Designing future peanut: the power of genomics-assisted breeding.

KEY MESSAGE: Integrating GAB methods with high-throughput phenotyping, genome editing, and speed breeding hold great potential in designing future smart peanut cultivars to meet market and food supply demands. Cultivated peanut (Arachis hypogaea L.), a legume crop greatly valued for its nourishing food, cooking oil, and fodder, is extensively grown worldwide. Despite decades of classical breeding efforts, the actual on-farm yield of peanut remains below its potential productivity due to the complicated interplay of genotype, environment, and management factors, as well as their intricate interactions. Integrating modern genomics tools into crop breeding is necessary to fast-track breeding efficiency and rapid progress. When combined with speed breeding methods, this integration can substantially accelerate the breeding process, leading to faster access of improved varieties to farmers. Availability of high-quality reference genomes for wild diploid progenitors and cultivated peanuts has accelerated the process of gene/quantitative locus discovery, developing markers and genotyping assays as well as a few molecular breeding products with improved resistance and oil quality. The use of new breeding tools, e.g., genomic selection, haplotype-based breeding, speed breeding, high-throughput phenotyping, and genome editing, is probable to boost genetic gains in peanut. Moreover, renewed attention to efficient selection and exploitation of targeted genetic resources is also needed to design high-quality and high-yielding peanut cultivars with main adaptation attributes. In this context, the combination of genomics-assisted breeding (GAB), genome editing, and speed breeding hold great potential in designing future improved peanut cultivars to meet market and food supply demands.

Genome-Wide Investigation of Apyrase (APY) Genes in Peanut (Arachis hypogaea L.) and Functional Characterization of a Pod-Abundant Expression Promoter AhAPY2-1p.

Peanut (Arachis hypogaea L.) is an important food and feed crop worldwide and is affected by various biotic and abiotic stresses. The cellular ATP levels decrease significantly during stress as ATP molecules move to extracellular spaces, resulting in increased ROS production and cell apoptosis. Apyrases (APYs) are the nucleoside phosphatase (NPTs) superfamily members and play an important role in regulating cellular ATP levels under stress. We identified 17 APY homologs in A. hypogaea (AhAPYs), and their phylogenetic relationships, conserved motifs, putative miRNAs targeting different AhAPYs, cis-regulatory elements, etc., were studied in detail. The transcriptome expression data were used to observe the expression patterns in different tissues and under stress conditions. We found that the AhAPY2-1 gene showed abundant expression in the pericarp. As the pericarp is a key defense organ against environmental stress and promoters are the key elements regulating gene expression, we functionally characterized the AhAPY2-1 promoter for its possible use in future breeding programs. The functional characterization of AhAPY2-1P in transgenic Arabidopsis plants showed that it effectively regulated GUS gene expression in the pericarp. GUS expression was also detected in flowers of transgenic Arabidopsis plants. Overall, these results strongly suggest that APYs are an important future research subject for peanut and other crops, and AhPAY2-1P can be used to drive the resistance-related genes in a pericarp-specific manner to enhance the defensive abilities of the pericarp.

ATP-binding cassette transporters expression profiling revealed its role in the development and regulating stress response in Solanum tuberosum.

The ATP-binding cassette (ABC) transporter gene family plays a vital role in substance transportation, including secondary metabolites, and phytohormones across membranous structures. It is still uncovered in potato (Solanum tuberosum), grown worldwide as a 3rd important food crop. The current study identified a total of 54 Stabc genes in potato genome. The accumulative phylogenetic tree of Stabc with arabidopsis, divided into eight groups (ABCA to ABCH). ABCG was the most prominent group covering 90% of Stabc genes, followed by ABCB group. The number and architecture of exon-intron varied from gene to gene. In addition, the presence of stress-responsive elements in the regulatory regions depicted their role in environmental stress. Furthermore, the tissue-specific and stress-specific expression profiling of Stabc genes and their validation through real-time-qPCR analysis revealed their role in development and stress. The presented results provided useful information for further functional analysis of Stabc genes and can also use as a reference study for other important crops.

Expression profiling of pathogenesis-related Protein-1 (PR-1) genes from Solanum tuberosum reveals its critical role in phytophthora infestans infection.

