Characterization of Two Novel Single-Stranded RNA Viruses from Agroathelia rolfsii, the Causal Agent of Peanut Stem Rot.

Peanut stem rot is a soil-borne disease caused by Agroathelia rolfsii. It occurs widely and seriously affects the peanut yield in most peanut-producing areas. The mycoviruses that induce the hypovirulence of some plant pathogenic fungi are potential resources for the biological control of fungal diseases in plants. Thus far, few mycoviruses have been found in A. rolfsii. In this study, two mitoviruses, namely, Agroathelia rolfsii mitovirus 1 (ArMV1) and Agroathelia rolfsii mitovirus 2 (ArMV2), were identified from the weakly virulent A. rolfsii strain GP3-1, and they were also found in other A. rolfsii isolates. High amounts of ArMV1 and ArMV2in the mycelium could reduce the virulence of A. rolfsii strains. This is the first report on the existence of mitoviruses in A. rolfsii. The results of this study may provide insights into the classification and evolution of mitoviruses in A. rolfsii and enable the exploration of the use of mycoviruses as biocontrol agents for the control of peanut stem rot.

Genome-Wide Analysis of Trehalose-6-Phosphate Phosphatase Gene Family and Their Expression Profiles in Response to Abiotic Stress in Groundnut.

Trehalose-6-phosphate phosphatase (TPP) is a pivotal enzyme in trehalose biosynthesis which plays an essential role in plant development and in the abiotic stress response. However, little is currently known about TPPs in groundnut. In the present study, a total of 16 AhTPP genes were identified, and can be divided into three phylogenetic subgroups. AhTPP members within the same subgroups generally displayed similar exon-intron structures and conserved motifs. Gene collinearity analysis revealed that segmental duplication was the primary factor driving the expansion of the AhTPP family. An analysis of the upstream promoter region of AhTPPs revealed eight hormone- and four stress-related responsive cis-elements. Transcriptomic analysis indicated high expression levels of AhTPP genes in roots or flowers, while RT-qPCR analysis showed upregulation of the six tested genes under different abiotic stresses, suggesting that AhTPPs play roles in growth, development, and response to various abiotic stresses. Subcellular localization analysis showed that AhTPP1A and AhTPP5A were likely located in both the cytoplasm and the nucleus. To further confirm their functions, the genes AhTPP1A and AhTPP5A were individually integrated into yeast expression vectors. Subsequent experiments demonstrated that yeast cells overexpressing these genes displayed increased tolerance to osmotic and salt stress compared to the control group. This study will not only lay the foundation for further study of AhTPP gene functions, but will also provide valuable gene resources for improving abiotic stress tolerance in groundnut and other crops.

Genome-wide identification of SWEET genes reveals their roles during seed development in peanuts.

Sugar Will Eventually be Exported Transporter (SWEET) proteins are highly conserved in various organisms and play crucial roles in sugar transport processes. However, SWEET proteins in peanuts, an essential leguminous crop worldwide, remain lacking in systematic characterization. Here, we identified 94 SWEET genes encoding the conservative MtN3/saliva domains in three peanut species, including 47 in Arachis hypogea, 23 in Arachis duranensis, and 24 in Arachis ipaensis. We observed significant variations in the exon-intron structure of these genes, while the motifs and domain structures remained highly conserved. Phylogenetic analysis enabled us to categorize the predicted 286 SWEET proteins from eleven species into seven distinct groups. Whole genome duplication/segment duplication and tandem duplication were the primary mechanisms contributing to the expansion of the total number of SWEET genes. In addition, an investigation of cis-elements in the potential promoter regions and expression profiles across 22 samples uncovered the diverse expression patterns of AhSWEET genes in peanuts. AhSWEET24, with the highest expression level in seeds from A. hypogaea Tifrunner, was observed to be localized on both the plasma membrane and endoplasmic reticulum membrane. Moreover, qRT-PCR results suggested that twelve seed-expressed AhSWEET genes were important in the regulation of seed development across four different peanut varieties. Together, our results provide a foundational basis for future investigations into the functions of SWEET genes in peanuts, especially in the process of seed development.

Full-length transcriptome sequencing provides insights into alternative splicing under cold stress in peanut.

