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.

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.

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.

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.

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.

Draft genome of the peanut A-genome progenitor (Arachis duranensis) provides insights into geocarpy, oil biosynthesis, and allergens.

Peanut or groundnut (Arachis hypogaea L.), a legume of South American origin, has high seed oil content (45-56%) and is a staple crop in semiarid tropical and subtropical regions, partially because of drought tolerance conferred by its geocarpic reproductive strategy. We present a draft genome of the peanut A-genome progenitor, Arachis duranensis, and 50,324 protein-coding gene models. Patterns of gene duplication suggest the peanut lineage has been affected by at least three polyploidizations since the origin of eudicots. Resequencing of synthetic Arachis tetraploids reveals extensive gene conversion in only three seed-to-seed generations since their formation by human hands, indicating that this process begins virtually immediately following polyploid formation. Expansion of some specific gene families suggests roles in the unusual subterranean fructification of Arachis For example, the S1Fa-like transcription factor family has 126 Arachis members, in contrast to no more than five members in other examined plant species, and is more highly expressed in roots and etiolated seedlings than green leaves. The A. duranensis genome provides a major source of candidate genes for fructification, oil biosynthesis, and allergens, expanding knowledge of understudied areas of plant biology and human health impacts of plants, informing peanut genetic improvement and aiding deeper sequencing of Arachis diversity.

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.

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.

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.

Identification of the Candidate Proteins Related to Oleic Acid Accumulation during Peanut (Arachis hypogaea L.) Seed Development through Comparative Proteome Analysis.

Peanuts (Arachis hypogaea L.) are an important oilseed crop, containing high contents of protein and fatty acids (FA). The major components of FA found in peanut oil are unsaturated FAs, including oleic acid (OA, C18:1) and linoleic acid (LOA, C18:2). Moreover, the high content of OA in peanut oil is beneficial for human health and long-term storage due to its antioxidant activity. However, the dynamic changes in proteomics related to OA accumulation during seed development still remain largely unexplored. In the present study, a comparative proteome analysis based on iTRAQ (isobaric Tags for Relative and Absolute Quantification) was performed to identify the critical candidate factors involved in OA formation. A total of 389 differentially expressed proteins (DEPs) were identified between high-oleate cultivar Kainong176 and low-oleate cultivar Kainong70. Among these DEPs, 201 and 188 proteins were upregulated and downregulated, respectively. In addition, these DEPs were categorized into biosynthesis pathways of unsaturated FAs at the early stage during the high-oleic peanut seed development, and several DEPs involved in lipid oxidation pathway were found at the stage of seed maturation. Meanwhile, 28 DEPs were sporadically distributed in distinct stages of seed formation, and their molecular functions were directly correlated to FA biosynthesis and degradation. Fortunately, the expression of FAB2 (stearoyl-acyl carrier protein desaturase), the rate-limiting enzyme in the upstream biosynthesis process of OA, was significantly increased in the early stage and then decreased in the late stage of seed development in the high-oleate cultivar Kainong176. Furthermore, real-time PCR verified the expression pattern of FAB2 at the mRNA level, which was consistent with its protein abundance. However, opposite results were found for the low-oleate cultivar Kainong70. Overall, the comparative proteome analysis provided valuable insight into the molecular dynamics of OA accumulation during peanut seed development.

Transcriptome-wide sequencing provides insights into geocarpy in peanut (Arachis hypogaea L.).

A characteristic feature of peanut is the subterranean fructification, geocarpy, in which the gynophore ('peg'), a specialized organ that transitions from upward growth habit to downward outgrowth upon fertilization, drives the developing pod into the soil for subsequent development underground. As a step towards understanding this phenomenon, we explore the developmental dynamics of the peanut pod transcriptome at 11 successive stages. We identified 110 217 transcripts across developmental stages and quantified their abundance along a pod developmental gradient in pod wall. We found that the majority of transcripts were differentially expressed along the developmental gradient as well as identified temporal programs of gene expression, including hundreds of transcription factors. Thought to be an adaptation to particularly harsh subterranean environments, both up- and down-regulated gene sets in pod wall were enriched for response to a broad array of stimuli, like gravity, light and subterranean environmental factors. We also identified hundreds of transcripts associated with gravitropism and photomorphogenesis, which may be involved in the geocarpy. Collectively, this study forms a transcriptional baseline for geocarpy in peanut as well as provides a considerable body of evidence that transcriptional regulation in peanut aerial and subterranean fruits is complex.

Comparative transcriptome analysis of aerial and subterranean pods development provides insights into seed abortion in peanut.

The peanut is a special plant for its aerial flowering but subterranean fructification. The failure of peg penetration into the soil leads to form aerial pod and finally seed abortion. However, the mechanism of seed abortion during aerial pod development remains obscure. Here, a comparative transcriptome analysis between aerial and subterranean pods at different developmental stages was produced using a customized NimbleGen microarray representing 36,158 unigenes. By comparing 4 consecutive time-points, totally 6,203 differentially expressed genes, 4,732 stage-specific expressed genes and 2,401 specific expressed genes only in aerial or subterranean pods were identified in this study. Functional annotation showed their mainly involvement in biosynthesis, metabolism, transcription regulation, transporting, stress response, photosynthesis, signal transduction, cell division, apoptosis, embryonic development, hormone response and light signaling, etc. Emphasis was focused on hormone response, cell apoptosis, embryonic development and light signaling relative genes. These genes might function as potential candidates to provide insights into seed abortion during aerial pod development. Ten candidate genes were validated by Real-time RT-PCR. Additionally, consistent with up-regulation of auxin response relative genes in aerial pods, endogenous IAA content was also significantly increased by HPLC analysis. This study will further provide new molecular insight that auxin and auxin response genes potentially contribute to peanut seed and pod development.