Pathogen-related (PR) proteins are an integral part of plants' defense mechanisms against various types of biotic and abiotic stresses. A little is known about the importance of these PR proteins in potato defense mechanisms. In the current study, a total of 22 pathogenesis-related 1 genes were identified in the potato genome. All identified proteins possessed the CAP superfamily domain with some other motifs. The cis-acting elements analysis identified several stress-responsive elements, including MYB, ABRE, and MeJRE. The gene duplication events demonstrated purifying and positive selection pressure. Expression profiling showed high transcripts level in root compared to other tissues; however, some genes have tissue-specific expression. Furthermore, the PR-1-5 gene is transcriptionally induced under Phytophthora infestans stress and hormonal (ABA and IAA) treatments. The Real-Time qPCR analysis also validated the RNA-seq data results of genes with maximum expression in roots compared to leaves and stems. The current study results provided basic data for functional characterization and can also use as a reference study for other important crops.

CRISPR/Cas9 to generate plant immunity against pathogen.

Different types of molecular approaches have been used for improving resistance against pathogens to secure food. Efficient and advanced genome editing tool as paralleled to earlier techniques like Zinc Finger Nuclease (ZFN), transcription activator-like effector nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). The approach of CRISPR/Cas9 has updated our abilities of genetic manipulation in many crops. The assembly of purposes that can be achieved through CRISPR/Cas9 and its related products make it a powerful system that will expose novel prospects in the complex domain of plant-pathogen interactions and will help to develop crop resistance against pathogens. CRISPR/Cas9 engineering permits DNA endonuclease guided by an RNA for a range of genome engineering applications across various eukaryotic species and provides an effective platform to create resistance against bacteria, viruses, insects, and fungi. In this review, we discuss CRISPR-Cas9 engineered crop plants resistant to specific pathogens.

Role of primary metabolites in plant defense against pathogens.

Plants under natural environment facing various pathogens, tend to produce defense to maintain their fitness and minimize pathogenic damage. Plant-pathogens interaction is gaining more importance by researches as, their means of the fight are primary metabolites. The ultimate result of either means of defense is pathogenesis or resistance. Plant defense mechanisms can be grouped either into inducible and constitutive defense or chemical, structural and morphological defense. Majority of defense mechanisms have a passive role, i.e. only defensive against pathogens, but a few are very active. Plant primary metabolites are catching interest in their immunity role. Deep information of molecular mechanisms involved during the plant-pathogen system is need of the day for future disease control. This review will highlight the role of primary metabolites and their mechanism of action in plant defense.

Role of secondary metabolites in plant defense against pathogens.

Pathogens get entry into host cell, reproduce there and use biological machinery of host plants which is threat to global crop production. Integrated management strategies based upon minimizing population and use of resistant cultivars can address this potential problem. In developing world farmers are less likely to adopt these approaches instead they prefer the use of chemical pesticides. Reckless use of chemical pesticides is destroying our ecosystem. That's why it is required to explore ecofriendly alternatives, like plant based metabolites to control pathogens. Studies conducted on different plant-metabolites reported that these metabolite can potentially combat plant pathogens. In this study we have also discussed some of plant secondary metabolites including alkaloids, flavonoids and phenolics. In this review we tried to highlight the new trends in utilizing secondary metabolites for controlling bacterial, viral and fungal pathogens with the hope that upcoming drugs will be human and ecosystem friendly.

Long non-coding RNAs as molecular players in plant defense against pathogens.

Long non-coding RNAs (lncRNAs) has significant role in of gene expression and silencing pathways for several biological processes in eukaryotes. lncRNAs has been reported as key player in remodeling chromatin and genome architecture, RNA stabilization and transcription regulation, including enhancer-associated activity. Host lncRNAs are reckoned as compulsory elements of plant defense. In response to pathogen attack, plants protect themselves with the help of lncRNAs -dependent immune systems in which lncRNAs regulate pathogen-associated molecular patterns (PAMPs) and other effectors. Role of lncRNAs in plant microbe interaction has been studied extensively but regulations of several lncRNAs still need extensive research. In this study we discussed and provide as overview the topical advancements and findings relevant to pathogen attack and plant defense mediated by lncRNAs. It is hoped that lncRNAs would be exploited as a mainstream player to achieve food security by tackling different plant diseases.

