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.

Global Transcriptome and Co-Expression Network Analyses Revealed Hub Genes Controlling Seed Size/Weight and/or Oil Content in Peanut.

Cultivated peanut (Arachis hypogaea L.) is an important economic and oilseed crop worldwide, providing high-quality edible oil and high protein content. Seed size/weight and oil content are two important determinants of yield and quality in peanut breeding. To identify key regulators controlling these two traits, two peanut cultivars with contrasting phenotypes were compared to each other, one having a larger seed size and higher oil content (Zhonghua16, ZH16 for short), while the second cultivar had smaller-sized seeds and lower oil content (Zhonghua6, ZH6). Whole transcriptome analyses were performed on these two cultivars at four stages of seed development. The results showed that ~40% of the expressed genes were stage-specific in each cultivar during seed development, especially at the early stage of development. In addition, we identified a total of 5356 differentially expressed genes (DEGs) between ZH16 and ZH6 across four development stages. Weighted gene co-expression network analysis (WGCNA) based on DEGs revealed multiple hub genes with potential roles in seed size/weight and/or oil content. These hub genes were mainly involved in transcription factors (TFs), phytohormones, the ubiquitin-proteasome pathway, and fatty acid synthesis. Overall, the candidate genes and co-expression networks detected in this study could be a valuable resource for genetic breeding to improve seed yield and quality traits in peanut.

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.

Gene expression and DNA methylation altering lead to the high oil content in wild allotetraploid peanut (A. monticola).

Introduction: The wild allotetraploid peanut Arachis monticola contains a higher oil content than the cultivated allotetraploid Arachis hypogaea. Besides the fact that increasing oil content is the most important peanut breeding objective, a proper understanding of its molecular mechanism controlling oil accumulation is still lacking. Methods: We investigated this aspect by performing comparative transcriptomics from developing seeds between three wild and five cultivated peanut varieties. Results: The analyses not only showed species-specific grouping transcriptional profiles but also detected two gene clusters with divergent expression patterns between two species enriched in lipid metabolism. Further analysis revealed that expression alteration of lipid metabolic genes with co-expressed transcription factors in wild peanut led to enhanced activity of oil biogenesis and retarded the rate of lipid degradation. In addition, bisulfite sequencing was conducted to characterize the variation of DNA methylation between wild allotetraploid (245, WH 10025) and cultivated allotetraploid (Z16, Zhh 7720) genotypes. CG and CHG context methylation was found to antagonistically correlate with gene expression during seed development. Differentially methylated region analysis and transgenic assay further illustrated that variations of DNA methylation between wild and cultivated peanuts could affect the oil content via altering the expression of peroxisomal acyl transporter protein (Araip.H6S1B). Discussion: From the results, we deduced that DNA methylation may negatively regulate lipid metabolic genes and transcription factors to subtly affect oil accumulation divergence between wild and cultivated peanuts. Our work provided the first glimpse on the regulatory mechanism of gene expression altering for oil accumulation in wild peanut and gene resources for future breeding applications.

Key Regulators of Sucrose Metabolism Identified through Comprehensive Comparative Transcriptome Analysis in Peanuts.

Sucrose content is a crucial indicator of quality and flavor in peanut seed, and there is a lack of clarity on the molecular basis of sucrose metabolism in peanut seed. In this context, we performed a comprehensive comparative transcriptome study on the samples collected at seven seed development stages between a high-sucrose content variety (ICG 12625) and a low-sucrose content variety (Zhonghua 10). The transcriptome analysis identified a total of 8334 genes exhibiting significantly different abundances between the high- and low-sucrose varieties. We identified 28 differentially expressed genes (DEGs) involved in sucrose metabolism in peanut and 12 of these encoded sugars will eventually be exported transporters (SWEETs). The remaining 16 genes encoded enzymes, such as cell wall invertase (CWIN), vacuolar invertase (VIN), cytoplasmic invertase (CIN), cytosolic fructose-bisphosphate aldolase (FBA), cytosolic fructose-1,6-bisphosphate phosphatase (FBP), sucrose synthase (SUS), cytosolic phosphoglucose isomerase (PGI), hexokinase (HK), and sucrose-phosphate phosphatase (SPP). The weighted gene co-expression network analysis (WGCNA) identified seven genes encoding key enzymes (CIN, FBA, FBP, HK, and SPP), three SWEET genes, and 90 transcription factors (TFs) showing a high correlation with sucrose content. Furthermore, upon validation, six of these genes were successfully verified as exhibiting higher expression in high-sucrose recombinant inbred lines (RILs). Our study suggested the key roles of the high expression of SWEETs and enzymes in sucrose synthesis making the genotype ICG 12625 sucrose-rich. This study also provided insights into the molecular basis of sucrose metabolism during seed development and facilitated exploring key candidate genes and molecular breeding for sucrose content in peanuts.

