Mutagenesis in the Age of Next-Generation-Sequencing and Genome Editing

A special issue of Plants (ISSN 2223-7747). This special issue belongs to the section "Plant Genetics, Genomics and Biotechnology".

Deadline for manuscript submissions: closed (30 June 2023) | Viewed by 27840

Special Issue Editor

USDA Agricultural Research Service, Washington, DC 20250, USA
Interests: mutation analyses in sorghum

Special Issue Information

Dear Colleagues,

Mutagenesis is a proven technique for inducing a broad spectrum of DNA variations and has created thousands of improved varieties in many crops. With the advent of molecular biology, mutagenesis has played a key role in the elucidation of the mechanisms of plant adaptation, growth and development, metabolic pathways, and signal transduction. Mutagenesis, especially that chemically induced, can introduce thousands of random mutations in a genome and create many scorable phenotypes. Map-based cloning is the conventional way to link a mutation (genotype) to phenotype, which can be a lengthy and expensive process. The advancement in next-generation sequencing has enabled the simultaneous generation and mapping of millions of DNA markers to identify the causal mutations for the phenotype (trait) of interest. The rapid and exciting progress in genome editing in the last few years promises to revolutionize plant breeding. However, it is still unfeasible to create multitudes of mutations in each gene in a genome to determine the mutations that are beneficial to plants. Genome editing can be more efficient if the targeted mutations are known. With the continuous improvement in sequencing technologies and decrease in sequencing costs, it has already become feasible to rapidly screen useful traits and efficiently and affordably identify the causal mutations from a limited number of mutant lines, providing informative targets for genome editing.

This Special Issue focuses on screening mutant populations for beneficial traits in crops and identifying the causal mutations underlying the relevant agronomic traits to provide targets for genome editing.

Dr. Zhanguo Xin
Guest Editor

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Keywords

  • mutagenesis
  • genome editing
  • agronomic trait
  • plant breeding
  • next-generation sequencing

Published Papers (11 papers)

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Editorial

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3 pages, 189 KiB  
Editorial
Mutagenesis in the Age of Next-Generation-Sequencing and Genome Editing
by Zhanguo Xin
Plants 2023, 12(19), 3403; https://0-doi-org.brum.beds.ac.uk/10.3390/plants12193403 - 27 Sep 2023
Viewed by 583
Abstract
Mutagenesis is a proven, classical technique for inducing a broad spectrum of DNA variations and has led to the creation of thousands of improved varieties in many crop species [...] Full article

