AhNPR3 regulates the expression of WRKY and PR genes, and mediates the immune response of the peanut (Arachis hypogaea L.).

Systemic acquired resistance is an essential immune response that triggers a broad-spectrum disease resistance throughout the plant. In the present study, we identified a peanut lesion mimic mutant m14 derived from an ethyl methane sulfonate-mutagenized mutant pool of peanut cultivar "Yuanza9102." Brown lesions were observed in the leaves of an m14 mutant from seedling stage to maturity. Using MutMap together with bulked segregation RNA analysis approaches, a G-to-A point mutation was identified in the exon region of candidate gene Arahy.R60CUW, which is the homolog of AtNPR3 (Nonexpresser of PR genes) in Arabidopsis. This point mutation caused a transition from Gly to Arg within the C-terminal transactivation domain of AhNPR3A. The mutation of AhNPR3A showed no effect in the induction of PR genes when treated with salicylic acid. Instead, the mutation resulted in upregulation of WRKY genes and several PR genes, including pathogenesis-related thaumatin- and chitinase-encoding genes, which is consistent with the resistant phenotype of m14 to leaf spot disease. Further study on the AhNPR3A gene will provide valuable insights into understanding the molecular mechanism of systemic acquired resistance in peanut. Moreover, our results indicated that a combination of MutMap and bulked segregation RNA analysis is an effective method for identifying genes from peanut mutants.

Development and evaluation of the utility of GenoBaits Peanut 40K for a peanut MAGIC population.

Population and genotype data are essential for genetic mapping. The multi-parent advanced generation intercross (MAGIC) population is a permanent mapping population used for precisely mapping quantitative trait loci. Moreover, genotyping-by-target sequencing (GBTS) is a robust high-throughput genotyping technology characterized by its low cost, flexibility, and limited requirements for information management and support. In this study, an 8-way MAGIC population was constructed using eight elite founder lines. In addition, GenoBaits Peanut 40K was developed and utilized for the constructed MAGIC population. A subset (297 lines) of the MAGIC population at the S2 stage was genotyped using GenoBaits Peanut 40K. Furthermore, these lines and the eight parents were analyzed in terms of pod length, width, area, and perimeter. A total of 27 single nucleotide polymorphisms (SNPs) were revealed to be significantly associated with peanut pod size-related traits according to a genome-wide association study. The GenoBaits Peanut 40K provided herein and the constructed MAGIC population will be applicable for future research to identify the key genes responsible for important peanut traits. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01417-w.

Physical mapping of repetitive oligonucleotides facilitates the establishment of a genome map-based karyotype to identify chromosomal variations in peanut.

BACKGROUND: Chromosomal variants play important roles in crop breeding and genetic research. The development of single-stranded oligonucleotide (oligo) probes simplifies the process of fluorescence in situ hybridization (FISH) and facilitates chromosomal identification in many species. Genome sequencing provides rich resources for the development of oligo probes. However, little progress has been made in peanut due to the lack of efficient chromosomal markers. Until now, the identification of chromosomal variants in peanut has remained a challenge. RESULTS: A total of 114 new oligo probes were developed based on the genome-wide tandem repeats (TRs) identified from the reference sequences of the peanut variety Tifrunner (AABB, 2n = 4x = 40) and the diploid species Arachis ipaensis (BB, 2n = 2x = 20). These oligo probes were classified into 28 types based on their positions and overlapping signals in chromosomes. For each type, a representative oligo was selected and modified with green fluorescein 6-carboxyfluorescein (FAM) or red fluorescein 6-carboxytetramethylrhodamine (TAMRA). Two cocktails, Multiplex #3 and Multiplex #4, were developed by pooling the fluorophore conjugated probes. Multiplex #3 included FAM-modified oligo TIF-439, oligo TIF-185-1, oligo TIF-134-3 and oligo TIF-165. Multiplex #4 included TAMRA-modified oligo Ipa-1162, oligo Ipa-1137, oligo DP-1 and oligo DP-5. Each cocktail enabled the establishment of a genome map-based karyotype after sequential FISH/genomic in situ hybridization (GISH) and in silico mapping. Furthermore, we identified 14 chromosomal variants of the peanut induced by radiation exposure. A total of 28 representative probes were further chromosomally mapped onto the new karyotype. Among the probes, eight were mapped in the secondary constrictions, intercalary and terminal regions; four were B genome-specific; one was chromosome-specific; and the remaining 15 were extensively mapped in the pericentric regions of the chromosomes. CONCLUSIONS: The development of new oligo probes provides an effective set of tools which can be used to distinguish the various chromosomes of the peanut. Physical mapping by FISH reveals the genomic organization of repetitive oligos in peanut chromosomes. A genome map-based karyotype was established and used for the identification of chromosome variations in peanut following comparisons with their reference sequence positions.

