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International Journal of Molecular... Oct 2022Mutation is a source of genetic diversity widely used in breeding programs for the acquisition of agronomically interesting characters in commercial varieties of the... (Review)
Review
Mutation is a source of genetic diversity widely used in breeding programs for the acquisition of agronomically interesting characters in commercial varieties of the species, as well as in the rest of crop species. Mutation can occur in nature at a very low frequency or can be induced artificially. Spontaneous or bud sport mutations in somatic cells can be vegetatively propagated to get an individual with the mutant phenotype. Unlike animals, plants have unlimited growth and totipotent cells that let somatic mutations to be transmitted to the progeny. On the other hand, in vitro tissue culture makes it possible to induce mutation in plant material and perform large screenings for mutant's selection and cleaning of chimeras. Finally, targeted mutagenesis has been boosted by the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 and Transcription activator-like effector nuclease (TALEN) editing technologies. Over the last few decades, environmental stressors such as global warming have been threatening the supply of global demand for food based on population growth in the near future. For this purpose, the release of new varieties adapted to such changes is a requisite, and selected or generated mutants by properly regulated mechanisms could be helpful to this task. In this work, we reviewed the most relevant mutations for breeding traits in species such as flowering time, self-compatibility, fruit quality, and disease tolerance, including new molecular perspectives in the present postgenomic era including CRISPR/Cas9 and TALEN editing technologies.
Topics: Animals; Gene Editing; CRISPR-Cas Systems; Transcription Activator-Like Effector Nucleases; Prunus; Plant Breeding; Mutation; Endonucleases; Genome, Plant
PubMed: 36362061
DOI: 10.3390/ijms232113273 -
PloS One 2022Lens culinaris is a proteinaceous food crop that is consumed worldwide for protein requirements. Mutation breeding has been used to improve protein content, yield, and...
Lens culinaris is a proteinaceous food crop that is consumed worldwide for protein requirements. Mutation breeding has been used to improve protein content, yield, and related traits, as well as to select highly desirable mutants that are economically significant. An investigation of genotypic variation in lentil germplasm was carried out using induced mutagenesis, with caffeine, ethyl methane sulfonate (EMS), lead nitrate, and cadmium nitrate as mutagens that resulted in 18 mutant lines in the M3 generation. For the present study, we analyzed the genetic diversity of lentil mutant lines using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and random amplified polymorphic DNA markers (RAPD). The heterozygosity of RAPD markers per primer ranged from 50.00-90.90% with an average of 71.04%. The genetic divergent analysis was performed using hierarchical clustering (UPGMA), exhibited that these mutant lines were classified mainly into five subpopulation or clusters. A close resemblance with highest genetic coefficient similarity (1.00) were observed between control and mutant H; between mutant M and E; between mutant Q and J2, while more divergent mutants were N2 with mutant B; and mutant R with mutant J1with least genetic coefficient similarity (0.22). Protein and mineral content (Fe, Zn and Cu) were increased significantly in some high yielding mutant lines concerning to the control plant, and showed polymorphic variations in polypeptide chains in terms of banding pattern. Stomatal morphology in high yielding mutants were perceived utilizing scanning electron microscopy (SEM), exhibiting variations in stomatal size, stomatal opening and number of stomata. The present study's promising mutant lines' biological, physiological, and molecular profiles provide a foundation for forthcoming preservation and consumption strategies to broaden the genetic diversity of the breeding population of lentil.
Topics: Lens Plant; Random Amplified Polymorphic DNA Technique; Genetic Markers; Caffeine; Sodium Dodecyl Sulfate; Plant Breeding; Mutagens; Methane; Genetic Variation
PubMed: 36279277
DOI: 10.1371/journal.pone.0274937 -
Journal of the American Society of... Jun 2022C3 glomerulopathy (C3G) is a heterogeneous group of chronic renal diseases characterized predominantly by glomerular C3 deposition and complement dysregulation....
BACKGROUND
C3 glomerulopathy (C3G) is a heterogeneous group of chronic renal diseases characterized predominantly by glomerular C3 deposition and complement dysregulation. Mutations in factor H-related (FHR) proteins resulting in duplicated dimerization domains are prototypical of C3G, although the underlying pathogenic mechanism is unclear.
METHODS
Using and assays, we performed extensive characterization of an FHR-1 mutant with a duplicated dimerization domain. To assess the FHR-1 mutant's association with disease susceptibility and renal prognosis, we also analyzed copy number variations and FHR-1 plasma levels in two Spanish C3G cohorts and in a control population.