Tannins as antimicrobial agents: Understanding toxic effects on pathogens.

"Tannins" are compounds that belong to a group of secondary metabolites found in plants. They have a polyphenolic nature and exhibit active actions as first line defenses against invading pathogens. Several studies have demonstrated the multiple activities of tannins, highlighting their effectiveness as broad-spectrum antimicrobial agents. Tannins have reported as antibacterial, antifungal, and antiviral compounds by preventing enzymatic activities and inhibiting the synthesis of nucleic acids. Additionally, tannins primarily strengthen the plant cell wall, making it almost impenetrable to harmful pathogens. Most tannins are synthesized via the phenylpropanoid pathway to become secondary metabolites. Increased uptake of tannins has the potential to provide permanent immunity to subsequent infections by strengthening cell walls and producing antimicrobial compounds. Tannins also demonstrate a synergistic response with other defense-related molecules, such as phytoalexins and pathogenesis-related proteins, including antimicrobial peptides. Studying the mechanisms mediated by tannins on pathogen behaviors would be beneficial in stimulating plant defense against pathogens. This understanding could help explain the occurrence of diseases and outbreaks and enable potential mitigation in both natural and agricultural ecosystems.

Molecular cloning and functional characterization of the promoter of a novel Aspergillus flavus inducible gene (AhOMT1) from peanut.

Peanut is an important oil and food legume crop grown in more than one hundred countries, but the yield and quality are often impaired by different pathogens and diseases, especially aflatoxins jeopardizing human health and causing global concerns. For better management of aflatoxin contamination, we report the cloning and characterization of a novel A. flavus inducible promoter of the O-methyltransferase gene (AhOMT1) from peanut. The AhOMT1 gene was identified as the highest inducible gene by A. flavus infection through genome-wide microarray analysis and verified by qRT-PCR analysis. AhOMT1 gene was studied in detail, and its promoter, fussed with the GUS gene, was introduced into Arabidopsis to generate homozygous transgenic lines. Expression of GUS gene was studied in transgenic plants under the infection of A. flavus. The analysis of AhOMT1 gene characterized by in silico assay, RNAseq, and qRT-PCR revealed minute expression in different organs and tissues with trace or no response to low temperature, drought, hormones, Ca2+, and bacterial stresses, but highly induced by A. flavus infection. It contains four exons encoding 297 aa predicted to transfer the methyl group of S-adenosyl-L-methionine (SAM). The promoter contains different cis-elements responsible for its expression characteristics. Functional characterization of AhOMT1P in transgenic Arabidopsis plants demonstrated highly inducible behavior only under A. flavus infection. The transgenic plants did not show GUS expression in any tissue(s) without inoculation of A. flavus spores. However, GUS activity increased significantly after inoculation of A. flavus and maintained a high level of expression after 48 hours of infection. These results provided a novel way for future management of peanut aflatoxins contamination through driving resistance genes in A. flavus inducible manner.

Genome-Wide Analysis and Expression Profiling of DUF668 Genes in Glycine max under Salt Stress.

The DUF668 gene performs a critical role in mitigating the impact of abiotic stress factors. In this study, we identified 30 DUF668 genes in a soybean genome, distributed across fifteen chromosomes. The phylogenetic analysis classified the DUF668 genes into three groups (group I, group II, and group III). Interestingly, gene structure analysis illustrated that several GmDUF668 genes were without introns. Furthermore, the subcellular localization results suggested that GmDUF668 proteins were present in the nucleus, mitochondria, cytoplasm, and plasma membrane. GmDUF668 promoters were analyzed in silico to gain insight into the presence of regulatory sequences for TFs binding. The expression profiling illustrated that GmDUF668 genes showed expression in leaves, roots, nodules, and flowers. To investigate their response to salt stress, we utilized the RNA sequencing data of GmDUF668 genes. The results unveiled that GmDUF668-8, GmDUF668-20, and GmDUF668-30 genes were upregulated against salt stress treatment. We further validated these findings using qRT-PCR analysis. These findings provide a scientific basis to explore the functions of GmDUF668 genes against different stress conditions.

In-silico identification and characterization of O-methyltransferase gene family in peanut (Arachis hypogaea L.) reveals their putative roles in development and stress tolerance.