Introduction: Peanut (Arachis hypogaea L.), also called groundnut is an important oil and cash crop grown widely in the world. The annual global production of groundnuts has increased to approximately 50 million tons, which provides a rich source of vegetable oils and proteins for humans. Low temperature (non-freezing) is one of the major factors restricting peanut growth, yield, and geographic distribution. Since the complexity of cold-resistance trait, the molecular mechanism of cold tolerance and related gene networks were largely unknown in peanut. Methods: In this study, comparative transcriptomic analysis of two peanut cultivars (SLH vs. ZH12) with differential cold tolerance under low temperature (10°C) was performed using Oxford Nanopore Technology (ONT) platform. Results and discussion: As a result, we identified 8,949 novel gene loci and 95,291 new/novel isoforms compared with the reference database. More differentially expressed genes (DEGs) were discovered in cold-sensitive cultivar (ZH12) than cold-tolerant cultivar (SLH), while more alternative splicing events were found in SLH compared to ZH12. Gene Ontology (GO) analyses of the common DEGs showed that the "response to stress", "chloroplast part", and "transcription factor activity" were the most enriched GO terms, indicating that photosynthesis process and transcription factors play crucial roles in cold stress response in peanut. We also detected a total of 708 differential alternative splicing genes (DASGs) under cold stress compared to normal condition. Intron retention (IR) and exon skipping (ES) were the most prevalent alternative splicing (AS) events. In total, 4,993 transcription factors and 292 splicing factors were detected, many of them had differential expression levels and/or underwent AS events in response to cold stress. Overexpression of two candidate genes (encoding trehalose-6-phosphatephosphatases, AhTPPs) in yeast improves cold tolerance. This study not only provides valuable resources for the study of cold resistance in peanut but also lay a foundation for genetic modification of cold regulators to enhance stress tolerance in crops.

Detection of two homologous major QTLs and development of diagnostic molecular markers for sucrose content in peanut.

KEY MESSAGE: We identified two stable and homologous major QTLs for sucrose content in peanut, and developed breeder-friendly molecular markers for marker-assisted selection breeding. Sucrose content is a crucial quality trait for edible peanuts, and increasing sucrose content is a key breeding objective. However, the genetic basis of sucrose content in peanut remains unclear, and major quantitative trait loci (QTLs) for sucrose content have yet to be identified. In this study, a high-density genetic map was constructed based on whole-genome re-sequencing data from a peanut RIL population. This map consisted of 2,042 bins and 24,142 SNP markers, making it one of the most comprehensive maps to date in terms of marker density. Two major QTLs (qSCA06.2 and qSCB06.2) were identified, explaining 31.41% and 24.13% of the phenotypic variance, respectively. Notably, these two QTLs were located in homologous genomic regions between the A and B subgenomes. The elite allele of qSCA06.2 was exclusive to Valencia-type, while the elite allele of qSCB06.2 existed in other peanut types. Importantly, the distribution of alleles from two homologous QTLs in the RIL population and diverse germplasm accessions consistently demonstrated that only the combination of elite allelic genotypes from both QTLs/genes resulted in a significantly dominant phenotype, accompanied by a substantial increase in sucrose content. The newly developed diagnostic markers for these QTLs were confirmed to be reliable and could facilitate future breeding efforts to enhance sucrose content using marker-assisted selection techniques. Overall, this study highlights the co-regulation of sucrose content by two major homologous QTLs/genes and provides valuable insights into the genetic basis of sucrose in peanuts.

Unveiling the molecular regulatory mechanisms underlying sucrose accumulation and oil reduction in peanut kernels through genetic mapping and transcriptome analysis.