RESULTS
Duplication of the dimerization domain conferred FHR-1 with an increased capacity to interact with C3-opsonized surfaces, which resulted in an excessive activation of the alternative pathway. This activation does not involve C3b binding competition with factor H. These findings support a scenario in which mutant FHR-1 binds to C3-activated fragments and recruits native C3 and C3b; this leads to formation of alternative pathway C3 convertases, which increases deposition of C3b molecules, overcoming FH regulation. This suggests that a balanced FHR-1/FH ratio is crucial to control complement amplification on opsonized surfaces. Consistent with this conceptual framework, we show that the genetic deficiency of FHR-1 or decreased FHR-1 in plasma confers protection against developing C3G and associates with better renal outcome.
CONCLUSIONS
Our findings explain how FHR-1 mutants with duplicated dimerization domains result in predisposition to C3G. They also provide a pathogenic mechanism that may be shared by other diseases, such as IgA nephropathy or age-related macular degeneration, and identify FHR-1 as a potential novel therapeutic target in C3G.
Topics: Blood Proteins; Complement C3; Complement C3b Inactivator Proteins; Complement Factor H; DNA Copy Number Variations; Disease Susceptibility; Glomerulonephritis, IGA; Humans; Prognosis
PubMed: 35545301
DOI: 10.1681/ASN.2021101318 -
International Journal of Molecular... Dec 2019The p53 family of proteins has grown substantially over the last 40 years. It started with p53, then p63, p73, isoforms and mutants of these proteins. The function of... (Review)
Review
The p53 family of proteins has grown substantially over the last 40 years. It started with p53, then p63, p73, isoforms and mutants of these proteins. The function of p53 as a tumour suppressor has been thoroughly investigated, but the functions of all isoforms and mutants and the interplay between them are still poorly understood. Mutant p53 proteins lose p53 function, display dominant-negative (DN) activity and display gain-of-function (GOF) to varying degrees. GOF was originally attributed to mutant p53's inhibitory function over the p53 family members p63 and p73. It has become apparent that this is not the only way in which mutant p53 operates as a large number of transcription factors that are not related to p53 are activated on mutant p53 binding. This raises the question to what extent mutant p53 binding to p63 and p73 plays a role in mutant p53 GOF. In this review, we discuss the literature around the interaction between mutant p53 and family members, including other binding partners, the functional consequences and potential therapeutics.
Topics: Humans; Mutant Proteins; Mutation; Neoplasms; Protein Binding; Protein Isoforms; Transcription Factors; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins
PubMed: 31817935
DOI: 10.3390/ijms20246188 -
BMC Plant Biology May 2022Leaf color mutants have reduced photosynthetic efficiency, which has severely negative impacts on crop growth and economic product yield. There are different chlorophyll...
BACKGROUND
Leaf color mutants have reduced photosynthetic efficiency, which has severely negative impacts on crop growth and economic product yield. There are different chlorophyll mutants in Arabidopsis and crops that can be used for genetic control and molecular mechanism studies of chlorophyll biosynthesis, chloroplast development and photoefficiency. Chlorophyll mutants in Brassica napus are mostly used for mapping and location research but are rarely used for physiological research. The chlorophyll-deficient mutant in this experiment were both genetically mapped and physiologically analyzed.
RESULTS
In this study, yellow leaf mutant of Brassica napus L. mutated by ethyl methyl sulfone (EMS) had significantly lower chlorophyll a, b and carotenoid contents than the wild type, and the net photosynthetic efficiency, stomatal conductance and transpiration rate were all significantly reduced. The mutant had sparse chloroplast distribution and weak autofluorescence. The granule stacks were reduced, and the shape was extremely irregular, with more broken stromal lamella. Transcriptome data analysis enriched the differentially expressed genes mainly in phenylpropane and sugar metabolism. The mutant was mapped to a 2.72 Mb region on A01 by using BSA-Seq, and the region was validated by SSR markers.
CONCLUSIONS
The mutant chlorophyll content and photosynthetic efficiency were significantly reduced compared with those of the wild type. Abnormal chloroplasts and thylakoids less connected to the stroma lamella appeared in the mutant. This work on the mutant will facilitate the process of cloning the BnaA01.cd gene and provide more genetic and physiological information concerning chloroplast development in Brassica napus.
Topics: Arabidopsis; Brassica napus; Chlorophyll; Chlorophyll A; Chromosome Mapping; Photosynthesis; Plant Leaves
PubMed: 35585493
DOI: 10.1186/s12870-022-03630-9 -
BMC Plant Biology Jul 2022Metals such as Zn or Cd are toxic to plant and humans when they are exposed in high quantities through contaminated soil or food. Noccaea caerulescens, an extraordinary...
