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.

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.

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.

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.

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.

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.

Genome-wide survey of the amino acid transporter gene family in wheat (Triticum aestivum L.): Identification, expression analysis and response to abiotic stress.

Amino acid transporters (AATs), which transport amino acids across cell membranes, play important roles in alleviating plant damage under stresses and in plant growth. To data, little is known about the AAT genes in wheat because of its complex genome. In this study, a total of 296 AAT genes were identified from the latest wheat genome sequence (IWGSC v1.1) and classified into 12 distinct subfamilies based upon their sequence composition and phylogenetic relationship. The expansion of the wheat AAT family was mainly the results of whole-genome duplication (WGD) and tandem events. The unequal expansion of different subfamilies brought new features to TaAATs. TaAATs were highly expressed and exhibited distinct expression patterns in different tissues. On the basis of homology and expression pattern analysis, we identified several wheat AAT family members that may affect grain quality. In addition, TaAAP3, TaATLa2 and TaATLb13 exhibited sustained expression in response to drought and high-temperature stress. These genes are involved in the response of wheat to abiotic stress by regulating the transport and distribution of amino acids. Overall, our results help to understand the complexity of TaAATs and provide a theoretical basis for further identification and utilization of AATs in wheat and other crop species.

Gene Expression Profiling Indicated Diverse Functions and Characteristics of Core Genes in Pea Aphid.

The pea aphid is a global insect pest, and variable phenotypes can be produced by pea aphids in the same genotype in response to changes in external environmental factors. However, detailed dynamic gene regulation networks and the core markers involved in different biological processes of pea aphids have not yet been reported. In this study, we obtained the published genomic and transcriptomic data, and performed transcriptome profiling of five pea aphid morphs (winged asexual female, wingless asexual female, wingless sexual female, winged male and wingless male) from each of three pea aphid genotypes, i.e., the transcriptomes from a total of 15 types of pea aphids were analyzed and the type-specific expression of genes in five different morphs was identified. The expression profiling was verified by quantitative real-time PCR (qPCR) analysis. Moreover, we determined the expression features and co-expression networks of highly variable genes. We also used the ARACNe method to obtain 263 core genes related to different biological pathways. Additionally, eight of the identified genes were aligned with transcription factor families, indicating that they act as transcription factors and regulate downstream genes. Furthermore, we found reliable markers using random forest methodology to distinguish different morphs of pea aphids. Our study provides a systematic and comprehensive approach for analyzing the core genes that may play important roles in a multitude of biological processes from the insect transcriptomes.

A Novel Software and Method for the Efficient Development of Polymorphic SSR Loci Based on Transcriptome Data.

Traditional methods for developing polymorphic microsatellite loci without reference sequences are time-consuming and labor-intensive, and the polymorphisms of simple sequence repeat (SSR) loci developed from expressed sequence tag (EST) databases are generally poor. To address this issue, in this study, we developed a new software (PSSRdt) and established an effective method for directly obtaining polymorphism details of SSR loci by analyzing diverse transcriptome data. The new method includes three steps, raw data processing, PSSRdt application, and loci extraction and verification. To test the practicality of the method, we successfully obtained 1940 potential polymorphic SSRs from the transcript dataset combined with 44 pea aphid transcriptomes. Fifty-two SSR loci obtained by the new method were selected for validating the polymorphic characteristics by genotyping in pea aphid individuals. The results showed that over 92% of SSR loci were polymorphic and 73.1% of loci were highly polymorphic. Our new software and method provide an innovative approach to microsatellite development based on RNA-seq data, and open a new path for the rapid mining of numerous loci with polymorphism to add to the body of research on microsatellites.

A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.

Identification, Expression, and Regulation of an Omega Class Glutathione S-transferase in Rhopalosiphum padi (L.) (Hemiptera: Aphididae) Under Insecticide Stress.

Glutathione S-transferases (GSTs) play an essential role in the detoxification of xenobiotic toxins in insects, including insecticides. However, few data are available for the bird cherry-oat aphid, Rhopalosiphum padi (L.). In this study, we cloned and sequenced the full-length cDNA of an omega GST gene (RpGSTO1) from R. padi, which contains 720 bp in length and encodes 239 amino acids. A phylogenetic analysis revealed that RpGSTO1 belongs to the omega class of insect GSTs. RpGSTO1 gene was highly expressed in transformed Escherichia coli and the protein was purified by affinity chromatography. The recombinant RpGSTO1 displayed reduced glutathione (GSH)-dependent conjugating activity toward the substrate 1-chloro-2, 4-dinitrobenzene (CDNB) substrate. The recombinant RpGSTO1 protein exhibited optimal activity at pH 7.0 and 30°C. In addition, a disk diffusion assay showed that E. coli overexpressing RpGSTO1 increased resistance to cumene hydroperoxide-induced oxidative stress. Real-time quantitative PCR analysis showed that the relative expression level of RpGSTO1 was different in response to different insecticides, suggesting that the enzyme could contribute to insecticide metabolism in R. padi. These findings indicate that RpGSTO1 may play a crucial role in counteracting oxidative stress and detoxifying the insecticides. The results of our study contribute to a better understanding the mechanisms of insecticide detoxification and resistance in R. padi.

De novo transcriptome analysis and microsatellite marker development for population genetic study of a serious insect pest, Rhopalosiphum padi (L.) (Hemiptera: Aphididae).

The bird cherry-oat aphid, Rhopalosiphum padi (L.), is one of the most abundant aphid pests of cereals and has a global distribution. Next-generation sequencing (NGS) is a rapid and efficient method for developing molecular markers. However, transcriptomic and genomic resources of R. padi have not been investigated. In this study, we used transcriptome information obtained by RNA-Seq to develop polymorphic microsatellites for investigating population genetics in this species. The transcriptome of R. padi was sequenced on an Illumina HiSeq 2000 platform. A total of 114.4 million raw reads with a GC content of 40.03% was generated. The raw reads were cleaned and assembled into 29,467 unigenes with an N50 length of 1,580 bp. Using several public databases, 82.47% of these unigenes were annotated. Of the annotated unigenes, 8,022 were assigned to COG pathways, 9,895 were assigned to GO pathways, and 14,586 were mapped to 257 KEGG pathways. A total of 7,936 potential microsatellites were identified in 5,564 unigenes, 60 of which were selected randomly and amplified using specific primer pairs. Fourteen loci were found to be polymorphic in the four R. padi populations. The transcriptomic data presented herein will facilitate gene discovery, gene analyses, and development of molecular markers for future studies of R. padi and other closely related aphid species.