Sucrose content is a key factor for the flavor of edible peanut, which determines the sweet taste of fresh peanut and also attribute to pleasant flavor of roasted peanut. To explore the genetic mechanism of the sucrose content in peanut, an F2 population was created by crossing the sweet cultivar Zhonghuatian 1 (ZHT1) with Nanyangbaipi (NYBP). A genomic region spanning 28.26 kb on chromosome A06 was identified for the sucrose content through genetic mapping, elucidating 47.5% phenotypic variance explained. As the sucrose content had a significantly negative correlation with the oil content, this region was also found to be related to the oil content explaining 37.2% of phenotype variation. In this region, Arahy.42CAD1 was characterized as the most likely candidate gene through a comprehensive analysis. The nuclear localization of Arahy.42CAD1 suggests its potential involvement in the regulation of gene expression for sucrose and oil contents in peanut. Transcriptome analysis of the developing seeds in both parents revealed that genes involved in glycolysis and triacylglycerol biosynthesis pathways were not significantly down-regulated in ZHT1, indicating that the sucrose accumulation was not attributed to the suppression of triacylglycerol biosynthesis. Based on the WGCNA analysis, Arahy.42CAD1 was co-expressed with the genes involved in vesicle transport and oil body assembly, suggesting that the sucrose accumulation may be caused by disruptions in TAG transportation or storage mechanisms. These findings offer new insights into the molecular mechanisms governing sucrose accumulation in peanut, and also provide a potential gene target for enhancing peanut flavor.

Identification of QTLs associated with very-long chain fatty acid (VLCFA) content via linkage mapping and BSA-seq in peanut.

KEY MESSAGE: Three major QTLs qA01, qB04.1 and qB05 for VLCFA content and their corresponding allele-specific markers will benefit peanut low VLCFA breeding, and a candidate gene Arahy.IF1JV3 was predicted. Peanut is a globally significant oilseed crop worldwide, and contains a high content (20%) of saturated fatty acid (SFA) in its seeds. As high level SFA intake in human dietary may increase the cardiovascular disease risk, reducing the SFA content in peanut is crucial for improving its nutritional quality. Half of the SFAs in peanut are very long-chain fatty acids (VLCFA), so reducing the VLCFA content is a feasible strategy to decrease the total SFA content. Luoaowan with extremely low VLCFA (4.80%) was crossed with Jihua16 (8.00%) to construct an F2:4 population. Three major QTLs including qA01, qB04.1 and qB05 for VLCFA content were detected with 4.43 ~ 14.32% phenotypic variation explained through linkage mapping. Meanwhile, three genomic regions on chromosomes B03, B04 and B05 were identified via BSA-seq approach. Two co-localized intervals on chromosomes B04 (100.10 ~ 103.97 Mb) and B05 (6.39 ~ 10.90 Mb) were identified. With markers developed based on SNP/InDel variations in qA01 between the two parents, the remaining interval was refined to 103.58 ~ 111.14 Mb. A candidate gene Arahy.IF1JV3 encoding a ß-ketoacyl-CoA synthase was found in qA01, and its expression level in Luoaowan was significantly lower than that in Jihua16. Allele-specific markers targeting qA01, qB04.1 and qB05 were developed and validated in F4 population, and an elite line with high oleic, low VLCFA (5.05%) and low SFA (11.48%) contents was selected. This study initially revealed the genetic mechanism of VLCFA content, built a marker-assisted selection system for low VLCFA breeding, and provided an effective method to decrease the SFA content in peanut.

Genome-wide analysis of UDP-glycosyltransferase gene family and identification of a flavonoid 7-O-UGT (AhUGT75A) enhancing abiotic stress in peanut (Arachis hypogaea L.).

BACKGROUND: Glycosylation, catalyzed by UDP-glycosyltransferase (UGT), was important for enhancing solubility, bioactivity, and diversity of flavonoids. Peanut (Arachis hypogaea L.) is an important oilseed and cash crop worldwide. In addition to provide high quality of edible oils and proteins, peanut seeds contain a rich source of flavonoid glycosides that benefit human health. However, information of UGT gene family was quite limited in peanut. RESULTS: In present study, a total of 267 AhUGTs clustered into 15 phylogenetic groups were identified in peanut genome. Group I has greatly expanded to contain the largest number of AhUGT genes. Segmental duplication was the major driving force for AhUGT gene family expansion. Transcriptomic analysis of gene expression profiles in various tissues and under different abiotic stress treatments indicated AhUGTs were involved in peanut growth and abiotic stress response. AhUGT75A (UGT73CG33), located in mitochondria, was characterized as a flavonoid 7-O-UGT by in vitro enzyme assays. The transcript level of AhUGT75A was strongly induced by abiotic stress. Overexpression of AhUGT75A resulted in accumulating less amount of malondialdehyde (MDA) and superoxide, and enhancing tolerance against drought and/or salt stress in transgenic Arabidopsis. These results indicated AhUGT75A played important roles in conferring abiotic stress tolerance through reactive oxygen species scavenging. CONCLUSIONS: Our research only not provides valuable information for functional characterization of UGTs in peanut, but also gives new insights into potential applications in breeding new cultivars with both desirable stress tolerance and health benefits.