BACKGROUND
Metals such as Zn or Cd are toxic to plant and humans when they are exposed in high quantities through contaminated soil or food. Noccaea caerulescens, an extraordinary Zn/Cd/Ni hyperaccumulating species, is used as a model plant for metal hyperaccumulation and phytoremediation studies. Current reverse genetic techniques to generate mutants based on transgenesis is cumbersome due to the low transformation efficiency of this species. We aimed to establish a mutant library for functional genomics by a non-transgenic approach, to identify mutants with an altered mineral profiling, and to screen for mutations in bZIP19, a regulator of Zn homeostasis in N. caerulescens.
RESULTS
To generate the N. caerulescens mutant library, 3000 and 5000 seeds from two sister plants of a single-seed recurrent inbred descendant of the southern French accession Saint-Félix-de-Pallières (SF) were mutagenized respectively by 0.3 or 0.4% ethyl methane sulfonate (EMS). Two subpopulations of 5000 and 7000 M2 plants were obtained after 0.3 or 0.4% EMS treatment. The 0.4% EMS treatment population had a higher mutant frequency and was used for TILLING. A High Resolution Melting curve analysis (HRM) mutation screening platform was optimized and successfully applied to detect mutations for NcbZIP19, encoding a transcription factor controlling Zn homeostasis. Of four identified point mutations in NcbZIP19, two caused non-synonymous substitutions, however, these two mutations did not alter the ionome profile compared to the wild type. Forward screening of the 0.4% EMS treatment population by mineral concentration analysis (ionomics) in leaf material of each M2 plant revealed putative mutants affected in the concentration of one or more of the 20 trace elements tested. Several of the low-Zn mutants identified in the ionomic screen did not give progeny, illustrating the importance of Zn for the species. The mutant frequency of the population was evaluated based on an average of 2.3 knockout mutants per tested monogenic locus.
CONCLUSIONS
The 0.4% EMS treatment population is effectively mutagenized suitable for forward mutant screens and TILLING. Difficulties in seed production in low Zn mutants, obtained by both forward and reverse genetic approach, hampered further analysis of the nature of the low Zn phenotypes.
Topics: Biodegradation, Environmental; Brassicaceae; Cadmium; Ethyl Methanesulfonate; Humans; Metals; Zinc
PubMed: 35869423
DOI: 10.1186/s12870-022-03739-x -
Blood Coagulation & Fibrinolysis : An... Jul 2022The aim of this study is to model classical Bernard Soulier Syndrome in the zebrafish by targeting Gp1ba. We obtained gp1ba mutant embryos from Zebrafish International...
The aim of this study is to model classical Bernard Soulier Syndrome in the zebrafish by targeting Gp1ba. We obtained gp1ba mutant embryos from Zebrafish International Resource Center and grew them to adulthood. The tail clips from these fish were used to prepare DNA and sequenced to identify heterozygotes. They were then bred to obtain homozygotes. The mutation was confirmed by DNA sequencing as a termination codon UAA in place of AAA codon at position 886 in the gp1ba transcript. Thus, at the Pro-295, the Gp1ba protein could be terminated. The blood from gp1ba homozygous and heterozygous mutants showed decreased ristocetin-mediated agglutination in the whole blood agglutination assay. The gp1ba heterozygous and homozygous larvae were subjected to a laser-assisted arterial thrombosis assay, and the results showed the prolonged occlusion in the caudal artery. These results suggested that the gp1ba mutant had a bleeding phenotype. The blood smears from the adult gp1ba, heterozygous and homozygous mutants, showed macrothrombocytes, similar to the human GP1BA deficiency that showed giant platelets. The bleeding assay on these heterozygous and homozygous mutants showed greater bleeding than wildtype, confirming the above findings. Taken together, the characterization of gp1ba zebrafish mutant suggested an autosomal dominant mode of inheritance. The zebrafish gp1ba mutant models classical Bernard Soulier Syndrome and could be used for reversing this phenotype to identify novel factors by the genome-wide piggyback knockdown method.
Topics: Animals; Bernard-Soulier Syndrome; Blood Platelets; Hemorrhage; Heterozygote; Homozygote; Platelet Glycoprotein GPIb-IX Complex; Zebrafish
PubMed: 35802508
DOI: 10.1097/MBC.0000000000001135 -
Methods in Molecular Biology (Clifton,... 2021Genes that play a role in stress response mechanisms and other phenotypes of Listeria monocytogenes can be identified by construction and screening of mutant libraries....