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.

Deep sequencing analysis of the transcriptomes of peanut aerial and subterranean young pods identifies candidate genes related to early embryo abortion.

The failure of peg penetration into the soil leads to seed abortion in peanut. Knowledge of genes involved in these processes is comparatively deficient. Here, we used RNA-seq to gain insights into transcriptomes of aerial and subterranean pods. More than 2 million transcript reads with an average length of 396 bp were generated from one aerial (AP) and two subterranean (SP1 and SP2) pod libraries using pyrosequencing technology. After assembly, sets of 49 632, 49 952 and 50 494 from a total of 74 974 transcript assembly contigs (TACs) were identified in AP, SP1 and SP2, respectively. A clear linear relationship in the gene expression level was observed between these data sets. In brief, 2194 differentially expressed TACs with a 99.0% true-positive rate were identified, among which 859 and 1068 TACs were up-regulated in aerial and subterranean pods, respectively. Functional analysis showed that putative function based on similarity with proteins catalogued in UniProt and gene ontology term classification could be determined for 59 342 (79.2%) and 42 955 (57.3%) TACs, respectively. A total of 2968 TACs were mapped to 174 KEGG pathways, of which 168 were shared by aerial and subterranean transcriptomes. TACs involved in photosynthesis were significantly up-regulated and enriched in the aerial pod. In addition, two senescence-associated genes were identified as significantly up-regulated in the aerial pod, which potentially contribute to embryo abortion in aerial pods, and in turn, to cessation of swelling. The data set generated in this study provides evidence for some functional genes as robust candidates underlying aerial and subterranean pod development and contributes to an elucidation of the evolutionary implications resulting from fruit development under light and dark conditions.

De novo assembly and characterisation of the transcriptome during seed development, and generation of genic-SSR markers in peanut (Arachis hypogaea L.).

BACKGROUND: The peanut (Arachis hypogaea L.) is an important oilseed crop in tropical and subtropical regions of the world. However, little about the molecular biology of the peanut is currently known. Recently, next-generation sequencing technology, termed RNA-seq, has provided a powerful approach for analysing the transcriptome, and for shedding light on the molecular biology of peanut. RESULTS: In this study, we employed RNA-seq to analyse the transcriptomes of the immature seeds of three different peanut varieties with different oil contents. A total of 26.1-27.2 million paired-end reads with lengths of 100 bp were generated from the three varieties and 59,077 unigenes were assembled with N50 of 823 bp. Based on sequence similarity search with known proteins, a total of 40,100 genes were identified. Among these unigenes, only 8,252 unigenes were annotated with 42 gene ontology (GO) functional categories. And 18,028 unigenes mapped to 125 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). In addition, 3,919 microsatellite markers were developed in the unigene library, and 160 PCR primers of SSR loci were used for validation of the amplification and the polymorphism. CONCLUSION: We completed a successful global analysis of the peanut transcriptome using RNA-seq, a large number of unigenes were assembled, and almost four thousand SSR primers were developed. These data will facilitate gene discovery and functional genomic studies of the peanut plant. In addition, this study provides insight into the complex transcriptome of the peanut and established a biotechnological platform for future research.

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.

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.

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.

Chromosome-length genome assemblies of six legume species provide insights into genome organization, evolution, and agronomic traits for crop improvement.

INTRODUCTION: Legume crops are an important source of protein and oil for human health and in fixing atmospheric N2 for soil enrichment. With an objective to accelerate much-needed genetic analyses and breeding applications, draft genome assemblies were generated in several legume crops; many of them are not high quality because they are mainly based on short reads. However, the superior quality of genome assembly is crucial for a detailed understanding of genomic architecture, genome evolution, and crop improvement. OBJECTIVES: Present study was undertaken with an objective of developing improved chromosome-length genome assemblies in six different legumes followed by their systematic investigation to unravel different aspects of genome organization and legume evolution. METHODS: We employed in situ Hi-C data to improve the existing draft genomes and performed different evolutionary and comparative analyses using improved genome assemblies. RESULTS: We have developed chromosome-length genome assemblies in chickpea, pigeonpea, soybean, subterranean clover, and two wild progenitor species of cultivated groundnut (A. duranensis and A. ipaensis). A comprehensive comparative analysis of these genome assemblies offered improved insights into various evolutionary events that shaped the present-day legume species. We highlighted the expansion of gene families contributing to unique traits such as nodulation in legumes, gravitropism in groundnut, and oil biosynthesis in oilseed legume crops such as groundnut and soybean. As examples, we have demonstrated the utility of improved genome assemblies for enhancing the resolution of "QTL-hotspot" identification for drought tolerance in chickpea and marker-trait associations for agronomic traits in pigeonpea through genome-wide association study. Genomic resources developed in this study are publicly available through an online repository, 'Legumepedia'. CONCLUSION: This study reports chromosome-length genome assemblies of six legume species and demonstrates the utility of these assemblies in crop improvement. The genomic resources developed here will have significant role in accelerating genetic improvement applications of legume crops.

Resolubilization of TCA precipitated plant proteins for 2-D electrophoresis.

In this short communication, a novel protocol for resolubilization of TCA-precipitated plant proteins for 2-DE is described. Guanidine hydrochloride (Gdn-HCl) is used as an intermediate for protein solubilization in which proteins are reduced and alkylated with tributylphosphane (TBP) and 2-vinylpyridine (2-VP). The blocking of -SH groups at Cys residues can greatly improve the solubility of TCA-precipitated proteins and obtain more high-quality protein spots in the 2-DE gel. This protocol is compatible with silver stain and MS identification.