Peanut, a globally significant food crop, is highly valued for its nutritional and economic importance. However, breeding efforts to enhance its protein content have been limited. In this study, a two-step chemical mutagenesis strategy was utilized on the normal-protein Spanish-type peanut cultivar Fuhua 19 (< 25% protein) to generate high-protein mutants. Through successive rounds of mutagenesis combined with near-infrared spectroscopy screening, eight mutants with protein levels exceeding 30% were identified. Among these, the mutant C-Za-454-2 consistently exhibited stable and elevated protein content across different locations and developmental stages. This mutant also displayed distinct ultrastructural changes, including an increased protein body area and reduced starch grain content, compared to the wild-type Fuhua 19. Biochemical analyses revealed enhanced activities of nitrogen metabolism enzymes, such as nitrate reductase and glutamate synthetase, which positively correlated with protein accumulation. Conversely, certain carbon metabolism enzyme activities were inversely correlated with protein content, highlighting a trade-off between carbohydrate and protein synthesis. These findings demonstrate the efficacy of iterative mutagenesis in producing high-protein peanut lines and provide a foundation for future genetic studies aimed at improving protein yield in peanut cultivars.

Peanut (Arachis hypogaea L.) production is critically constrained by soil-borne diseases, notably bacterial wilt (Ralstonia solanacearum), southern blight (Sclerotium rolfsii), and pod rot (Pythium myriotylum). Developing cultivars with multi-disease resistance and high kernel quality is essential for sustainable cultivation. In this study, 30 peanut genotypes were evaluated for resistance to these three pathogens and profiled for key biochemical quality traits. Genetic diversity was assessed using 32 transposable element-based molecular markers. The results revealed significant phenotypic variation. Genotype 23S6 emerged as an elite multi-resistant germplasm, exhibiting immunity to bacterial wilt and resistance to both southern blight and pod rot. Correlation analysis revealed significant biological trade-offs: bacterial wilt survival rate was positively correlated with protein content (r = 0.338) but also positively correlated with pod rot disease index (r = 0.366). Furthermore, sucrose content showed a significant positive correlation with southern blight susceptibility (r = 0.363). Genetic diversity analysis indicated a broad genetic base for bacterial wilt resistance (h = 0.228) but a critically narrow base for southern blight resistance (h = 0.101). These findings highlight specific genetic constraints and trade-offs in multi-trait breeding, positioning 23S6 as a rare donor for breaking these unfavorable assocations.

Peanut is a vital cash crop globally, and enhancing its seed protein content is essential for nutritional security. However, the genetic basis of seed protein content in peanut remains unclear. In this study, bulked segregant analysis combined with next-generation sequencing (BSA-Seq) was employed to isolate a candidate gene, AhSPC, associated with peanut seed protein content. Its function was then characterized in Arabidopsis thaliana through CRISPR/Cas9-mediated gene knockout and overexpression analysis. Gene editing of the homologous gene in Arabidopsis significantly reduced seed protein content in six lines, while two lines showed no obvious change. Overexpression of the AhSPC gene in Arabidopsis led to an increase in seed protein content in three transgenic lines, but some lines showed no significant change, or even a decrease. These inconsistent results might be attributed to functional redundancy, epigenetic modifications, resource competition, or feedback regulation mechanisms within the protein synthesis pathway. This study provides insights into the molecular mechanisms of peanut seed protein content regulation and offers a potential genetic target for molecular breeding.

