The RIL population, comprising 248 individual lines derived from Silihong (A. hypogaea var. fastigiate) ×Jinonghei3 (A. hypogaea var. hypogaea), was used for QTL mapping. The population was developed at the experimental station of Hebei Agriculture University, Baoding, China. The RIL population and its parental lines were planted in experimental fields in Baoding (38.79°N, 115.56°E), Hebei Province (planted in May and harvested in September of 2018 (E1), 2020 (E2), and 2021 (E3)) and Fuxin (N42°01′, E121°40′), Liaoning Province (planted in May and harvested in September of 2021 (E4)). The parental lines and their 248 RILs were grown as previously described (Li et al., 2024). The browning of the inner fruit shell color signals the optimal timing for peanut harvest (Liang et al., 2018). At physiological maturity, seeds (E1 and E2) from eight middle-row plants in each plot were harvested and air-dried. 50% of the seeds harvested from E3 and E4 were stored at -20°C for FS sugar content analysis, and 50% were naturally air dried for DMS sugar content analysis. Store for future use once moisture content drops below 8% (Qu et al., 2022).

The total sugar content was determined using the 3, 5-dinitrosalicylic acid method according to Hou et al. (2017). Soluble sugar content was determined by the anthrone colorimetric method, as improved by Chen et al. (2022). A 0.1 g tissue sample was extracted from the middle section of the peanut cotyledon and homogenized using a tissue grinder (JXFSTPRP-24 A, Shanghai Jing Xin Co., LTD.). Subsequent procedures were performed in strict accordance with the manufacturer’s instructions of the sugar content assay kit (Gris Biotechnology Co., LTD.).

Statistical analyses, including analysis of variance (ANOVA), frequency distribution, and the independent sample t-test analysis, were performed using the IBM SPSS Statistics version 25 software (IBM SPSS, USA).

A high-density genetic map previously constructed in our laboratory was used for QTL mapping (Li et al., 2024). The inclusive composite interval mapping (ICIM) method in QTL IciMapping V4.2 software was used for scanning the phenotypic value and sugar content data in every environment. To validate the authenticity of detected QTLs and obtain more information, LOD≥2.5 and 1000 permutation test (PT) were employed as threshold methods. The PT method was used for detecting additive QTL. For individual environment analyses, as well as multi-environment joint and epistatic analyses, the BIP and MET functional modules of QTL IciMapping v4.2 were applied. QTLs were named as q + the abbreviated trait name + linkage group number, designating one of multiple QTL in a single linkage group following the international Rules of Genetic Nomenclature (Li et al., 2024).

Candidate gene expression profiles related to sugar content were generated from transcriptome data (Deng et al., 2025). Transcriptome sequencing were used to analyze the regulatory mechanism during the drying process at five time points in parents. The transcriptome data of Silihong deposited in the NCBI SRA database (accession: PRJNA1212227). The transcriptome data of Jinonghei3 was unpublished.

The SNPS in the QTL interval of Silihong and Jinonghei3 were resequenced to develop KASP markers and subsequently screened. The primers were designed based on the differential SNP sequences identified. The primer sequences of KASPs are presented in Table 1. KASP assays were performed in 10 μL reaction volumes comprising 2.0 μL of genomic DNA (25–50 ng), 5 μL of 2×AQP master mix, 0.15 μmol L-1 of each forward primer, 0.4 μmol L-1 of the common reverse primer, and double-distilled water (JasonGen Biotech Co., Ltd., Beijing, China). PCR was performed on an ABI QuantStudio6 machine (ABI Life Technologies, CA, USA) using the following PCR conditions: 95°C for 10 min; 95°C for 20 s, 61°C (−0.6°C per cycle) for 40 s for 10 cycles; 95°C for 20 s, 55°C for 40 s for 31 cycles. Fluorescence was detected, and data analysis was manually performed using the built-in ABI QuantStudio6 software. Then, the KASP markers were assessed in a peanut panel comprising 13 lines and 19 peanut varieties and landraces cultivated in China.

In both fresh seeds (FS) and dried mature seeds (DMS), the TSC of Jinonghei3 was higher than that of Silihong, and the SSC and SC contents of Silihong exceeded those of Jinonghei3. However, across conditions, TSC, SSC, and SC of lines in DMS were higher than in FS (Figure 1). This indicates that the drying process affects the sugar content and that there are genetic differences.

The sugar contents of FS and DMS were affected by the environment. The TSC of DMS in E1 was lower than in other environments. The coefficients of variation for SC in DMS and FS were higher than those of TSC and SSC in the corresponding environment (Table 2), and transgressive segregation for SC was more pronounced in the offspring population.

TSC, SSC, and SC were all affected by both environmental and genetic factors. Heritability estimates indicated that genetic control predominated. TSC, SSC, and SC exhibited relatively high broad-sense heritability (h²), ranging from 0.66 to 0.89 (Table 3). Through genetic analysis of multi-environmental populations, the loci associated with phenotypic variation could be analyzed, and then the genetic mechanism of TSC, SSC, and SC in peanut was studied.

