Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization.

As an economic crop, pepper satisfies people's spicy taste and has medicinal uses worldwide. To gain a better understanding of Capsicum evolution, domestication, and specialization, we present here the genome sequence of the cultivated pepper Zunla-1 (C. annuum L.) and its wild progenitor Chiltepin (C. annuum var. glabriusculum). We estimate that the pepper genome expanded ∼0.3 Mya (with respect to the genome of other Solanaceae) by a rapid amplification of retrotransposons elements, resulting in a genome comprised of ∼81% repetitive sequences. Approximately 79% of 3.48-Gb scaffolds containing 34,476 protein-coding genes were anchored to chromosomes by a high-density genetic map. Comparison of cultivated and wild pepper genomes with 20 resequencing accessions revealed molecular footprints of artificial selection, providing us with a list of candidate domestication genes. We also found that dosage compensation effect of tandem duplication genes probably contributed to the pungent diversification in pepper. The Capsicum reference genome provides crucial information for the study of not only the evolution of the pepper genome but also, the Solanaceae family, and it will facilitate the establishment of more effective pepper breeding programs.

Genome-wide identification and analysis of drought-responsive genes and microRNAs in tobacco.

Drought stress response is a complex trait regulated at transcriptional and post-transcriptional levels in tobacco. Since the 1990s, many studies have shown that miRNAs act in many ways to regulate target expression in plant growth, development and stress response. The recent draft genome sequence of Nicotiana benthamiana has provided a framework for Digital Gene Expression (DGE) and small RNA sequencing to understand patterns of transcription in the context of plant response to environmental stress. We sequenced and analyzed three Digital Gene Expression (DGE) libraries from roots of normal and drought-stressed tobacco plants, and four small RNA populations from roots, stems and leaves of control or drought-treated tobacco plants, respectively. We identified 276 candidate drought responsive genes (DRGs) with sequence similarities to 64 known DRGs from other model plant crops, 82 were transcription factors (TFs) including WRKY, NAC, ERF and bZIP families. Of these tobacco DRGs, 54 differentially expressed DRGs included 21 TFs, which belonged to 4 TF families such as NAC (6), MYB (4), ERF (10), and bZIP (1). Additionally, we confirmed expression of 39 known miRNA families (122 members) and five conserved miRNA families, which showed differential regulation under drought stress. Targets of miRNAs were further surveyed based on a recently published study, of which ten targets were DRGs. An integrated gene regulatory network is proposed for the molecular mechanisms of tobacco root response to drought stress using differentially expressed DRGs, the changed expression profiles of miRNAs and their target transcripts. This network analysis serves as a reference for future studies on tobacco response stresses such as drought, cold and heavy metals.

The dynamics of DNA methylation in maize roots under Pb stress.

Plants adapt to adverse conditions through a series of physiological, cellular, and molecular processes, culminating in stress tolerance. However, little is known about the associated regulatory mechanisms at the epigenetic level in maize under lead (Pb) stress. Therefore, in this study, we aimed to compare DNA methylation profiles during the dynamic development of maize roots following Pb treatment to identify candidate genes involved in the response to Pb stress. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) was used to investigate the genome-wide DNA methylation patterns in maize roots under normal condition (A1) and 3 mM Pb(NO3)2 stress for 12 h (K2), 24 h (K3) and 48 h (K4). The results showed that the average methylation density was the highest in CpG islands (CGIs), followed by the intergenic regions. Within the gene body, the methylation density of the introns was higher than those of the UTRs and exons. In total, 3857 methylated genes were found in 4 tested samples, including 1805 differentially methylated genes for K2 versus A1, 1508 for K3 versus A1, and 1660 for K4 versus A1. Further analysis showed that 140 genes exhibited altered DNA methylation in all three comparisons, including some well-known stress-responsive transcription factors and proteins, such as MYB, AP2/ERF, bZIP, serine-threonine/tyrosine-proteins, pentatricopeptide repeat proteins, RING zinc finger proteins, F-box proteins, leucine-rich repeat proteins and tetratricopeptide repeat proteins. This study revealed the genome-scale DNA methylation patterns of maize roots in response to Pb exposure and identified candidate genes that potentially regulate root dynamic development under Pb stress at the methylation level.

Heterosis in early maize ear inflorescence development: a genome-wide transcription analysis for two maize inbred lines and their hybrid.

