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To cope with manganese (Mn) deficiency, plants have evolved an efficient transport system to uptake and redistribute Mn. However, the underlying molecular mechanisms remain to be demonstrated. We carried out a forward genetic screen in a root high-affinity Mn transporter nramp1 mutant background in Arabidopsis thaliana and identified an uncharacterized Mn transport NRAMP2. We investigated the effect of nramp2 mutation on root growth and reactive oxygen species (ROS) accumulation and we also examined the NRAMP2 expression pattern, and the subcellular localization and transport activity of NRAMP2. Mutation of NRAMP2 impaired plant growth, while overexpression of NRAMP2 improved plant growth under low Mn conditions. In the nramp2-1nramp1 double mutant, Mn deficiency inhibited root cell elongation and root hair development, which was associated with increased hydrogen peroxide (H2O2) accumulation. NRAMP2 is preferentially localized to the trans-Golgi network. NRAMP2 has Mn influx transport activity in yeast, and mutation of NRAMP2 led to greater Mn retention in roots. Our results suggest that under Mn-deficient conditions, increased accumulation of H2O2 is partially responsible for the root growth inhibition and NRAMP2 is involved in remobilization of Mn in Golgi for root growth.

Ataxia‐telangiectasia (A‐T) is an autosomal recessive neurological disorder caused by mutations in the ATM gene. Classical splicing mutations (type I) delete entire exons during pre‐mRNA splicing. In this report, we describe nine examples of nonclassical splicing mutations in 12 A‐T patients and compare cDNA changes to estimates of splice junction strengths based on maximum entropy modeling. These mutations fall into three categories: pseudoexon insertions (type II), single nucleotide changes within the exon (type III), and intronic changes that disrupt the conserved 3′ splice sequence and lead to partial exon deletion (type IV). Four patients with a previously reported type II (pseudoexon) mutation all shared a common founder haplotype. Three patients with apparent missense or silent mutations actually had type III aberrant splicing and partial deletion of an exon. Five patients had type IV mutations that could have been misinterpreted as classical splicing mutations. Instead, their mutations disrupt a splice site and use another AG splice site located nearby within the exon; they lead to partial deletions at the beginning of exons. These nonclassical splicing mutations create frameshifts that result in premature termination codons. Without screening cDNA or using accurate models of splice site strength, the consequences of these genomic mutations cannot be reliably predicted. This may lead to further misinterpretation of genotype–phenotype correlations and may subsequently impact upon gene‐based therapeutic approaches. Hum Mutat 23:67–76, 2004. © 2003 Wiley‐Liss, Inc.

Heavy ion beam (HIB) is an effective physical mutagen that has been widely used in plant mutational breeding. Systemic knowledge of the effects caused by different HIB doses at developmental and genomic levels will facilitate efficient breeding for crops. Here we examined the effects of HIB systematically. Kitaake rice seeds were irradiated by ten doses of carbon ion beams (CIB, 25 – 300 Gy), which is the most widely used HIB. We initially examined the growth, development and photosynthetic parameters of the M 1 population and found that doses exceeding 125 Gy caused significant physiological damages to rice. Subsequently, we analyzed the genomic variations in 179 M 2 individuals from six treatments (25 – 150 Gy) via whole-genome sequencing (WGS). The mutation rate peaks at 100 Gy (2.66×10 -7 /bp). Importantly, we found that mutations shared among different panicles of the same M 1 individual are at low ratios, validating the hypothesis that different panicles may be derived from different progenitor cells. Furthermore, we isolated 129 mutants with distinct phenotypic variations, including changes in agronomic traits, from 11,720 M 2 plants, accounting for a 1.1% mutation rate. Among them, about 50% possess stable inheritance in M 3 . WGS data of 11 stable M 4 mutants, including three lines with higher yields, reveal their genomic mutational profiles and candidate genes. Our results demonstrate that HIB is an effective tool that facilitates breeding, that the optimal dose range for rice is 67 – 90% median lethal dose (LD 50 ), and that the mutants isolated here can be further used for functional genomic research, genetic analysis, and breeding.