Aflatoxin contamination, primarily caused by Aspergillus flavus invasion, poses significant risks to food and seed safety in peanut. This study aimed to evaluate the resistance of various peanut genotypes to A. flavus infection and subsequent aflatoxin production, addressing the need for effective breeding strategies to enhance resistance. A total of 227 peanut germplasm materials, including 172 cultivars, 29 breeding lines, and 26 wild accessions, were assessed for their response to A. flavus infection. The materials underwent artificial inoculation with a standard strain of A. flavus, followed by quantification of aflatoxin B1 (AFT B1) levels. Infection indices were calculated based on visual assessments of spore coverage, and genetic diversity was analyzed among selected materials. The evaluation identified one resistant entry (OPLX-86) (0.44

This study challenges conventional approaches to authentication of intersectional hybrids between the cultivated peanut (Arachis hypogaea L.) and the wild species A. glabrata Benth., achieved through an innovative in situ embryo rescue technique. Systematic phenotyping and genotyping of F₁ hybrids revealed unexpected segregation in key traits: plant architecture (erect vs. prostrate) and seed oleic acid content (high-oleic vs. normal-oleic). Notably, some F₁ hybrids displayed high-oleic phenotypes despite heterozygosity at FAD2 or lacked diagnostic transposable element (TE) markers from the wild parent, thereby indicating potential genomic instability or epigenetic silencing. Our findings underscore the limitations of single-method hybrid authentication, advocating instead for integrated multi-locus molecular, phenotypic, and biochemical approaches. These results not only enhance our understanding of trait expression in distant hybrids but also provide insights for optimizing peanut distal hybridization breeding strategies.

The cultivated peanut is a globally important crop, valued for its oil, food, and feed uses, but has a narrow genetic base. High stress resistance, good-quality and high-yield factors residing in wild species constitute valuable resources for genetic improvement of the peanut cultigen. Some wild species are used as groundcovers, while others utilized as potted plants. Previous studies have focused on compatible wild relatives, but there is a lack of research on the use of incompatible Arachis species. This study aimed to investigate the hybrids produced from a cross between the peanut cultivar Huayu 665 and the incompatible species Arachis paraguariensis, enhancing our understanding of distant hybridization. Ture F1 intersectional hybrids were identified by transposon element marker pairs. Leaf stomata were observed, and main agronomic traits were investigated. The F1 hybrids exhibited significantly fewer large stomata (14.78 per mm2) and longer stomata (26.52 μm) on the abaxial leaf epidermis compared to the female parent, Huayu 665, which had 20.56 per mm2 and 16.86 μm, respectively. Compared to Huayu 665, the F1 hybrids exhibited a longer first pair of lateral branches, a wider range of seed set, and more branches, but produced fewer pods per plant. A hybrid with a plant type similar to the female parent was identified. The authenticity of the hybrids was confirmed through molecular, anatomical, and morphological analyses. The hybrid resembling the cultigen may accelerate the utilization of incompatible wild species in peanut breeding. However, its chromosome composition is yet to be determined. To avoid missing true hybrid identification in peanut remote crosses, use of molecular markers distributed across different chromosomes of the wild species was proposed.

Peanut is a key cash crop worldwide, yet the limited availability of functional genes and markers for breeding hinders further progress, largely due to the lack of an efficient and user-friendly transformation system. This study aimed to comprehensively evaluate the effectiveness of nodal agroinjection, a novel transformation technique we developed for peanut, by introducing the soybean cold-tolerance gene SCTF-1. Putative transgenic seeds and seedlings were screened using genomic DNA PCR, while transgene expression was analyzed via qRT-PCR and phenotypic assessments. Southern blotting confirmed the stable integration of SCTF-1. The transgenic seedlings displayed enhanced chilling tolerance, characterized by increased proline accumulation, reduced malondialdehyde (MDA), and elevated peroxidase (POD) activity. These findings demonstrate that nodal agroinjection is an efficient and reliable approach for generating transgenic peanut and analyzing gene function. This method offers a promising alternative to conventional tissue culture-based transformation strategies.