QTL mapping of web blotch resistance in peanut by high-throughput genome-wide sequencing.

BACKGROUND: Web blotch is one of the most important foliar diseases worldwide in peanut (Arachis hypogaea L.). The identification of quantitative trait loci (QTLs) for peanut web blotch resistance represents the basis for gene mining and the application of molecular breeding technologies. RESULTS: In this study, a peanut recombinant inbred line (RIL) population was used to map QTLs for web blotch resistance based on high-throughput genome-wide sequencing. Frequency distributions of disease grade and disease index in five environments indicated wide phenotypic variations in response to web blotch among RILs. A high-density genetic map was constructed, containing 3634 bin markers distributed on 20 peanut linkage groups (LGs) with an average genetic distance of 0.5 cM. In total, eight QTLs were detected for peanut web blotch resistance in at least two environments, explaining from 2.8 to 15.1% of phenotypic variance. Two major QTLs qWBRA04 and qWBRA14 were detected in all five environments and were linked to 40 candidate genes encoding nucleotide-binding site leucine-rich repeat (NBS-LRR) or other proteins related to disease resistances. CONCLUSIONS: The results of this study provide a basis for breeding peanut cultivars with web blotch resistance.

Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing.

BACKGROUND: Increasing the content of oleic acid in peanut seeds is one of the major goals in peanut breeding due to consumer and industry benefits, such as anti-oxidation and long shelf-life. Homeologous ahFAD2A and ahFAD2B genes encode fatty acid desaturases, which are the key enzymes for converting oleic acid to linoleic acid that oxidizes readily. To date, all high oleic acid peanut varieties result from natural mutations occurred in both genes. A method to induce mutations in the genes of other elite cultivars could speed introgression of this valuable trait. The gene-editing approach utilizing CRISPR/Cas9 technology was employed to induce de novo mutations in the ahFAD2 genes using peanut protoplasts and hairy root cultures as models. RESULTS: The hot spot of natural mutation in these genes was selected as the target region. Appropriate sgRNAs were designed and cloned into a CRISPR/Cas9 expression plasmid. As a result of CRISPR/Cas9 activity, three mutations were identified - G448A in ahFAD2A, and 441_442insA and G451T in ahFAD2B. The G448A and 441_442insA mutations are the same as those seen in existing high oleate varieties and the G451T is new mutation. Because natural mutations appear more often in the ahFAD2A gene than in the ahFAD2B gene in subspecies A. hypogaea var. hypogaea, the mutations induced in ahFAD2B by gene editing may be useful in developing high oleate lines with many genetic backgrounds after validation of oleic acid content in the transformed lines. The appearance of the G448A mutation in ahFAD2A is a further benefit for high oleic acid oil content. CONCLUSIONS: Overall, these results showed that mutations were, for the first time, induced by CRISPR-based gene editing approach in peanut. This research demonstrated the potential application of gene editing for mutagenesis in peanut and suggested that CRISPR/Cas9 technology may be useful in the peanut breeding programs.

High-resolution chromosome painting with repetitive and single-copy oligonucleotides in Arachis species identifies structural rearrangements and genome differentiation.

BACKGROUND: Arachis contains 80 species that carry many beneficial genes that can be utilized in the genetic improvement of peanut (Arachis hypogaea L. 2n = 4x = 40, genome AABB). Chromosome engineering is a powerful technique by which these genes can be transferred and utilized in cultivated peanut. However, their small chromosomes and insufficient cytological markers have made chromosome identification and studies relating to genome evolution quite difficult. The development of efficient cytological markers or probes is very necessary for both chromosome engineering and genome discrimination in cultivated peanut. RESULTS: A simple and efficient oligonucleotide multiplex probe to distinguish genomes, chromosomes, and chromosomal aberrations of peanut was developed based on eight single-stranded oligonucleotides (SSONs) derived from repetitive sequences. High-resolution karyotypes of 16 Arachis species, two interspecific F1 hybrids, and one radiation-induced M1 plant were then developed by fluorescence in situ hybridization (FISH) using oligonucleotide multiplex, 45S and 5S rDNAs, and genomic in situ hybridization (GISH) using total genomic DNA of A. duranensis (2n = 2x = 20, AA) and A. ipaënsis (2n = 2x = 20, BB) as probes. Genomes, chromosomes, and aberrations were clearly identifiable in the established karyotypes. All eight cultivars had similar karyotypes, whereas the eight wild species exhibited various chromosomal variations. In addition, a chromosome-specific SSON library was developed based on the single-copy sequence of chromosome 6A of A. duranensis. In combination with repetitive SSONs and rDNA FISH, the single-copy SSON library was applied to identify the corresponding A3 chromosome in the A. duranensis karyotype. CONCLUSIONS: The development of repetitive and single-copy SSON probes for FISH and GISH provides useful tools for the differentiation of chromosomes and identification of structural chromosomal rearrangement. It facilitates the development of high-resolution karyotypes and detection of chromosomal variations in Arachis species. To our knowledge, the methodology presented in this study demonstrates for the first time the correlation between a sequenced chromosome region and a cytologically identified chromosome in peanut.

