Beyond NGS data sharing for plant ecological resilience and improvement of agronomic traits.

Decoding complex plant omics is essential for advancing our understanding of plant biology, evolution, and breeding as well as for practical applications in agriculture, conservation, and biotechnology. The advent of Next-Generation Sequencing (NGS) has revolutionized global plant genomic research, offering high-throughput, cost-effective, and accurate methods for generating genomic data. However, challenges still exist that suggest an entirely unresolved genome characterized by high heterozygosity, extensive repetitive sequences, and complex ploidy features. In addition, individual investigation of genomic information from various genetic resources is essential for omics research, as there are differences in traits within a single breed beyond a species due to the uniqueness of sequence variation. This article provides high-quality genomic and transcriptomic insights targeted at the agronomical background.

The landscape of abiotic and biotic stress-responsive splice variants with deep RNA-seq datasets in hot pepper.

Alternative splicing (AS) is a widely observed phenomenon in eukaryotes that plays a critical role in development and stress responses. In plants, the large number of RNA-seq datasets in response to different environmental stressors can provide clues for identification of condition-specific and/or common AS variants for preferred agronomic traits. We report RNA-seq datasets (350.7 Gb) from Capsicum annuum inoculated with one of three bacteria, one virus, or one oomycete and obtained additional existing transcriptome datasets. In this study, we investigated the landscape of AS in response to environmental stressors, signaling molecules, and tissues from 425 total samples comprising 841.49 Gb. In addition, we identified genes that undergo AS under specific and shared stress conditions to obtain potential genes that may be involved in enhancing tolerance to stressors. We uncovered 1,642,007 AS events and identified 4,354 differential alternative splicing genes related to environmental stressors, tissues, and signaling molecules. This information and approach provide useful data for basic-research focused on enhancing tolerance to environmental stressors in hot pepper or establishing breeding programs.

Global co-expression network for key factor selection on environmental stress RNA-seq dataset in Capsicum annuum.

Environmental stresses significantly affect plant growth, development, and productivity. Therefore, a deeper understanding of the underlying stress responses at the molecular level is needed. In this study, to identify critical genetic factors associated with environmental stress responses, the entire 737.3 Gb clean RNA-seq dataset across abiotic, biotic stress, and phytohormone conditions in Capsicum annuum was used to perform individual differentially expressed gene analysis and to construct gene co-expression networks for each stress condition. Subsequently, gene networks were reconstructed around transcription factors to identify critical factors involved in the stress responses, including the NLR gene family, previously implicated in resistance. The abiotic and biotic stress networks comprise 233 and 597 hubs respectively, with 10 and 89 NLRs. Each gene within the NLR groups in the network exhibited substantial expression to particular stresses. The integrated analysis strategy of the transcriptome network revealed potential key genes for complex environmental conditions. Together, this could provide important clues to uncover novel key factors using high-throughput transcriptome data in other species as well as plants.

High-Resolution Melting (HRM) Genotyping.

High-resolution melting (HRM) analysis is a simple, fast, and inexpensive real-time polymerase chain reaction (PCR)-based method used to identify genetic variation between populations and detect single-nucleotide polymorphisms (SNPs) in nucleic acid sequences. HRM is a powerful technique that detects the differences between SNP allele melting temperatures by using a fluorescent dye inserted into the duplex deoxyribonucleic acid (DNA) structure. Prior to performing HRM analysis, optimizing the primer design, PCR mixture, and software settings is essential to obtain accurate and reliable results. In this chapter, we describe a detailed SNP genotyping method that includes primer design and the analysis of the shapes and positions of the melt curve of the luminescence intensity of the fluorescent dye attached to amplified DNA using software of qPCR instruments. This protocol is applicable for genotyping germplasm, genetic mapping, and marker-assisted breeding in plants.

Universal gene co-expression network reveals receptor-like protein genes involved in broad-spectrum resistance in pepper (Capsicum annuum L.).

