Physiological and biochemical mechanisms underlying the role of anthocyanin in acquired tolerance to salt stress in peanut (Arachis hypogaea L.).

Anthocyanin is an important pigment that prevents oxidative stress and mediates adaptation of plants to salt stress. Peanuts with dark red and black testa are rich in anthocyanin. However, correlation between salt tolerance and anthocyanin content in black and dark red testa peanuts is unknown. In this study, three peanut cultivars namely YZ9102 (pink testa), JHR1 (red testa) and JHB1 (black testa) were subjected to sodium chloride (NaCl) stress. The plant growth, ion uptake, anthocyanin accumulation, oxidation resistance and photosynthetic traits were comparatively analyzed. We observed that the plant height, leaf area and biomass under salt stress was highly inhibited in pink color testa (YZ9102) as compare to black color testa (JHB1). JHB1, a black testa colored peanut was identified as the most salt-tolerance cultivar, followed by red (JHR1) and pink(YZ9102). During salt stress, JHB1 exhibited significantly higher levels of anthocyanin and flavonoid accumulation compared to JHR1 and YZ9102, along with increased relative activities of antioxidant protection and photosynthetic efficiency. However, the K+/Na+ and Ca2+/Na+ were consistently decreased among three cultivars under salt stress, suggesting that the salt tolerance of black testa peanut may not be related to ion absorption. Therefore, we predicted that salt tolerance of JHB1 may be attributed to the accumulation of the anthocyanin and flavonoids, which activated antioxidant protection against the oxidative damage to maintain the higher photosynthetic efficiency and plant growth. These findings will be useful for improving salt tolerance of peanuts.

High-density bin-based genetic map reveals a 530-kb chromosome segment derived from wild peanut contributing to late leaf spot resistance.

KEY MESSAGE: Twenty-eight QTLs for LLS disease resistance were identified using an amphidiploid constructed mapping population, a favorable 530-kb chromosome segment derived from wild species contributes to the LLS resistance. Late leaf spot (LLS) is one of the major foliar diseases of peanut, causing serious yield loss and affecting the quality of kernel and forage. Some wild Arachis species possess higher resistance to LLS as compared with cultivated peanut; however, ploidy level differences restrict utilization of wild species. In this study, a synthetic amphidiploid (Ipadur) of wild peanuts with high LLS resistance was used to cross with Tifrunner to construct TI population. In total, 200 recombinant inbred lines were collected for whole-genome resequencing. A high-density bin-based genetic linkage map was constructed, which includes 4,809 bin markers with an average inter-bin distance of 0.43 cM. The recombination across cultivated and wild species was unevenly distributed, providing a novel recombination landscape for cultivated-wild Arachis species. Using phenotyping data collected across three environments, 28 QTLs for LLS disease resistance were identified, explaining 4.35-20.42% of phenotypic variation. The major QTL located on chromosome 14, qLLS14.1, could be consistently detected in 2021 Jiyang and 2022 Henan with 20.42% and 12.12% PVE, respectively. A favorable 530-kb chromosome segment derived from Ipadur was identified in the region of qLLS14.1, in which 23 disease resistance proteins were located and six of them showed significant sequence variations between Tifrunner and Ipadur. Allelic variation analysis indicating the 530-kb segment of wild species might contribute to the disease resistance of LLS. These associate genomic regions and candidate resistance genes are of great significance for peanut breeding programs for bringing durable resistance through pyramiding such multiple LLS resistance loci into peanut cultivars.

Ling-Gui-Zhu-Gan decoction protects against doxorubicin-induced myocardial injury by downregulating ferroptosis.

OBJECTIVE: To investigate the mechanism of Ling-Gui-Zhu-Gan decoction (LGZGD) protects against doxorubicin (DOX)-induced myocardial injury. METHODS: In vivo experiment, rats were divided into six groups: normal group, model group (15 mg/kg, DOX), Dex group(150 mg/kg, Dex), LGZGD-L group (2.1 g/kg), LGZGD-M group (4.2 g/kg), and LGZGD-H group (8.4 g/kg). We used HE and Masson staining to observe the histopathological changes, echocardiography to assess the cardiac function, and western blot and RT-qPCR to detect the expressions of Nrf2, GPX4, Fpn1, and Ptgs2. In vitro experiment, we used immunofluorescence to detect ROS production, and RT-qPCR to detect gene expression of GPX4, Fpn1, and Ptgs2. KEY FINDINGS: In vivo, LGZGD improved cardiac systolic function. LGZGD significantly reduced MDA, LDH, and CK levels, increased SOD activity, enhanced the protein expression of Nrf2, GPX4, and Fpn1, and decreased Ptgs2 levels. In vitro, LGZGD-containing serum significantly reduced ROS, increased the gene expression of GPX4 and Fpn1, and decreased the gene expression of Ptgs2. Furthermore, compared with the LGZGD (si-NC) group, the LGZGD (si-Nrf2) group had decreased gene expression of Nrf2, GPX4, and Fpn1 and increased gene expression of Ptgs2. CONCLUSIONS: LGZGD can ameliorate DOX-cardiotoxicity by activating the Nrf2 signaling pathway and inhibiting ferroptosis in cardiomyocytes.

