guo and Chengshe Wang

Plants 2020, 9(6), 698; https://doi.org/10.3390/plants9060698 - 30 May 2020

Cited by 8 | Viewed by 2478

Abstract

Early leaf senescence negatively impacts the grain yield in wheat (Triticum aestivum L.). Induced mutants provide an important resource for mapping and cloning of genes for early leaf senescence. In our previous study, Els2, a single incomplete dominance gene, that caused [...] Read more.

Early leaf senescence negatively impacts the grain yield in wheat (Triticum aestivum L.). Induced mutants provide an important resource for mapping and cloning of genes for early leaf senescence. In our previous study, Els2, a single incomplete dominance gene, that caused early leaf senescence phenotype in the wheat mutant LF2099, had been mapped on the long arm of chromosome 2B. The objective of this study was to develop molecular markers tightly linked to the Els2 gene and construct a high-resolution map surrounding the Els2 gene. Three tightly linked single-nucleotide polymorphism (SNP) markers were obtained from the Illumina Wheat 90K iSelect SNP genotyping array and converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers. To saturate the Els2 region, the Axiom® Wheat 660K SNP array was used to screen bulked extreme phenotype DNA pools, and 9 KASP markers were developed. For fine mapping of the Els2 gene, these KASP markers and previously identified polymorphic markers were analyzed in a large F₂ population of the LF2099 × Chinese Spring cross. The Els2 gene was located in a 0.24-cM genetic region flanked by the KASP markers AX-111643885 and AX-111128667, which corresponded to a physical interval of 1.61 Mb in the Chinese Spring chromosome 2BL containing 27 predicted genes with high confidence. The study laid a foundation for a map-based clone of the Els2 gene controlling the mutation phenotype and revealing the molecular regulatory mechanism of wheat leaf senescence. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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14 pages, 2861 KiB

Open AccessArticle

Characterization of Gene Isoforms Related to Cellulose and Lignin Biosynthesis in Kenaf (Hibiscus cannabinus L.) Mutant

by Jae Il Lyu, Rahul Ramekar, Dong-Gun Kim, Jung Min Kim, Min-Kyu Lee, Nguyen Ngoc Hung, Jin-Baek Kim, Joon-Woo Ahn, Si-Yong Kang, Ik-Young Choi, Kyoung-Cheul Park and Soon-Jae Kwon

Plants 2020, 9(5), 631; https://doi.org/10.3390/plants9050631 - 14 May 2020

Cited by 4 | Viewed by 3424

Abstract

Kenaf is a source of fiber and a bioenergy crop that is considered to be a third world crop. Recently, a new kenaf cultivar, "Jangdae," was developed by gamma irradiation. It exhibited distinguishable characteristics such as higher biomass, higher seed yield, and earlier [...] Read more.

Kenaf is a source of fiber and a bioenergy crop that is considered to be a third world crop. Recently, a new kenaf cultivar, "Jangdae," was developed by gamma irradiation. It exhibited distinguishable characteristics such as higher biomass, higher seed yield, and earlier flowering than the wild type. We sequenced and analyzed the transcriptome of apical leaf and stem using Pacific Biosciences single-molecule long-read isoform sequencing platform. De novo assembly yielded 26,822 full-length transcripts with a total length of 59 Mbp. Sequence similarity against protein sequence allowed the functional annotation of 11,370 unigenes. Among them, 10,100 unigenes were assigned gene ontology terms, the majority of which were associated with the metabolic and cellular process. The Kyoto encyclopedia of genes and genomes (KEGG) analysis mapped 8875 of the annotated unigenes to 149 metabolic pathways. We also identified the majority of putative genes involved in cellulose and lignin-biosynthesis. We further evaluated the expression pattern in eight gene families involved in lignin-biosynthesis at different growth stages. In this study, appropriate biotechnological approaches using the information obtained for these putative genes will help to modify the desirable content traits in mutants. The transcriptome data can be used as a reference dataset and provide a resource for molecular genetic studies in kenaf. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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15 pages, 1773 KiB

Open AccessArticle

High-Throughput Phenotyping (HTP) Data Reveal Dosage Effect at Growth Stages in Arabidopsis thaliana Irradiated by Gamma Rays

by Sungyul Chang, Unseok Lee, Min Jeong Hong, Yeong Deuk Jo and Jin-Baek Kim

Plants 2020, 9(5), 557; https://doi.org/10.3390/plants9050557 - 27 Apr 2020

Cited by 15 | Viewed by 4057

Abstract

The effects of radiation dosages on plant species are quantitatively presented as the lethal dose or the dose required for growth reduction in mutation breeding. However, lethal dose and growth reduction fail to provide dynamic growth behavior information such as growth rate after [...] Read more.