First Report of Peanut Black Pod Rot Caused by Berkeleyomyces rouxiae in China.

Peanut (Arachis hypogaea L.) is an important oilseed and cash crop cultivated in over 100 countries worldwide. The major producers are China, India and USA (Ding et al. 2022). In September 2022, peanut pods exhibiting black necrotic symptoms on the shell surface were observed in Puyang, Henan Province, China. These black spots often merged to form larger necrotic spots on the shell. Disease incidence was 100% in susceptible varieties. Symptomatic shell pieces were surface sterilized with 75% ethanol for 3 min, rinsed three times with sterile water, and then transferred onto PDA medium supplemented with 25 µg/ml chloramphenicol (Long et al. 2022). Isolation frequency of a fungus with similar-appearing colonies from symptomatic pods was 81.7%. A pure culture of a representative isolate, PYHB, was obtained through single-sporing and maintained on PDA plates at 25℃ in darkness. The colony initially appeared white but turned black within 2 days. The isolate produced dark brown, unicellular chlamydospores, which were arranged in club-shaped chains consisting of two to seven cells. The size of the unicellular chlamydospores varied from 3.34 to 15.27 µm (average:6.81, n = 100) in length and 8.30 to 15.51 µm (average:11.29, n = 100) in width. The endoconidia were hyaline and cylindrical, measuring 7.91-22.94 × 1.69-4.81 µm (average: 12.16 × 3.13, n = 100). Based on morphological characteristics, the isolate was tentatively identified as a Berkeleyomyces sp. (Nel et al. 2018; Long et al. 2022). The ITS region of r-DNA, the ribosomal large subunit (LSU), the minichromosome maintenance complex component 7 (MCM7), and the 60S ribosomal protein RPL10 (60S) genes were amplified using ITS1/ITS4, LR0R/LR5, rouxMCM7-F/rouxMCM7-R and roux60s-F/roux60s-R primers, respectively (White et al. 1990; Vilgalys and Hester 1990; Nakane and Usami 2020). The sequences were deposited in GenBank (ITS: OR053803; LSU: OR053818; MCM7: OR058549; 60S: OR060656). Through BLASTn analysis of the NCBI GenBank database, the generated ITS and LSU sequences showed 100% identity to Berkeleyomyces rouxiae (GenBank MF952418.1 and MF948662.1, respectively) and B. basicola (GenBank MT221585.1 and MH868639.1, respectively). Importantly, the MCM7 and 60S sequences were 100% identical to B. rouxiae (GenBank MF967114.1 and MF967077.1, respectively). Phylogenetic analysis combining ITS, LSU, MCM7, and 60S sequences showed that the isolate PYHB clustered with B. rouxiae. To evaluate pathogenicity, surface-sterilized healthy peanut pods (n = 90) were immersed in a 1×106 spore/ml conidial suspension obtained from isolate PYHB for 5 min and placed in Petri dishes containing moistened cotton at 25°C for 10 days. Pods (n = 90) inoculated with sterile water served as controls. Inoculated pods displayed black necrosis 10 days after inoculation (dai), whereas no symptoms were observed on the control pods at 21 dai. The reisolated pathogen was shown to be identical to the original inoculum through morphological and phylogenetic analysis. Black root rot is a fungal disease caused by Berkeleyomyces spp. (syn. Thielaviopsis spp.) and affects various crops and ornamentals, such as cotton, tobacco, carrot, holly, and pansy (Rahnama et al. 2022). The causal agents B. rouxiae and B. basicola have similar morphological characteristics but can be differentiated through molecular characterization (Nel et al. 2018). To our knowledge, this is the first report of black pod rot in peanut caused by B. rouxiae in China. The finding from this study will contribute to the development of monitoring and management strategies to combat this destructive disease in peanut cultivation.

scRNA-seq Reveals the Mechanism of Fatty Acid Desaturase 2 Mutation to Repress Leaf Growth in Peanut (Arachis hypogaea L.).