Genes that play a role in stress response mechanisms and other phenotypes of Listeria monocytogenes can be identified by construction and screening of mutant libraries. In this chapter, we describe the construction and screening of mutant libraries of L. monocytogenes using the plasmid pMC38, carrying a mariner-based transposon system (TC1/mariner) and constructed by Cao et al. (Appl Environ Microbiol 73:2758-2761, 2007). Following screening of mutant libraries, putative mutants are identified and the transposon is localized, leading to identification of the genes responsible for the phenotype of interest. To confirm the role of the transposon-harboring gene in the relevant phenotype, transposon mutants are genetically complemented with the wild-type gene using the site-specific temperature-sensitive integration vector pPL2, constructed by Lauer et al. (J Bacteriol 184:4177-4186, 2002).
Topics: Electroporation; Genetic Vectors; Humans; Listeria monocytogenes; Listeriosis; Mutagenesis, Insertional; Mutation; Plasmids; Polymerase Chain Reaction; Retroelements
PubMed: 32975775
DOI: 10.1007/978-1-0716-0982-8_14 -
Methods in Molecular Biology (Clifton,... 2020Reverse genetics approaches for characterizing phenotypes of mutants in a gene of interest (GOI) require thorough genotyping and phenotypic analysis. However, special...
Reverse genetics approaches for characterizing phenotypes of mutants in a gene of interest (GOI) require thorough genotyping and phenotypic analysis. However, special challenges are encountered when a GOI is expressed in reproductive tissues: a variety of assays are required to characterize the phenotype and a mutant may show sporophytic and/or gametophytic defects in male and/or female reproductive tissues, which are structurally and functionally intertwined. Here, we present a streamlined workflow to characterize mutants with reproductive defects, primarily using Arabidopsis as a model, which can also be adapted to characterize mutants in other flowering plants. Procedures described here can be used to distinguish different kinds of reproductive defects and pinpoint the defective reproductive step(s) in a mutant. Although our procedures emphasize the characterization of mutants with male reproductive defects, they can nevertheless be used to identify female reproductive defects, as those defects could manifest alongside, and sometimes require, male reproductive tissues.
Topics: Arabidopsis; Genetic Techniques; Mutation; Ovule; Plant Breeding; Plant Infertility; Pollen; Workflow
PubMed: 32529432
DOI: 10.1007/978-1-0716-0672-8_8 -
Current Protocols Nov 2022Forward genetics is used to identify the genetic basis for a phenotype. The approach involves identifying a mutant organism exhibiting a phenotype of interest and then...
Forward genetics is used to identify the genetic basis for a phenotype. The approach involves identifying a mutant organism exhibiting a phenotype of interest and then mapping the causative locus or gene. Bulked-segregant analysis (BSA) is a quick and effective approach to map mutants using pools of mutants and wild-type plants from a segregating population to identify linkage of the mutant phenotype, and this approach has been successfully used in plants. Traditional linkage mapping approaches are outdated and time intensive, and can be very difficult. With the highly evolved development and reduction in cost of high-throughput sequencing, this new approach combined with BSA has become extremely effective in multiple plant species, including Zea mays (maize). While the approach is incredibly powerful, careful experimental design, bioinformatic mapping techniques, and interpretation of results are important to obtain the desired results in an effective and timely manner. Poor design of a mapping population, limitations in bioinformatic experience, and inadequate understanding of sequence data are limitations of these approaches for the researcher. Here, we describe a straightforward protocol for mapping mutations responsible for a phenotype of interest in maize, using high-throughput sequencing and BSA. Specifically, we discuss relevant aspects of developing a mutant mapping population. This is followed by a detailed protocol for DNA preparation and analysis of short-read sequences to map and identify candidate causative mutations responsible for the mutant phenotype of interest. We provide command-line and perl scripts to complete the bioinformatic analysis of the mutant sequence data. This protocol lays out the design of the BSA, bioinformatic approaches, and interpreting the sequencing data. These methods are very adaptable to any forward genetics experiment and provide a step-by-step approach to identifying the genetic basis of a maize mutant phenotype. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Bulked-segregant analysis and high-throughput sequencing to map maize mutants.
Topics: Zea mays; High-Throughput Nucleotide Sequencing; Chromosome Mapping; Genetic Linkage; Phenotype
PubMed: 36350247
DOI: 10.1002/cpz1.591