Receptor-like proteins (RLPs) on plant cells have been implicated in immune responses and developmental processes. Although hundreds of RLP genes have been identified in plants, only a few RLPs have been functionally characterized in a limited number of plant species. Here, we identified RLPs in the pepper (Capsicum annuum) genome and performed comparative transcriptomics coupled with the analysis of conserved gene co-expression networks (GCNs) to reveal the role of core RLP regulators in pepper-pathogen interactions. A total of 102 RNA-seq datasets of pepper plants infected with four pathogens were used to construct CaRLP-targeted GCNs (CaRLP-GCNs). Resistance-responsive CaRLP-GCNs were merged to construct a universal GCN. Fourteen hub CaRLPs, tightly connected with defense-related gene clusters, were identified in eight modules. Based on the CaRLP-GCNs, we evaluated whether hub CaRLPs in the universal GCN are involved in the biotic stress response. Of the nine hub CaRLPs tested by virus-induced gene silencing, three genes (CaRLP264, CaRLP277, and CaRLP351) showed defense suppression with less hypersensitive response-like cell death in race-specific and non-host resistance response to viruses and bacteria, respectively, and consistently enhanced susceptibility to Ralstonia solanacearum and/or Phytophthora capsici. These data suggest that key CaRLPs are involved in the defense response to multiple biotic stresses and can be used to engineer a plant with broad-spectrum resistance. Together, our data show that generating a universal GCN using comprehensive transcriptome datasets can provide important clues to uncover genes involved in various biological processes.

Tissue-Specific RNA-Seq Analysis and Identification of Receptor-Like Proteins Related to Plant Growth in Capsicum annuum.

Receptor-like proteins (RLPs) are a gene family of cell surface receptors that are involved in plant growth, development, and disease resistance. In a recent study, 438 pepper RLP genes were identified in the Capsicum annuum genome (CaRLPs) and determined to be present in response to multiple biotic stresses. To further understand the role of CaRLPs in plant growth and development, we analyzed expression patterns of all CaRLPs from various pepper tissues and developmental stages using RNA-seq. Ten CaRLP genes were selected for further analysis according to transcript levels with hierarchical clustering. The selected CaRLP genes displayed similarity of motifs within the same groups and structures typical of RLPs. To examine RLP function in growth and development, we performed loss-of-function analysis using a virus-induced gene silencing system. Three of the ten tested CaRLPs (CaRLP238, 253, and 360) in silenced plants exhibited phenotypic alteration with growth retardation compared to controls. All three gene-silenced peppers showed significant differences in root dry weight. Only CaRLP238 had significant differences in both root and shoot dry weight. Our results suggest that CaRLPs may play important roles in regulation of plant growth and development as well as function in defense responses to biotic stresses in the RLP gene family.

Leaf-to-Whole Plant Spread Bioassay for Pepper and Ralstonia solanacearum Interaction Determines Inheritance of Resistance to Bacterial Wilt for Further Breeding.

Bacterial wilt (BW) disease from Ralstonia solanacearum is a serious disease and causes severe yield losses in chili peppers worldwide. Resistant cultivar breeding is the most effective in controlling BW. Thus, a simple and reliable evaluation method is required to assess disease severity and to investigate the inheritance of resistance for further breeding programs. Here, we developed a reliable leaf-to-whole plant spread bioassay for evaluating BW disease and then, using this, determined the inheritance of resistance to R. solanacearum in peppers. Capsicum annuum 'MC4' displayed a completely resistant response with fewer disease symptoms, a low level of bacterial cell growth, and significant up-regulations of defense genes in infected leaves compared to those in susceptible 'Subicho'. We also observed the spreading of wilt symptoms from the leaves to the whole susceptible plant, which denotes the normal BW wilt symptoms, similar to the drenching method. Through this, we optimized the evaluation method of the resistance to BW. Additionally, we performed genetic analysis for resistance inheritance. The parents, F1 and 90 F2 progenies, were evaluated, and the two major complementary genes involved in the BW resistance trait were confirmed. These could provide an accurate evaluation to improve resistant pepper breeding efficiency against BW.

Comprehensive transcriptome resource for response to phytohormone-induced signaling in Capsicum annuum L.

OBJECTIVES: Phytohormones are small signaling molecules with crucial roles in plant growth, development, and environmental adaptation to biotic and abiotic stress responses. Despite several previously published molecular studies focused on plant hormones, our understanding of the transcriptome induced by phytohormones remains unclear, especially in major crops. Here, we aimed to provide transcriptome dataset using RNA sequencing for phytohormone-induced signaling in plant. DATA DESCRIPTION: We used high-throughput RNA sequencing profiling to investigate the pepper plant response to treatment with four major phytohormones (salicylic acid, jasmonic acid, ethylene, and abscisic acid). This dataset yielded 78 samples containing three biological replicates per six different time points for each treatment and the control, constituting 187.8 Gb of transcriptome data (2.4 Gb of each sample). This comprehensive parallel transcriptome data provides valuable information for understanding the relationships and molecular networks that regulate the expression of phytohormone-related genes involved in plant developments and environmental stress adaptation.

Transcriptome profiling of abiotic responses to heat, cold, salt, and osmotic stress of Capsicum annuum L.

