ScRNA-seq reveals dark- and light-induced differentially expressed gene atlases of seedling leaves in Arachis hypogaea L.

Although the regulatory mechanisms of dark and light-induced plant morphogenesis have been broadly investigated, the biological process in peanuts has not been systematically explored on single-cell resolution. Herein, 10 cell clusters were characterized using scRNA-seq-identified marker genes, based on 13 409 and 11 296 single cells from 1-week-old peanut seedling leaves grown under dark and light conditions. 6104 genes and 50 transcription factors (TFs) displayed significant expression patterns in distinct cell clusters, which provided gene resources for profiling dark/light-induced candidate genes. Further pseudo-time trajectory and cell cycle evidence supported that dark repressed the cell division and perturbed normal cell cycle, especially the PORA abundances correlated with 11 TFs highly enriched in mesophyll to restrict the chlorophyllide synthesis. Additionally, light repressed the epidermis cell developmental trajectory extending by inhibiting the growth hormone pathway, and 21 TFs probably contributed to the different genes transcriptional dynamic. Eventually, peanut AHL17 was identified from the profile of differentially expressed TFs, which encoded protein located in the nucleus promoted leaf epidermal cell enlargement when ectopically overexpressed in Arabidopsis through the regulatory phytohormone pathway. Overall, our study presents the different gene atlases in peanut etiolated and green seedlings, providing novel biological insights to elucidate light-induced leaf cell development at the single-cell level.

A genomic variation map provides insights into peanut diversity in China and associations with 28 agronomic traits.

Peanut (Arachis hypogaea L.) is an important allotetraploid oil and food legume crop. China is one of the world's largest peanut producers and consumers. However, genomic variations underlying the migration and divergence of peanuts in China remain unclear. Here we reported a genome-wide variation map based on the resequencing of 390 peanut accessions, suggesting that peanuts might have been introduced into southern and northern China separately, forming two cultivation centers. Selective sweep analysis highlights asymmetric selection between the two subgenomes during peanut improvement. A classical pedigree from South China offers a context for the examination of the impact of artificial selection on peanut genome. Genome-wide association studies identified 22,309 significant associations with 28 agronomic traits, including candidate genes for plant architecture and oil biosynthesis. Our findings shed light on peanut migration and diversity in China and provide valuable genomic resources for peanut improvement.

Silica nanoparticles conferring resistance to bacterial wilt in peanut (Arachis hypogaea L.).

Peanut bacterial wilt (PBW) caused by the pathogen Ralstonia solanacearum severely affects the growth and yield potential of peanut crop. In this study, we synthesized silica nanoparticles (SiO2 NPs), a prospective efficient management approach to control PBW, and conducted a hydroponic experiment to investigate the effects of different SiO2 NPs treatments (i.e., 0, 100, and 500 mg L-1 as NP0, NP100, and NP500, respectively) on promoting plant growth and resistance to R. solanacearum. Results indicated that the disease indices of NP100 and NP500 decreased by 51.5 % and 55.4 % as compared with NP0 under R. solanacearum inoculation, respectively, while the fresh and dry weights and shoot length of NP100 and NP500 increased by 7.62-42.05 %, 9.45-32.06 %, and 2.37-17.83 %, respectively. Furthermore, SiO2 NPs induced an improvement in physio-biochemical enzymes (superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, and lipoxygenase) which eliminated the excess production of hydrogen peroxide, superoxide anions, and malondialdehyde to alleviate PBW stress. Notably, the targeted metabolomic analysis indicated that SiO2 NPs enhanced salicylic acid (SA) contents, which involved the induction of systemic acquired resistance (SAR). Moreover, the transcriptomic analysis revealed that SiO2 NPs modulated the expression of multiple transcription factors (TFs) involved in the hormone pathway, such as AHLs, and the identification of hormone pathways related to plant defense responses, such as the SA pathway, which activated SA-dependent defense mechanisms. Meanwhile, the up-regulated expression of the SA-metabolism gene, salicylate carboxymethyltransferase (SAMT), initiated SAR to promote PBW resistance. Overall, our findings revealed that SiO2 NPs, functioning as a plant elicitor, could effectively modulate physiological enzyme activities and enhance SA contents through the regulation of SA-metabolism genes to confer the PBW resistance in peanuts, which highlighted the potential of SiO2 NPs for sustainable agricultural practices.

scRNA-seq Reveals the Mechanism of Fatty Acid Desaturase 2 Mutation to Repress Leaf Growth in Peanut (Arachis hypogaea L.).