Heterosis, or hybrid vigor, contributes to superior agronomic performance of hybrids compared to their inbred parents. Despite its importance, little is known about the genetic and molecular basis of heterosis. Early maize ear inflorescences formation affects grain yield, and are thus an excellent model for molecular mechanisms involved in heterosis. To determine the parental contributions and their regulation during maize ear-development-genesis, we analyzed genome-wide digital gene expression profiles in two maize elite inbred lines (B73 and Mo17) and their F1 hybrid using deep sequencing technology. Our analysis revealed 17,128 genes expressed in these three genotypes and 22,789 genes expressed collectively in the present study. Approximately 38% of the genes were differentially expressed in early maize ear inflorescences from heterotic cross, including many transcription factor genes and some presence/absence variations (PAVs) genes, and exhibited multiple modes of gene action. These different genes showing differential expression patterns were mainly enriched in five cellular component categories (organelle, cell, cell part, organelle part and macromolecular complex), five molecular function categories (structural molecule activity, binding, transporter activity, nucleic acid binding transcription factor activity and catalytic activity), and eight biological process categories (cellular process, metabolic process, biological regulation, regulation of biological process, establishment of localization, cellular component organization or biogenesis, response to stimulus and localization). Additionally, a significant number of genes were expressed in only one inbred line or absent in both inbred lines. Comparison of the differences of modes of gene action between previous studies and the present study revealed only a small number of different genes had the same modes of gene action in both maize seedlings and ear inflorescences. This might be an indication that in different tissues or developmental stages, different global expression patterns prevail, which might nevertheless be related to heterosis. Our results support the hypotheses that multiple molecular mechanisms (dominance and overdominance modes) contribute to heterosis.

Key candidate genes for male sterility in peppers unveiled via transcriptomic and proteomic analyses.

This study aimed to enhance the use of male sterility in pepper to select superior hybrid generations. Transcriptomic and proteomic analyses of fertile line 1933A and nucleic male sterility line 1933B of Capsicum annuum L. were performed to identify male sterility-related proteins and genes. The phylogenetic tree, physical and chemical characteristics, gene structure characteristics, collinearity and expression characteristics of candidate genes were analyzed. The study identified 2,357 differentially expressed genes, of which 1,145 and 229 were enriched in the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases, respectively. A total of 7,628 quantifiable proteins were identified and 29 important proteins and genes were identified. It is worth noting that the existence of CaPRX genes has been found in both proteomics and transcriptomics, and 3 CaPRX genes have been identified through association analysis. A total of 66 CaPRX genes have been identified at the genome level, which are divided into 13 subfamilies, all containing typical CaPRX gene conformal domains. It is unevenly distributed across 12 chromosomes (including the virtual chromosome Chr00). Salt stress and co-expression analysis show that male sterility genes are expressed to varying degrees, and multiple transcription factors are co-expressed with CaPRXs, suggesting that they are involved in the induction of pepper salt stress. The study findings provide a theoretical foundation for genetic breeding by identifying genes, metabolic pathways, and molecular mechanisms involved in male sterility in pepper.

Genome-wide analysis of OFP gene family in pepper (Capsicum annuum L.).

Ovate family proteins (OFPs) are transcriptional inhibitors that regulate plant growth and development and play important roles in the synthesis of secondary cell walls during pollen development. This study identified the pepper OFP gene family based on the genome-wide analysis and used bioinformatics methods to provide a fundamental profile of the gene family. 74 OFP genes with typical Ovate domain were identified in cultivated pepper Zunla-1, wild pepper Chiltepin and CM334. Chromosome mapping revealed that CazOFP genes were unevenly distributed on 11 chromosomes and Chr00 in Zunla-1, CacOFP genes on 12 chromosomes in Chiltepin, and CamOFP genes on 12 chromosomes and two Scaffflods in CM334. Gene structure analysis revealed that CaOFP genes possessed 1-3 exons, and the analysis of physicochemical properties suggested that CaOFPs were hydrophilic. Many cis-acting elements were identified in the promoter region of CaOFP genes, including ABRE, ARE, Box 4, G-box, TC-rich, and TCT-motif. The expression patterns of pepper at different growth stages showed that CaOFP genes were actively involved in the growth and fruit development of pepper, and CazOFP16 and CazOFP17 were actively involved in response to multiple hormones and stress events. qRT-PCR was also used to verify the expression of CazOFP gene in two developmental stages of seven pepper varieties with different fruit shapes, and it was found that CaOFP genes may be involved in the formation of fruit type in pepper. This study provides theoretical and practical evidence for future research on the OFP gene family.