Cultivated peanut (Arachis hypogaea) is a leading protein and oil-providing crop and food source in many countries. At the same time, it is affected by a number of biotic and abiotic stresses. O-methyltransferases (OMTs) play important roles in secondary metabolism, biotic and abiotic stress tolerance. However, the OMT genes have not been comprehensively analyzed in peanut. In this study, we performed a genome-wide investigation of A. hypogaea OMT genes (AhOMTs). Gene structure, motifs distribution, phylogenetic history, genome collinearity and duplication of AhOMTs were studied in detail. Promoter cis-elements, protein-protein interactions, and micro-RNAs targeting AhOMTs were also predicted. We also comprehensively studied their expression in different tissues and under different stresses. We identified 116 OMT genes in the genome of cultivated peanut. Phylogenetically, AhOMTs were divided into three groups. Tandem and segmental duplication events played a role in the evolution of AhOMTs, and purifying selection pressure drove the duplication process. AhOMT promoters were enriched in several key cis-elements involved in growth and development, hormones, light, and defense-related activities. Micro-RNAs from 12 different families targeted 35 AhOMTs. GO enrichment analysis indicated that AhOMTs are highly enriched in transferase and catalytic activities, cellular metabolic and biosynthesis processes. Transcriptome datasets revealed that AhOMTs possessed varying expression levels in different tissues and under hormones, water, and temperature stress. Expression profiling based on qRT-PCR results also supported the transcriptome results. This study provides the theoretical basis for further work on the biological roles of AhOMT genes for developmental and stress responses.

Genome-Wide Characterization of Ascorbate Peroxidase Gene Family in Peanut (Arachis hypogea L.) Revealed Their Crucial Role in Growth and Multiple Stress Tolerance.

Ascorbate peroxidase (APX), an important antioxidant enzyme, plays a significant role in ROS scavenging by catalyzing the decrease of hydrogen peroxide under various environmental stresses. Nevertheless, information about the APX gene family and their evolutionary and functional attributes in peanut (Arachis hypogea L.) was not reported. Therefore, a comprehensive genome-wide study was performed to discover the APX genes in cultivated peanut genome. This study identified 166 AhAPX genes in the peanut genome, classified into 11 main groups. The gene duplication analysis showed that AhAPX genes had experienced segmental duplications and purifying selection pressure. Gene structure and motif investigation indicated that most of the AhAPX genes exhibited a comparatively well-preserved exon-intron pattern and motif configuration contained by the identical group. We discovered five phytohormones-, six abiotic stress-, and five growth and development-related cis-elements in the promoter regions of AhAPX. Fourteen putative ah-miRNAs from 12 families were identified, targeting 33 AhAPX genes. Furthermore, we identified 3,257 transcription factors from 38 families (including AP2, ARF, B3, bHLH, bZIP, ERF, MYB, NAC, WRKY, etc.) in 162 AhAPX genes. Gene ontology and KEGG enrichment analysis confirm the role of AhAPX genes in oxidoreductase activity, catalytic activity, cell junction, cellular response to stimulus and detoxification, biosynthesis of metabolites, and phenylpropanoid metabolism. Based on transcriptome datasets, some genes such as AhAPX4/7/17/77/82/86/130/133 and AhAPX160 showed significantly higher expression in diverse tissues/organs, i.e., flower, leaf, stem, roots, peg, testa, and cotyledon. Likewise, only a few genes, including AhAPX4/17/19/55/59/82/101/102/137 and AhAPX140, were significantly upregulated under abiotic (drought and cold), and phytohormones (ethylene, abscisic acid, paclobutrazol, brassinolide, and salicylic acid) treatments. qRT-PCR-based expression profiling presented the parallel expression trends as generated from transcriptome datasets. Our discoveries gave new visions into the evolution of APX genes and provided a base for further functional examinations of the AhAPX genes in peanut breeding programs.

Corrigendum: Genome-Wide Identification and Expression Profiling of Germin-Like Proteins Reveal Their Role in Regulating Abiotic Stress Response in Potato.

[This corrects the article DOI: 10.3389/fpls.2021.831140.].

Comprehensive genome sequence analysis of the devastating tobacco bacterial phytopathogen Ralstonia solanacearum strain FJ1003.