Physiological and Proteomics Analyses Reveal Low-Phosphorus Stress Affected the Regulation of Photosynthesis in Soybean.

Previous studies have revealed a significant genetic relationship between phosphorus (P)-efficiency and photosynthesis-related traits in soybean. In this study, we used proteome profiling in combination with expression analysis, biochemical investigations, and leaf ultrastructural analysis to identify the underlying physiological and molecular responses. The expression analysis and ultrastructural analysis showed that the photosynthesis key genes were decreased at transcript levels and the leaf mesophyll and chloroplast were severely damaged after low-P stress. Approximately 55 protein spots showed changes under low-P condition by mass spectrometry, of which 17 were involved in various photosynthetic processes. Further analysis revealed the depression of photosynthesis caused by low-P stress mainly involves the regulation of leaf structure, adenosine triphosphate (ATP) synthesis, absorption and transportation of CO2, photosynthetic electron transport, production of assimilatory power, and levels of enzymes related to the Calvin cycle. In summary, our findings indicated that the existence of a stringent relationship between P supply and the genomic control of photosynthesis in soybean. As an important strategy to protect soybean photosynthesis, P could maintain the stability of cell structure, up-regulate the enzymes' activities, recover the process of photosystem II (PSII), and induce the expression of low-P responsive genes and proteins.

Genome-Wide Characterization of the Phenylalanine Ammonia-Lyase Gene Family and Their Potential Roles in Response to Aspergillus flavus L. Infection in Cultivated Peanut (Arachis hypogaea L.).

Phenylalanine ammonia-lyase (PAL) is an essential enzyme in the phenylpropanoid pathway, in which numerous aromatic intermediate metabolites play significant roles in plant growth, adaptation, and disease resistance. Cultivated peanuts are highly susceptible to Aspergillus flavus L. infection. Although PAL genes have been characterized in various major crops, no systematic studies have been conducted in cultivated peanuts, especially in response to A. flavus infection. In the present study, a systematic genome-wide analysis was conducted to identify PAL genes in the Arachis hypogaea L. genome. Ten AhPAL genes were distributed unevenly on nine A. hypogaea chromosomes. Based on phylogenetic analysis, the AhPAL proteins were classified into three groups. Structural and conserved motif analysis of PAL genes in A. hypogaea revealed that all peanut PAL genes contained one intron and ten motifs in the conserved domains. Furthermore, synteny analysis indicated that the ten AhPAL genes could be categorized into five pairs and that each AhPAL gene had a homologous gene in the wild-type peanut. Cis-element analysis revealed that the promoter region of the AhPAL gene family was rich in stress- and hormone-related elements. Expression analysis indicated that genes from Group I (AhPAL1 and AhPAL2), which had large number of ABRE, WUN, and ARE elements in the promoter, played a strong role in response to A. flavus stress.

Chloroplast and whole-genome sequencing shed light on the evolutionary history and phenotypic diversification of peanuts.

Cultivated peanut (Arachis hypogaea L.) is a widely grown oilseed crop worldwide; however, the events leading to its origin and diversification are not fully understood. Here by combining chloroplast and whole-genome sequence data from a large germplasm collection, we show that the two subspecies of A. hypogaea (hypogaea and fastigiata) likely arose from distinct allopolyploidization and domestication events. Peanut genetic clusters were then differentiated in relation to dissemination routes and breeding efforts. A combination of linkage mapping and genome-wide association studies allowed us to characterize genes and genomic regions related to main peanut morpho-agronomic traits, namely flowering pattern, inner tegument color, growth habit, pod/seed weight and oil content. Together, our findings shed light on the evolutionary history and phenotypic diversification of peanuts and might be of broad interest to plant breeders.