Research

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14 pages, 2768 KiB  
Article
Variations in Total Protein and Amino Acids in the Sequenced Sorghum Mutant Library
by Adil Khan, Nasir Ali Khan, Scott R. Bean, Junping Chen, Zhanguo Xin and Yinping Jiao
Plants 2023, 12(8), 1662; https://0-doi-org.brum.beds.ac.uk/10.3390/plants12081662 - 15 Apr 2023
Cited by 4 | Viewed by 1401
Abstract
Sorghum (Sorghum bicolor) is the fifth most important cereal crop worldwide; however, its utilization in food products can be limited due to reduced nutritional quality related to amino acid composition and protein digestibility in cooked products. Low essential amino acid levels [...] Read more.
Sorghum (Sorghum bicolor) is the fifth most important cereal crop worldwide; however, its utilization in food products can be limited due to reduced nutritional quality related to amino acid composition and protein digestibility in cooked products. Low essential amino acid levels and digestibility are influenced by the composition of the sorghum seed storage proteins, kafirins. In this study, we report a core collection of 206 sorghum mutant lines with altered seed storage proteins. Wet lab chemistry analysis was conducted to evaluate the total protein content and 23 amino acids, including 19 protein-bound and 4 non-protein amino acids. We identified mutant lines with diverse compositions of essential and non-essential amino acids. The highest total protein content in these lines was almost double that of the wild-type (BTx623). The mutants identified in this study can be used as a genetic resource to improve the sorghum grain quality and determine the molecular mechanisms underlying the biosynthesis of storage protein and starch in sorghum seeds. Full article
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12 pages, 4601 KiB  
Article
Generation of Sesame Mutant Population by Mutagenesis and Identification of High Oleate Mutants by GC Analysis
by Ming Li Wang, Brandon Tonnis, Xianran Li and John Bradly Morris
Plants 2023, 12(6), 1294; https://0-doi-org.brum.beds.ac.uk/10.3390/plants12061294 - 13 Mar 2023
Cited by 1 | Viewed by 1174
Abstract
Sesame is one of the important oilseed crops in the world. Natural genetic variation exists in the sesame germplasm collection. Mining and utilizing the genetic allele variation from the germplasm collection is an important approach for seed quality improvement. The sesame germplasm accession, [...] Read more.
Sesame is one of the important oilseed crops in the world. Natural genetic variation exists in the sesame germplasm collection. Mining and utilizing the genetic allele variation from the germplasm collection is an important approach for seed quality improvement. The sesame germplasm accession, PI 263470, which has a significantly higher level of oleic acid (54.0%) than the average (39.5%), was identified by screening the entire USDA germplasm collection. The seeds from this accession were planted in a greenhouse. Leaf tissues and seeds were harvested from individual plants. DNA sequencing of the coding region of the fatty acid desaturase gene (FAD2) confirmed that this accession contained a natural mutation of G425A which may correspond to the deduced amino acid substitution of R142H leading to the high level of oleic acid, but it was a mixed accession with three genotypes (G/G, G/A, and A/A at the position). The genotype with A/A was selected and self-crossed for three generations. The purified seeds were used for EMS-induced mutagenesis to further enhance the level of oleic acid. A total of 635 M2 plants were generated from mutagenesis. Some mutant plants had significant morphological changes including leafy flat stems and others. M3 seeds were used for fatty acid composition analysis by gas chromatography (GC). Several mutant lines were identified with high oleic acid (70%). Six M3 mutant lines plus one control line were advanced to M7 or M8 generations. Their high oleate traits from M7 or M8 seeds harvested from M6 or M7 plants were further confirmed. The level of oleic acid from one mutant line (M7 915-2) was over 75%. The coding region of FAD2 was sequenced from these six mutants, but no mutation was identified. Additional loci may contribute to the high level of oleic acid. The mutants identified in this study can be used as breeding materials for sesame improvement and as genetic materials for forward genetic studies. Full article
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12 pages, 3011 KiB  
Article
Genome Survey Sequencing and Genetic Background Characterization of Ilex chinensis Sims (Aquifoliaceae) Based on Next-Generation Sequencing
by Peng Zhou, Jiao Li, Jing Huang, Fei Li, Qiang Zhang and Min Zhang
Plants 2022, 11(23), 3322; https://doi.org/10.3390/plants11233322 - 01 Dec 2022
Cited by 2 | Viewed by 1300
Abstract
Ilex chinensis Sims. is an evergreen arbor species with high ornamental and medicinal value that is widely distributed in China. However, there is a lack of molecular and genomic data for this plant, which severely restricts the development of its relevant research. To [...] Read more.
