Targeted genome editing in polyploids: lessons from Brassica.

CRISPR-mediated genome editing has emerged as a powerful tool for creating targeted mutations in the genome for various applications, including studying gene functions, engineering resilience against biotic and abiotic stresses, and increasing yield and quality. However, its utilization is limited to model crops for which well-annotated genome sequences are available. Many crops of dietary and economic importance, such as wheat, cotton, rapeseed-mustard, and potato, are polyploids with complex genomes. Therefore, progress in these crops has been hampered due to genome complexity. Excellent work has been conducted on some species of Brassica for its improvement through genome editing. Although excellent work has been conducted on some species of Brassica for genome improvement through editing, work on polyploid crops, including U's triangle species, holds numerous implications for improving other polyploid crops. In this review, we summarize key examples from genome editing work done on Brassica and discuss important considerations for deploying CRISPR-mediated genome editing more efficiently in other polyploid crops for improvement.

Functional and evolutionary study of MLO gene family in the regulation of Sclerotinia stem rot resistance in Brassica napus L.

BACKGROUND: Oilseed rape (Brassica napus L.) is known as one of the most important oilseed crops cultivated around the world. However, its production continuously faces a huge challenge of Sclerotinia stem rot (SSR), a destructive disease caused by the fungus Sclerotinia sclerotiorum, resulting in huge yield loss annually. The SSR resistance in B. napus is quantitative and controlled by a set of minor genes. Identification of these genes and pyramiding them into a variety are a major strategy for SSR resistance breeding in B. napus. RESULTS: Here, we performed a genome-wide association study (GWAS) using a natural population of B. napus consisting of 222 accessions to identify BnaA08g25340D (BnMLO2_2) as a candidate gene that regulates the SSR resistance. BnMLO2_2 was a member of seven homolog genes of Arabidopsis Mildew Locus O 2 (MLO2) and the significantly SNPs were mainly distributed in the promoter of BnMLO2_2, suggesting a role of BnMLO2_2 expression level in the regulation of SSR resistance. We expressed BnMLO2_2 in Arabidopsis and the transgenic plants displayed an enhanced SSR resistance. Transcriptome profiling of different tissues of B. napus revealed that BnMLO2_2 had the most expression level in leaf and silique tissues among all the 7 BnMLO2 members and also expressed higher in the SSR resistant accession than in the susceptible accession. In Arabidopsis, mlo2 plants displayed reduced resistance to SSR, whereas overexpression of MLO2 conferred plants an enhanced SSR resistance. Moreover, a higher expression level of MLO2 showed a stronger SSR resistance in the transgenic plants. The regulation of MLO2 in SSR resistance may be associated with the cell death. Collinearity and phylogenetic analysis revealed a large expansion of MLO family in Brassica crops. CONCLUSION: Our study revealed an important role of BnMLO2 in the regulation of SSR resistance and provided a new gene candidate for future improvement of SSR resistance in B. napus and also new insights into understanding of MLO family evolution in Brassica crops.

New possibilities for trait improvement via mobile CRISPR-RNA.

CRISPR/Cas9 gene technology is transported as RNA from transgenic roots to distal parts of unmodified grafted scion, where it is translated into proteins to induce heritable mutagenesis at desired loci. This technique has the potential to produce transgene-free and genetically stable plants in difficult-to-propagate and near-extinct species.

High-Throughput Association Mapping in Brassica napus L.: Methods and Applications.