Cytokinins (CKs) are key factors in modulating the growth and development of peanut pods during the rapid-expansion stage, which determines the final pod size. To explore the effect of exogenous CKs on pod development, two peanut varieties with contrasting pod sizes were used for the comparison of differences in the phenotype, cell number, endogenous CK level, and CK-related gene expression of pods under different concentrations of 6-benzyladenine (6-BA) treatment. Developing peanut pods were sampled at 10, 15, 20, 25, and 30 days after pegging (DAP). The results indicated that the 6-BA treatment increased pod length and width and promoted dry matter accumulation, with 20 mg L−1 6-BA generally having the most significant effect. Cytological analysis confirmed that 6-BA increased pod size by increasing the cell number during the rapid-expansion stage. Additionally, 6-BA treatment enhanced the expression of CK biosynthesis-related genes (AhIPT1 and AhIPT2), resulting in elevated endogenous CK content. Furthermore, the upregulation of CK signal transduction-related genes (AhHRS) coincided with the increase in cell number, suggesting potential involvement in cell proliferation regulation. This coordinated response enhanced the maximum growth rate (Vmax), earlier initiation of the rapid-expansion stage, and ultimately increased the pod size.

The cultivated peanut (Arachis hypogaea L.) is a globally important oilseed and economic crop, but its narrow genetic base limits breeding progress. Wild Arachis species represent valuable genetic resources for enhancing the resilience of the peanut cultigen. While wild species from section Arachis are widely used in breeding programs, the detection of alien chromosomes in hybrids remains challenging due to limited molecular tools. In this study, a cost-effective and efficient system was established for generating species-specific molecular markers using low-coverage next-generation sequencing data, bypassing the need for whole-genome assembly. Utilizing the Chorus2 software, specific alien-chromosome oligo (SAO) markers were developed for four wild species, A. duranensis (accession A19), A. pusilla (A10), A. appresipilla (A33), and A. glabrata (G2 and G3). A total of 1166 primer pairs were designed, resulting in 220 SAO markers specific to A. duranensis, 77 to A. pusilla, 112 to A. appresipilla, 69 to A. glabrata G2, and 59 to A. glabrata G3, with the highest development efficiency observed in A. duranensis (55.0%). These markers span all chromosomes of the five wild accessions. Genome-wide, chromosome-specific SAO markers enable the efficient detection of introgressed alien chromosomes and provide insight into syntenic relationships among homoeologous chromosomes. These markers offer an effective tool for identifying favorable genes and facilitating targeted introgression for the genetic improvement of the cultivated peanut.

The cultivated peanut (Arachis hypogaea L.) is a significant edible oilseed crop. Although substantial progress has been made in breeding new peanut varieties through conventional methods, the narrow gene base of the cultivated peanut (stemming from six evolutionary bottlenecks) poses growing challenges for developing breakthrough varieties via intraspecific hybridization alone. Consequently, extensive efforts have been directed towards the utilization of incompatible wild Arachis species. In this study, we present the first genetic diversity assessment of 30 progenies derived from intersectional crosses between the high-oleic peanut cultivar Huayu 665 and two wild species, A. paraguariensis and A. appresipilla, alongside 32 cultivated materials and 29 wild accessions, using transposon element markers. The hybrids demonstrated greater genetic diversity than the cultivars, expanding the gene base for peanut breeding. Hybrid progenies with enhanced insect pest resistance and elevated levels of both oleic acid and oil/protein content were tentatively obtained. This work lays the groundwork for the future development of high-yielding, high-quality peanut varieties through the utilization of crop wild relatives.

Peanut is a major cash crop in the world.In recent years,peanut pod rot has become increasingly prevalent,resulting in significant yield and quality losses.Resistance breeding is considered a cost-effective approach for managing pod rot.Previous research in the United States identified significant anatomical differences in leaves and shells among peanut cultivars with varying responses to pod rot caused by Pythium myriotylum Drechs.and Rhizoctonia solani Kuhn.However,whether similar anatomical features correlate with resistance to peanut pod rot in Laixi,Qingdao,remained unknown,where the primary causal pathogen for the disease was F.solani.In this study,nine peanut varieties with differing disease reactions to peanut pod rot were planted in the same plots.Paraffin sections of leaves and shells were prepared and stained to evaluate anatomical features,and the main agronomic traits were assessed alongside leaf spot disease ratings.All the four anatomical features,leaf palisade cell number,palisade cell width,index(palisade cell number per mm × cell width in μm),and shell lignin staining area,were measured before the onset of pod rot and were found to be negatively correlated with pod rot scores.Given the consistent and strong correlation of these anatomical features with pod rot resistance and their high heritability estimates,the pre-existing resistance could be identified even in the absence of the disease,which is particularly valuable for fields where pod rot may not occur every year.This study provided useful anatomical indicators for selecting resistance to peanut pod rot,predominantly caused by F.solani.