A new N-containing phenolic glycoside from Capsicum annuum L.

A new N-containing phenolic glycoside (1) and seven known compounds were isolated from the fruits of Capsicum annuum L. Their structures were elucidated by extensive spectroscopic methods including MS, 1D and 2D NMR (HSQC, HMBC, 1H-1H COSY, etc.). The stereo configuration of the new compound was determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. Compounds 1-8 were evaluated in vitro for their cytotoxic activities against HCT-116 cell line using Sulforhodamine B (SRB) assay, and the result showed only compound 6 displayed a weak inhibitory activity against HCT-116 (IC50 = 22.5 ± 3.3 mmol/mL).

Molecular Characterization and Expression Analysis of the Na+/H+ Exchanger Gene Family in Capsicum annuum L.

The Na+/H+ exchangers (NHXs) are a class of transporters involved in ion balance during plant growth and abiotic stress. We performed systematic bioinformatic identification and expression-characteristic analysis of CaNHX genes in pepper to provide a theoretical basis for pepper breeding and practical production. At the whole-genome level, the members of the CaNHX gene family of cultivated and wild pepper were systematically identified using bioinformatics methods. Sequence alignment and phylogenetic tree construction were performed using MEGA X software, and the gene functional domain, conserved motif, and gene structure were analyzed and visualized. At the same time, the co-expression network of CaNHX genes was analyzed, and salt-stress analysis and fluorescence quantitative verification of the Zunla-1 cultivar under stress conditions were performed. A total of 9 CaNHX genes were identified, which have typical functional domains of the Na+/H+ exchanger gene. The physical and chemical properties of the protein showed that the protein was hydrophilic, with a size of 503-1146 amino acids. Analysis of the gene structure showed that Chr08 was the most localized chromosome, with 8-24 exons. Cis-acting element analysis showed that it mainly contains cis-acting elements such as light response, salicylic acid response, defense, and stress response. Transcriptom and co-expression network analysis showed that under stress, the co-expressed genes of CaNHX genes in roots and leaves were more obvious than those in the control group, including ABA, IAA, and salt. The transcriptome and co-expression were verified by qRT-PCR. In this study, the CaNHX genes were identified at the genome level of pepper, which provides a theoretical foundation for improving the stress resistance, production, development, and utilization of pepper in genetic breeding.

Homogenate-Ultrasound-Assisted Ionic Liquid Extraction of Total Flavonoids from Selaginella involven: Process Optimization, Composition Identification, and Antioxidant Activity.

In this paper, an efficient approach to extract total flavonoids (TFs) from Selaginella involvens (Sw.) Spring using homogenate-ultrasound-assisted ionic liquid (IL) extraction (HUA-ILE) was first developed. The results indicated that EPyBF4 was selected as the suitable extractant. According to the single factor experiment and response surface methodology, the IL concentration of 0.10 mol/L, the extraction time of 160 s, the liquid/solid ratio of 13:1 mL/g, and the extraction power of 300 W were concluded as the best conditions. A yield of 8.48 ± 0.27 mg/g TF content was obtained. Compared with HUA ethanol extraction, ultrasound-assisted IL extraction, and percolation extraction, the TF content obtained by the HUA-ILE method could be increased by 2 to 4 times, and the extraction time could be reduced by 100 times. Furthermore, 16 compounds of the TF extract were finally identified through ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry, among which 11 compounds were first discovered in S. involven. The contents of six biflavonoids in S. involven were determined simultaneously adopting high-performance liquid chromatography, including amentoflavone, hinokiflavone, bilobetin, ginkgetin, isoginkgetin, and heveaflavone. The TF extract in S. involven was proved to have potent antioxidant activity through the four antioxidant experiments. In conclusion, HUA-ILE was applied for the first time to exploit a green, efficient, and novel approach to extract TFs, and the research also provided promising prospects for applications of S. involven.

Identification of MicroRNA Targets of Capsicum spp. Using MiRTrans-a Trans-Omics Approach.