Diatoms are major components of phytoplankton and play a key role in the ecology of aquatic ecosystems. These algae are of great scientific importance for a wide variety of research areas, ranging from marine ecology and oceanography to biotechnology. During the last 20 years, the availability of genomic information on selected diatom species and a substantial progress in genetic manipulation, strongly contributed to establishing diatoms as molecular model organisms for marine biology research. Recently, tailored TALEN endonucleases and the CRISPR/Cas9 system were utilized in diatoms, allowing targeted genetic modifications and the generation of knockout strains. These approaches are extremely valuable for diatom research because breeding, forward genetic screens by random insertion, and chemical mutagenesis are not applicable to the available model species Phaeodactylum tricornutum and Thalassiosira pseudonana, which do not cross sexually in the lab. Here, we provide an overview of the genetic toolbox that is currently available for performing stable genetic modifications in diatoms. We also discuss novel challenges that need to be addressed to fully exploit the potential of these technologies for the characterization of diatom biology and for metabolic engineering.

Background The CRISPR/Cas9 system is being used for genome editing purposes by many research groups in multiple plant species. Traditional sequencing methods to identify homozygous mutants are time-consuming, laborious and expensive. Results We have developed a method to screen CRISPR/Cas9-induced mutants through Mutation Sites Based Specific Primers Polymerase Chain Reaction (MSBSP-PCR). The MSBSP-PCR method was successfully used to identify homozygous/biallelic mutants in Nicotiana tabacum and Arabidopsis thaliana , and we speculate that it can be used for the identification of CRISPR/Cas9-induced mutants in other plant species. Compared to traditional sequencing methods, MSBSP-PCR is simpler, faster and cheaper. Conclusions The MSBSP-PCR method is simple to implement and can save time and cost in the screening of CRISPR/Cas9-induced homozygous/biallelic mutants.

Identifying mutant traits is essential for improving crop yield, quality, and stress resistance in plant breeding. Historically, the efficiency of breeding has been constrained by throughput and accuracy. Recent significant advancements have been made through the development of automated, high-accuracy, and high-throughput equipment. However, challenges remain in the post-processing of large-scale image data and its practical application and evaluation in breeding. This study presents a comparative analysis of human and machine recognition, with validation of a randomly selected mutant at the physiological level performed on wild-type Arabidopsis thaliana and a candidate mutant of the M 3 generation, which was generated through mutagenesis with heavy ion beams (HIBs) and 60 Co-γ radiation. The mutant populations were subjected to image acquisition and automated screening using the High-throughput Plant Imaging System (HTPIS), generating approximately 10 GB of data (4,635 image datasets). We performed Principal Components Analysis (PCA), scatter matrix clustering, and Logistic Growth Curve (LGC) analyses, and compared these results with those obtained from traditional manual screening based on human visual assessment, and randomly selected #197 candidate mutants for validation in terms of growth and development, chlorophyll fluorescence, and subcellular structure. Our findings demonstrate that as the confidence interval level increases from 75 to 99.9%, the accuracy of machine-based mutant identification decreases from 1 to 0.446, while the false positive rate decreases from 0.817 to 0.118, and the false negative rate increases from 0 to 0.554. Nevertheless, machine-based screening remains more accurate and efficient than human assessment. This study evaluated and validated the efficiency (greater than 80%) of high-throughput techniques for screening mutants in complex populations of radiation-induced progeny, and presented a graphical data processing procedure for high-throughput screening of mutants, providing a basis for breeding techniques utilizing HIBs and γ-ray radiation, and offering innovative approaches and methodologies for radiation-induced breeding in the context of high-throughput big data.

The Leguminosae constitutes the second largest family of plants in the world. In the face of global climate change and reduction of cultivated land area, legumes have been attached more importance to feed the future in light of their nutritional values and environmental benefits. However, the yields of legume crops have thus far been far below their theoretical potentials. Despite that hybrid breeding and gene editing technologies have hold great promises to the improvement of crops, they are generally difficult to apply in legumes because of the anatomical constraints for making hybrids in these species and the low transformation efficiency impeding gene modifications. Mutation technologies, as an alternative, can speed up breeding and facilitate functional genomic studies in legume species, yet mutant screening technique remains a major challenge in this field. In this review, we detail the currently available mutant library resources in major legume crops following a brief introduction of the methods of mutation induction. We then revisit the current mainstream technologies for mutant screening, with focus on the new emerging next-generation sequencing-based approaches. Overall, this review provides a collection of information that dedicates to assisting legume researchers and breeders to more easily find and better utilize mutant resources for the improvement of these crops.