Transcriptome analysis of pod mutant reveals plant hormones are important regulators in controlling pod size in peanut (Arachis hypogaea L.).

Pod size is an important yield-influencing trait in peanuts. It is affected by plant hormones and identifying the genes related to these hormones may contribute to pod-related trait improvements in peanut breeding programs. However, there is limited information on the molecular mechanisms of plant hormones that regulate pod size in peanuts. We identified a mutant with an extremely small pod (spm) from Yuanza 9102 (WT) by 60Co γ-radiation mutagenesis. The length and width of the natural mature pod in spm were only 71.34% and 73.36% of those in WT, respectively. We performed comparative analyses for morphological characteristics, anatomy, physiology, and global transcriptome between spm and WT pods. Samples were collected at 10, 20, and 30 days after peg elongation into the soil, representing stages S1, S2, and S3, respectively. The differences in pod size between WT and spm were seen at stage S1 and became even more striking at stages S2 and S3. The cell sizes of the pods were significantly smaller in spm than in WT at stages S1, S2, and S3. These results suggested that reduced cell size may be one of the important contributors for the small pod in spm. The contents of indole-3-acetic acid (IAA), gibberellin (GA), and brassinosteroid (BR) were also significantly lower in spm pods than those in WT pods at all three stages. RNA-Seq analyses showed that 1,373, 8,053, and 3,358 differently expressed genes (DEGs) were identified at stages S1, S2, and S3, respectively. Functional analyses revealed that a set of DEGs was related to plant hormone biosynthesis, plant hormone signal transduction pathway, and cell wall biosynthesis and metabolism. Furthermore, several hub genes associated with plant hormone biosynthesis and signal transduction pathways were identified through weighted gene co-expression network analysis. Our results revealed that IAA, GA, and BR may be important regulators in controlling pod size by regulating cell size in peanuts. This study provides helpful information for the understanding of the complex mechanisms of plant hormones in controlling pod size by regulating the cell size in peanuts and will facilitate the improvement of peanut breeding.

Gene Co-expression Network Analysis of the Comparative Transcriptome Identifies Hub Genes Associated With Resistance to Aspergillus flavus L. in Cultivated Peanut (Arachis hypogaea L.).

Cultivated peanut (Arachis hypogaea L.), a cosmopolitan oil crop, is susceptible to a variety of pathogens, especially Aspergillus flavus L., which not only vastly reduce the quality of peanut products but also seriously threaten food safety for the contamination of aflatoxin. However, the key genes related to resistance to Aspergillus flavus L. in peanuts remain unclear. This study identifies hub genes positively associated with resistance to A. flavus in two genotypes by comparative transcriptome and weighted gene co-expression network analysis (WGCNA) method. Compared with susceptible genotype (Zhonghua 12, S), the rapid response to A. flavus and quick preparation for the translation of resistance-related genes in the resistant genotype (J-11, R) may be the drivers of its high resistance. WGCNA analysis revealed that 18 genes encoding pathogenesis-related proteins (PR10), 1-aminocyclopropane-1-carboxylate oxidase (ACO1), MAPK kinase, serine/threonine kinase (STK), pattern recognition receptors (PRRs), cytochrome P450, SNARE protein SYP121, pectinesterase, phosphatidylinositol transfer protein, and pentatricopeptide repeat (PPR) protein play major and active roles in peanut resistance to A. flavus. Collectively, this study provides new insight into resistance to A. flavus by employing WGCNA, and the identification of hub resistance-responsive genes may contribute to the development of resistant cultivars by molecular-assisted breeding.

Mapping of a QTL associated with sucrose content in peanut kernels using BSA-seq.

As an important factor affecting the edible quality of peanut kernels, sucrose content is a complex quantitative trait regulated by multiple factors. In this study, an F2 segregating population and a recombinant inbred line (RIL) population, derived from a cross between the high sucrose content variety Jihuatian 1 and the low sucrose content line PI478819, were used as materials to map a quantitative trait locus (QTL) associated with sucrose content in peanut kernels. Four QTLs were initially located on chromosomes A03 and A06 based on BSA-seq technology, and multiple kompetitive allele-specific PCR markers were developed based on single-nucleotide polymorphisms (SNPs) in the intervals. The markers were genotyped in the RIL population and finely mapped to a stable QTL, qSUCA06, located on chromosome A06 within a 0.29-Mb physical genomic interval (112367085-112662675 bp), which accounted for 31.95%-41.05% of the phenotypic variance explained. SNP and insertion/deletion annotations were performed on genes in the candidate interval, and having screened out those genes with mutations in exons, candidate genes were verified by qRT-PCR. The results revealed that Arahy.Y2LWD9 may be the main gene regulating sucrose content. The QTL identified in this study will not only contribute to marker-assisted breeding for improvement of peanut sucrose content but also paves the way for identifying gene function.