Due to its high genetic diversity and broad host range, Ralstonia solanacearum, the causative phytopathogen of the bacterial wilt (BW) disease, is considered a "species complex". The R. solanacearum strain FJ1003 belonged to phylotype I, and was isolated from the Fuzhou City in Fujian Province of China. The pathogen show host specificity and infects tobacco, especially in the tropical and subtropical regions. To elucidate the pathogenic mechanisms of FJ1003 infecting tobacco, a complete genome sequencing of FJ1003 using single-molecule real-time (SMRT) sequencing technology was performed. The full genome size of FJ1003 was 5.90 Mb (GC%, 67%), containing the chromosome (3.7 Mb), megaplasmid (2.0 Mb), and small plasmid (0.2 Mb). A total of 5133 coding genes (3446 and 1687 genes for chromosome and megaplasmid, respectively) were predicted. A comparative genomic analysis with other strains having the same and different hosts showed that the FJ1003 strain had 90 specific genes, possibly related to the host range of R. solanacearum. Horizontal gene transfer (HGT) was widespread in the genome. A type Ⅲ effector protein (Rs_T3E_Hyp14) was present on both the prophage and genetic island (GI), suggesting that this gene might have been acquired from other bacteria via HGT. The Rs_T3E_Hyp14 was proved to be a virulence factor in the pathogenic process of R. solanacearum through gene knockout strategy, which affects the pathogenicity and colonization ability of R. solanacearum in the host. Therefore, this study will improve our understanding of the virulence of R. solanacearum and provide a theoretical basis for tobacco disease resistance breeding.

Genome-wide identification of germin-like proteins in peanut (Arachis hypogea L.) and expression analysis under different abiotic stresses.

Peanut is an important food and feed crop, providing oil and protein nutrients. Germins and germin-like proteins (GLPs) are ubiquitously present in plants playing numerous roles in defense, growth and development, and different signaling pathways. However, the GLP members have not been comprehensively studied in peanut at the genome-wide scale. We carried out a genome-wide identification of the GLP genes in peanut genome. GLP members were identified comprehensively, and gene structure, genomic positions, motifs/domains distribution patterns, and phylogenetic history were studied in detail. Promoter Cis-elements, gene duplication, collinearity, miRNAs, protein-protein interactions, and expression were determined. A total of 84 GLPs (AhGLPs ) were found in the genome of cultivated peanut. These GLP genes were clustered into six groups. Segmental duplication events played a key role in the evolution of AhGLPs, and purifying selection pressure was underlying the duplication process. Most AhGLPs possessed a well-maintained gene structure and motif organization within the same group. The promoter regions of AhGLPs contained several key cis-elements responsive to 'phytohormones', 'growth and development', defense, and 'light induction'. Seven microRNAs (miRNAs) from six families were found targeting 25 AhGLPs. Gene Ontology (GO) enrichment analysis showed that AhGLPs are highly enriched in nutrient reservoir activity, aleurone grain, external encapsulating structure, multicellular organismal reproductive process, and response to acid chemicals, indicating their important biological roles. AhGLP14, AhGLP38, AhGLP54, and AhGLP76 were expressed in most tissues, while AhGLP26, AhGLP29, and AhGLP62 showed abundant expression in the pericarp. AhGLP7, AhGLP20, and AhGLP21, etc., showed specifically high expression in embryo, while AhGLP12, AhGLP18, AhGLP40, AhGLP78, and AhGLP82 were highly expressed under different hormones, water, and temperature stress. The qRT-PCR results were in accordance with the transcriptome expression data. In short, these findings provided a foundation for future functional investigations on the AhGLPs for peanut breeding programs.

Proteomics analysis of Cyclobalanopsis gilva provides new insights of low seed germination.