Though crossing wild relatives to modern cultivars is a usual means to introduce alleles of stress tolerance, an alternative is domesticating wild species that are already tolerant to various kinds of stresses. As a test case, we chose Kuntze, which has fast growth, short vegetative stage, and broad resistance to pests and diseases. We developed an ethyl methanesulfonate-mutagenized population and obtained three mutants with reduced seed dormancy and one with reduced pod shattering. We crossed one of the mutants of less seed dormancy to the wild type and confirmed that the phenotype was inherited in a Mendelian manner. assembly of genome, and the following resequencing of the F2 progenies successfully identified a Single Nucleotide Polymorphism (SNP) associated with seed dormancy. By crossing and pyramiding the mutant phenotypes, we will be able to turn into a crop which is yet primitive but can be cultivated without pesticides.

ObjectiveIt remains controversial whether tumour mutational burden (TMB) or neoantigens are prognostic markers in hepatocellular carcinoma (HCC). This study aimed to define the function of TMB or neoantigens in antitumour immunotherapy.DesignNeoantigens of patients (n=56) were analysed by pVAC tools with major histocompatibility complex-1 (MHC-I) algorithms based on whole exome sequencing and neoantigens with mutant type IC50 <50 nM were defined as high-affinity neoantigens (HANs). Patients were segregated into HAN-high/low groups by median of HAN value, and overall survival (OS) was analysed. Autologous organoid killing model was developed to clarify the antitumour activity of HANs.ResultsThe value of HAN showed a better correlation with OS (p=0.0199) than TMB (p=0.7505) or neoantigens (p=0.2297) in patients with HCC and positively correlated with the frequency of CD39+CD8+ tumour infiltrating lymphocytes (TILs). Furthermore, HAN-specific CD8+ T cells were identified in CD39+CD8+ TILs, which showed better antitumour activity in HAN-high versus HAN-low group. In addition, more effective HAN peptides were identified in HAN-high versus HAN-low group. Besides, flow cytometry data showed that in fresh tumour, CD39+PD-1intCD8+ TILs displayed an effector phenotype and stronger antitumour activity in HAN-high versus HAN-low group. More importantly, patients in HAN-high versus HAN-low group showed a better prognosis after anti-PD-1 therapy.ConclusionsOur study first demonstrates that HAN value positively correlates with better OS in patients with HCC. HANs trigger antitumour activity by activating tumour-reactive CD39+CD8+ T cells, and patients in HAN-high group benefited more from anti-PD-1 therapy than HAN-low group. These findings may provide a novel strategy for personalised antitumour therapies for HCC.

Background: Several variants of the SARS-CoV-2 have been documented globally during the current COVID-19 pandemic. The N501Y, 69-70del, K417N, and E484K SARS-CoV-2 mutations have been documented among the most relevant due to their potential pathogenic biological effects. This study aimed to design, validate, and propose a fast real-time RT-qPCR assay to detect SARS-CoV-2 mutations with possible clinical and epidemiological relevance in the Mexican population.Methods: Targeting spike (S) gene mutations of SARS-CoV-2 (N501Y, 69-70del, K417N, and E484K), specific primers, and probes for three specific quantitative reverse transcription PCR (RT-qPCR) assays were designed, and validated using Sanger sequencing. These assays were applied in clinical samples of 1060 COVID-19 patients from Jalisco Mexico.Results: In silico analyzes showed high specificity of the three assays. Amplicons of samples were confirmed through sequencing. The screening of samples of COVID-19 patients allowed the identification of the E484K mutation in nine individuals and the identification of P.2 Brazilian variant in Mexico.Conclusion: This work provides low-cost RT-qPCR assays for rapid screening and molecular surveillance of mutations with potential clinical impact. This strategy allowed the detection of E484K mutation and P.2 variant for the first time in samples from the Mexican population.