Oil seed rape (Braasica napus L.) is ranked second among oil seed crops cultivated globally for edible oil for human, and seed cake for animal consumption. Recent genetic and genomics advancements highlighted the diversity that exists within B. napus, which is largely discovered using the most promising genetic markers called single nucleotide polymorphism (SNP). Their calling rate is also enhanced to ~100 folds after the continuous advancements in the next generation sequencing (NGS) technologies. As the high throughput of NGS resulted in multi-Giga bases data, the detailed quality control (QC) prior to downstream analyses is a pre-requisite. It mainly involved the removal of false positives, missing proportions, filtering of low-quality SNPs, and adjustments of minor-allele frequency and heterozygosity. After marker-trait association, for conformation of target SNPs, validations of SNPs can be performed using various methods, especially allele-specific PCR assay-based methods have been utilized for SNP genotyping of genes targeting agronomic traits and somaclonal variations occurred during transgenic studies. In the present study, the authors mainly argue on the genotypic progress, and pipelines/methods that are being used for detection, calling, filtering, and validation of SNPs. Also, insight is provided into the application of SNPs in linkage and association mapping, including QTL mapping and genome-wide association studies targeting mainly developmental traits related to the root system and plant architecture, flowering time, silique, and oil quality. Briefly, the present study provides the recent information and recommendations on the SNP genotyping methods and its applications, which can be useful for marker-assisted breeding in B. napus and other crops.

Computational Tools and Resources for CRISPR/Cas Genome Editing.

The past decade has witnessed a rapid evolution in identifying more versatile clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) nucleases and their functional variants, as well as in developing precise CRISPR/Cas-derived genome editors. The programmable and robust features of the genome editors provide an effective RNA-guided platform for fundamental life science research and subsequent applications in diverse scenarios, including biomedical innovation and targeted crop improvement. One of the most essential principles is to guide alterations in genomic sequences or genes in the intended manner without undesired off-target impacts, which strongly depends on the efficiency and specificity of single guide RNA (sgRNA)-directed recognition of targeted DNA sequences. Recent advances in empirical scoring algorithms and machine learning models have facilitated sgRNA design and off-target prediction. In this review, we first briefly introduce the different features of CRISPR/Cas tools that should be taken into consideration to achieve specific purposes. Secondly, we focus on the computer-assisted tools and resources that are widely used in designing sgRNAs and analyzing CRISPR/Cas-induced on- and off-target mutations. Thirdly, we provide insights into the limitations of available computational tools that would help researchers of this field for further optimization. Lastly, we suggest a simple but effective workflow for choosing and applying web-based resources and tools for CRISPR/Cas genome editing.

Role of phytomelatonin responsive to metal stresses: An omics perspective and future scenario.

A pervasive melatonin (N-acetyl-5-methoxytryptamine) reveals a crucial role in stress tolerance and plant development. Melatonin (MT) is a unique molecule with multiple phenotypic expressions and numerous actions within the plants. It has been extensively studied in crop plants under different abiotic stresses such as drought, salinity, heat, cold, and heavy metals. Mainly, MT role is appraised as an antioxidant molecule that deals with oxidative stress by scavenging reactive oxygen species (ROS) and modulating stress related genes. It improves the contents of different antioxidant enzyme activities and thus, regulates the redox hemostasis in crop plants. In this comprehensive review, regulatory effects of melatonin in plants as melatonin biosynthesis, signaling pathway, modulation of stress related genes and physiological role of melatonin under different heavy metal stress have been reviewed in detail. Further, this review has discussed how MT regulates different genes/enzymes to mediate defense responses and overviewed the context of transcriptomics and phenomics followed by the metabolomics pathways in crop plants.

Analysis of Tissue-Specific Defense Responses to Sclerotinia sclerotiorum in Brassica napus.

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum (S. sclerotiorum) is the main disease threat of oilseed rape (Brassica napus), resulting in huge economic losses every year. SSR resistance manifests as quantitative disease resistance (QDR), and no gene with complete SSR resistance has been cloned or reported so far. Transcriptome analysis has revealed a large number of defense-related genes and response processes. However, the similarities and differences in the defense responses of different tissues are rarely reported. In this study, we analyzed the similarities and differences of different tissues in response to S. sclerotiorum at 24 h post inoculation (hpi) by using the published transcriptome data for respective leaf and stem inoculation. At 24 hpi, large differences in gene expression exist in leaf and stem, and there are more differentially expressed genes and larger expression differences in leaf. The leaf is more sensitive to S. sclerotiorum and shows a stronger response than stem. Different defense responses appear in the leaf and stem, and the biosynthesis of lignin, callose, lectin, chitinase, PGIP, and PR protein is activated in leaf. In the stem, lipid metabolism-mediated defense responses are obviously enhanced. For the common defense responses in both leaf and stem, the chain reactions resulting from signal transduction and biological process take the primary responsibility. This research will be beneficial to exploit the potential of different tissues in plant defense and find higher resistance levels of genotypic variability in different environments. Our results are significant in the identification of resistance genes and analysis of defense mechanisms.