Dissection of the genetic basis of oil content in Chinese peanut cultivars through association mapping.

BACKGROUND: Peanut is one of the primary sources for vegetable oil worldwide, and enhancing oil content is the main objective in several peanut breeding programs of the world. Tightly linked markers are required for faster development of high oil content peanut varieties through genomics-assisted breeding (GAB), and association mapping is one of the promising approaches for discovery of such associated markers. RESULTS: An association mapping panel consisting of 292 peanut varieties extensively distributed in China was phenotyped for oil content and genotyped with 583 polymorphic SSR markers. These markers amplified 3663 alleles with an average of 6.28 alleles per locus. The structure, phylogenetic relationship, and principal component analysis (PCA) indicated two subgroups majorly differentiating based on geographic regions. Genome-wide association analysis identified 12 associated markers including one (AGGS1014_2) highly stable association controlling up to 9.94% phenotypic variance explained (PVE) across multiple environments. Interestingly, the frequency of the favorable alleles for 12 associated markers showed a geographic difference. Two associated markers (AGGS1014_2 and AHGS0798) with 6.90-9.94% PVE were verified to enhance oil content in an independent RIL population and also indicated selection during the breeding program. CONCLUSION: This study provided insights into the genetic basis of oil content in peanut and verified highly associated two SSR markers to facilitate marker-assisted selection for developing high-oil content breeding peanut varieties.

Identification of Two Novel Peanut Genotypes Resistant to Aflatoxin Production and Their SNP Markers Associated with Resistance.

Aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) are the most common aflatoxins produced by Aspergillus flavus in peanuts, with high carcinogenicity and teratogenicity. Identification of DNA markers associated with resistance to aflatoxin production is likely to offer breeders efficient tools to develop resistant cultivars through molecular breeding. In this study, seeds of 99 accessions of a Chinese peanut mini-mini core collection were investigated for their reaction to aflatoxin production by a laboratory kernel inoculation assay. Two resistant accessions (Zh.h0551 and Zh.h2150) were identified, with their aflatoxin content being 8.11%-18.90% of the susceptible control. The 99 peanut accessions were also genotyped by restriction site-associated DNA sequencing (RAD-Seq) for a genome-wide association study (GWAS). A total of 60 SNP (single nucleotide polymorphism) markers associated with aflatoxin production were detected, and they explained 16.87%-31.70% of phenotypic variation (PVE), with SNP02686 and SNP19994 possessing 31.70% and 28.91% PVE, respectively. Aflatoxin contents of accessions with "AG" (existed in Zh.h0551 and Zh.h2150) and "GG" genotypes of either SNP19994 or SNP02686 were significantly lower than that of "AA" genotypes in the mean value of a three-year assay. The resistant accessions and molecular markers identified in this study are likely to be helpful for deployment in aflatoxin resistance breeding in peanuts.

High-resolution mapping of a major and consensus quantitative trait locus for oil content to a ~ 0.8-Mb region on chromosome A08 in peanut (Arachis hypogaea L.).

KEY MESSAGE: ddRAD-seq-based high-density genetic map comprising 2595 loci identified a major and consensus QTL with a linked marker in a 0.8-Mb physical interval for oil content in peanut. Enhancing oil content is an important breeding objective in peanut. High-resolution mapping of quantitative trait loci (QTLs) with linked markers could facilitate marker-assisted selection in breeding for target traits. In the present study, a recombined inbred line population (Xuhua 13 × Zhonghua 6) was used to construct a genetic map based on double-digest restriction-site-associated DNA sequencing (ddRAD-seq). The resulting high-density genetic map contained 2595 loci, and spanned a length of 2465.62 cM, with an average distance of 0.95 cM/locus. Seven QTLs for oil content were identified on five linkage groups, including the major and stable QTL qOCA08.1 on chromosome A08 with 10.14-27.19% phenotypic variation explained. The physical interval of qOCA08.1 was further delimited to a ~ 0.8-Mb genomic region where two genes affecting oil synthesis had been annotated. The marker SNPOCA08 was developed targeting the SNP loci associated with oil content and validated in peanut cultivars with diverse oil contents. The major and stable QTL identified in the present study could be further dissected for gene discovery. Furthermore, the tightly linked marker for oil content would be useful in marker-assisted breeding in peanut.

Mitigating Aflatoxin Contamination in Groundnut through A Combination of Genetic Resistance and Post-Harvest Management Practices.

Aflatoxin is considered a "hidden poison" due to its slow and adverse effect on various biological pathways in humans, particularly among children, in whom it leads to delayed development, stunted growth, liver damage, and liver cancer. Unfortunately, the unpredictable behavior of the fungus as well as climatic conditions pose serious challenges in precise phenotyping, genetic prediction and genetic improvement, leaving the complete onus of preventing aflatoxin contamination in crops on post-harvest management. Equipping popular crop varieties with genetic resistance to aflatoxin is key to effective lowering of infection in farmer's fields. A combination of genetic resistance for in vitro seed colonization (IVSC), pre-harvest aflatoxin contamination (PAC) and aflatoxin production together with pre- and post-harvest management may provide a sustainable solution to aflatoxin contamination. In this context, modern "omics" approaches, including next-generation genomics technologies, can provide improved and decisive information and genetic solutions. Preventing contamination will not only drastically boost the consumption and trade of the crops and products across nations/regions, but more importantly, stave off deleterious health problems among consumers across the globe.