Genome-Wide Identification and Expression Analysis of AP2/ERF Transcription Factor Related to Drought Stress in Cultivated Peanut (Arachis hypogaea L.).

APETALA2/ethylene response element-binding factor (AP2/ERF) transcription factors (TFs) have been found to regulate plant growth and development and response to various abiotic stresses. However, detailed information of AP2/ERF genes in peanut against drought has not yet been performed. Herein, 185 AP2/ERF TF members were identified from the cultivated peanut (A. hypogaea cv. Tifrunner) genome, clustered into five subfamilies: AP2 (APETALA2), ERF (ethylene-responsive-element-binding), DREB (dehydration-responsive-element-binding), RAV (related to ABI3/VP), and Soloist (few unclassified factors)). Subsequently, the phylogenetic relationship, intron-exon structure, and chromosomal location of AhAP2/ERF were further characterized. All of these AhAP2/ERF genes were distributed unevenly across the 20 chromosomes, and 14 tandem and 85 segmental duplicated gene pairs were identified which originated from ancient duplication events. Gene evolution analysis showed that A. hypogaea cv. Tifrunner were separated 64.07 and 66.44 Mya from Medicago truncatula L. and Glycine max L., respectively. Promoter analysis discovered many cis-acting elements related to light, hormones, tissues, and stress responsiveness process. The protein interaction network predicted the exitance of functional interaction among families or subgroups. Expression profiles showed that genes from AP2, ERF, and dehydration-responsive-element-binding subfamilies were significantly upregulated under drought stress conditions. Our study laid a foundation and provided a panel of candidate AP2/ERF TFs for further functional validation to uplift breeding programs of drought-resistant peanut cultivars.

Transcriptomic analyses reveal the expression and regulation of genes associated with resistance to early leaf spot in peanut.

OBJECTIVE: Early leaf spot (ELS) caused by Cercospora arachidicola (Hori) is a serious foliar disease in peanut worldwide, which causes considerable reduction of yield. Identification of resistance genes is important for both conventional and molecular breeding. Few resistance genes have been identified and the mechanism of defense responses to this pathogen remains unknown. RESULTS: We detected several genes involved in disease resistance to ELS through transcriptome analysis. Using RNA-seq technology, one hundred thirty-three differentially expressed genes (DEGs) were identified between resistant and susceptible lines. Among these DEGs, coiled coil-nucleotide binding-leucine rich repeat (NLR) type resistance genes were identified as duplicated R genes on the chromosome B2. Peanut phytoalexin deficient 4 (PAD4) regulator of effector-triggered immunity mediated by NLR resistance proteins and polyphenol oxidase (PPO) genes play important roles in early leaf spot resistance. Our study provides the useful information on plant response to C. arachidicola infection in peanut. The results suggest that a few major genes and several factors mediate the resistance to ELS disease, showing the characteristics of quantitative trait in defense responses.

Development of an oligonucleotide dye solution facilitates high throughput and cost-efficient chromosome identification in peanut.

BACKGROUND: Development of oligonucleotide probes facilitates chromosome identification via fluorescence in situ hybridization (FISH) in many organisms. RESULTS: We report a high throughput and economical method of chromosome identification based on the development of a dye solution containing 2 × saline-sodium citrate (SSC) and oligonucleotide probes. Based on the concentration, staining time, and sequence effects of oligonucleotides, an efficient probe dye of peanut was developed for chromosome identification. To validate the effects of this solution, 200 slides derived from 21 accessions of the cultivated peanut and 30 wild Arachis species were painted to identify Arachis genomes and establish karyotypes. The results showed that one jar of dye could be used to paint 10 chromosome preparations and recycled at least 10 times to efficiently dye more than 100 slides. The A, B, K, F, E, and H genomes showed unique staining karyotype patterns and signal colors. CONCLUSIONS: Based on the karyotype patterns of Arachis genomes, we revealed the relationships among the A, B, K, F, E, and H genomes in genus Arachis, and demonstrated the potential for adoption of this oligonucleotide dye solution in practice.