A co-localized region between markers 3330–3331 on chromosome B06 with PVE values of 7.72% -19.46% was associated with TSC, SSC, and SC in DMS, as well as TSC and SSC in FS. The co-localized region was designated qSB06. To further investigate the genetic basis of sugar content in peanut seeds, genes within the qSB06 interval were examined. The genome of Tifrunner used as the reference genome (Bhat et al., 2023). qSB06 spanned approximately 722.71kb interval and contained 56 genes (Figure 3A). Resequencing of the two parental lines revealed 319 SNPs within this region, including nine within coding sequences. Among these, 1 SNP resulted in a synonymous mutation, 5 SNPs were missense mutations, and 1 SNP was missing (Table 5). Gene Ontology (GO) enrichment analysis indicated that most genes were related to cellular components (Figure 3B), with 5 genes involved in ATP binding. Among them, arahy.B6Y69S encodes the MYB transcription factor MYB92; Arahy.XM8JCA encodes an ascorbic acid transporter protein involved in transmembrane transport. Arahy.3URM83 and arahy.P7PTW7 encode exo70 family proteins G1 and Syntaxin/t-SNARE family proteins, respectively, involved in vesicle transport.

The candidate genes within the qSB06 interval, defined as arahy.3URM83, arahy. 41Y8R9, and arahy.P7PTW7, were tightly associated with sugar synthesis. Arahy.3URM83, which encodes an exo70 family proteins of the extracellular vesicle subunit, exhibited an expression pattern that increased initially and then declined during the drying and maturation stages in both parental lines (Figure 4). Arahy.41Y8R9, encoding growth regulatory factor, showed the opposite expression trend compared to arahy.3URM83 during maturation, decreasing initially and subsequently increasing (Figure 4). Therefore, it can be inferred that this gene is also involved in sugar biosynthesis in peanuts and positively regulates sugar synthesis and accumulation. arahy. P7PTW7 is a Syntaxin/t-SNARE family protein involved in vesicle-mediated transport; however, in the transcriptome analysis, it showed no significant changes between the two parents (Figure 4).

KASP markers developed based on the arahy.PKWB63 within the qB06 interval (physical interval 147.74Mb-148.46Mb, marker interval 3330-3331) were used to validate the association between genotype and sucrose content. Results showed that genotypes carrying the CC allele exhibited significantly higher sucrose content than those with the TT genotype (Figure 5). The genotypes of each material and the statistical results of sucrose content are shown in Table 6. For the materials with the genotype CC, SC ranged from 53.41mg/g to 88.52mg/g, with an average of 68.35mg/g. For the material with the genotype TT, SC ranged from 11.04mg/g to 38.78mg/g, with an average of 24.63mg/g (Table 6). The SC of the materials with the CC genotype was significantly different from that of materials with the TT genotype, and the genotype–phenotype correspondence was consistent. These results indicate that the developed KASPs can effectively screen TSC, SSC, and SC in peanuts.

The sugar content in peanut is substantially influenced by environmental factors such as temperature, light, and soil. Specifically, low-temperature environments in cold regions have positively affected sugar accumulation in peanut seeds (Wang et al., 2021). Furthermore, extending light exposure can significantly increase the accumulation of photosynthetic products in leaves, thereby supplying more substrates for sugar synthesis in the seeds (Chen et al., 2020). Soil potassium enrichment also contributes positively, leading to a significant increase in seed sugar content (Abo El-Kasem et al., 2025). Consequently, peanuts cultivated in cold regions typically exhibit higher sugar levels.

Despite experiencing lower temperatures and more favorable light conditions during kernel maturation than Baoding, Fuxin consistently showed lower TSC, SSC, and SC in fresh peanut seeds. This indicates that the relationship between these environmental factors and sugar accumulation in peanuts is complex and may involve other influencing variables. Soil conditions may exert a greater influence than temperature and light. Future studies should integrate meteorological conditions, soil, and other factors to clarify the environmental factors that affect sugar content and identify the differential responses of peanut germplasm with varying sugar levels to temperature and light. This will also provide a theoretical basis for region-specific peanut cultivation aimed at optimizing sugar content.

Interestingly, while lines with low TSC, SSC, and SC of DMS in Fuxin exhibited higher levels than in Baoding, those with higher sugar content displayed the opposite trend. Previous studies have shown that the drying process affects the quality of peanuts and involves genotypic differences (Deng et al., 2025). According to the historical meteorological data, temperatures in Fuxin are lower than those in Baoding during mid-to-late September. During this period of peanut dehydration and drying, the physiological and biochemical activity inside the seeds is vigorous, and it is the key period for sugars to be converted into storage substances such as oil and protein. Low temperature reduces the physiological and biochemical metabolism rate, and the conversion process is hindered. This may be the reason why the DMS sugar content in Fuxin was found to be higher than that in Baoding. Germplasm with high sugar content may be predominantly affected by genetic factors and less by environmental factors. The genetic differences in sugar content during the drying period warrant further investigation.