The effects of radiation dosages on plant species are quantitatively presented as the lethal dose or the dose required for growth reduction in mutation breeding. However, lethal dose and growth reduction fail to provide dynamic growth behavior information such as growth rate after irradiation. Irradiated seeds of Arabidopsis were grown in an environmentally controlled high-throughput phenotyping (HTP) platform to capture growth images that were analyzed with machine learning algorithms. Analysis of digital phenotyping data revealed unique growth patterns following treatments below LD₅₀ value at 641 Gy. Plants treated with 100-Gy gamma irradiation showed almost identical growth pattern compared with wild type; the hormesis effect was observed >21 days after sowing. In 200 Gy-treated plants, a uniform growth pattern but smaller rosette areas than the wild type were seen (p < 0.05). The shift between vegetative and reproductive stages was not retarded by irradiation at 200 and 300 Gy although growth inhibition was detected under the same irradiation dose. Results were validated using 200 and 300 Gy doses with HTP in a separate study. To our knowledge, this is the first study to apply a HTP platform to measure and analyze the dosage effect of radiation in plants. The method enabled an in-depth analysis of growth patterns, which could not be detected previously due to a lack of time-series data. This information will improve our knowledge about the effects of radiation in model plant species and crops. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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17 pages, 3910 KiB

Open AccessArticle

Mutagenic Effect of Three Ion Beams on Rice and Identification of Heritable Mutations by Whole Genome Sequencing

by Yunchao Zheng, Shan Li, Jianzhong Huang, Haowei Fu, Libin Zhou, Yoshiya Furusawa and Qingyao Shu

Plants 2020, 9(5), 551; https://doi.org/10.3390/plants9050551 - 26 Apr 2020

Cited by 15 | Viewed by 3618

Abstract

High-energy ion beams are known to be an effective and unique type of physical mutagen in plants. However, no study on the mutagenic effect of argon (Ar) ion beam radiation on rice has been reported. Genome-wide studies on induced mutations are important to [...] Read more.

High-energy ion beams are known to be an effective and unique type of physical mutagen in plants. However, no study on the mutagenic effect of argon (Ar) ion beam radiation on rice has been reported. Genome-wide studies on induced mutations are important to comprehend their characteristics for establishing knowledge-based protocols for mutation induction and breeding, which are still very limited in rice. The present study aimed to investigate the mutagenic effect of three ion beams, i.e., Ar, carbon (C) and neon (Ne) on rice and identify and characterize heritable induced mutations by the whole genome sequencing of six M₄ plants. Dose-dependent damage effects were observed on M₁ plants, which were developed from ion beam irradiated dry seeds of two indica (LH15, T23) and two japonica (DS551, DS48) rice lines. High frequencies of chlorophyll-deficient seedlings and male-sterile plants were observed in all M₂ populations (up to ~30% on M₁ plant basis); plants from the seeds of different panicles of a common M₁ plant appeared to have different mutations; the whole genome-sequencing demonstrated that there were 236–453 mutations in each of the six M₄ plants, including single base substitutions (SBSs) and small insertion/deletions (InDels), with the number of SBSs ~ 4–8 times greater than that of InDels; SBS and InDel mutations were distributed across different genomic regions of all 12 chromosomes, however, only a small number of mutations (0–6) were present in exonic regions that might have an impact on gene function. In summary, the present study demonstrates that Ar, C and Ne ion beam radiation are all effective for mutation induction in rice and has revealed at the genome level the characteristics of the mutations induced by the three ion beams. The findings are of importance to the efficient use of ion beam radiation for the generation and utilization of mutants in rice. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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11 pages, 1245 KiB

Open AccessArticle

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

by Sang Hoon Kim, Se Won Kim, Joon-Woo Ahn, Jaihyunk Ryu, Soon-Jae Kwon, Byoung-Cheorl Kang and Jin-Baek Kim

Plants 2020, 9(4), 546; https://doi.org/10.3390/plants9040546 - 23 Apr 2020

Cited by 11 | Viewed by 3320

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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12 pages, 2685 KiB