Nitrogen application in pod zone improves yield and quality of two peanut cultivars by modulating nitrogen accumulation and metabolism.

Cultivated peanut (Arachis hypogaea L.) represents one of the most important oil and cash crops world-widely. Unlike many other legumes, peanuts absorb nitrogen through their underground pods. Despite this unique feature, the relationship between yield and nitrogen uptake within the pod zone remains poorly understood. In our pot experiment, we divided the underground peanut part into two zones-pod and root-and investigated the physiological and agronomic traits of two peanut cultivars, SH11 (large seeds, LS) and HY23 (small seeds, SS), at 10 (S1), 20 (S2), and 30 (S3) days after gynophores penetrated the soil, with nitrogen application in the pod zone. Results indicated that nitrogen application increased pod yield, kernel protein content, and nitrogen accumulation in plants. For both LS and SS peanut cultivars, optimal nitrogen content was 60 kg·hm- 2, leading to maximum yield. LS cultivar exhibited higher yield and nitrogen accumulation increases than SS cultivar. Nitrogen application up-regulated the expression of nitrogen metabolism-related genes in the pod, including nitrate reductase (NR), nitrite reductase (NIR), glutamine synthetase (GS), glutamate synthase (NADH-GOGAT), ATP binding cassette (ABC), and nitrate transporter (NRT2). Additionally, nitrogen application increased enzyme activity in the pod, including NR, GS, and GOGAT, consistent with gene expression levels. These nitrogen metabolism traits exhibited higher up-regulations in the large-seeded cultivar than in the small-seeded one and showed a significant correlation with yield in the large-seeded cultivar at S2 and S3. Our findings offer a scientific basis for the judicious application and efficient utilization of nitrogen fertilization in peanuts, laying the groundwork for further elucidating the molecular mechanisms of peanut nitrogen utilization.

Genome-wide identification and expression analysis of GA20ox and GA3ox genes during pod development in peanut.

Background: Gibberellins (GAs) play important roles in regulating peanut growth and development. GA20ox and GA3ox are key enzymes involved in GA biosynthesis. These enzymes encoded by a multigene family belong to the 2OG-Fe (II) oxygenase superfamily. To date, no genome-wide comparative analysis of peanut AhGA20ox and AhGA3ox-encoding genes has been performed, and the roles of these genes in peanut pod development are not clear. Methods: A whole-genome analysis of AhGA20ox and AhGA3ox gene families in peanut was carried out using bioinformatic tools. The expression of these genes at different stage of pod development was analyzed using qRT-PCR. Results: In this study, a total of 15 AhGA20ox and five AhGA3ox genes were identified in peanut genome, which were distributed on 14 chromosomes. Phylogenetic analysis divided the GA20oxs and GA3oxs into three groups, but AhGA20oxs and AhGA3oxs in two groups. The conserved pattern of gene structure, cis-elements, and protein motifs further confirmed their evolutionary relationship in peanut. AhGA20ox and AhGA3ox genes were differential expressed at different stages of pod development. The strong expression of AhGA20ox1/AhGA20ox4, AhGA20ox12/AhGA20ox15, AhGA3ox1 and AhGA3ox4/AhGA3ox5 in S1-stage indicated that these genes could have a key role in controlling peg elongation. Furthermore, AhGA20ox and AhGA3ox also showed diverse expression patterns in different peanut tissues including leaves, main stems, flowers and inflorescences. Noticeably, AhGA20ox9/AhGA20ox11 and AhGA3o4/AhGA3ox5 were highly expressed in the main stem, whereas the AhGA3ox1 and AhGA20ox10 were strongly expressed in the inflorescence. The expression levels of AhGA20ox2/AhGA20ox3, AhGA20ox5/AhGA20ox6, AhGA20ox7/AhGA20ox8, AhGA20ox13/AhGA20ox14 and AhGA3ox2/AhGA3ox3 were high in the flowers, suggesting their involvement in flower development. These results provide a basis for deciphering the roles of AhGA20ox and AhGA3ox in peanut growth and development, especially in pod development.