Pod length (PL) is one of the major traits determining pod size and yield of peanut. Discovering the quantitative trait loci (QTL) and identifying candidate genes associated with PL are essential for breeding high-yield peanut. In this study, quantitative trait loci sequencing (QTL-seq) was performed using the F2 population constructed by a short-pod variety Tifrunner (Tif) and a long-pod line Lps, and a 0.77 Mb genomic region on chromosome 07 was identified as the candidate region for PL. Then, the candidate region was narrowed to a 265.93 kb region by traditional QTL approach. RNA-seq analysis showed that there were four differentially expressed genes (DEGs) in the candidate region, among which Arahy.PF2L6F (AhCDC48) and Arahy.P4LK2T (AhTAA1) were speculated to be PL-related candidate genes. These results were informative for the elucidation of the underlying regulatory mechanism in peanut pod length and would facilitate further identification of valuable target genes.

The cultivated peanut, Arachis hypogaea L., is an important source of edible oil and highly digestible protein. Wild incompatible Arachis species outside section Arachis are ideal gene reservoirs for genetic improvement of the peanut crop. Among these, A. glabrata Benth. stands out for its noted resistance to various stresses. Traditional in vitro embryo rescue techniques have been fraught with challenges, including time consumption, resource intensiveness, late intervention timing, and limited effectiveness. In this study, we employed three hormone formulations in an innovative in situ embryo rescue approach to facilitate the production of intersectional Arachis hybrids. Through this method, hybrid seeds resulting from the crossing of two incompatible species, namely A. glabrata and A. paraguariensis, with four high-oleic peanut varieties were successfully obtained. Molecular marker analysis and observation of plant characteristics confirmed the hybrid nature of these seeds. This breakthrough represents a significant advancement in expediting the utilization of incompatible wild Arachis species in peanut breeding programs. Moreover, the in situ embryo rescue technique showcased in this study holds promise for application in other plant species characterized by postzygotic cross-incompatibility.

High-oleic peanuts have attracted the attention of food processors and consumers for their good keeping quality and purported multiple health benefits. It is of interest to investigate whether high-oleic peanuts can replace their normal-oleic counterparts in Asian style foods. After long-term storage, Huayu 668 garlic-flavored peanuts exhibited a much lower peroxide value, and a comparable or lower acid value as compared with normal-oleic peanuts; the peroxide value of Huayu 668 salted crispy peanuts was on a par with salted dried normal-oleic Tianfu 3 prepared with special shelf life extension processing, while the acid value was more than twice as high. The peroxide value and acid value of all the peanut products herein did not exceed the upper limits specified in China national standard. Significant genotype × storage interactions (garlic flavor and total) and genotype effects (roasted peanut flavor, crunchiness and total) were detected in sensory quality indicators for garlic and salted crispy/dried peanut products, respectively. High-oleic Huayu 668 did not show any disadvantages in terms of organoleptic quality compared to normal-oleic peanuts. The outcome of this study suggested that Huayu 668 could be used for garlic-flavored peanut production and had potential in salted crispy peanut and salted dried peanut processing.

The cultivated peanut (Arachis hypogaea L.) is a main cash crop globally, providing oil, protein, and various beneficial phytochemicals, with high-oleic peanut offering enhanced health benefits and oxidative stability. Despite these advantages, many widely cultivated peanut varieties remain normal-oleic, and the conversion of these varieties to high-oleic types without compromising yield and adaptability is of significant interest. This study evaluated the feasibility of using Pingyangmycin, a chemical mutagen, to induce high-oleic mutations in the popular peanut variety 308 through floral organ injection. The results showed that this method effectively generated high-oleic mutants with oleic acid content exceeding 75%. The mutants yielded more pods and kernels than the parental variety. Genotypic analysis confirmed mutations in the FAD2A and FAD2B genes, associated with the high-oleic phenotype. This novel approach, which reduces seed and reagent requirements and accelerates the breeding timeline, holds promise for enhancing peanut breeding programs and the development of high-oleic cultivars with superior quality and yield.