Fatty Acid Desaturase 2 (FAD2) controls the conversion of oleic acids into linoleic acids. Mutations in FAD2 not only increase the high-oleic content, but also repress the leaf growth. However, the mechanism by which FAD2 regulates the growth pathway has not been elucidated in peanut leaves with single-cell resolution. In this study, we isolated fad2 mutant leaf protoplast cells to perform single-cell RNA sequencing. Approximately 24,988 individual cells with 10,249 expressed genes were classified into five major cell types. A comparative analysis of 3495 differentially expressed genes (DEGs) in distinct cell types demonstrated that fad2 inhibited the expression of the cytokinin synthesis gene LOG in vascular cells, thereby repressing leaf growth. Further, pseudo-time trajectory analysis indicated that fad2 repressed leaf cell differentiation, and cell-cycle evidence displayed that fad2 perturbed the normal cell cycle to induce the majority of cells to drop into the S phase. Additionally, important transcription factors were filtered from the DEG profiles that connected the network involved in high-oleic acid accumulation (WRKY6), activated the hormone pathway (WRKY23, ERF109), and potentially regulated leaf growth (ERF6, MYB102, WRKY30). Collectively, our study describes different gene atlases in high-oleic and normal peanut seedling leaves, providing novel biological insights to elucidate the molecular mechanism of the high-oleic peanut-associated agronomic trait at the single-cell level.

Prospects for developing allergen-depleted food crops.

In addition to the challenge of meeting global demand for food production, there are increasing concerns about food safety and the need to protect consumer health from the negative effects of foodborne allergies. Certain bio-molecules (usually proteins) present in food can act as allergens that trigger unusual immunological reactions, with potentially life-threatening consequences. The relentless working lifestyles of the modern era often incorporate poor eating habits that include readymade prepackaged and processed foods, which contain additives such as peanuts, tree nuts, wheat, and soy-based products, rather than traditional home cooking. Of the predominant allergenic foods (soybean, wheat, fish, peanut, shellfish, tree nuts, eggs, and milk), peanuts (Arachis hypogaea) are the best characterized source of allergens, followed by tree nuts (Juglans regia, Prunus amygdalus, Corylus avellana, Carya illinoinensis, Anacardium occidentale, Pistacia vera, Bertholletia excels), wheat (Triticum aestivum), soybeans (Glycine max), and kidney beans (Phaseolus vulgaris). The prevalence of food allergies has risen significantly in recent years including chance of accidental exposure to such foods. In contrast, the standards of detection, diagnosis, and cure have not kept pace and unfortunately are often suboptimal. In this review, we mainly focus on the prevalence of allergies associated with peanut, tree nuts, wheat, soybean, and kidney bean, highlighting their physiological properties and functions as well as considering research directions for tailoring allergen gene expression. In particular, we discuss how recent advances in molecular breeding, genetic engineering, and genome editing can be used to develop potential low allergen food crops that protect consumer health.

Dissection of the Genetic Basis of Resistance to Stem Rot in Cultivated Peanuts (Arachis hypogaea L.) through Genome-Wide Association Study.

Peanut (Arachis hypogaea) is an important oilseed and cash crop worldwide, contributing an important source of edible oil and protein for human nutrition. However, the incidence of stem rot disease caused by Athelia rolfsii poses a major challenge to peanut cultivation, resulting in significant yield losses. In this study, a panel of 202 peanut accessions was evaluated for their resistance to stem rot by inoculating plants in the field with A. rolfsii-infested oat grains in three environments. The mean disease index value of each environment for accessions in subsp. fasitigiate and subsp. hypogaea showed no significant difference. Accessions from southern China displayed the lowest disease index value compared to those from other ecological regions. We used whole-genome resequencing to analyze the genotypes of the accessions and to identify significant SNPs associated with stem rot resistance through genome-wide association study (GWAS). A total of 121 significant SNPs associated with stem rot resistance in peanut were identified, with phenotypic variation explained (PVE) ranging from 12.23% to 15.51%. A total of 27 candidate genes within 100 kb upstream and downstream of 23 significant SNPs were annotated, which have functions related to recognition, signal transduction, and defense response. These significant SNPs and candidate genes provide valuable information for further validation and molecular breeding to improve stem rot resistance in peanut.