Phylogenomic Analysis of Cytochrome P450 Gene Superfamily and Their Association with Flavonoids Biosynthesis in Peanut (Arachis hypogaea L.).

Cytochrome P450s (CYPs) constitute extensive enzyme superfamilies in the plants, playing pivotal roles in a multitude of biosynthetic and detoxification pathways essential for growth and development, such as the flavonoid biosynthesis pathway. However, CYPs have not yet been systematically studied in the cultivated peanuts (Arachis hypogaea L.), a globally significant cash crop. This study addresses this knowledge deficit through a comprehensive genome-wide analysis, leading to the identification of 589 AhCYP genes in peanuts. Through phylogenetic analysis, all AhCYPs were systematically classified into 9 clans, 43 gene families. The variability in the number of gene family members suggests specialization in biological functions. Intriguingly, both tandem duplication and fragment duplication events have emerged as pivotal drivers in the evolutionary expansion of the AhCYP superfamily. Ka/Ks analysis underscored the substantial influence of strong purifying selection on the evolution of AhCYPs. Furthermore, we selected 21 genes encoding 8 enzymes associated with the flavonoid pathway. The results of quantitative real-time PCR (qRT-PCR) experiments unveiled stage-specific expression patterns during the development of peanut testa, with discernible variations between pink and red testa. Importantly, we identified a direct correlation between gene expression levels and the accumulation of metabolites. These findings offer valuable insights into elucidating the comprehensive functions of AhCYPs and the underlying mechanisms governing the divergent accumulation of flavonoids in testa of different colors.

Transcriptional networks orchestrating red and pink testa color in peanut.

BACKGROUND: Testa color is an important trait of peanut (Arachis hypogaea L.) which is closely related with the nutritional and commercial value. Pink and red are main color of peanut testa. However, the genetic mechanism of testa color regulation in peanut is not fully understood. To elucidate a clear picture of peanut testa regulatory model, samples of pink cultivar (Y9102), red cultivar (ZH12), and two RNA pools (bulk red and bulk pink) constructed from F4 lines of Y9102 x ZH12 were compared through a bulk RNA-seq approach. RESULTS: A total of 2992 differential expressed genes (DEGs) were identified among which 317 and 1334 were up-regulated and 225 and 1116 were down-regulated in the bulk red-vs-bulk pink RNA pools and Y9102-vs-ZH12, respectively. KEGG analysis indicates that these genes were divided into significantly enriched metabolic pathways including phenylpropanoid, flavonoid/anthocyanin, isoflavonoid and lignin biosynthetic pathways. Notably, the expression of the anthocyanin upstream regulatory genes PAL, CHS, and CHI was upregulated in pink and red testa peanuts, indicating that their regulation may occur before to the advent of testa pigmentation. However, the differential expression of down-stream regulatory genes including F3H, DFR, and ANS revealed that deepening of testa color not only depends on their gene expression bias, but also linked with FLS inhibition. In addition, the down-regulation of HCT, IFS, HID, 7-IOMT, and I2'H genes provided an alternative mechanism for promoting anthocyanin accumulation via perturbation of lignin and isoflavone pathways. Furthermore, the co-expression module of MYB, bHLH, and WRKY transcription factors also suggested a fascinating transcriptional activation complex, where MYB-bHLH could utilize WRKY as a co-option during the testa color regulation by augmenting anthocyanin biosynthesis in peanut. CONCLUSIONS: These findings reveal candidate functional genes and potential strategies for the manipulation of anthocyanin biosynthesis to improve peanut varieties with desirable testa color.

Effect of Salt Stress on Growth and Physiological Properties of Asparagus Seedlings.