Fatty Acid Desaturase 2 (FAD2) controls the conversion of oleic acids into linoleic acids. Mutations in FAD2 not only increase the high-oleic content, but also repress the leaf growth. However, the mechanism by which FAD2 regulates the growth pathway has not been elucidated in peanut leaves with single-cell resolution. In this study, we isolated fad2 mutant leaf protoplast cells to perform single-cell RNA sequencing. Approximately 24,988 individual cells with 10,249 expressed genes were classified into five major cell types. A comparative analysis of 3495 differentially expressed genes (DEGs) in distinct cell types demonstrated that fad2 inhibited the expression of the cytokinin synthesis gene LOG in vascular cells, thereby repressing leaf growth. Further, pseudo-time trajectory analysis indicated that fad2 repressed leaf cell differentiation, and cell-cycle evidence displayed that fad2 perturbed the normal cell cycle to induce the majority of cells to drop into the S phase. Additionally, important transcription factors were filtered from the DEG profiles that connected the network involved in high-oleic acid accumulation (WRKY6), activated the hormone pathway (WRKY23, ERF109), and potentially regulated leaf growth (ERF6, MYB102, WRKY30). Collectively, our study describes different gene atlases in high-oleic and normal peanut seedling leaves, providing novel biological insights to elucidate the molecular mechanism of the high-oleic peanut-associated agronomic trait at the single-cell level.

Silicon Application for the Modulation of Rhizosphere Soil Bacterial Community Structures and Metabolite Profiles in Peanut under Ralstonia solanacearum Inoculation.

Silicon (Si) has been shown to promote peanut growth and yield, but whether Si can enhance the resistance against peanut bacterial wilt (PBW) caused by Ralstonia solanacearum, identified as a soil-borne pathogen, is still unclear. A question regarding whether Si enhances the resistance of PBW is still unclear. Here, an in vitro R. solanacearum inoculation experiment was conducted to study the effects of Si application on the disease severity and phenotype of peanuts, as well as the microbial ecology of the rhizosphere. Results revealed that Si treatment significantly reduced the disease rate, with a decrement PBW severity of 37.50% as compared to non-Si treatment. The soil available Si (ASi) significantly increased by 13.62-44.87%, and catalase activity improved by 3.01-3.10%, which displayed obvious discrimination between non-Si and Si treatments. Furthermore, the rhizosphere soil bacterial community structures and metabolite profiles dramatically changed under Si treatment. Three significantly changed bacterial taxa were observed, which showed significant abundance under Si treatment, whereas the genus Ralstonia genus was significantly suppressed by Si. Similarly, nine differential metabolites were identified to involve into unsaturated fatty acids via a biosynthesis pathway. Significant correlations were also displayed between soil physiochemical properties and enzymes, the bacterial community, and the differential metabolites by pairwise comparisons. Overall, this study reports that Si application mediated the evolution of soil physicochemical properties, the bacterial community, and metabolite profiles in the soil rhizosphere, which significantly affects the colonization of the Ralstonia genus and provides a new theoretical basis for Si application in PBW prevention.

Genome-Wide Identification and Expression of FAR1 Gene Family Provide Insight Into Pod Development in Peanut (Arachis hypogaea).