Open AccessCommunication

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

by Sang Hoon Kim, Se Won Kim, Jaihyunk Ryu, Si-Yong Kang, Byoung-Cheorl Kang and Jin-Baek Kim

Plants 2020, 9(4), 532; https://doi.org/10.3390/plants9040532 - 20 Apr 2020

Cited by 7 | Viewed by 2952

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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13 pages, 2768 KiB

Open AccessArticle

Colchicine-Induced Polyploidy in Rhododendron fortunei Lindl

by Lan Mo, Junhao Chen, Xiongzhen Lou, Qiangwei Xu, Renhui Dong, Zaikang Tong, Huahong Huang and Erpei Lin

Plants 2020, 9(4), 424; https://doi.org/10.3390/plants9040424 - 31 Mar 2020

Cited by 33 | Viewed by 5333

Abstract

Polyploidy in Rhododendron fortunei has great potential to improve its horticultural and commercial value, and to also meet market demands. In this study, a feasible method for polyploid induction in R. fortunei via colchicine treatment was established, and the obtained polyploid plants were [...] Read more.

Polyploidy in Rhododendron fortunei has great potential to improve its horticultural and commercial value, and to also meet market demands. In this study, a feasible method for polyploid induction in R. fortunei via colchicine treatment was established, and the obtained polyploid plants were identified and characterized. As a result, the stem bases of tissue-cultured plantlets treated with 0.1% colchicine for 24 h showed the highest polyploid induction with a rate of 36.67%. By flow cytometric analysis, 69 tetraploids and 29 octoploids were identified in the regenerated plants that were examined. Phenotypic analysis indicated that the leaves of tetraploid and octoploid plants were smaller, rounder and thicker with more abundant and longer epidermal hairs than those of diploids. Furthermore, the stomata of polyploids were larger and sparser than those of diploids. An increase in chlorophyll content was also detected in polyploids, which resulted in darker green leaves. In conclusion, our study established an effective method to induce polyploidy in R. fortunei, which could be used to develop new genetic resources for breeding R. fortunei and other Rhododendron species in the future. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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16 pages, 4468 KiB

Open AccessFeature PaperArticle

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

by Muhammad Irfan Siddique, Seungki Back, Joung-Ho Lee, Jinkwan Jo, Siyoung Jang, Koeun Han, Jelli Venkatesh, Jin-Kyung Kwon, Yeong Deuk Jo and Byoung-Cheorl Kang

Plants 2020, 9(3), 396; https://doi.org/10.3390/plants9030396 - 23 Mar 2020

Cited by 27 | Viewed by 9021

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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10 pages, 1397 KiB

Open AccessArticle

Mutations of OsPLDa1 Increase Lysophospholipid Content and Enhance Cooking and Eating Quality in Rice

by Muhammad Saad Shoaib Khan, Rasbin Basnet, Sulaiman Ahmed, Jinsong Bao and Qingyao Shu

Plants 2020, 9(3), 390; https://doi.org/10.3390/plants9030390 - 21 Mar 2020

Cited by 12 | Viewed by 3187

Abstract

Phospholipids belong to a significant class of lipids and comprise ~10% of total lipids in rice grains. Lysophospholipid (LPL) is derived from the hydrolysis of phospholipids and plays an important role in rice grain quality. Our previous study demonstrated that mutations in a [...] Read more.

Phospholipids belong to a significant class of lipids and comprise ~10% of total lipids in rice grains. Lysophospholipid (LPL) is derived from the hydrolysis of phospholipids and plays an important role in rice grain quality. Our previous study demonstrated that mutations in a phospholipase D gene (OsPLDα1) significantly altered lipid metabolites and reduced phytic acid content. In the present study, the effect of two ospldα1 mutations on LPL and other physicochemical prosperities of brown rice was further investigated, with the aim of assessing the overall importance of ospldα1 mutations in rice grain quality. Metabolite profiling revealed a ~15% increase in LPL level in both ospldα1 mutants as compared with their wild-type (WT) parent. Both ospldα1 mutations significantly lowered the apparent amylose content in brown rice flour (~1.9%) and altered viscosity profiles with significantly increased breakdown (+12.4%) and significantly reduced setback viscosity (−6.2%). Moreover, both ospldα1 mutations significantly lowered the gelatinization onset, peak temperature and retrogradation percentage of brown rice flour. This study demonstrated that OsPLDα1 plays a crucial role in rice grain quality and its mutation could, in general, improve the cooking and eating quality and nourishment of brown rice. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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14 pages, 3502 KiB