Salinity Inhibits Rice Seed Germination by Reducing α-Amylase Activity via Decreased Bioactive Gibberellin Content.

Seed germination plays important roles in the establishment of seedlings and their subsequent growth; however, seed germination is inhibited by salinity, and the inhibitory mechanism remains elusive. Our results indicate that NaCl treatment inhibits rice seed germination by decreasing the contents of bioactive gibberellins (GAs), such as GA1 and GA4, and that this inhibition can be rescued by exogenous bioactive GA application. To explore the mechanism of bioactive GA deficiency, the effect of NaCl on GA metabolic gene expression was investigated, revealing that expression of both GA biosynthetic genes and GA-inactivated genes was up-regulated by NaCl treatment. These results suggest that NaCl-induced bioactive GA deficiency is caused by up-regulated expression of GA-inactivated genes, and the up-regulated expression of GA biosynthetic genes might be a consequence of negative feedback regulation of the bioactive GA deficiency. Moreover, we provide evidence that NaCl-induced bioactive GA deficiency inhibits rice seed germination by decreasing α-amylase activity via down-regulation of α-amylase gene expression. Additionally, exogenous bioactive GA rescues NaCl-inhibited seed germination by enhancing α-amylase activity. Thus, NaCl treatment reduces bioactive GA content through promotion of bioactive GA inactivation, which in turn inhibits rice seed germination by decreasing α-amylase activity via down-regulation of α-amylase gene expression.

A SNP-Based Linkage Map Revealed QTLs for Resistance to Early and Late Leaf Spot Diseases in Peanut (Arachis hypogaea L.).

Cultivated peanut (Arachis hypogaea L.) is an important oilseed crop that is grown extensively in Africa, Asia and America. The diseases early and late leaf spot severely constrains peanut production worldwide. Because multiple genes control resistance to leaf spot diseases, conventional breeding is a time-consuming approach for pyramiding resistance genes into a single genotype. Marker-assisted selection (MAS) would complement and accelerate conventional breeding once molecular markers tightly associated with the resistance genes are identified. In this study, we have generated a large number of SNPs through genotyping by sequencing (GBS) and constructed a high-resolution map with an average distance of 1.34 cM among 2,753 SNP markers distributed on 20 linkage groups. QTL mapping has revealed that major QTL within a confidence interval could provide an efficient way to detect putative resistance genes. Analysis of the interval sequences has indicated that a major QTL for resistance to late leaf spot anchored by two NBS-LRR resistance genes on chromosome B05. Two major QTLs located on chromosomes A03 and B04 were associated with resistance genes for early leaf spot. Sequences within the confidence interval would facilitate identifying resistance genes and applying marker-assisted selection for resistance.

Genetic Diversity, Population Structure, and Botanical Variety of 320 Global Peanut Accessions Revealed Through Tunable Genotyping-by-Sequencing.

Cultivated peanut (Arachis hypogaea L.) were classified into six botanical varieties according to the morphological characteristics. However, their genetic evolutionary relationships at the genome-wide level were still unclear. A total of 320 peanut accessions, including four of the six botanical varieties, and 37,128 high-quality single nucleotide polymorphisms (SNPs) detected by tunable genotyping-by-sequencing (tGBS) were used to reveal the evolutionary relationships among different botanical varieties and verify the phenotypic classification. A phylogenetic tree indicated that the tested accessions were grouped into three clusters. Almost all of the peanut accessions in cluster C1 belong to var. fastigiata, and clusters C2 and C3 mainly consisted of accessions from var. vulgaris and subsp. hypogaea, respectively. The results of a principal component analysis were consistent with relationships revealed in the phylogenetic tree. Population structure analysis showed that var. fastigiata and var. vulgaris were not separated when K = 2 (subgroup number), whereas they were clearly divided when K = 3. However, var. hypogaea and var. hirsuta could not be distinguished from each other all the way. The nucleotide diversity (π) value implied that var. vulgaris exhibited the highest genetic diversity (0.048), followed by var. fastigiata (0.035) and subsp. hypogaea (0.012), which is consistent with the result of phylogenetic tree. Moreover, the fixation index (FST) value confirmed that var. fastigiata and var. vulgaris were closely related to each other (FST = 0.284), while both of them were clearly distinct from var. hypogaea (FST > 0.4). The present study confirmed the traditional botanical classifications of cultivated peanut at the genome-wide level. Furthermore, the reliable SNPs identified in this study may be a valuable resource for peanut breeders.