The far-red-impaired response 1 (FAR1) transcription family were initially identified as important factors for phytochrome A (phyA)-mediated far-red light signaling in Arabidopsis; they play crucial roles in controlling the growth and development of plants. The reported reference genome sequences of Arachis, including A. duranensis, A. ipaensis, A. monticola, and A. hypogaea, and its related species Glycine max provide an opportunity to systematically perform a genome-wide identification of FAR1 homologous genes and investigate expression patterns of these members in peanut species. Here, a total of 650 FAR1 genes were identified from four Aarchis and its closely related species G. max. Of the studied species, A. hypogaea contained the most (246) AhFAR1 genes, which can be classified into three subgroups based on phylogenic relationships. The synonymous (Ks) and non-synonymous (Ka) substitution rates, phylogenetic relationship and synteny analysis of the FAR1 family provided deep insight into polyploidization, evolution and domestication of peanut AhFAR1 genes. The transcriptome data showed that the AhFAR1 genes exhibited distinct tissue- and stage-specific expression patterns in peanut. Three candidate genes including Ahy_A10g049543, Ahy_A06g026579, and Ahy_A10g048401, specifically expressed in peg and pod, might participate in pod development in the peanut. The quantitative real-time PCR (qRT-PCR) analyses confirmed that the three selected genes were highly and specifically expressed in the peg and pod. This study systematically analyzed gene structure, evolutionary characteristics and expression patterns of FAR1 gene family, which will provide a foundation for the study of genetic and biological function in the future.

Lipid profile variations in high olecic acid peanuts by following different cooking processes.

High oleic acid (OA) peanut seed (PS), contains a higher ratio of oleic acid (C18:1) compared to general PS, which is favored by consumers due to its health benefits. However, comprehensive lipid metabolite profiles of high-OA PS, once they have been processed via domestic cooking methods, have never been produced. To establish a scientific guide for the selection of the most appropriate processing method for high-OA PS, lipidomics was performed to identify 706 lipid metabolites in high-OA PS following boiling, baking and frying, between the three groups, 75, 175 and 242 lipid metabolites were differentially expressed respectively. Additionally, 46 glycerolipids with C18:1 molecular were observed in the lipid profiles of the treatment groups compared to the raw sample. Further evaluation of seven lipid peroxides and six antioxidant status of each testing group suggested that boiled PS retained the highest levels of lipids and antioxidant activity. Following these findings, boiling appears to be an appropriate processing method when attempting to conserve the beneficial substances found in the PS. Finally, the levels of major free fatty acids present in high-OA PS, were jointly quantified by conventional methods (GC-MS) and lipidomic analysis. FA/C16:0 levels were similar, FA/C18:0, FA/C18:1 displayed opposite results, FA/C18:2 levels increased following frying and FA/C18:3 levels were down regulated once the PS was boiled. This indicates that GC-MS is a potential method of validation for the results of lipidomic analysis. Conclusively, this in depth understanding of lipid content in relation to domestic cooking methods has provided a foundation for the processing of high-OA peanut products.

Impact of different cooking methods on the chemical profile of high-oleic acid peanut seeds.

High oleic acid (OA) peanut seeds (PS) can be beneficial for human health. However, chemical variations in high-OA PS after domestic cooking are not fully understood. In order to investigate the impact of different cooking methods on the chemical profile of high-OA PS, widely established metabolomics approach was employed to identify the relative contents of PS metabolites. Herein, 630 metabolites within 27 categories were characterized in PS, of which 141, 157, 402 differential metabolites were observed in each treatment group (boiling, baking, and frying) when compared to the raw seed. Accordingly, bioactive substances were maximally preserved in baked high-OA PS. Further conventional methods (HPLC-UV/GC-MS) quantified the absolute composition of amino and fatty acids, verifying the reliability of metabolomic analysis. Collectively, the understanding of the phytochemical substances in relation to the domestic cooking method established a foundation for future high-OA PS processing.

Single-cell RNA-seq describes the transcriptome landscape and identifies critical transcription factors in the leaf blade of the allotetraploid peanut (Arachis hypogaea L.).