The Role of Membrane Transporters in Plant Growth and Development, and Abiotic Stress Tolerance.

The proteins of membrane transporters (MTs) are embedded within membrane-bounded organelles and are the prime targets for improvements in the efficiency of water and nutrient transportation. Their function is to maintain cellular homeostasis by controlling ionic movements across cellular channels from roots to upper plant parts, xylem loading and remobilization of sugar molecules from photosynthesis tissues in the leaf (source) to roots, stem and seeds (sink) via phloem loading. The plant's entire source-to-sink relationship is regulated by multiple transporting proteins in a highly sophisticated manner and driven based on different stages of plant growth and development (PG&D) and environmental changes. The MTs play a pivotal role in PG&D in terms of increased plant height, branches/tiller numbers, enhanced numbers, length and filled panicles per plant, seed yield and grain quality. Dynamic climatic changes disturbed ionic balance (salt, drought and heavy metals) and sugar supply (cold and heat stress) in plants. Due to poor selectivity, some of the MTs also uptake toxic elements in roots negatively impact PG&D and are later on also exported to upper parts where they deteriorate grain quality. As an adaptive strategy, in response to salt and heavy metals, plants activate plasma membranes and vacuolar membrane-localized MTs that export toxic elements into vacuole and also translocate in the root's tips and shoot. However, in case of drought, cold and heat stresses, MTs increased water and sugar supplies to all organs. In this review, we mainly review recent literature from Arabidopsis, halophytes and major field crops such as rice, wheat, maize and oilseed rape in order to argue the global role of MTs in PG&D, and abiotic stress tolerance. We also discussed gene expression level changes and genomic variations within a species as well as within a family in response to developmental and environmental cues.

A Critical Review: Recent Advancements in the Use of CRISPR/Cas9 Technology to Enhance Crops and Alleviate Global Food Crises.

Genome editing (GE) has revolutionized the biological sciences by creating a novel approach for manipulating the genomes of living organisms. Many tools have been developed in recent years to enable the editing of complex genomes. Therefore, a reliable and rapid approach for increasing yield and tolerance to various environmental stresses is necessary to sustain agricultural crop production for global food security. This critical review elaborates the GE tools used for crop improvement. These tools include mega-nucleases (MNs), such as zinc-finger nucleases (ZFNs), and transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Specifically, this review addresses the latest advancements in the role of CRISPR/Cas9 for genome manipulation for major crop improvement, including yield and quality development of biotic stress- and abiotic stress-tolerant crops. Implementation of this technique will lead to the production of non-transgene crops with preferred characteristics that can result in enhanced yield capacity under various environmental stresses. The CRISPR/Cas9 technique can be combined with current and potential breeding methods (e.g., speed breeding and omics-assisted breeding) to enhance agricultural productivity to ensure food security. We have also discussed the challenges and limitations of CRISPR/Cas9. This information will be useful to plant breeders and researchers in the thorough investigation of the use of CRISPR/Cas9 to boost crops by targeting the gene of interest.

SNP- and Haplotype-Based GWAS of Flowering-Related Traits in Brassica napus.