Ilex chinensis Sims. is an evergreen arbor species with high ornamental and medicinal value that is widely distributed in China. However, there is a lack of molecular and genomic data for this plant, which severely restricts the development of its relevant research. To obtain the whole reference genome, we first conducted a genome survey of I. chinensis by next-generation sequencing (NGS) to perform de novo whole-genome sequencing. As a result, our estimates using k-mer and flow cytometric analysis suggested the genome size of I. chinensis to be around 618–655 Mb, with the GC content, heterozygous rate, and repeat sequence rate of 37.52%, 1.1%, and 38%, respectively. A total of 334,649 microsatellite motifs were detected from the I. chinensis genome data, which will provide basic molecular markers for germplasm characterization, genetic diversity, and QTL mapping studies for I. chinensis. In summary, the I. chinensis genome is complex with high heterozygosity and few repeated sequences. Overall, this is the first report on the genome features of I. chinensis, and the information may lay a strong groundwork for future whole-genome sequencing and molecular breeding studies of this species. Full article
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18 pages, 1933 KiB  
Article
Spectrum and Density of Gamma and X-ray Induced Mutations in a Non-Model Rice Cultivar
by Joanna Jankowicz-Cieslak, Bernhard J. Hofinger, Luka Jarc, Sini Junttila, Bence Galik, Attila Gyenesei, Ivan L. Ingelbrecht and Bradley J. Till
Plants 2022, 11(23), 3232; https://0-doi-org.brum.beds.ac.uk/10.3390/plants11233232 - 25 Nov 2022
Cited by 3 | Viewed by 1353
Abstract
Physical mutagens are a powerful tool used for genetic research and breeding for over eight decades. Yet, when compared to chemical mutagens, data sets on the effect of different mutagens and dosages on the spectrum and density of induced mutations remain lacking. To [...] Read more.
Physical mutagens are a powerful tool used for genetic research and breeding for over eight decades. Yet, when compared to chemical mutagens, data sets on the effect of different mutagens and dosages on the spectrum and density of induced mutations remain lacking. To address this, we investigated the landscape of mutations induced by gamma and X-ray radiation in the most widely cultivated crop species: rice. A mutant population of a tropical upland rice, Oryza sativa L., was generated and propagated via self-fertilization for seven generations. Five dosages ranging from 75 Gy to 600 Gy in both X-ray and gamma-irradiated material were applied. In the process of a forward genetic screens, 11 unique rice mutant lines showing phenotypic variation were selected for mutation analysis via whole-genome sequencing. Thousands of candidate mutations were recovered in each mutant with single base substitutions being the most common, followed by small indels and structural variants. Higher dosages resulted in a higher accumulation of mutations in gamma-irradiated material, but not in X-ray-treated plants. The in vivo role of all annotated rice genes is yet to be directly investigated. The ability to induce a high density of single nucleotide and structural variants through mutagenesis will likely remain an important approach for functional genomics and breeding. Full article
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9 pages, 803 KiB  
Communication
Soybean Protein and Oil Variants Identified through a Forward Genetic Screen for Seed Composition
by Karen Hudson
Plants 2022, 11(21), 2966; https://0-doi-org.brum.beds.ac.uk/10.3390/plants11212966 - 03 Nov 2022
Cited by 3 | Viewed by 1648
Abstract
Mutagenesis remains an important tool in soybean biology. In classical plant mutation breeding, mutagenesis has been a trusted approach for decades, creating stable non-transgenic variation, and many mutations have been incorporated into germplasm for several crops, especially to introduce favorable seed composition traits. [...] Read more.
Mutagenesis remains an important tool in soybean biology. In classical plant mutation breeding, mutagenesis has been a trusted approach for decades, creating stable non-transgenic variation, and many mutations have been incorporated into germplasm for several crops, especially to introduce favorable seed composition traits. We performed a genetic screen for aberrant oil or protein composition of soybean seeds, and as a result isolated over 100 mutant lines for seed composition phenotypes, with particular interest in high protein or high oil phenotypes. These lines were followed for multiple seasons and generations to select the most stable traits for further characterization. Through backcrossing and outcrossing experiments, we determined that a subset of the lines showed recessive inheritance, while others showed a dominant inheritance pattern that suggests the involvement of multiple loci and genetic mechanisms. These lines can be used as a resource for future studies of the genetic control of seed protein and oil content in soybean. Full article
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19 pages, 2998 KiB  
Article
Targeted Mutagenesis of the Multicopy Myrosinase Gene Family in Allotetraploid Brassica juncea Reduces Pungency in Fresh Leaves across Environments
by Dale Karlson, Julius P. Mojica, Thomas J. Poorten, Shai J. Lawit, Sathya Jali, Raj Deepika Chauhan, Gina M. Pham, Pradeep Marri, Sharon L. Guffy, Justin M. Fear, Cherie A. Ochsenfeld, Tracey A. (Lincoln) Chapman, Bruno Casamali, Jorge P. Venegas, Hae Jin Kim, Ashley Call, William L. Sublett, Lolita G. Mathew, Aabid Shariff, Joseph M. Watts, Mike Mann, Aaron Hummel and Ryan Rappadd Show full author list remove Hide full author list
Plants 2022, 11(19), 2494; https://0-doi-org.brum.beds.ac.uk/10.3390/plants11192494 - 23 Sep 2022
Cited by 10 | Viewed by 7374
Abstract
Recent breeding efforts in Brassica have focused on the development of new oilseed feedstock crop for biofuels (e.g., ethanol, biodiesel, bio-jet fuel), bio-industrial uses (e.g., bio-plastics, lubricants), specialty fatty acids (e.g., erucic acid), and producing low glucosinolates levels for oilseed and feed meal [...] Read more.
