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.