Genetic mapping of AhVt1, a novel genetic locus that confers the variegated testa color in cultivated peanut (Arachis hypogaea L.) and its utilization for marker-assisted selection.

Introduction: Peanut (Arachis hypogaea L.) is an important cash crop worldwide. Compared with the ordinary peanut with pure pink testa, peanut with variegated testa color has attractive appearance and a higher market value. In addition, the variegated testa represents a distinct regulation pattern of anthocyanin accumulation in integument cells. Methods: In order to identify the genetic locus underlying variegated testa color in peanut, two populations were constructed from the crosses between Fuhua 8 (pure-pink testa) and Wucai (red on white variegated testa), Quanhonghua 1 (pure-red testa) and Wucai, respectively. Genetic analysis and bulked sergeant analysis sequencing were applied to detect and identify the genetic locus for variegated testa color. Marker-assisted selection was used to develop new variegated testa peanut lines. Results: As a result, all the seeds harvested from the F1 individuals of both populations showed the variegated testa type with white trace. Genetic analysis revealed that the pigmentation of colored region in red on white variegated testa was controlled by a previous reported gene AhRt1, while the formation of white region (un-pigmented region) in variegated testa was controlled by another single genetic locus. This locus, named as AhVt1 (Arachis hypogaea Variegated Testa 1), was preliminary mapped on chromosome 08 through bulked sergeant analysis sequencing. Using a secondary mapping population derived from the cross between Fuhua 8 and Wucai, AhVt1 was further mapped to a 1.89-Mb genomic interval by linkage analysis, and several potential genes associated with the uneven distribution of anthocyanin, such as MADS-box, MYB, and Chalcone synthase-like protein, were harbored in the region. Moreover, the molecular markers closely linked to the AhVt1 were developed, and the new variegated testa peanut lines were obtained with the help of marker-assisted selection. Conclusion: Our findings will accelerate the breeding program for developing new peanut varieties with "colorful" testa colors and laid a foundation for map-based cloning of gene responsible for variegated testa.

Red fluorescence protein (DsRed2) promotes the screening efficiency in peanut genetic transformation.

Peanut (Arachis hypogaea L.), one of the leading oilseed crops worldwide, is an important source of vegetable oil, protein, minerals and vitamins. Peanut is widely cultivated in Asia, Africa and America, and China is the largest producer and consumer of peanut. Genetic engineering has shown great potential to alter the DNA makeup of an organism which is largely hindered by the low transformation and screening efficiency including in peanut. DsRed2 is a reporter gene widely utilized in genetic transformation to facilitate the screening of transformants, but never used in peanut genetic transformation. In this study, we have demonstrated the potential of the red fluorescence protein DsRed2 as a visual reporter to improve screening efficiency in peanut. DsRed2 was firstly expressed in protoplasts isolated from peanut cultivar Zhonhua 12 by PEG, and red fluorescence was successfully detected. Then, DsRed2 was expressed in peanut plants Zhonghua 12 driven by 35S promoter via Agrobacterium tumefaciens-mediated transformation. Red fluorescence was visually observed in calli and regenerated shoots, as well as in roots, leaves, flowers, fresh pod shells and mature seeds, suggesting that transgenic screening could be initiated at the early stage of transformation, and continued to the progeny. Upon screening with DsRed2, the positive plant rate was increased from 56.9% to 100%. The transgenic line was then used as the male parent to be crossed with Zhonghua 24, and the hybrid seeds showed red fluorescence as well, indicating that DsRed2 could be applied to hybrid plant identification very efficiently. DsRed2 was also expressed in hairy roots of Huayu 23 via Agrobacterium rhizogenes-mediated transformation, and the transgenic roots were easily selected by red fluorescence. In summary, the DsRed2 is an ideal reporter to achieve maximum screening efficiency and accuracy in peanut genetic transformation.

Identification and Pyramiding Major QTL Loci for Simultaneously Enhancing Aflatoxin Resistance and Yield Components in Peanut.