Peppers (Capsicum annuum L.), belonging to the Solanaceae family, are one of the most economically important crops globally. Like other crops, peppers are threatened by diverse environmental conditions due to different pathogens and abiotic stresses. High-quality reference genomes with massive datasets of transcriptomes from various conditions can provide clues to preferred agronomic traits for breeding. However, few global gene expression profiling datasets have been published to examine the environmental stress-resistant mechanisms in peppers. In this study, we report the RNA-seq analyses of peppers treated with heat, cold, salinity, and osmotic stress at six different time points. RNA-seq libraries from 78 RNA samples containing three biological replicates per time point for each of the abiotic stresses and a mock control were constructed. A total of 204.68 Gb of transcriptome data were verified by differentially expressed genes and gene ontology enrichment analysis. Analyses of the transcriptome data in this study will provide useful information for basic studies of various stimuli to facilitate the development of stress-resistant pepper cultivars.

Development of Clustered Resistance Gene Analogs-Based Markers of Resistance to Phytophthora capsici in Chili Pepper.

The soil-borne pathogen Phytophthora capsici causes severe destruction of Capsicum spp. Resistance in Capsicum against P. capsici is controlled by numerous minor quantitative trait loci (QTLs) and a consistent major QTL on chromosome 5. Molecular markers on Capsicum chromosome 5 have been developed to identify the predominant genetic contributor to resistance but have achieved little success. In this study, previously reported molecular markers were used to reanalyze the major QTL region on chromosome 5 (6.2 Mbp to 139.2 Mbp). Candidate resistance gene analogs (RGAs) were identified in the extended major QTL region including 14 nucleotide binding site leucine-rich repeats, 3 receptor-like kinases, and 1 receptor-like protein. Sequence comparison of the candidate RGAs was performed between two Capsicum germplasms that are resistant and susceptible, respectively, to P. capsici. 11 novel RGA-based markers were developed through high-resolution melting analysis which were closely linked to the major QTL for P. capsici resistance. Among the markers, CaNB-5480 showed the highest cosegregation rate at 86.9% and can be applied to genotyping of the germplasms that were not amenable by previous markers. With combination of three markers such as CaNB-5480, CaRP-5130 and CaNB-5330 increased genotyping accuracy for 61 Capsicum accessions. These could be useful to facilitate high-throughput germplasm screening and further characterize resistance genes against P. capsici in pepper.

Genome-wide Identification, Classification, and Expression Analysis of the Receptor-Like Protein Family in Tomato.

Receptor-like proteins (RLPs) are involved in plant development and disease resistance. Only some of the RLPs in tomato (Solanum lycopersicum L.) have been functionally characterized though 176 genes encoding RLPs, which have been identified in the tomato genome. To further understand the role of RLPs in tomato, we performed genome-guided classification and transcriptome analysis of these genes. Phylogenic comparisons revealed that the tomato RLP members could be divided into eight subgroups and that the genes evolved independently compared to similar genes in Arabidopsis. Based on location and physical clustering analyses, we conclude that tomato RLPs likely expanded primarily through tandem duplication events. According to tissue specific RNA-seq data, 71 RLPs were expressed in at least one of the following tissues: root, leaf, bud, flower, or fruit. Several genes had expression patterns that were tissue specific. In addition, tomato RLP expression profiles after infection with different pathogens showed distinguish gene regulations according to disease induction and resistance response as well as infection by bacteria and virus. Notably, Some RLPs were highly and/or unique expressed in susceptible tomato to pathogen, suggesting that the RLP could be involved in disease response, possibly as a host-susceptibility factor. Our study could provide an important clues for further investigations into the function of tomato RLPs involved in developmental and response to pathogens.

Genome-wide comparative analysis in Solanaceous species reveals evolution of microRNAs targeting defense genes in Capsicum spp.

MicroRNAs (miRNAs) play roles in various biological processes in plants including growth, development, and disease resistance. Previous studies revealed that some plant miRNAs produce secondary small interfering RNAs (siRNAs) such as phased, secondary siRNAs (phasiRNAs), and they regulate a cascade of gene expression. We performed a genome-wide comparative analysis of miRNAs in Solanaceous species (pepper, tomato, and potato), from an evolutionary perspective. Microsynteny of miRNAs was analysed based on the genomic loci and their flanking genes and most of the well-conserved miRNA genes maintained microsynteny in Solanaceae. We identified target genes of the miRNAs via degradome analysis and found that several miRNAs target many genes encoding nucleotide-binding leucine-rich repeat (NLR) or receptor-like proteins (RLPs), which are known to be major players in defense responses. In addition, disease-resistance-associated miRNAs trigger phasiRNA production in pepper, indicating amplification of the regulation of disease-resistance gene families. Among these, miR-n033a-3p, whose target NLRs have been duplicated in pepper, targets more NLRs belonging to specific subgroup in pepper than those in potato. miRNAs targeting resistance genes might have evolved to regulate numerous targets in Solanaceae, following expansion of target resistance genes. This study provides an insight into evolutionary relationship between miRNAs and their target defense genes in plants.

