Low temperature is the most common abiotic stress factor in peanut cultivation. Chitooligosaccharide (COS) plays an important role in the low-temperature resistance in plants, however, the role of COS in regulating the cold tolerance in peanuts is not clear. This research investigated the effects of exogenous COS on peanut seedlings in response to low temperatures. The results showed that exogenous COS can significantly alleviate the cooling symptoms of seedlings by reducing the content of malondialdehyde (MDA) and reactive oxygen species (ROS) under simulated low-temperature conditions (8 °C). These reductions may be related to the elevation of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and polyphenoloxidase (PPO) activities and the increased contents of osmotic substances such as soluble sugars (SS), soluble protein (SP), proline (Pro), and total phenols (TP) compared with those in untreated seedlings. Meanwhile, the contents of chlorophyll a and b in a peanut leaf also increased, as well as the net photosynthetic rate (Pn), resulted in an increased plant height, a heavier fresh weight, and an increased dry weight. Notably, the treatment of 100 mg·L⁻¹ COS had maximum gain effects compared with those of other treatments. In summary, this study revealed the usage of COS for chilling stress alleviation, and 100 mg L⁻¹ foliar spraying is recommended.

High oleic acid peanut (HOAP) is extensively embraced in China because of its high nutritional value and enhanced oxidative stability. However, its dissemination has been severely constrained in high altitude and high latitude areas due to chilling stress during sowing, resulting in significant yield loss in these regions. Despite the lack of understanding of the molecular mechanisms underlying low temperature germination (LTG) in HOAP, discovering the quantitative trait loci (QTL) that confer this trait will undoubtedly benefit breeding efforts. In the present study, we identified putative genomic regions and single nucleotide polymorphisms (SNPs) that govern LTG tolerance of HOAP in an F₂ population derived from the cross of chilling-tolerant YH65 and chilling-sensitive FL14 using bulk segregant analysis (BSA). Analysis of ΔSNP-index and Euclidean distance (ED) value association pinpointed the overlapped region to a 2.29 Mb interval on chromosome A05. The candidate interval showed that 122 genes were significantly related to response to abiotic stress and plant–pathogen interaction. Furthermore, an SNP site associated with LTG tolerance was discovered. The SNP site was employed as a Kompetitive Allele Specific PCR (KASP) marker and validated in a universal peanut panel. These findings may provide valuable insight into the molecular mechanism underpinning LTG tolerance and facilitate marker-assisted selective breeding in HOAP.

The capability of embryogenic callus induction is a prerequisite for in vitro plant regeneration. However, embryogenic callus induction is strongly genotype-dependent, thus hindering the development of in vitro plant genetic engineering technology. In this study, to examine the genetic variation in embryogenic callus induction rate (CIR) in peanut (Arachis hypogaea L.) at the seventh, eighth, and ninth subcultures (T7, T8, and T9, respectively), we performed genome-wide association studies (GWAS) for CIR in a population of 353 peanut accessions. The coefficient of variation of CIR among the genotypes was high in the T7, T8, and T9 subcultures (33.06%, 34.18%, and 35.54%, respectively), and the average CIR ranged from 1.58 to 1.66. A total of 53 significant single-nucleotide polymorphisms (SNPs) were detected (based on the threshold value −log₁₀(p) = 4.5). Among these SNPs, SNPB03-83801701 showed high phenotypic variance and neared a gene that encodes a peroxisomal ABC transporter 1. SNPA05-94095749, representing a nonsynonymous mutation, was located in the Arahy.MIX90M locus (encoding an auxin response factor 19 protein) at T8, which was associated with callus formation. These results provide guidance for future elucidation of the regulatory mechanism of embryogenic callus induction in peanut.

The gibberellic acid-stimulated Arabidopsis (GASA) gene family is essential for plant growth and development, hormone level control, and phytohormone signal transmission. Different plants have been shown to contain numerous GASA homologs. However, there is no knowledge about these proteins in peanuts. In the current study, we performed a thorough bioinformatics and expression analysis and found 20, 22, and 40 GASA genes by genome-wide analyses of A. hypogaea L., A. duranensis, and A. ipaensis, respectively. We analyzed and predicted the physical properties of these genes. Based on the results of our phylogenetic analysis, the evolutionary tree constructed from the 40 AhGASA proteins was divided into seven categories, forming a total of 14 gene pairs. According to our observations, tandem duplication is a significant factor in the expansion of the GASA gene family. AhGASA was unevenly distributed on 20 chromosomes, and 17 tandem duplicated genes were identified. A co-lineage analysis with the A/B subgenome identified 69 linear/parallel homologous gene pairs. A cis-element analysis revealed that the AhGASA protein is crucial for hormone responsiveness. In materials with different size traits at various stages of peanut pod development, transcriptomics and RT-qPCR analyses revealed that AhGASA genes are expressed at various levels and are tissue-specific. This finding suggests that some AhGASA genes may be involved in controlling peanut pod size. This study suggests that GASA genes are crucial for controlling the development of peanut pods and provides the first systematic identification and analysis of GASA genes in peanut. These findings will help future research into the function of the GASA gene in the cultivated peanut.

Prolonged expression of gene-editing components in CRISPR-modified plants can interfere with phenotypic analysis of target traits, increase the risk of off-target mutations, and lead to unnecessary metabolic burden. To mitigate these issues in peanut (Arachis hypogaea L.), callus-specific promoters were screened to restrict Cas9 expression to the callus stage, minimizing its activity in regenerated plants. In this study, six callus-specific genes in peanut were identified by mining RNA sequencing datasets and validating their expression profiles using quantitative reverse transcriptase PCR. The promoters of Arahy.H0FE8D, Arahy.WT3AEF, Arahy.I20Q6X, Arahy.ELJ55T, and Arahy.N9CMH4 were cloned and assessed for their expression activity. Beta-glucuronidase (GUS) histochemical staining confirmed that all five promoters were functional in peanut callus. Further investigation revealed their ability to drive cytosine base editing via a deaminase-nCas9 fusion protein, with all promoters successfully inducing precise base substitutions in peanut. Notably, P_Ah-H0FE8D, P_Ah-WT3AEF, P_Ah-ELJ55T, and P_Ah-N9CMH4 exhibited comparable or higher editing efficiencies than the commonly used cauliflower mosaic virus 35S promoter. These findings provide valuable tools for improving the biosafety of CRISPR-based genome editing in peanut breeding programs.

Peanut rust, caused by Puccinia arachidis Speg., is one of the most significant leaf diseases globally, and has a severe impact on peanut yield and quality. The development of disease-resistant varieties is recognized as an effective strategy to mitigate the damage caused by peanut rust. However, the research foundation for understanding peanut rust remains relatively limited. In this study, we identified significant single nucleotide polymorphisms (SNPs) associated with peanut rust resistance using a natural population consisting of 353 peanut germplasm accessions. These accessions were analyzed based on resequencing data and rust disease phenotypes across one laboratory test and three field trials. A total of 18 significant SNPs were identified on chromosomes A05 (5 SNPs), A08 (7 SNPs), and A12 (6 SNPs). Notably, three SNPs—Arahy.05_93085395, Arahy.05_93114354, and Arahy.12_4097252—were consistently detected across multiple environments. Within their confidence intervals, 48 genes were annotated, including 9 NLR domain-containing genes functionally related to plant disease resistance, which may serve as candidate genes for peanut rust resistance. This study provides insights into the regulatory mechanisms underlying peanut rust resistance.