Single-cell RNA-seq (scRNA-seq) has been highlighted as a powerful tool for the description of human cell transcriptome, but the technology has not been broadly applied in plant cells. Herein, we describe the successful development of a robust protoplast cell isolation system in the peanut leaf. A total of 6,815 single cells were divided into eight cell clusters based on reported marker genes by applying scRNA-seq. Further, a pseudo-time analysis was used to describe the developmental trajectory and interaction network of transcription factors (TFs) of distinct cell types during leaf growth. The trajectory enabled re-investigation of the primordium-driven development processes of the mesophyll and epidermis. These results suggest that palisade cells likely differentiate into spongy cells, while the epidermal cells originated earlier than the primordium. Subsequently, the developed method integrated multiple technologies to efficiently validate the scRNA-seq result in a homogenous cell population. The expression levels of several TFs were strongly correlated with epidermal ontogeny in accordance with obtained scRNA-seq values. Additionally, peanut AHL23 (AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 23), which is localized in nucleus, promoted leaf growth when ectopically expressed in Arabidopsis by modulating the phytohormone pathway. Together, our study displays that application of scRNA-seq can provide new hypotheses regarding cell differentiation in the leaf blade of Arachis hypogaea. We believe that this approach will enable significant advances in the functional study of leaf blade cells in the allotetraploid peanut and other plant species.

A proteomic analysis of peanut seed at different stages of underground development to understand the changes of seed proteins.

In order to obtain more valuable insights into the protein dynamics and accumulation of allergens in seeds during underground development, we performed a proteomic study on developing peanut seeds at seven different stages. A total of 264 proteins with altered abundance and contained at least one unique peptide was detected by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). All identified proteins were classified into five functional categories as level 1 and 20 secondary functional categories as level 2. Among them, 88 identified proteins (IPs) were related to carbohydrate/ amino acid/ lipid transport and metabolism, indicating that carbohydrate/amino acid/ lipid metabolism played a key role in the underground development of peanut seeds. Hierarchical cluster analysis showed that all IPs could be classified into eight cluster groups according to the abundance profiles, suggesting that the modulatory patterns of these identified proteins were complicated during seed development. The largest group contained 41 IPs, the expression of which decreased at R 2 and reached a maximum at R3 but gradually decreased from R4. A total of 14 IPs were identified as allergen-like proteins by BLAST with A genome (Arachis duranensis) or B genome (Arachis ipaensis) translated allergen sequences. Abundance profile analysis of 14 identified allergens showed that the expression of all allergen proteins was low or undetectable by 2-DE at the early stages (R1 to R4), and began to accumulate from the R5 stage and gradually increased. Network analysis showed that most of the significant proteins were involved in active metabolic pathways in early development. Real time RT-PCR analysis revealed that transcriptional regulation was approximately consistent with expression at the protein level for 8 selected identified proteins. In addition, some amino acid sequences that may be associated with new allergens were also discussed.

Global transcriptome analysis of subterranean pod and seed in peanut (Arachis hypogaea L.) unravels the complexity of fruit development under dark condition.

Peanut pods develop underground, which is the most salient characteristic in peanut. However, its developmental transcriptome remains largely unknown. In the present study, we sequenced over one billion transcripts to explore the developmental transcriptome of peanut pod using Illumina sequencing. Moreover, we identified and quantified the abundances of 165,689 transcripts in seed and shell tissues along with a pod developmental gradient. The dynamic changes of differentially expressed transcripts (DETs) were described in seed and shell. Additionally, we found that photosynthetic genes were not only pronouncedly enriched in aerial pod, but also played roles in developing pod under dark condition. Genes functioning in photomorphogenesis showed distinct expression profiles along subterranean pod development. Clustering analysis unraveled a dynamic transcriptome, in which transcripts for DNA synthesis and cell division during pod expansion were transitioning to transcripts for cell expansion and storage activity during seed filling. Collectively, our study formed a transcriptional baseline for peanut fruit development under dark condition.

Improving Gene Annotation of the Peanut Genome by Integrated Proteogenomics Workflow.