The root-knot nematode (RKN) disease is a highly destructive soilborne disease that significantly affects peanut yield in Northern China. The composition of the soil microbiome plays a crucial role in plant disease resistance, particularly for soilborne diseases like RKN. However, the relationship between the occurrence of RKN disease and the structure and diversity of bacterial communities in peanut fields remains unclear. To investigate bacterial diversity and the community structure of peanut fields with severe RKN disease, we applied 16S full-length amplicon sequencing based on the third high-throughput sequencing technology. The results indicated no significant differences in soil bacterial α-diversity between resistant and susceptible plants at the same site. However, the Simpson index of resistant plants was higher at the site of peanut-wheat-maize rotation (Ro) than that at the site of peanut continuous cropping (Mo), showing an increase of 21.92%. The dominant phyla identified in the peanut bulk soil included Proteobacteria, Acidobacteria, Actinobacteria, Planctomycetes, Chloroflexi, Firmicutes, and Bacteroidetes. Further analysis using LEfSe (Linear discriminant analysis effect size) revealed that Sulfuricellaceae at the family level was a biomarker in the bulk soil of susceptible peanut compared to resistant peanut. Additionally, Singulisphaera at the genus level was significantly more enriched in the bulk soil of resistant peanut than that of susceptible peanut. Soil properties were found to contribute to the abundance of bacterial operational taxonomic units (OTUs). Available phosphorus (AP), available nitrogen (AN), organic matter (OM), and pH made a positive contribution to the bacterial OTUs, while available potassium (AK) made a negative contribution. The metabolic pathway of novobiocin biosynthesis was only enriched in soil samples from resistant peanut plants. Eleven candidate beneficial bacteria and ten candidate harmful strains were identified in resistant and susceptible peanut, respectively. The identification of these beneficial bacteria provides a resource for potential biocontrol agents that can help improve peanut resistance to RKN disease. Overall, the study demonstrated that severe RKN disease could reduce the abundance and diversity of bacterial communities in peanut bulk soil. The identification of beneficial bacteria associated with resistant peanut offered the possibility for developing biocontrol strategies to enhance peanut resistance to RKN disease.

Peanut is a major food crop prone to enrichment in toxic cadmium (Cd). To control Cd levels in peanut, this study aimed to develop a near infrared spectroscopy (NIRS) model for determining Cd concentration in peanut kernels as an alternative to traditional wet chemistry techniques, which can be expensive, time-consuming, and destructive. Near-infrared (NIR) diffuse reflectance spectra of 110 bulk peanut kernel samples were collected, and Cd concentration of the kernel samples was determined by inductively coupled plasma mass spectrometry. A robust quantitative NIRS prediction model for Cd concentration in peanut kernels was developed for the first time. In the calibration set, the best model had a high coefficient of determination (R cal 2 = 0.9194) and a low root mean square error of cross-validation (RMSECV = 0.0388). In the prediction set of 105 additional peanut kernel samples not involved in the model development, the coefficient of determination (R p 2 ) was as high as 0.9539 and the root mean square error of prediction was as low as 0.0341. This study provides a rapid and low-cost screening tool for low-Cd breeding and Cd management in peanut.