The sugar content of FS is mainly influenced by the expression of metabolic genes active during different stages of seed development. The sugar content of DMS is not only affected by the expression of genes related to each developmental stage, but also by the expression of genes during the drying process. The results of this study demonstrated that sugar content in both FS and DMS is genotype-dependent. DMS represents the physiological maturation of FS after drying; therefore, the QTLs affecting DMS sugar content are more complex. This study conducted QTL analysis on the sugar content of FS and DMS using an RIL population. The qSB06 affecting SC-DMS, TFC, and SSC in both FS and DMS was a key locus for sugar synthesis. The QTLs of SC-FS differed completely from those in DMS, likely reflecting the distinct physiological role of sucrose in the source–sink dynamics of plants. Sucrose is a product of photosynthesis and the principal transported carbohydrate. After reaching the sink, it can be hydrolyzed into hexoses, which enter the tricarboxylic acid pathway for protein and oil conversion. It may also be converted into polysaccharides such as cellulose and starch (Xu et al., 2025). The final content in FS is therefore largely determined by the genotype. Future research should further dissect the functional differences among QTLs in FS and DMS in order to clarify the key sugar metabolism pathways in FS and DMS.

Recent studies have explored sugar content traits in mature peanut kernels, using QTL mapping and GWAS analyses to explore the regions and genes related to sugar content traits in peanuts (Zhang et al., 2023). This study identified 22 and 11 QTLs in DMS and FS, respectively, through QTL mapping. The main QTL locus qSA06, located on chromosome A06, associated with total sugar, soluble sugar, and sucrose content, overlaps with the QTL located by Huai et al. (2024), which were associated with sucrose and oil content. The other major QTL, qSB06, identified in this study, is a newly identified locus associated with total sugar content, soluble sugar content, and sucrose content, and has been detected in DMS and FS across multiple environments. This interval includes a 722 kb region and 56 genes. A major QTL linked to SC was identified in FS, located on chromosome A02, explaining11.67% of the phenotypic variation. Collectively, these QTLs provide valuable targets for breeders to simultaneously or separately optimize sugar content in FS and DMS.

Although the major QTL region on chromosome B06 spanned a relatively large physical interval (~0.72 Mb), candidate genes could still be preliminarily identified. Using reference genome annotations and existing transcriptome data (Wang et al., 2024), three genes (arahy.3URM83, arahy.41Y8R9, arahy.P7PTW7) were identified as potential candidate genes influencing sugar content. Arahy.3URM83 encodes an exo70 family protein of the outer capsule subunit, and its expression pattern during seed drying exhibits an initial increase followed by a decrease. Dong et al. (2023) identified and performed a genome-wide characterization of the cotton EXO70 gene family, and found that EXO70 in cotton is related to fiber quality, and upland cotton with a greater EXCO70 gene copy number is more sensitive to environmental stress. Wang et al. (2016) found that silencing certain EXCO70 genes in soybean could inhibit the formation of root nodules and promote premature aging of leaves. Overexpression of EXCO70 in Arabidopsis thaliana revealed that it could affect leaf and flower development. Therefore, it is speculated that it plays an important role in growth and development in leguminous plants by affecting protein and nutrient transport in vesicles. Homologous genes have also been implicated in the negative regulation of sucrose content.

Arahy.41Y8R9 is a growth regulatory factor (GRF) gene whose expression level during peanut drying decreases initially and then increases, opposite to arahy.3URM83. The resequencing results revealed allelic variation between the parents, with a C-to-A substitution at the 460th base in Silihong. Zhao et al. (2019) identified the GRF gene family in cultivated peanuts, which were distributed on 20 chromosomes. The expression levels of AhGRF5a and AhGRF5b genes in pod tissues are higher than those in root, leaf, and stem tissues. Multiple genes of the peanut GRF family were found to be involved in grain growth and development. Therefore, it can be inferred that this gene may positively regulate sugar accumulation.

Arahy.P7PTW7 is annotated as a Syntaxin/t-SNARE family protein involved in vesicle-mediated transport. Rui et al. (2022) conducted functional studies on the Arabidopsis thaliana T-SNARE protein SYP31/SYP32 gene and found that this gene is involved in regulating COPI vesicle-mediated reverse transport and maintaining Golgi apparatus morphology, and plays a role in the development of Arabidopsis male gametophytes. Zhang et al. (2016) identified and examined the biological function of the SNARE protein FgVam7 binding protein in wheat, and found that the SNARE protein FgPep12 participates in regulating the growth, development, and pathogenicity of Fusarium graminearum by participating in vesicle transport. In this study, a base deletion at position 725 was identified in Jinonghei3. The above three genes are closely associated with the transportation and storage of sugars and exhibit sequence variation between the parents. The specific genetic mechanisms involved in regulating sugar synthesis and transport in peanuts need to be further explored.