Peanut (Arachis hypogaea L.) is a staple crop in semiarid tropical and subtropical regions. Although the genome of peanut has been fully sequenced, the current gene annotations are still incomplete. New technologies in genomics and proteomics have resulted in the emergence of proteogenomics, which can integrate genomic, transcriptomic, and proteomic data for improving gene annotation. In the present study, we collected RNA-seq and proteomic data from multiple tissues such as seed, shell, and gynophore of peanut and utilized a proteogenomic approach to improve the gene annotation of peanut based on these data. A total of 1 935 655 904 RNA-seq reads and 7 490 280 MS/MS spectra were collected. Ultimately, 13 767 annotated genes were found with evidence at the protein level, and seven novel protein-coding genes were found with both RNA-seq and proteomics evidence. In addition, 35 gene models were updated based on proteomics data. Proteogenomic approaches improved the gene annotation in certain aspects by integrating both RNA-seq and proteomic data. We expect that these approaches could help improve existing genome annotations of other species.

Integrated Analysis of Comparative Lipidomics and Proteomics Reveals the Dynamic Changes of Lipid Molecular Species in High-Oleic Acid Peanut Seed.

Modern peanut contains fatty acid desaturase 2 (FAD2) mutation, which is capable of producing high oleic acid for human health. However, the dynamic changes of the lipidome regarding fad2 remain elusive in peanut seed. In the present study, 547 lipid features were identified in high- and normal-oleic peanut seeds by utilizing the mass spectrometric approach. The fad2-induced differently expressed lipids (DELs) were polarly distributed at early and maturation stages during high-oleic acid (OA) seed development. Subsequently, integration of previously published proteomic data and lipidomic data revealed that 21 proteins and 149 DELs were annotated into the triacylglycerol assembly map, of which nine enzymes and 31 lipid species shared similar variation tendencies. Additionally, the variation tendencies of 17 acyl fatty acids were described in a hypothetical biosynthetic pathway. Collectively, the understanding of the lipid composition correlated with fad2 established a foundation for future high-OA peanut breeding based on lipidomic data.

Genome-wide identification of microsatellite markers from cultivated peanut (Arachis hypogaea L.).

BACKGROUND: Microsatellites, or simple sequence repeats (SSRs), represent important DNA variations that are widely distributed across the entire plant genome and can be used to develop SSR markers, which can then be used to conduct genetic analyses and molecular breeding. Cultivated peanut (A. hypogaea L.), an important oil crop worldwide, is an allotetraploid (AABB, 2n = 4× = 40) plant species. Because of its complex genome, genomic marker development has been very challenging. However, sequencing of cultivated peanut genome allowed us to develop genomic markers and construct a high-density physical map. RESULTS: A total of 8,329,496 SSRs were identified, including 3,772,653, 4,414,961, and 141,882 SSRs that were distributed in subgenome A, B, and nine scaffolds, respectively. Based on the flanking sequences of the identified SSRs, a total of 973,984 newly developed SSR markers were developed in subgenome A (462,267), B (489,394), and nine scaffolds (22,323), with an average density of 392.45 markers per Mb. In silico PCR evaluation showed that an average of 88.32% of the SSR markers generated only one in silico-specific product in two tetraploid A. hypogaea varieties, Tifrunner and Shitouqi. A total of 39,599 common SSR markers were identified among the two A. hypogaea varieties and two progenitors, A. duranensis and A. ipaensis. Additionally, an amplification effectiveness of 44.15% was observed by real PCR validation. Moreover, a total of 1276 public SSR loci were integrated with the newly developed SSR markers. Finally, a previously known leaf spot quantitative trait locus (QTL), qLLS_T13_A05_7, was determined to be in a 1.448-Mb region on chromosome A05. In this region, a total of 819 newly developed SSR markers were located and 108 candidate genes were detected. CONCLUSIONS: The availability of these newly developed and public SSR markers both provide a large number of molecular markers that could potentially be used to enhance the process of trait genetic analyses and improve molecular breeding strategies for cultivated peanut.

Transcriptomic Analysis Reveals the High-Oleic Acid Feedback Regulating the Homologous Gene Expression of Stearoyl-ACP Desaturase 2 (SAD2) in Peanuts.