The microRNA (miRNA) can regulate the transcripts that are involved in eukaryotic cell proliferation, differentiation, and metabolism. Especially for plants, our understanding of miRNA targets, is still limited. Early attempts of prediction on sequence alignments have been plagued by enormous false positives. It is helpful to improve target prediction specificity by incorporating the other data sources such as the dependency between miRNA and transcript expression or even cleaved transcripts by miRNA regulations, which are referred to as trans-omics data. In this paper, we developed MiRTrans (Prediction of MiRNA targets by Trans-omics data) to explore miRNA targets by incorporating miRNA sequencing, transcriptome sequencing, and degradome sequencing. MiRTrans consisted of three major steps. First, the target transcripts of miRNAs were predicted by scrutinizing their sequence characteristics and collected as an initial potential targets pool. Second, false positive targets were eliminated if the expression of miRNA and its targets were weakly correlated by lasso regression. Third, degradome sequencing was utilized to capture the miRNA targets by examining the cleaved transcripts that regulated by miRNAs. Finally, the predicted targets from the second and third step were combined by Fisher's combination test. MiRTrans was applied to identify the miRNA targets for Capsicum spp. (i.e., pepper). It can generate more functional miRNA targets than sequence-based predictions by evaluating functional enrichment. MiRTrans identified 58 miRNA-transcript pairs with high confidence from 18 miRNA families conserved in eudicots. Most of these targets were transcription factors; this lent support to the role of miRNA as key regulator in pepper. To our best knowledge, this work is the first attempt to investigate the miRNA targets of pepper, as well as their regulatory networks. Surprisingly, only a small proportion of miRNA-transcript pairs were shared between degradome sequencing and expression dependency predictions, suggesting that miRNA targets predicted by a single technology alone may be prone to report false negatives.

Genome-Wide Identification and Expression Profile of Dof Transcription Factor Gene Family in Pepper (Capsicum annuum L.).

Dof (DNA-binding One Zinc Finger) transcription factor family is unique to plants and has diverse roles associated with plant-specific phenomena, such as light, phytohormone and defense responses as well as seed development and germination. Although, genome-wide analysis of this family has been performed in many species, information regarding Dof genes in the pepper, Capsicum annuum L., is extremely limited. In this study, exhaustive searches of pepper genome revealed 33 potential CaDofs that were phylogenetically clustered into four subgroups. Twenty-nine of the 33 Dof genes could be mapped on 11 chromosomes, except for chromosome 7. The intron/exon organizations and conserved motif compositions of these genes were also analyzed. Additionally, phylogenetic analysis and classification of the Dof transcription factor family in eight plant species revealed that S. lycopersicum and C. annuum as well as O. sativa and S. bicolor Dof proteins may have evolved conservatively. Moreover, comprehensive expression analysis of CaDofs using a RNA-seq atlas and quantitative real-time polymerase chain reaction (qRT-PCR) revealed that these genes exhibit a variety of expression patterns. Most of the CaDofs were expressed in at least one of the tissues tested, whereas several genes were identified as being highly responsive to heat and salt stresses. Overall, this study describes the first genome-wide analysis of the pepper Dof family, whose genes exhibited different expression patterns in all primary fruit developmental stages and tissue types, as in response to abiotic stress. In particular, some Dof genes might be used as biomarkers for heat and salt stress. The results could expand our understanding of the roles of Dof genes in pepper.

A comprehensive characterization of simple sequence repeats in pepper genomes provides valuable resources for marker development in Capsicum.

The sequences of the full set of pepper genomes including nuclear, mitochondrial and chloroplast are now available for use. However, the overall of simple sequence repeats (SSR) distribution in these genomes and their practical implications for molecular marker development in Capsicum have not yet been described. Here, an average of 868,047.50, 45.50 and 30.00 SSR loci were identified in the nuclear, mitochondrial and chloroplast genomes of pepper, respectively. Subsequently, systematic comparisons of various species, genome types, motif lengths, repeat numbers and classified types were executed and discussed. In addition, a local database composed of 113,500 in silico unique SSR primer pairs was built using a homemade bioinformatics workflow. As a pilot study, 65 polymorphic markers were validated among a wide collection of 21 Capsicum genotypes with allele number and polymorphic information content value per marker raging from 2 to 6 and 0.05 to 0.64, respectively. Finally, a comparison of the clustering results with those of a previous study indicated the usability of the newly developed SSR markers. In summary, this first report on the comprehensive characterization of SSR motifs in pepper genomes and the very large set of SSR primer pairs will benefit various genetic studies in Capsicum.

Giant omental lipoma in children.

Lipoma is a rare benign neoplasm of mature fat cells. Asymptomatic abdominal mass, progressive abdominal distension, and intraperitoneal radiolucent fat density mass on computed tomography are the main clinical symptoms and signs. An unusual case is presented in this report. A complete excision was performed on an 11-month-old boy with a giant omental lipoma, and no recurrence was found in the 12-month follow-up.