Global gene expression profiling for fruit organs and pathogen infections in the pepper, Capsicum annuum L.

Hot pepper (Capsicum annuum) is one of the most consumed vegetable crops in the world and useful to human as it has many nutritional and medicinal values. Genomic resources of pepper are publically available since the pepper genomes have been completed and massive data such as transcriptomes have been deposited. Nevertheless, global transcriptome profiling is needed to identify molecular mechanisms related to agronomic traits in pepper, but limited analyses are published. Here, we report the comprehensive analysis of pepper transcriptomes during fruit ripening and pathogen infection. For the ripening, transcriptome data were obtained from placenta and pericarp at seven developmental stages. To reveal global transcriptomic landscapes during infection, leaves at six time points post-infection by one of three pathogens (Phytophthora infestans, Pepper mottle virus, and Tobacco mosaic virus P0 strain) were profiled. The massive parallel transcriptome profiling in this study will serve as a valuable resource for detection of molecular networks of fruit development and disease resistance in Capsicum annuum.

Bacillus velezensis YC7010 Enhances Plant Defenses Against Brown Planthopper Through Transcriptomic and Metabolic Changes in Rice.

Brown planthopper (BPH; Nilaparvata lugens Stål) is one of the most serious insect pests, which reduce rice yield remarkably in many rice-growing areas. A few plant growth-promoting rhizobacteria induce systemic resistance against herbivorous insects. Here we show that root drenching of rice seedlings with an endophytic strain Bacillus velezensis YC7010 enhanced defenses against BPH. Based on high-throughput transcriptome analysis, systemic resistance against BPH was induced by B. velezensis YC7010 via salicylic acid (SA)- and jasmonic acid (JA)-dependent pathways. Increased leaf contents of secondary metabolites, tricin and C-glycosyl flavone and cell-wall contents of lignin and cellulose were the key defense mechanisms inducing resistance against BPH during the three-way interaction. This study shows for the first time that chemical changes and strengthening of physical barriers play important roles simultaneously in plant defense against BPH in rice by the endophytic bacteria. This defense was induced by lipopeptides including a novel bacillopeptin X.

New reference genome sequences of hot pepper reveal the massive evolution of plant disease-resistance genes by retroduplication.

BACKGROUND: Transposable elements are major evolutionary forces which can cause new genome structure and species diversification. The role of transposable elements in the expansion of nucleotide-binding and leucine-rich-repeat proteins (NLRs), the major disease-resistance gene families, has been unexplored in plants. RESULTS: We report two high-quality de novo genomes (Capsicum baccatum and C. chinense) and an improved reference genome (C. annuum) for peppers. Dynamic genome rearrangements involving translocations among chromosomes 3, 5, and 9 were detected in comparison between C. baccatum and the two other peppers. The amplification of athila LTR-retrotransposons, members of the gypsy superfamily, led to genome expansion in C. baccatum. In-depth genome-wide comparison of genes and repeats unveiled that the copy numbers of NLRs were greatly increased by LTR-retrotransposon-mediated retroduplication. Moreover, retroduplicated NLRs are abundant across the angiosperms and, in most cases, are lineage-specific. CONCLUSIONS: Our study reveals that retroduplication has played key roles for the massive emergence of NLR genes including functional disease-resistance genes in pepper plants.

Genome analysis of Hibiscus syriacus provides insights of polyploidization and indeterminate flowering in woody plants.

Hibiscus syriacus (L.) (rose of Sharon) is one of the most widespread garden shrubs in the world. We report a draft of the H. syriacus genome comprised of a 1.75 Gb assembly that covers 92% of the genome with only 1.7% (33 Mb) gap sequences. Predicted gene modeling detected 87,603 genes, mostly supported by deep RNA sequencing data. To define gene family distribution among relatives of H. syriacus, orthologous gene sets containing 164,660 genes in 21,472 clusters were identified by OrthoMCL analysis of five plant species, including H. syriacus, Arabidopsis thaliana, Gossypium raimondii, Theobroma cacao and Amborella trichopoda. We inferred their evolutionary relationships based on divergence times among Malvaceae plant genes and found that gene families involved in flowering regulation and disease resistance were more highly divergent and expanded in H. syriacus than in its close relatives, G. raimondii (DD) and T. cacao. Clustered gene families and gene collinearity analysis revealed that two recent rounds of whole-genome duplication were followed by diploidization of the H. syriacus genome after speciation. Copy number variation and phylogenetic divergence indicates that WGDs and subsequent diploidization led to unequal duplication and deletion of flowering-related genes in H. syriacus and may affect its unique floral morphology.