Salt stress could inhibit the growth and development of crops and negatively affect yield and quality. The objective of this study was to investigate the physiological responses of different asparagus cultivars to salt stress. Twenty days old seedlings ofasalt-tolerant Apollo andasalt-sensitive cultivar JL1 were subjected to 0 (CK) and120 mM NaCl stress for 20 d. Their changes in growth, ion contents, antioxidant enzyme activities and gene expression were analyzed. Salt stress significantly inhibited the growth of both cultivars, and JL1 showed a greater decrease than Apollo. The root development of Apollo was promoted by 120 mM NaCl treatment. The Na+ content in roots, stems, and leaves of both cultivars was increased under salt stress, while K+ content and K+/Na+ decreased. The salt-tolerant cultivar Apollo showed less extent of increase in Na+ and decrease in K+ content and kept a relatively high K+/Na+ ratio to compare with JL1. The contents of proline, soluble sugar and protein increased in Apollo, while thesesubstances changed differently in JL1 under salt stress. Activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were gradually increased under salt stress in Apollo, while the corresponding enzyme activities in JL1 were decreased at the late stage of salt stress. The expression of SOD, POD, and CAT genes of both cultivars changed in a similar way to the enzyme activities. Malondialdehyde (MDA) content was increased slightly in Apollo, while increased significantly in JL1. At the late stage of salt stress, Apollomaintained a relatively high K+/Na+, osmotic adjustment ability and antioxidant defense capability, and therefore exhibited higher tolerance to salt stress than that of JL1.

Insights into the Novel FAD2 Gene Regulating Oleic Acid Accumulation in Peanut Seeds with Different Maturity.

AhFAD2 is a key enzyme catalyzing the conversion of oleic acid into linoleic acid. The high oleic acid characteristic of peanut mainly comes from the homozygous recessive mutation of AhFAD2A and AhFAD2B genes (aabb). However, even in high-oleic-acid varieties with the aabb genotype, the oleic acid content of seeds with different maturity varies significantly. Therefore, in addition to AhFAD2A and AhFAD2B, other FAD2 members or regulators may be involved in this process. Which FAD2 genes are involved in the regulatory processes associated with seed maturity is still unclear. In this study, four stable lines with different genotypes (AABB, aaBB, AAbb, and aabb) were used to analyze the contents of oleic acid and linoleic acid at different stages of seed development in peanut. Three new AhFAD2 genes (AhFAD2-7, AhFAD2-8, and AhFAD2-9) were cloned based on the whole-genome sequencing results of cultivated peanuts. All peanut FAD2 genes showed tissue preference in expression; however, only the expression level of AhFAD2-7 was positively correlated with the linoleic acid concentration in peanut seeds. These findings provide new insights into the regulation of oleic acid accumulation by maturity, and AhFAD2-7 plays an important role in the maturity dependent accumulation of oleic acid and linoleic acid in peanut.

BSA­seq and genetic mapping identified candidate genes for branching habit in peanut.

KEY MESSAGE: The candidate gene AhLBA1 controlling lateral branch angel of peanut was fine-mapped to a 136.65-kb physical region on chromosome 15 using the BSA-seq and QTL mapping. Lateral branch angel (LBA) is an important plant architecture trait of peanut, which plays key role in lodging, peg soil penetration and pod yield. However, there are few reports of fine mapping and quantitative trait loci (QTLs)/cloned genes for LBA in peanut. In this project, a mapping population was constructed using a spreading variety Tifrunner and the erect variety Fuhuasheng. Through bulked segregant analysis sequencing (BSA-seq), a major gene related to LBA, named as AhLBA1, was preliminarily mapped at the region of Chr.15: 150-160 Mb. Then, using traditional QTL approach, AhLBA1 was narrowed to a 1.12 cM region, corresponding to a 136.65-kb physical interval of the reference genome. Of the nine genes housed in this region, three of them were involved in hormone metabolism and regulation, including one "F-box protein" and two "2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase (2OG oxygenase)" encoding genes. In addition, we found that the level of some classes of cytokinin (CK), auxin and ethylene showed significant differences between spreading and erect peanuts at the junction of main stem and lateral branch. These findings will aid further elucidation of the genetic mechanism of LBA in peanut and facilitating marker-assisted selection (MAS) in the future breeding program.

Targeted metabolome analysis reveals accumulation of metabolites in testa of four peanut germplasms.

Cultivated peanut (Arachis hypogaea L.) is an important source of edible oil and protein. Peanut testa (seed coat) provides protection for seeds and serves as a carrier for diversity metabolites necessary for human health. There is significant diversity available for testa color in peanut germplasms. However, the kinds and type of metabolites in peanut testa has not been comprehensively investigated. In this study, we performed metabolite profiling using UPLC-MS/MS for four peanut germplasm lines with different testa colors, including pink, purple, red, and white. A total of 85 metabolites were identified in four peanuts. Comparative metabolomics analysis identified 78 differentially accumulated metabolites (DAMs). Some metabolites showed significant correlation with other metabolites. For instance, proanthocyanidins were positively correlated with cyanidin 3-O-rutinoside and malvin, and negatively correlated with pelargonidin-3-glucoside. We observed that the total proanthocyanidins are most abundant in pink peanut variety WH10. The red testa accumulated more isoflavones, flavonols and anthocyanidins compared with that in pink testa. These results provided valuable information about differential accumulation of metabolites in testa with different color, which are helpful for further investigation of the molecular mechanism underlying biosynthesis and accumulation of these metabolites in peanut.