Peanut is susceptible to Aspergillus flavus infection, and the consequent aflatoxin contamination has been recognized as an important risk factor affecting food safety and industry development. Planting peanut varieties with resistance to aflatoxin contamination is regarded as an ideal approach to decrease the risk in food safety, but most of the available resistant varieties have not been extensively used in production because of their low yield potential mostly due to possessing small pods and seeds. Hence, it is highly necessary to integrate resistance to aflatoxin and large seed weight. In this study, an RIL population derived from a cross between Zhonghua 16 with high yield and J 11 with resistance to infection of A. flavus and aflatoxin production, was used to identify quantitative trait locus (QTL) for aflatoxin production (AP) resistance and hundred-seed weight (HSW). From combined analysis using a high-density genetic linkage map constructed, 11 QTLs for AP resistance with 4.61-11.42% phenotypic variation explanation (PVE) and six QTLs for HSW with 3.20-28.48% PVE were identified, including three major QTLs for AP resistance (qAFTA05.1, qAFTB05.2 and qAFTB06.3) and three for HSW (qHSWA05, qHSWA08 and qHSWB06). In addition, qAFTA05.1, qAFTB06.3, qHSWA05, qHSWA08 and qHSWB06 were detected in multiple environments. The aflatoxin contents under artificial inoculation were decreased by 34.77-47.67% in those segregated lines harboring qAFTA05.1, qAFTB05.2 and qAFTB06.3, while the HSWs were increased by 47.56-49.46 g in other lines harboring qHSWA05, qHSWA08 and qHSWB06. Conditional QTL mapping indicated that HSW and percent seed infection index (PSII) had no significant influence on aflatoxin content. Interestingly, the QT 1059 simultaneously harboring alleles of aflatoxin content including qAFTA05.1 and qAFTB05.2, alleles of PSII including qPSIIB03.1, qPSIIB03.2, and qPSIIB10 and alleles of HSW including qHSWA05, qHSWB06, qHSWA08 had better resistance to A. flavus infection and to toxin production and higher yield potential compared with the two parents of the RIL. The above identified major loci for AP resistance and HWS would be helpful for marker-assisted selection in peanut breeding.

Identification of a stable major sucrose-related QTL and diagnostic marker for flavor improvement in peanut.

KEY MESSAGE: An InDel marker closely linked with a major and stable quantitative trait locus (QTL) on chromosome A08, qSUCA08.2, controlling sucrose content will benefit peanut flavor improvement. Sucrose is the main soluble sugar in mature peanut kernel, and its content is a key determinant of flavor. However, the genetic basis of sucrose content in peanut remains poorly understood, which limits the progress of flavor improvement. In the present study, two genomic regions (qSUCA08a and qSUCB06a) for sucrose content on chromosomes A08 and B06 were identified by QTL-seq in a RIL population derived from a cross between Zhonghua 10 and ICG 12625. In the interval of qSUCB06a, QTL qSUCB06.2 was detected through QTL mapping in a single environment. The qSUCA08a was further dissected into 3 adjacent genomic regions using linkage analysis including a major QTL qSUCA08.2 explaining 5.43-17.84% phenotypic variation across five environments. A 61-bp insertion at position 35,099,320 in the higher sucrose parent ICG 12625 was found in qSUCA08.2. An InDel marker SUC.InDel.A08 based on the insertion/deletion polymorphism was developed and validated within a natural population containing 172 peanut cultivars in two environments. The mean sucrose content of 93 cultivars with ICG 12625 allele was significantly higher than that of 79 cultivars with Zhonghua 10 allele. The qSUCA08.2 corresponding to a 2.11 Mb interval harbored 110 genes. Among these genes, a total of 19 genes were considered as candidate genes including 5 non-synonymous mutation genes and 14 differentially expressed genes during seed development. These results provide new insights into the genetic basis of sucrose regulation in peanut and benefit the breeding program for developing new varieties with excellent flavor.

Visualizing the Distribution of Lipids in Peanut Seeds by MALDI Mass Spectrometric Imaging.