A valuable plant, Cyclobalanopsis gilva, (C. gilva) has a low germination rate (below 50%) under its natural habitations. In order to examine the reasons for the low germination rate, the seeds of C. gilva (germinated and non-germinated) were evaluated using comparative proteomics analysis. A total of 3078 differentially abundant proteins (DAPs) were identified through a label-free method; most DAPs up-accumulated in germinated seeds were related to carbohydrates metabolism. Furthermore the proteins related to the signals, stress, and protein metabolism showed up-accumulation in germinated and no abundance or down-accumulation in non-germinated seeds. Enzyme activity of HK, PGK, PFK, and PK from glycolysis in SG-Control samples were 1.7-, 1.1-, 1.4-, and 1.3-times higher compared with those in control ones while CS, NAD-MDH, α-KGDH, and ICDH from the TCA cycle in SG-Control samples were 3, 1.1, 1.2, and 1.2 times higher than those in NG-Control ones. The ß-amylase activity was 4-fold higher in successfully germinated seeds compared to non-germinated seeds. Interestingly, α-amylase did not show significant changes in protein abundance and enzyme activity among the three samples. The present findings reveal that unsuccessful germination of C. gilva seeds is due to lack of energy.

Mitogen-Activated Protein Kinase Expression Profiling Revealed Its Role in Regulating Stress Responses in Potato (Solanum tuberosum).

Mitogen-activated protein kinase (MAPK) cascades are the universal signal transduction networks that regulate cell growth and development, hormone signaling, and other environmental stresses. However, their essential contribution to plant tolerance is very little known in the potato (Solanum tuberosum) plant. The current study carried out a genome-wide study of StMAPK and provided a deep insight using bioinformatics tools. In addition, the relative expression of StMAPKs was also assessed in different plant tissues. The similarity search results identified a total of 22 StMAPK genes in the potato genome. The sequence alignment also showed conserved motif TEY/TDY in most StMAPKs with conserved docking LHDXXEP sites. The phylogenetic analysis divided all 22 StMAPK genes into five groups, i.e., A, B, C, D, and E, showing some common structural motifs. In addition, most of the StMAPKs were found in a cluster form at the terminal of chromosomes. The promoter analysis predicted several stress-responsive Cis-acting regulatory elements in StMAPK genes. Gene duplication under selection pressure also indicated several purifying and positive selections in StMAPK genes. In potato, StMAPK2, StMAPK6, and StMAPK19 showed a high expression in response to heat stress. Under ABA and IAA treatment, the expression of the total 20 StMAPK genes revealed that ABA and IAA played an essential role in this defense process. The expression profiling and real-time qPCR (RT-qPCR) exhibited their high expression in roots and stems compared to leaves. These results deliver primary data for functional analysis and provide reference data for other important crops.

Saponin toxicity as key player in plant defense against pathogens.

Microbial pathogens attack every plant tissue, including leaves, roots, shoots, and flowers during all growth stages. Thus, they cause several diseases resulting in a plant's failure or loss of the whole crop in severe cases. To combat the pathogens attack, plants produce some biologically active toxic compounds known as saponins. The saponins are secondary metabolic compounds produced in healthy plants with potential anti-pathogenic activity and serve as potential chemical barriers against pathogens. Saponins are classified into two major groups the steroidal and terpenoid saponins. Here, we reported the significance of saponin toxins in the war against insect pests, fungal, and bacterial pathogens. Saponins are present in both cultivated (chilies, spinach, soybean, quinoa, onion, oat, tea, etc.) and wild plant species. As they are natural toxic constituents of plant defense, breeders and plant researchers aiming to boost plant imm unity should focus on transferring these compounds in cash crops.

Establishment of a Novel and Efficient Agrobacterium-Mediated in Planta Transformation System for Passion Fruit (Passiflora edulis).

Passion fruit (Passiflora edulis) is an important fruit crop with high economic value. Genetic engineering plays an important role in crop improvement with desired traits and gene functional studies. The lack of a simple, efficient, and stable transformation system for passion fruit has greatly limited gene functional studies. In this study, a simple and efficient Agrobacterium-mediated in planta transformation system for passion fruit was established, using Agrobacterium virulent strain EHA105 harboring the binary vectors pCAMBIA1301 and pCAMBIA1302 with GUS and GFP reporter genes. The system requires less time and labor costs than conventional transformation systems, and no additional phytohormones and sterile conditions are required. Regeneration efficiency of 86% and transformation efficiency of 29% were achieved, when the wounds were wrapped with Parafilm and the plants were kept in darkness for 15 days. Approximately 75% of the regenerated plants had a single shoot and 26% multiple shoots. The transformation was confirmed at the DNA and RNA levels as well as by GUS staining and GFP fluorescent measurements. The developed protocol will contribute to the genetic improvement of passion fruit breeding.