Traits related to flowering time are the most promising agronomic traits that directly impact the seed yield and oil quality of rapeseed (Brassica napus L.). Developing early flowering and maturity rapeseed varieties is an important breeding objective in B. napus. Many studies have reported on days to flowering, but few have reported on budding, bolting, and the interval between bolting and DTF. Therefore, elucidating the genetic architecture of QTLs and genes regulating flowering time, we presented an integrated investigation on SNP and haplotype-based genome-wide association study of 373 diverse B. napus germplasm, which were genotyped by the 60K SNP array and were phenotyped in the four environments. The results showed that a total of 15 and 37 QTLs were detected from SNP and haplotype-based GWAS, respectively. Among them, seven QTL clusters were identified by haplotype-based GWAS. Moreover, three and eight environmentally stable QTLs were detected by SNP-GWAS and haplotype-based GWAS, respectively. By integrating the above two approaches and by co-localizing the four traits, ten (10) genomic regions were under selection on chromosomes A03, A07, A08, A10, C06, C07, and C08. Interestingly, the genomic regions FT.A07.1, FT.A08, FT.C06, and FT.C07 were identified as novel. In these ten regions, a total of 197 genes controlling FT were detected, of which 14 highly expressed DEGs were orthologous to 13 Arabidopsis thaliana genes after integration with transcriptome results. In a nutshell, the above results uncovered the genetic architecture of important agronomic traits related to flowering time and provided a basis for multiple molecular marker-trait associations in B. napus.

Biofortification of Cereals and Pulses Using New Breeding Techniques: Current and Future Perspectives.

Cereals and pulses are consumed as a staple food in low-income countries for the fulfillment of daily dietary requirements and as a source of micronutrients. However, they are failing to offer balanced nutrition due to deficiencies of some essential compounds, macronutrients, and micronutrients, i.e., cereals are deficient in iron, zinc, some essential amino acids, and quality proteins. Meanwhile, the pulses are rich in anti-nutrient compounds that restrict the bioavailability of micronutrients. As a result, the population is suffering from malnutrition and resultantly different diseases, i.e., anemia, beriberi, pellagra, night blindness, rickets, and scurvy are common in the society. These facts highlight the need for the biofortification of cereals and pulses for the provision of balanced diets to masses and reduction of malnutrition. Biofortification of crops may be achieved through conventional approaches or new breeding techniques (NBTs). Conventional approaches for biofortification cover mineral fertilization through foliar or soil application, microbe-mediated enhanced uptake of nutrients, and conventional crossing of plants to obtain the desired combination of genes for balanced nutrient uptake and bioavailability. Whereas, NBTs rely on gene silencing, gene editing, overexpression, and gene transfer from other species for the acquisition of balanced nutritional profiles in mutant plants. Thus, we have highlighted the significance of conventional and NBTs for the biofortification of cereals and pulses. Current and future perspectives and opportunities are also discussed. Further, the regulatory aspects of newly developed biofortified transgenic and/or non-transgenic crop varieties via NBTs are also presented.

Endogenous indole-3-acetic acid and nitric oxide are required for calcium-mediated alleviation of copper oxide nanoparticles toxicity in wheat seedlings.

The action of nanoparticles is increasingly being studied in recent years to minimize their toxic impacts. Besides this, efforts are also being made to minimize their toxicity in crop plants by using various chemicals, i.e. nutrients, donors of signaling molecules, plant hormones, and so on. However, associated alleviatory mechanisms are still not well known. Therefore, in the present study, we have investigated the toxicity of CuONPs and its mitigation by exogenously applied calcium (Ca). The focus was on whether indole-3-acetic acid (IAA) or endogenous nitric oxide (NO) has any role in accomplishing this task. CuONPs declined wheat growth due to increased accumulation of Cu and oxidative stress markers such as superoxide radicals, hydrogen peroxide, and lipid peroxidation (malondialdehyde) and it was also accompanied by a decline in endogenous NO. CuONPs also altered the redox status of ascorbate and glutathione by inhibiting the activity of their regenerating enzymes. This collectively leads to cell death in wheat seedlings. However, exogenous supplementation of Ca mitigated toxic effects of CuONPs by reducing the excess accumulation of Cu, which caused remarkable enhancement in growth, protein contents, photosynthetic pigments, and endogenous NO; altogether protecting wheat roots from cell death. Interestingly, addition of 2,3,5-triiodobenzoic acid (TIBA) further increased CuONPs toxicity even in the presence of Ca, but the addition of IAA rescued this effect of TIBA. These results clearly show that Ca mitigates CuONPs toxicity in wheat seedlings by involving IAA. Further, the results also showed that endogenous NO has a positive and indispensable role in Ca-mediated mitigation of CuONPs toxicity in wheat seedlings.