Peanut (also called groundnut, Arachis hypogaea L.) seeds are used for producing edible oils and functional foods, and offer a rich source of lipids, proteins and carbohydrates. However, the location of these metabolites has not yet been firmly established. In the present study, the matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) technique was applied to investigate spatial distribution of lipids and other key components in seeds of three peanut cultivars (ZH9, KQBH, HP). A total of 103 metabolites, including 34 lipid compounds, were putatively identified by MALDI-MSI. The abundance and spatial distribution of glycerolipids (GLs) and glycerophospholipids (GPs) were compared among the three peanut cultivars. All the identified lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE) and phosphatidylcholines (PCs) were distributed mainly in the inner part of seeds. The visualization of phosphatidic acids (PAs) and triacylglycerols (TGs) revealed a dramatic metabolic heterogeneity between the different tissues making up the seed. The non-homogeneous spatial distribution of metabolites appeared to be related to the different functions of particular tissue regions. These results indicated that MALDI-MSI could be useful for investigating the lipids of foodstuffs from a spatial perspective. The present study may contribute to the development of oil crops with higher oil yields, and to improvement of food processing.

Genetic dissection of fatty acid components in the Chinese peanut (Arachis hypogaea L.) mini-core collection under multi-environments.

Peanut (Arachis hypogaea L.) is an important source of edible oil and protein for human nutrition. The quality of peanut seed oil is mainly determined by the composition of fatty acids, especially the contents of oleic acid and linoleic acid. Improving the composition of fatty acids in the seed oil is one of the main objectives for peanut breeding globally. To uncover the genetic basis of fatty acids and broaden the genetic variation in future peanut breeding programs, this study used genome-wide association studies (GWAS) to identify loci associated with target traits and developed diagnostic marker. The contents of eight fatty acid components of the Chinese peanut mini-core collection were measured under four environments. Using the phenotypic information and over one hundred thousand single nucleotide polymorphisms (SNPs), GWAS were conducted to investigate the genetics basis of fatty acids under multi-environments. Overall, 75 SNPs were identified significant trait associations with fatty acid components. Nineteen associations were repeatedly identified in multiple environments, and 13 loci were co-associated with two or three traits. Three stable major associated loci were identified, including two loci for oleic acid and linoleic acid on chromosome A09 [mean phenotypic variation explained (PVE): 38.5%, 10.35%] and one for stearic acid on B06 (mean PVE: 23%). According to functional annotations, 21 putative candidate genes related to fatty acid biosynthesis were found underlying the three associations. The allelic effect of SNP A09-114690064 showed that the base variation was highly correlated with the phenotypic variation of oleic acid and linoleic acid contents, and a cost-effective Kompetitive allele-Specific PCR (KASP) diagnostic marker was developed. Furthermore, the SNP A09-114690064 was found to change the cis-element CAAT (-) in the promoter of ahFAD2A to YACT (+), leading dozens of times higher expression level. The enhancer-like activity of ahFAD2A promoter was identified that was valuable for enriching the regulation mechanism of ahFAD2A. This study improved our understanding on the genetic architecture of fatty acid components in peanut, and the new effective diagnostic marker would be useful for marker-assisted selection of high-oleic peanut breeding.

Genetic, Phenotypic, and Pathogenic Variation Among Athelia rolfsii, the Causal Agent of Peanut Stem Rot in China.

Peanut stem rot caused by Athelia rolfsii is a serious soilborne disease worldwide and is becoming increasingly important in China. A total of 293 A. rolfsii isolates were collected from four representative peanut producing provinces in northern, central, and southern China. These isolates were assigned to 45 mycelial compatibility groups (MCGs) through pairing testing. The MCG diversity among isolates was greater in the southern sampled provinces compared with the northern provinces. A high level of genetic variability was found among the isolates from Guangdong Province in southern China. Variations were found in mycelial growth rate and sclerotial number, size, and dry weight of isolates sampled from places in different latitudes. Size and dry weight of sclerotia were positively correlated with latitude (P < 0.01), but the number of sclerotia was negatively correlated with latitude (P < 0.01). All tester isolates were pathogenic on peanut but varied in disease index. Inter-simple sequence repeat analysis and unweighted pair-group method with arithmetic average clustering resulted in three distinct clusters that were associated with the geographical location of the collection sites and sclerotial traits but were not associated with virulence of these isolates. These findings imply that genetic diversity, morphological traits, and virulence among A. rolfsii isolates varied in diverse geographical regions in China, and genetic diversity and sclerotial traits might be affected by latitude.