Divergent evolution of multiple virus-resistance genes from a progenitor in Capsicum spp.

Plants have evolved hundreds of nucleotide-binding and leucine-rich domain proteins (NLRs) as potential intracellular immune receptors, but the evolutionary mechanism leading to the ability to recognize specific pathogen effectors is elusive. Here, we cloned Pvr4 (a Potyvirus resistance gene in Capsicum annuum) and Tsw (a Tomato spotted wilt virus resistance gene in Capsicum chinense) via a genome-based approach using independent segregating populations. The genes both encode typical NLRs and are located at the same locus on pepper chromosome 10. Despite the fact that these two genes recognize completely different viral effectors, the genomic structures and coding sequences of the two genes are strikingly similar. Phylogenetic studies revealed that these two immune receptors diverged from a progenitor gene of a common ancestor. Our results suggest that sequence variations caused by gene duplication and neofunctionalization may underlie the evolution of the ability to specifically recognize different effectors. These findings thereby provide insight into the divergent evolution of plant immune receptors.

Genome-wide analysis of Dof transcription factors reveals functional characteristics during development and response to biotic stresses in pepper.

The DNA-binding with one zinc finger proteins (Dofs) are a plant-specific family of transcription factors. The Dofs are involved in a variety of biological processes such as phytohormone production, seed development, and environmental adaptation. Dofs have been previously identified in several plants, but not in pepper. We identified 33 putative Dof genes in pepper (CaDofs). To gain an overview of the CaDofs, we analyzed phylogenetic relationships, protein motifs, and evolutionary history. We divided the 33 CaDofs, containing 25 motifs, into four major groups distributed on eight chromosomes. We discovered an expansion of the CaDofs dated to a recent duplication event. Segmental duplication that occurred before the speciation of the Solanaceae lineages was predominant among the CaDofs. The global gene-expression profiling of the CaDofs by RNA-seq analysis showed distinct temporal and pathogen-specific variation during development and response to biotic stresses (two TMV strains, PepMoV, and Phytophthora capsici), suggesting functional diversity among the CaDofs. These results will provide the useful clues into the responses of Dofs in biotic stresses and promote a better understanding of their multiple function in pepper and other species.

Genome-Wide Comparative Analyses Reveal the Dynamic Evolution of Nucleotide-Binding Leucine-Rich Repeat Gene Family among Solanaceae Plants.

Plants have evolved an elaborate innate immune system against invading pathogens. Within this system, intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors are known play critical roles in effector-triggered immunity (ETI) plant defense. We performed genome-wide identification and classification of NLR-coding sequences from the genomes of pepper, tomato, and potato using fixed criteria. We then compared genomic duplication and evolution features. We identified intact 267, 443, and 755 NLR-encoding genes in tomato, potato, and pepper genomes, respectively. Phylogenetic analysis and classification of Solanaceae NLRs revealed that the majority of NLR super family members fell into 14 subgroups, including a TIR-NLR (TNL) subgroup and 13 non-TNL subgroups. Specific subgroups have expanded in each genome, with the expansion in pepper showing subgroup-specific physical clusters. Comparative analysis of duplications showed distinct duplication patterns within pepper and among Solanaceae plants suggesting subgroup- or species-specific gene duplication events after speciation, resulting in divergent evolution. Taken together, genome-wide analysis of NLR family members provide insights into their evolutionary history in Solanaceae. These findings also provide important foundational knowledge for understanding NLR evolution and will empower broader characterization of disease resistance genes to be used for crop breeding.

Plant NB-LRR proteins: tightly regulated sensors in a complex manner.

As plants are sessile, they have evolved hundreds of resistance (R) genes to defend themselves against multiple pathogens. Most of plant R genes encode proteins with the nucleotide-binding and leucine-rich repeat (NB-LRR) domains that interact with pathogen effectors to induce defense responses. Recent findings describing R proteins structures, host interactors and transcriptional and posttranscriptional regulators have broadened our understanding of R gene activity regulation. Genome-wide analyses of NB-LRR genes are useful for identifying host and nonhost R genes and elucidating complex resistance mechanisms. This review provides an overview of the functions of identified NB-LRRs and intra- and intermolecular R gene regulators.