Health risk assessment of hexachlorocyclohexane in soil, water and plants in the agricultural area of Potohar region, Punjab, Pakistan.

In this study analysis of soil, water and plant residue samples is presented to evaluate the contamination levels and possible health risks. Hexachlorocyclohexane (HCH) is a persistent organic pollutant used as a pesticide in agricultural sector for pest control in order to obtain higher productivity. For analysis soil, water and crop residue samples were collected from different agricultural areas of the northern Punjab region of Pakistan. The investigation of the samples shows significant levels of HCH residues in all types of samples. Gas chromatography-mass spectrometry analysis was used to assess the higher residue levels of HCH in the samples. The concentration of HCH residues detected in samples ranged from 2.43 to 8.88 µg/g in soil, nd -5.87 µg/l in water and nd - 4.87 µg/g in plants. The presence of HCH residues in soil, water and plant samples was beyond the recommended quality guidelines. Human health risk was evaluated for cancer and non-cancer risks through dietary and non-dietary exposure routes. The hazard index was HI > 1 in children and HI < 1 in adults, while the non-dietary incremental lifetime cancer risks (ILCR) were beyond the internationally acceptable limit of 1 × 10-5. Hence, results of the present investigation concluded the presence of high levels of HCH residues in samples and pose high health risk to the inhabitants. These findings are alarming and apprise the concerned departments for the remediation of contamination and proper implementation of environmental laws in the area.

Genome-wide investigation and expression analysis of membrane-bound fatty acid desaturase genes under different biotic and abiotic stresses in sunflower (Helianthus annuus L.).

Membrane-bound fatty acid desaturase (FAD) gene family plays crucial roles in regulation of fatty acid (FA) compositions in plants. Sunflower (Helianthus annuus L.) is an important oilseed crop in the world; however, no comprehensive study on exploring the role of FAD family in relation to stress tolerance in sunflower has been performed yet. In this study, we identified 40 putative FAD genes in H. annuus (HaFAD), which were unevenly distributed across 13 of the total 17 chromosomes. Phylogenetic analysis indicated that HaFAD genes were divided into four subfamilies, as supported by highly conserved gene structures and motifs. Collinearity analysis showed that tandem duplication events played a crucial role in the expansion of HaFAD gene family. In addition, tissue-specific expression showed that 32 HaFAD genes were widely expressed in various tissues or organs of sunflower. Furthermore, qRT-PCR results revealed significant expression changes of HaFAD genes in response to abiotic (cadmium, drought) and biotic (Orobanche cumana) stresses, suggesting their important functions in response to different stresses. Therefore, our results provide insights into HaFAD gene family in response to different stresses, and some specific up-regulated genes such as HaFAD3.2, HaADS8, HaFAD2.1, and HaADS9 would be the potential candidate genes for the sunflower tolerance breeding.

Insights on SNP types, detection methods and their utilization in Brassica species: Recent progress and future perspectives.

The genus Brassica, family Brassicaceae (Cruciferae), comprises many important species of oil crops, vegetables and medicinal plants including B. rapa, B. oleracea, B. nigra, B. napus, B. juncea, B. carinata. Genomic researches in Brassica species is constrained by polyploidization, mainly due to its complicated genomic structure. However, rapid development of methods for detecting single nucleotide polymorphisms (SNP), such as next generation sequencing and SNP microarray, has accelerated release of reference Brassica species genomes as well as discovery of large numbers and genome-wide SNPs, thus intensifying forward genetics in this genus. In this review, we summarize biological characteristics, classification and various methods for detecting SNPs, focusing on high-throughput techniques. Moreover, we describe the pivotal roles of SNPs in genetic diversity, linkage map construction and QTL mapping, comparative genomics, linkage disequilibrium and genome-wide association studies. These insights are expected to deepen our understanding and guide further advancements in Brassica species research.

A global survey of the transcriptome of allopolyploid Brassica napus based on single-molecule long-read isoform sequencing and Illumina-based RNA sequencing data.