Peanuts with high oleic acid content are usually considered to be beneficial for human health and edible oil storage. In breeding practice, peanut lines with high monounsaturated fatty acids are selected using fatty acid desaturase 2 (FAD2), which is responsible for the conversion of oleic acid (C18:1) to linoleic acid (C18:2). Here, comparative transcriptomics were used to analyze the global gene expression profile of high- and normal-oleic peanut cultivars at six time points during seed development. First, the mutant type of FAD2 was determined in the high-oleic peanut (H176). The result suggested that early translation termination occurred simultaneously in the coding sequence of FAD2-A and FAD2-B, and the cultivar H176 is capable of utilizing a potential germplasm resource for future high-oleic peanut breeding. Furthermore, transcriptomic analysis identified 74 differentially expressed genes (DEGs) involved in lipid metabolism in high-oleic peanut seed, of which five DEGs encoded the fatty acid desaturase. Aradu.XM2MR belonged to the homologous gene of stearoyl-ACP (acyl carrier protein) desaturase 2 (SAD2) that converted the C18:0 into C18:1. Further subcellular localization studies indicated that FAD2 was located at the endoplasmic reticulum (ER), and Aradu.XM2MR was targeted to the plastid in Arabidopsis protoplast cells. To examine the dynamic mechanism of this finding, we focused on the peroxidase (POD)-mediated fatty acid (FA) degradation pathway. The fad2 mutant significantly increased the POD activity and H2O2 concentration at the early stage of seed development, implying that redox signaling likely acted as a messenger to connect the signaling transduction between the high-oleic content and Aradu.XM2MR transcription level. Taken together, transcriptome analysis revealed the feedback mechanism of SAD2 (Aradu.XM2MR) associated with FAD2 mutation during the seed developmental stage, which could provide a potential peanut breeding strategy based on identified candidate genes to improve the content of oleic acid.

Sequencing of Cultivated Peanut, Arachis hypogaea, Yields Insights into Genome Evolution and Oil Improvement.

Cultivated peanut (Arachis hypogaea) is an allotetraploid crop planted in Asia, Africa, and America for edible oil and protein. To explore the origins and consequences of tetraploidy, we sequenced the allotetraploid A. hypogaea genome and compared it with the related diploid Arachis duranensis and Arachis ipaensis genomes. We annotated 39 888 A-subgenome genes and 41 526 B-subgenome genes in allotetraploid peanut. The A. hypogaea subgenomes have evolved asymmetrically, with the B subgenome resembling the ancestral state and the A subgenome undergoing more gene disruption, loss, conversion, and transposable element proliferation, and having reduced gene expression during seed development despite lacking genome-wide expression dominance. Genomic and transcriptomic analyses identified more than 2 500 oil metabolism-related genes and revealed that most of them show altered expression early in seed development while their expression ceases during desiccation, presenting a comprehensive map of peanut lipid biosynthesis. The availability of these genomic resources will facilitate a better understanding of the complex genome architecture, agronomically and economically important genes, and genetic improvement of peanut.

Consensus map integration and QTL meta-analysis narrowed a locus for yield traits to 0.7 cM and refined a region for late leaf spot resistance traits to 0.38 cM on linkage group A05 in peanut (Arachis hypogaea L.).

BACKGROUND: Many large-effect quantitative trait loci (QTLs) for yield and disease resistance related traits have been identified in different mapping populations of peanut (Arachis hypogaea L.) under multiple environments. However, only a limited number of QTLs have been used in marker-assisted selection (MAS) because of unfavorable epistatic interactions between QTLs in different genetic backgrounds. Thus, it is essential to identify consensus QTLs across different environments and genetic backgrounds for use in MAS. Here, we used QTL meta-analysis to identify a set of consensus QTLs for yield and disease resistance related traits in peanut. RESULTS: A new integrated consensus genetic map with 5874 loci was constructed. The map comprised 20 linkage groups (LGs) and was up to a total length of 2918.62 cM with average marker density of 2.01 loci per centimorgan (cM). A total of 292 initial QTLs were projected on the new consensus map, and 40 meta-QTLs (MQTLs) for yield and disease resistance related traits were detected on four LGs. The genetic intervals of these consensus MQTLs varied from 0.20 cM to 7.4 cM, which is narrower than the genetic intervals of the initial QTLs, meaning they may be suitable for use in MAS. Importantly, a region of the map that previously co-localized multiple major QTLs for pod traits was narrowed from 3.7 cM to 0.7 cM using an overlap region of four MQTLs for yield related traits on LG A05, which corresponds to a physical region of about 630.3 kb on the A05 pseudomolecule of peanut, including 38 annotated candidate genes (54 transcripts) related to catalytic activity and metabolic process. Additionally, one major MQTL for late leaf spot (LLS) was identified in a region of about 0.38 cM. BLAST searches identified 26 candidate genes (30 different transcripts) in this region, some of which were annotated as related to regulation of disease resistance in different plant species. CONCLUSIONS: Combined with the high-density marker consensus map, all the detected MQTLs could be useful in MAS. The biological functions of the 64 candidate genes should be validated to unravel the molecular mechanisms of yield and disease resistance in peanut.