The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication.

High oil and protein content make tetraploid peanut a leading oil and food legume. Here we report a high-quality peanut genome sequence, comprising 2.54 Gb with 20 pseudomolecules and 83,709 protein-coding gene models. We characterize gene functional groups implicated in seed size evolution, seed oil content, disease resistance and symbiotic nitrogen fixation. The peanut B subgenome has more genes and general expression dominance, temporally associated with long-terminal-repeat expansion in the A subgenome that also raises questions about the A-genome progenitor. The polyploid genome provided insights into the evolution of Arachis hypogaea and other legume chromosomes. Resequencing of 52 accessions suggests that independent domestications formed peanut ecotypes. Whereas 0.42-0.47 million years ago (Ma) polyploidy constrained genetic variation, the peanut genome sequence aids mapping and candidate-gene discovery for traits such as seed size and color, foliar disease resistance and others, also providing a cornerstone for functional genomics and peanut improvement.

Transcriptome Analysis of a New Peanut Seed Coat Mutant for the Physiological Regulatory Mechanism Involved in Seed Coat Cracking and Pigmentation.

Seed-coat cracking and undesirable color of seed coat highly affects external appearance and commercial value of peanuts (Arachis hypogaea L.). With an objective to find genetic solution to the above problems, a peanut mutant with cracking and brown colored seed coat (testa) was identified from an EMS treated mutant population and designated as "peanut seed coat crack and brown color mutant line (pscb)." The seed coat weight of the mutant was almost twice of the wild type, and the germination time was significantly shorter than wild type. Further, the mutant had lower level of lignin, anthocyanin, proanthocyanidin content, and highly increased level of melanin content as compared to wild type. Using RNA-Seq, we examined the seed coat transcriptome in three stages of seed development in the wild type and the pscb mutant. The RNA-Seq analysis revealed presence of highly differentially expressed phenylpropanoid and flavonoid pathway genes in all the three seed development stages, especially at 40 days after flowering (DAF40). Also, the expression of polyphenol oxidases and peroxidase were found to be activated significantly especially in the late seed developmental stage. The genome-wide comparative study of the expression profiles revealed 62 differentially expressed genes common across all the three stages. By analyzing the expression patterns and the sequences of the common differentially expressed genes of the three stages, three candidate genes namely c36498_g1 (CCoAOMT1), c40902_g2 (kinesin), and c33560_g1 (MYB3) were identified responsible for seed-coat cracking and brown color phenotype. Therefore, this study not only provided candidate genes but also provided greater insights and molecular genetic control of peanut seed-coat cracking and color variation. The information generated in this study will facilitate further identification of causal gene and diagnostic markers for breeding improved peanut varieties with smooth and desirable seed coat color.

Abundant microsatellite diversity and oil content in wild Arachis species.

The peanut (Arachis hypogaea) is an important oil crop. Breeding for high oil content is becoming increasingly important. Wild Arachis species have been reported to harbor genes for many valuable traits that may enable the improvement of cultivated Arachis hypogaea, such as resistance to pests and disease. However, only limited information is available on variation in oil content. In the present study, a collection of 72 wild Arachis accessions representing 19 species and 3 cultivated peanut accessions were genotyped using 136 genome-wide SSR markers and phenotyped for oil content over three growing seasons. The wild Arachis accessions showed abundant diversity across the 19 species. A. duranensis exhibited the highest diversity, with a Shannon-Weaver diversity index of 0.35. A total of 129 unique alleles were detected in the species studied. A. rigonii exhibited the largest number of unique alleles (75), indicating that this species is highly differentiated. AMOVA and genetic distance analyses confirmed the genetic differentiation between the wild Arachis species. The majority of SSR alleles were detected exclusively in the wild species and not in A. hypogaea, indicating that directional selection or the hitchhiking effect has played an important role in the domestication of the cultivated peanut. The 75 accessions were grouped into three clusters based on population structure and phylogenic analysis, consistent with their taxonomic sections, species and genome types. A. villosa and A. batizocoi were grouped with A. hypogaea, suggesting the close relationship between these two diploid wild species and the cultivated peanut. Considerable phenotypic variation in oil content was observed among different sections and species. Nine alleles were identified as associated with oil content based on association analysis, of these, three alleles were associated with higher oil content but were absent in the cultivated peanut. The results demonstrated that there is great potential to increase the oil content in A. hypogaea by using the wild Arachis germplasm.