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.

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.

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.

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.

Comprehensive evaluation of Chinese peanut mini-mini core collection and QTL mapping for aflatoxin resistance.

BACKGROUND: Aflatoxin contamination caused by Aspergillus fungi has been a serious factor affecting food safety of peanut (Arachis hypogaea L.) because aflatoxins are highly harmful for human and animal health. As three mechanisms of resistance to aflatoxin in peanut including shell infection resistance, seed infection resistance and aflatoxin production resistance exist among naturally evolved germplasm stocks, it is highly crucial to pyramid these three resistances for promoting peanut industry development and protecting consumers' health. However, less research effort has been made yet to investigate the differentiation and genetic relationship among the three resistances in diversified peanut germplasm collections. RESULTS: In this study, the Chinese peanut mini-mini core collection selected from a large basic collection was systematically evaluated for the three resistances against A. flavus for the first time. The research revealed a wide variation among the diversified peanut accessions for all the three resistances. Totally, 14 resistant accessions were identified, including three with shell infection resistance, seven with seed infection resistance and five with aflatoxin production resistance. A special accession, Zh.h1312, was identified with both seed infection and aflatoxin production resistance. Among the five botanic types of A. hypogaea, the var. vulgaris (Spanish type) belonging to subspecies fastigiata is the only one which possessed all the three resistances. There was no close correlation between shell infection resistance and other two resistances, while there was a significant positive correlation between seed infection and toxin production resistance. All the three resistances had a significant negative correlation with pod or seed size. A total of 16 SNPs/InDels associated with the three resistances were identified through genome-wide association study (GWAS). Through comparative analysis, Zh.h1312 with seed infection resistance and aflatoxin production resistance was also revealed to possess all the resistance alleles of associated loci for seed infection index and aflatoxin content. CONCLUSIONS: This study provided the first comprehensive understanding of differentiation of aflatoxin resistance in diversified peanut germplasm collection, and would further contribute to the genetic enhancement for resistance to aflatoxin contamination.

Detection of a major QTL and development of KASP markers for seed weight by combining QTL-seq, QTL-mapping and RNA-seq in peanut.

KEY MESSAGE: Combining QTL-seq, QTL-mapping and RNA-seq identified a major QTL and candidate genes, which contributed to the development of KASP markers and understanding of molecular mechanisms associated with seed weight in peanut. Seed weight, as an important component of seed yield, is a significant target of peanut breeding. However, relatively little is known about the quantitative trait loci (QTLs) and candidate genes associated with seed weight in peanut. In this study, three major QTLs on chromosomes A05, B02, and B06 were determined by applying the QTL-seq approach in a recombinant inbred line (RIL) population. Based on conventional QTL-mapping, these three QTL regions were successfully narrowed down through newly developed single nucleotide polymorphism (SNP) and simple sequence repeat markers. Among these three QTL regions, qSWB06.3 exhibited stable expression, contributing mainly to phenotypic variance across environments. Furthermore, differentially expressed genes (DEGs) were identified at the three seed developmental stages between the two parents of the RIL population. It was found that the DEGs were widely distributed in the ubiquitin-proteasome pathway, the serine/threonine-protein pathway, signal transduction of hormones and transcription factors. Notably, DEGs at the early stage were mostly involved in regulating cell division, whereas DEGs at the middle and late stages were primarily involved in cell expansion during seed development. The expression patterns of candidate genes related to seed weight in qSWB06.3 were investigated using quantitative real-time PCR. In addition, the allelic diversity of qSWB06.3 was investigated in peanut germplasm accessions. The marker Ah011475 has higher efficiency for discriminating accessions with different seed weights, and it would be useful as a diagnostic marker in marker-assisted breeding. This study provided insights into the genetic and molecular mechanisms of seed weight in peanut.

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.

Genome sequencing and comparative genomic analysis of highly and weakly aggressive strains of Sclerotium rolfsii, the causal agent of peanut stem rot.