Transcriptome Analysis and Gene Expression Profiling of the Peanut Small Seed Mutant Identified Genes Involved in Seed Size Control.

Seed size is a key factor affecting crop yield and a major agronomic trait concerned in peanut (Arachis hypogaea L.) breeding. However, little is known about the regulation mechanism of peanut seed size. In the present study, a peanut small seed mutant1 (ssm1) was identified through irradiating peanut cultivar Luhua11 (LH11) using 60Coγ ray. Since the globular embryo stage, the embryo size of ssm1 was significantly smaller than that of LH11. The dry seed weight of ssm1 was only 39.69% of the wild type LH14. The seeds were wrinkled with darker seed coat. The oil content of ssm1 seeds were also decreased significantly. Seeds of ssm1 and LH11 were sampled 10, 20, and 40 days after pegging (DAP) and were used for RNA-seq. The results revealed that genes involved in plant hormones and several transcription factors related to seed development were differentially expressed at all three stages, especially at DAP10 and DAP20. Genes of fatty acid biosynthesis and late embryogenesis abundant protein were significantly decreased to compare with LH11. Interestingly, the gene profiling data suggested that PKp2 and/or LEC1 could be the key candidate genes leading to the small seed phenotype of the mutant. Our results provide valuable clues for further understanding the mechanisms underlying seed size control in peanut.

The Mechanisms Underlying Salt Resistance Mediated by Exogenous Application of 24-Epibrassinolide in Peanut.

Peanut is one of the most important oil crops in the world, the growth and productivity of which are severely affected by salt stress. 24-epibrassinolide (EBL) plays an important role in stress resistances. However, the roles of exogenous EBL on the salt tolerance of peanut remain unclear. In this study, peanut seedlings treated with 150 mM NaCl and with or without EBL spray were performed to investigate the roles of EBL on salt resistance. Under 150 mM NaCl conditions, foliar application of 0.1 µM EBL increased the activity of catalase and thereby could eliminate reactive oxygen species (ROS). Similarly, EBL application promoted the accumulation of proline and soluble sugar, thus maintaining osmotic balance. Furthermore, foliar EBL spray enhanced the total chlorophyll content and high photosynthesis capacity. Transcriptome analysis showed that under NaCl stress, EBL treatment up-regulated expression levels of genes encoding peroxisomal nicotinamide adenine dinucleotide carrier (PMP34), probable sucrose-phosphate synthase 2 (SPS2) beta-fructofuranosidase (BFRUCT1) and Na+/H+ antiporters (NHX7 and NHX8), while down-regulated proline dehydrogenase 2 (PRODH). These findings provide valuable resources for salt resistance study in peanut and lay the foundation for using BR to enhance salt tolerance during peanut production.

Mutation of an Essential 60S Ribosome Assembly Factor MIDASIN 1 Induces Early Flowering in Arabidopsis.

Ribosome biogenesis is tightly associated with plant growth and reproduction. Mutations in genes encoding ribosomal proteins (RPs) or ribosome biogenesis factors (RBFs) generally result in retarded growth and delayed flowering. However, the early-flowering phenotype resulting from the ribosome biogenesis defect is rarely reported. We previously identified that the AAA-ATPase MIDASIN 1 (MDN1) functions as a 60S RBF in Arabidopsis. Here, we found that its weak mutant mdn1-1 is early-flowering. Transcriptomic analysis showed that the expression of FLOWERING LOCUS C (FLC) is down-regulated, while that of some autonomous pathway genes and ABSCISIC ACID-INSENSITIVE 5 (ABI5) is up-regulated in mdn1-1. Phenotypic analysis revealed that the flowering time of mdn1-1 is severely delayed by increasing FLC expression, suggesting that the early flowering in mdn1-1 is likely associated with the downregulation of FLC. We also found that the photoperiod pathway downstream of CONSTANTS (CO) and FLOWERING LOCUS T (FT) might contribute to the early flowering in mdn1-1. Intriguingly, the abi5-4 allele completely blocks the early flowering in mdn1-1. Collectively, our results indicate that the ribosome biogenesis defect elicited by the mutation of MDN1 leads to early flowering by affecting multiple flowering regulation pathways.