Brassica napus is a recent allopolyploid derived from the hybridization of Brassica rapa (Ar Ar ) and Brassica oleracea (Co Co ). Because of the high sequence similarity between the An and Cn subgenomes, it is difficult to provide an accurate landscape of the whole transcriptome of B. napus. To overcome this problem, we applied a single-molecule long-read isoform sequencing (Iso-Seq) technique that can produce long reads to explore the complex transcriptome of B. napus at the isoform level. From the Iso-Seq data, we obtained 147 698 non-redundant isoforms, capturing 37 403 annotated genes. A total of 18.1% (14 934/82 367) of the multi-exonic genes showed alternative splicing (AS). In addition, we identified 549 long non-coding RNAs, the majority of which displayed tissue-specific expression profiles, and detected 7742 annotated genes that possessed isoforms containing alternative polyadenylation sites. Moreover, 31 591 AS events located in open reading frames (ORFs) lead to potential protein isoforms by in-frame or frameshift changes in the ORF. Illumina RNA sequencing of five tissues that were pooled for Iso-Seq was also performed and showed that 69% of the AS events were tissue-specific. Our data provide abundant transcriptome resources for a transcript isoform catalog of B. napus, which will facilitate genome reannotation, strengthen our understanding of the B. napus transcriptome and be applied for further functional genomic research.

Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook.

In most crop breeding programs, the rate of yield increment is insufficient to cope with the increased food demand caused by a rapidly expanding global population. In plant breeding, the development of improved crop varieties is limited by the very long crop duration. Given the many phases of crossing, selection, and testing involved in the production of new plant varieties, it can take one or two decades to create a new cultivar. One possible way of alleviating food scarcity problems and increasing food security is to develop improved plant varieties rapidly. Traditional farming methods practiced since quite some time have decreased the genetic variability of crops. To improve agronomic traits associated with yield, quality, and resistance to biotic and abiotic stresses in crop plants, several conventional and molecular approaches have been used, including genetic selection, mutagenic breeding, somaclonal variations, whole-genome sequence-based approaches, physical maps, and functional genomic tools. However, recent advances in genome editing technology using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated (Cas) proteins have opened the door to a new plant breeding era. Therefore, to increase the efficiency of crop breeding, plant breeders and researchers around the world are using novel strategies such as speed breeding, genome editing tools, and high-throughput phenotyping. In this review, we summarize recent findings on several aspects of crop breeding to describe the evolution of plant breeding practices, from traditional to modern speed breeding combined with genome editing tools, which aim to produce crop generations with desired traits annually.

Lead Toxicity in Cereals: Mechanistic Insight Into Toxicity, Mode of Action, and Management.

Cereals are the major contributors to global food supply, accounting for more than half of the total human calorie requirements. Sustainable availability of quality cereal grains is an important step to address the high-priority issue of food security. High concentrations of heavy metals specifically lead (Pb) in the soil negatively affect biochemical and physiological processes regulating grain quality in cereals. The dietary intake of Pb more than desirable quantity via food chain is a major concern for humans, as it can predispose individuals to chronic health issues. In plant systems, high Pb concentrations can disrupt several key metabolic processes such as electron transport chain, cellular organelles integrity, membrane stability index, PSII connectivity, mineral metabolism, oxygen-evolving complex, and enzymatic activity. Plant growth-promoting rhizobacteria (PGPR) has been recommended as an inexpensive strategy for remediating Pb-contaminated soils. A diverse group of Ascomycetes fungi, i.e., dark septate endophytes is successfully used for this purpose. A symbiotic relationship between endophytes and host cereal induces Pb tolerance by immobilizing Pb ions. Molecular and cellular modifications in plants under Pb-stressed environments are explained by transcription factor families such as bZIP, ERF, and GARP as a regulator. The role of metal tolerance protein (MTP), natural resistance-associated macrophage protein (NRAMP), and heavy metal ATPase in decreasing Pb toxicity is well known. In the present review, we provided the contemporary synthesis of existing data regarding the effects of Pb toxicity on morpho-physiological and biochemical responses of major cereal crops. We also highlighted the mechanism/s of Pb uptake and translocation in plants, critically discussed the possible management strategies and way forward to overcome the menace of Pb toxicity in cereals.