Open AccessArticle

Molecular Characterization and Identification of Calnexin 1 As a Radiation Biomarker from Tradescantia BNL4430

by Hye-Jeong Ha, Saminathan Subburaj, Young-Sun Kim, Jin-Baek Kim, Si-Yong Kang and Geung-Joo Lee

Plants 2020, 9(3), 387; https://doi.org/10.3390/plants9030387 - 20 Mar 2020

Cited by 3 | Viewed by 2901

Abstract

Calnexin (CNX) is an integral membrane protein that functions as a chaperone in the endoplasmic reticulum for the correct folding of proteins under stress conditions, rendering organisms tolerant under adverse conditions. Studies have investigated the cytogenetic effects of gamma irradiation (Ɣ-IR) on plants, [...] Read more.

Calnexin (CNX) is an integral membrane protein that functions as a chaperone in the endoplasmic reticulum for the correct folding of proteins under stress conditions, rendering organisms tolerant under adverse conditions. Studies have investigated the cytogenetic effects of gamma irradiation (Ɣ-IR) on plants, but information on the molecular response under Ɣ-IR remains limited. Previously, we constructed a cDNA library of an irradiation-sensitive bioindicator plant, Tradescantia BNL4430 (T-4430) under Ɣ-IR, in which the Calnexin-1 gene was highly upregulated at 50 mGy treatment. TrCNX1 encodes a 61.4 kDa protein with conserved signature motifs similar to already reported CNX1s. TrCNX1 expression was evaluated by semiquantitative reverse transcriptase PCR and quantitative real-time PCR and was ubiquitously expressed in various tissues and highly upregulated in flower petals under 50 mGy Ɣ-IR stress. The protective function of TrCNX1 was investigated by overexpression of TrCNX1 in an Escherichia coli BL21(DE3) heterologous system. Using plate assay, we showed that TrCNX1 increased the viability of E. coli transformants under both UV-B and Ɣ-IR compared with the control, demonstrating that TrCNX1 functions under irradiation stress. TrCNX1 may enhance irradiation stress tolerance in crops and act as a radio marker gene to monitor the effects of radiation. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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Review

Jump to: Research

47 pages, 3582 KiB

Open AccessReview

Next Generation Sequencing Based Forward Genetic Approaches for Identification and Mapping of Causal Mutations in Crop Plants: A Comprehensive Review

by Parmeshwar K. Sahu, Richa Sao, Suvendu Mondal, Gautam Vishwakarma, Sudhir Kumar Gupta, Vinay Kumar, Sudhir Singh, Deepak Sharma and Bikram K. Das

Plants 2020, 9(10), 1355; https://doi.org/10.3390/plants9101355 - 14 Oct 2020

Cited by 42 | Viewed by 10945

Abstract

The recent advancements in forward genetics have expanded the applications of mutation techniques in advanced genetics and genomics, ahead of direct use in breeding programs. The advent of next-generation sequencing (NGS) has enabled easy identification and mapping of causal mutations within a short [...] Read more.

The recent advancements in forward genetics have expanded the applications of mutation techniques in advanced genetics and genomics, ahead of direct use in breeding programs. The advent of next-generation sequencing (NGS) has enabled easy identification and mapping of causal mutations within a short period and at relatively low cost. Identifying the genetic mutations and genes that underlie phenotypic changes is essential for understanding a wide variety of biological functions. To accelerate the mutation mapping for crop improvement, several high-throughput and novel NGS based forward genetic approaches have been developed and applied in various crops. These techniques are highly efficient in crop plants, as it is relatively easy to grow and screen thousands of individuals. These approaches have improved the resolution in quantitative trait loci (QTL) position/point mutations and assisted in determining the functional causative variations in genes. To be successful in the interpretation of NGS data, bioinformatics computational methods are critical elements in delivering accurate assembly, alignment, and variant detection. Numerous bioinformatics tools/pipelines have been developed for such analysis. This article intends to review the recent advances in NGS based forward genetic approaches to identify and map the causal mutations in the crop genomes. The article also highlights the available bioinformatics tools/pipelines for reducing the complexity of NGS data and delivering the concluding outcomes. Full article

(This article belongs to the Special Issue Plant Mutation Breeding)

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