Recent breeding efforts in Brassica have focused on the development of new oilseed feedstock crop for biofuels (e.g., ethanol, biodiesel, bio-jet fuel), bio-industrial uses (e.g., bio-plastics, lubricants), specialty fatty acids (e.g., erucic acid), and producing low glucosinolates levels for oilseed and feed meal production for animal consumption. We identified a novel opportunity to enhance the availability of nutritious, fresh leafy greens for human consumption. Here, we demonstrated the efficacy of disarming the ‘mustard bomb’ reaction in reducing pungency upon the mastication of fresh tissue—a major source of unpleasant flavor and/or odor in leafy Brassica. Using gene-specific mutagenesis via CRISPR-Cas12a, we created knockouts of all functional copies of the type-I myrosinase multigene family in tetraploid Brassica juncea. Our greenhouse and field trials demonstrate, via sensory and biochemical analyses, a stable reduction in pungency in edited plants across multiple environments. Collectively, these efforts provide a compelling path toward boosting the human consumption of nutrient-dense, fresh, leafy green vegetables. Full article
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12 pages, 1546 KiB  
Article
Targeting TOR and SnRK1 Genes in Rice with CRISPR/Cas9
by Bhuvan Pathak, Chandan Maurya, Maria C. Faria, Zahra Alizada, Soumen Nandy, Shan Zhao, Muhammed Jamsheer K and Vibha Srivastava
Plants 2022, 11(11), 1453; https://0-doi-org.brum.beds.ac.uk/10.3390/plants11111453 - 30 May 2022
Cited by 3 | Viewed by 2692
Abstract
Genome targeting with CRISPR/Cas9 is a popular method for introducing mutations and creating knock-out effects. However, limited information is currently available on the mutagenesis of essential genes. This study investigated the efficiency of CRISPR/Cas9 in targeting rice essential genes: the singleton TARGET OF [...] Read more.
Genome targeting with CRISPR/Cas9 is a popular method for introducing mutations and creating knock-out effects. However, limited information is currently available on the mutagenesis of essential genes. This study investigated the efficiency of CRISPR/Cas9 in targeting rice essential genes: the singleton TARGET OF RAPAMYCIN (OsTOR) and the three paralogs of the Sucrose non-fermenting-1 (SNF1)-related kinase 1 (OsSnRK1α), OsSnRK1αA, OsSnRK1αB and OsSnRK1αC. Strong activity of constitutively expressed CRISPR/Cas9 was effective in creating mutations in OsTOR and OsSnRK1α genes, but inducible CRISPR/Cas9 failed to generate detectable mutations. The rate of OsTOR mutagenesis was relatively lower and only the kinase domain of OsTOR could be targeted, while mutations in the HEAT region were unrecoverable. OsSnRK1α paralogs could be targeted at higher rates; however, sterility or early senescence was observed in >50% of the primary mutants. Additionally, OsSnRK1αB and OsSnRK1αC, which bear high sequence homologies, could be targeted simultaneously to generate double-mutants. Further, although limited types of mutations were found in the surviving mutants, the recovered lines displayed loss-of-function or knockdown tor or snrk1 phenotypes. Overall, our data show that mutations in these essential genes can be created by CRISPR/Cas9 to facilitate investigations on their roles in plant development and environmental response in rice. Full article
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10 pages, 834 KiB  
Article
Application of CRISPR/Cas9 System for Efficient Gene Editing in Peanut
by Anjanasree K. Neelakandan, David A. Wright, Sy M. Traore, Xingli Ma, Binita Subedi, Suman Veeramasu, Martin H. Spalding and Guohao He
Plants 2022, 11(10), 1361; https://0-doi-org.brum.beds.ac.uk/10.3390/plants11101361 - 20 May 2022
Cited by 8 | Viewed by 2513
Abstract
Peanuts are an economically important crop cultivated worldwide. However, several limitations restrained its productivity, including biotic/abiotic stresses. CRISPR/Cas9-based gene-editing technology holds a promising approach to developing new crops with improved agronomic and nutritional traits. Its application has been successful in many important crops. [...] Read more.
Peanuts are an economically important crop cultivated worldwide. However, several limitations restrained its productivity, including biotic/abiotic stresses. CRISPR/Cas9-based gene-editing technology holds a promising approach to developing new crops with improved agronomic and nutritional traits. Its application has been successful in many important crops. However, the application of this technology in peanut research is limited, probably due to the lack of suitable constructs and protocols. In this study, two different constructs were generated to induce insertion/deletion mutations in the targeted gene for a loss of function study. The first construct harbors the regular gRNA scaffold, while the second construct has the extended scaffold plus terminator. The designed gRNA targeting the coding sequence of the FAD2 genes was cloned into both constructs, and their functionality and efficiency were validated using the hairy root transformation system. Both constructs displayed insertions and deletions as the types of edits. The construct harboring the extended plus gRNA terminator showed a higher editing efficiency than the regular scaffold for monoallelic and biallelic mutations. These two constructs can be used for gene editing in peanuts and could provide tools for improving peanut lines for the benefit of peanut breeders, farmers, and industry. Full article
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Review