Corrigendum: Genome Sequencing and Analysis of the Peanut B-Genome Progenitor (Arachis ipaensis).

[This corrects the article DOI: 10.3389/fpls.2018.00604.].

Genome Sequencing and Analysis of the Peanut B-Genome Progenitor (Arachis ipaensis).

Peanut (Arachis hypogaea L.), an important leguminous crop, is widely cultivated in tropical and subtropical regions. Peanut is an allotetraploid, having A and B subgenomes that maybe have originated in its diploid progenitors Arachis duranensis (A-genome) and Arachis ipaensis (B-genome), respectively. We previously sequenced the former and here present the draft genome of the latter, expanding our knowledge of the unique biology of Arachis. The assembled genome of A. ipaensis is ~1.39 Gb with 39,704 predicted protein-encoding genes. A gene family analysis revealed that the FAR1 family may be involved in regulating peanut special fruit development. Genomic evolutionary analyses estimated that the two progenitors diverged ~3.3 million years ago and suggested that A. ipaensis experienced a whole-genome duplication event after the divergence of Glycine max. We identified a set of disease resistance-related genes and candidate genes for biological nitrogen fixation. In particular, two and four homologous genes that may be involved in the regulation of nodule development were obtained from A. ipaensis and A. duranensis, respectively. We outline a comprehensive network involved in drought adaptation. Additionally, we analyzed the metabolic pathways involved in oil biosynthesis and found genes related to fatty acid and triacylglycerol synthesis. Importantly, three new FAD2 homologous genes were identified from A. ipaensis and one was completely homologous at the amino acid level with FAD2 from A. hypogaea. The availability of the A. ipaensis and A. duranensis genomic assemblies will advance our knowledge of the peanut genome.

TALEN-mediated targeted mutagenesis of fatty acid desaturase 2 (FAD2) in peanut (Arachis hypogaea L.) promotes the accumulation of oleic acid.

KEY MESSAGE: A first creation of high oleic acid peanut varieties by using transcription activator-like effecter nucleases (TALENs) mediated targeted mutagenesis of Fatty Acid Desaturase 2 (FAD2). Transcription activator like effector nucleases (TALENs), which allow the precise editing of DNA, have already been developed and applied for genome engineering in diverse organisms. However, they are scarcely used in higher plant study and crop improvement, especially in allopolyploid plants. In the present study, we aimed to create targeted mutagenesis by TALENs in peanut. Targeted mutations in the conserved coding sequence of Arachis hypogaea fatty acid desaturase 2 (AhFAD2) were created by TALENs. Genetic stability of AhFAD2 mutations was identified by DNA sequencing in up to 9.52 and 4.11% of the regeneration plants at two different targeted sites, respectively. Mutation frequencies among AhFAD2 mutant lines were significantly correlated to oleic acid accumulation. Genetically, stable individuals of positive mutant lines displayed a 0.5-2 fold increase in the oleic acid content compared with non-transgenic controls. This finding suggested that TALEN-mediated targeted mutagenesis could increase the oleic acid content in edible peanut oil. Furthermore, this was the first report on peanut genome editing event, and the obtained high oleic mutants could serve for peanut breeding project.