Bulk RNA-Seq Analysis Reveals Differentially Expressed Genes Associated with Lateral Branch Angle in Peanut.

Lateral branch angle (LBA), or branch habit, is one of the most important agronomic traits in peanut. To date, the underlying molecular mechanisms of LBA have not been elucidated in peanut. To acquire the differentially expressed genes (DEGs) related to LBA, a TI population was constructed through the hybridization of a bunch-type peanut variety Tifrunner and prostrate-type Ipadur. We report the identification of DEGs related to LBA by sequencing two RNA pools, which were composed of 45 F3 lines showing an extreme opposite bunch and prostrate phenotype. We propose to name this approach Bulk RNA-sequencing (BR-seq) as applied to several plant species. Through BR-seq analysis, a total of 3083 differentially expressed genes (DEGs) were identified, including 13 gravitropism-related DEGs, 22 plant hormone-related DEGs, and 55 transcription factors-encoding DEGs. Furthermore, we also identified commonly expressed alternatively spliced (AS) transcripts, of which skipped exon (SE) and retained intron (RI) were most abundant in the prostrate and bunch-type peanut. AS isoforms between prostrate and bunch peanut highlighted important clues to further understand the post-transcriptional regulatory mechanisms of branch angle regulation. Our findings provide not only important insights into the landscape of the regulatory pathway involved in branch angle formation but also present practical information for peanut molecular breeding in the future.

High-Density Genetic Variation Map Reveals Key Candidate Loci and Genes Associated With Important Agronomic Traits in Peanut.

Peanut is one of the most important cash crops with high quality oil, high protein content, and many other nutritional elements, and grown globally. Cultivated peanut (Arachis hypogaea L.) is allotetraploid with a narrow genetic base, and its genetics and molecular mechanisms controlling the agronomic traits are poorly understood. Here, we report a comprehensive genome variation map based on the genotyping of a panel of 178 peanut cultivars using Axiom_Arachis2 SNP array, including 163 representative varieties of different provinces in China, and 15 cultivars from 9 other countries. According to principal component analysis (PCA) and phylogenetic analysis, the peanut varieties were divided into 7 groups, notable genetic divergences between the different areas were shaped by environment and domestication. Using genome-wide association study (GWAS) analysis, we identified several marker-trait associations (MTAs) and candidate genes potentially involved in regulating several agronomic traits of peanut, including one MTA related with hundred seed weight, one MTA related with total number of branches, and 14 MTAs related with pod shape. This study outlines the genetic basis of these peanut cultivars and provides 13,125 polymorphic SNP markers for further distinguishing and utility of these elite cultivars. In addition, the candidate loci and genes provide valuable information for further fine mapping of QTLs and improving the quality and yield of peanut using a genomic-assisted breeding method.

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.

BSA­seq and genetic mapping reveals AhRt2 as a candidate gene responsible for red testa of peanut.

KEY MESSAGE: The candidate recessive gene AhRt2 responsible for red testa of peanut was identified through combined BSA-seq and linkage mapping approaches. The testa color of peanuts (Arachis hypogaea L.) is an important trait, and those with red testa are particularly popular owing to the high-anthocyanin content. However, the identification of genes underlying the regulation of the red testa trait in peanut are rarely reported. In order to fine map red testa gene, two F2:4 populations were constructed through the cross of YZ9102 (pink testa) with ZH12 (red testa) and ZH2 (red testa). Genetic analysis indicated that red testa was controlled by a single recessive gene named as AhRt2 (Red testa gene 2). Using BSA-seq approach, AhRt2 was preliminary identified on chromosome 12, which was further mapped to a 530-kb interval using 220 recombinant lines through linkage mapping. Furthermore, functional annotation, expression profiling, and the analyses of sequence variation confirmed that the anthocyanin reductase namely (Arahy.IK60LM) was the most likely candidate gene for AhRt2. It was found that a SNP in the third exon of AhRt2 altered the encoding amino acids, and was associated with red testa in peanut. In addition, a closely linked molecular marker linked with red testa trait in peanut was also developed for future studies. Our results provide valuable insight into the molecular mechanism underlying peanut testa color and present significant diagnostic marker resources for marker-assisted selected breeding in peanut.