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22 pages, 2425 KiB  
Review
Genes Impacting Grain Weight and Number in Wheat (Triticum aestivum L. ssp. aestivum)
by Brandon J. Tillett, Caleb O. Hale, John M. Martin and Michael J. Giroux
Plants 2022, 11(13), 1772; https://0-doi-org.brum.beds.ac.uk/10.3390/plants11131772 - 04 Jul 2022
Cited by 6 | Viewed by 2420
Abstract
The primary goal of common wheat (T. aestivum) breeding is increasing yield without negatively impacting the agronomic traits or product quality. Genetic approaches to improve the yield increasingly target genes that impact the grain weight and number. An energetic trade-off exists [...] Read more.
The primary goal of common wheat (T. aestivum) breeding is increasing yield without negatively impacting the agronomic traits or product quality. Genetic approaches to improve the yield increasingly target genes that impact the grain weight and number. An energetic trade-off exists between the grain weight and grain number, the result of which is that most genes that increase the grain weight also decrease the grain number. QTL associated with grain weight and number have been identified throughout the hexaploid wheat genome, leading to the discovery of numerous genes that impact these traits. Genes that have been shown to impact these traits will be discussed in this review, including TaGNI, TaGW2, TaCKX6, TaGS5, TaDA1, WAPO1, and TaRht1. As more genes impacting the grain weight and number are characterized, the opportunity is increasingly available to improve common wheat agronomic yield by stacking the beneficial alleles. This review provides a synopsis of the genes that impact grain weight and number, and the most beneficial alleles of those genes with respect to increasing the yield in dryland and irrigated conditions. It also provides insight into some of the genetic mechanisms underpinning the trade-off between grain weight and number and their relationship to the source-to-sink pathway. These mechanisms include the plant size, the water soluble carbohydrate levels in plant tissue, the size and number of pericarp cells, the cytokinin and expansin levels in developing reproductive tissue, floral architecture and floral fertility. Full article
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18 pages, 1840 KiB  
Review
CRISPR/Cas9 in Planta Hairy Root Transformation: A Powerful Platform for Functional Analysis of Root Traits in Soybean
by Mohsen Niazian, François Belzile and Davoud Torkamaneh
Plants 2022, 11(8), 1044; https://0-doi-org.brum.beds.ac.uk/10.3390/plants11081044 - 12 Apr 2022
Cited by 14 | Viewed by 4039
Abstract
Sequence and expression data obtained by next-generation sequencing (NGS)-based forward genetics methods often allow the identification of candidate causal genes. To provide true experimental evidence of a gene’s function, reverse genetics techniques are highly valuable. Site-directed mutagenesis through transfer DNA (T-DNA) delivery is [...] Read more.
Sequence and expression data obtained by next-generation sequencing (NGS)-based forward genetics methods often allow the identification of candidate causal genes. To provide true experimental evidence of a gene’s function, reverse genetics techniques are highly valuable. Site-directed mutagenesis through transfer DNA (T-DNA) delivery is an efficient reverse screen method in plant functional analysis. Precise modification of targeted crop genome sequences is possible through the stable and/or transient delivery of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) reagents. Currently, CRISPR/Cas9 is the most powerful reverse genetics approach for fast and precise functional analysis of candidate genes/mutations of interest. Rapid and large-scale analyses of CRISPR/Cas-induced mutagenesis is achievable through Agrobacterium rhizogenes-mediated hairy root transformation. The combination of A. rhizogenes hairy root-CRISPR/Cas provides an extraordinary platform for rapid, precise, easy, and cost-effective “in root” functional analysis of genes of interest in legume plants, including soybean. Both hairy root transformation and CRISPR/Cas9 techniques have their own complexities and considerations. Here, we discuss recent advancements in soybean hairy root transformation and CRISPR/Cas9 techniques. We highlight the critical factors required to enhance mutation induction and hairy root transformation, including the new generation of reporter genes, methods of Agrobacterium infection, accurate gRNA design strategies, Cas9 variants, gene regulatory elements of gRNAs and Cas9 nuclease cassettes and their configuration in the final binary vector to study genes involved in root-related traits in soybean. Full article
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