Using individual single peanut kernels from 80 varieties/lines,near-infrared models were developed based on the MPA Fourier transform infrared spectrometer （Bruker Optics,Germany）,predictive of oleic acid,linoleic acid,palmitic acid,erucic acid,and predictive of eicosenoic acid,palmitoleic acid,behenic acid,myristic acid,α-linolenic acid,and long-chain saturated fatty acids and unsaturated fatty acids for the first time.Through automatic optimization with OPUS 7.5 software,the optimal spectral pretreatment mode for oleic acid,linoleic acid,palmitic acid,behenic acid,myristic acid,long-chain saturated fatty acids and unsaturated fatty acids was"first derivative+vector normalization",with dimensions of 10,10,8,10,5,10,10,the decision coefficients （R~2） were0.98,0.98,0.97,0.87,0.88,0.93,0.98,and 0.98,respectively,and the root mean square error of cross validation（RMSECV） was 2.87%,2.18%,0.461%,0.133%,0.00416%,0.741%,and 0.19%,respectively.The optimal spectral pretreatment method for erucic acid was"first derivative+Multiplicative Scatter Correction",the dimension was 9,R~2 was 0.92,and RMSECV was 0.00628%;the optimal spectral pretreatment for palmitoleic acid was"first derivative+minus a straight line",with the dimension of 10,R~2 of 0.80 and RMSECV of 0.00 602%.The optimal spectral pretreatment for eicosenoic acid and α-linolenic acid was"vector normalization",with dimensions of 8 and10,R~2 of 0.88 and 0.77,respectively,and RMSECV of 0.0731% and 0.026%,respectively.The average relative deviation between the true value of each index and the predicted value of the model ranged from 0.12% to 2.50%.The 11 near-infrared models for fatty acid in peanut kernels had good prediction power.They could be used in rapid quality screening and evaluation for peanut breeding and processing.

Compared to its normal-oleic counterpart, high-oleic peanut has better keeping quality and much more health benefits. Breeding high-oleic peanut through conventional means is a tedious process that typically takes several years. Genome editing, however, may shorten the duration. This study aimed to test the effectiveness of the node injection method coupled with CRISPR/Cas9 technology in inducing FAD2B mutations and high-oleic phenotype in peanut. Huayu 23, a popular normal-oleic runner type peanut cultivar having dysfunctional FAD2A and functional FAD2B , was transformed with CRISPR/Cas9 construct targeting FAD2B , resulting in two T 1 seeds with over 80% oleic acid and a 442 A insertion in FAD2B . The high-oleic phenotype in T 2 seeds was inheritable from the T 1 generation. As a genotype-independent, simple and easy method for peanut genetic transformation, node injection has great potential in functional analysis of genes and peanut varietal improvement. This method is of reference value to other seed plant species.

Background Pod size is an important yield target trait for peanut breeding. However, the molecular mechanism underlying the determination of peanut pod size still remains unclear. Results In this study, two peanut varieties with contrasting pod sizes were used for comparison of differences on the transcriptomic and endogenous hormonal levels. Developing peanut pods were sampled at 10, 15, 20, 25 and 30 days after pegging (DAP). Our results showed that the process of peanut pod-expansion could be divided into three stages: the gradual-growth stage, the rapid-growth stage and the slow-growth stage. Cytological analysis confirmed that the faster increase of cell-number during the rapid-growth stage was the main reason for the formation of larger pod size in Lps. Transcriptomic analyses showed that the expression of key genes related to the auxin, the cytokinin (CK) and the gibberellin (GA) were mostly up-regulated during the rapid-growth stage. Meanwhile, the cell division-related differentially expressed genes (DEGs) were mostly up-regulated at 10DAP which was consistent with the cytological-observation. Additionally, the absolute quantification of phytohormones were carried out by liquid-chromatography coupled with the tandem-mass-spectrometry (LC–MS/MS), and results supported the findings from comparative transcriptomic studies. Conclusions It was speculated that the differential expression levels of TAA1 and ARF (auxin-related), IPT and B-ARR (CK-related), KAO , GA20ox and GA3ox (GA-related), and certain cell division-related genes ( gene-LOC112747313 and gene-LOC112754661 ) were important participating factors of the determination-mechanism of peanut pod sizes. These results were informative for the elucidation of the underlying regulatory network in peanut pod-growth and would facilitate further identification of valuable target genes.