BACKGROUND: Stem rot caused by Sclerotium rolfsii is a very important soil-borne disease of peanut. S. rolfsii is a necrotrophic plant pathogenic fungus with an extensive host range and worldwide distribution. It can infect peanut stems, roots, pegs and pods, leading to varied yield losses. S. rolfsii strains GP3 and ZY collected from peanut in different provinces of China exhibited a significant difference in aggressiveness on peanut plants by artificial inoculation test. In this study, de-novo genome sequencing of these two distinct strains was performed aiming to reveal the genomic basis of difference in aggressiveness. RESULTS: Scleotium rolfsii strains GP3 and ZY, with weak and high aggressiveness on peanut plants, exhibited similar growth rate and oxalic acid production in laboratory. The genomes of S. rolfsii strains GP3 and ZY were sequenced by Pacbio long read technology and exhibited 70.51 Mb and 70.61 Mb, with contigs of 27 and 23, and encoded 17,097 and 16,743 gene models, respectively. Comparative genomic analysis revealed that the pathogenicity-related gene repertoires, which might be associated with aggressiveness, differed between GP3 and ZY. There were 58 and 45 unique pathogen-host interaction (PHI) genes in GP3 and ZY, respectively. The ZY strain had more carbohydrate-active enzymes (CAZymes) in its secretome than GP3, especially in the glycoside hydrolase family (GH), the carbohydrate esterase family (CBM), and the polysaccharide lyase family (PL). GP3 and ZY also had different effector candidates and putative secondary metabolite synthetic gene clusters. These results indicated that differences in PHI, secreted CAZymes, effectors and secondary metabolites may play important roles in aggressive difference between these two strains. CONCLUSIONS: The data provided a further understanding of the S. rolfsii genome. Genomic comparison provided clues to the difference in aggressiveness of S. rolfsii strains.

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.

Transcriptome and metabolome reveal redirection of flavonoids in a white testa peanut mutant.

BACKGROUND: Coat color determines both appearance and nutrient quality of peanut. White seed coat in peanut can enhance the processing efficiency and quality of peanut oil. An integrative analysis of transcriptomes, metabolomes and histocytology was performed on wsc mutant and its wild type to investigate the regulatory mechanisms underlying color pigmentation. RESULT: Metabolomes revealed flavonoids were redirected in wsc, while multi-omics analyses of wsc mutant seeds and testae uncovered WSC influenced the flavonoids biosynthesis in testa as well as suberin formation, glycolysis, the TCA cycle and amino acid metabolism. The mutation also enhanced plant hormones synthesis and signaling. Further, co-expression analysis showed that FLS genes co-expressed with MBW complex member genes. Combining tissue expression patterns, genetic analyses, and the annotation of common DEGs for these three stages revealed that three testa specific expressed candidate genes, Araip.M7RY3, Aradu.R8PMF and Araip.MHR6K were likely responsible for the white testa phenotype. WSC might be regulated expression competition between FLS and DFR by controlling hormone synthesis and signaling as well as the MBW complex. CONCLUSIONS: The results of this study therefore provide both candidate genes and novel approaches that can be applied to improve peanut with desirable seed coat color and flavonoid quality.

Arachis hypogaea gene expression atlas for fastigiata subspecies of cultivated groundnut to accelerate functional and translational genomics applications.

Spatio-temporal and developmental stage-specific transcriptome analysis plays a crucial role in systems biology-based improvement of any species. In this context, we report here the Arachis hypogaea gene expression atlas (AhGEA) for the world's widest cultivated subsp. fastigiata based on RNA-seq data using 20 diverse tissues across five key developmental stages. Approximately 480 million paired-end filtered reads were generated followed by identification of 81 901 transcripts from an early-maturing, high-yielding, drought-tolerant groundnut variety, ICGV 91114. Further, 57 344 genome-wide transcripts were identified with ≥1 FPKM across different tissues and stages. Our in-depth analysis of the global transcriptome sheds light into complex regulatory networks namely gravitropism and photomorphogenesis, seed development, allergens and oil biosynthesis in groundnut. Importantly, interesting insights into molecular basis of seed development and nodulation have immense potential for translational genomics research. We have also identified a set of stable expressing transcripts across the selected tissues, which could be utilized as internal controls in groundnut functional genomics studies. The AhGEA revealed potential transcripts associated with allergens, which upon appropriate validation could be deployed in the coming years to develop consumer-friendly groundnut varieties. Taken together, the AhGEA touches upon various important and key features of cultivated groundnut and provides a reference for further functional, comparative and translational genomics research for various economically important traits.

Genome-wide expression quantitative trait locus analysis in a recombinant inbred line population for trait dissection in peanut.

The transcriptome connects genome to the gene function and ultimate phenome in biology. So far, transcriptomic approach was not used in peanut for performing trait mapping in bi-parental populations. In this research, we sequenced the whole transcriptome in immature seeds in a peanut recombinant inbred line (RIL) population and explored thoroughly the landscape of transcriptomic variations and its genetic basis. The comprehensive analysis identified total 49 691 genes in RIL population, of which 92 genes followed a paramutation-like expression pattern. Expression quantitative trait locus (eQTL) analysis identified 1207 local eQTLs and 15 837 distant eQTLs contributing to the whole-genome transcriptomic variation in peanut. There were 94 eQTL hot spot regions detected across the genome with the dominance of distant eQTL. By integrating transcriptomic profile and annotation analyses, we unveiled a putative candidate gene and developed a linked marker InDel02 underlying a major QTL responsible for purple testa colour in peanut. Our result provided a first understanding of genetic basis of whole-genome transcriptomic variation in peanut and illustrates the potential of the transcriptome-aid approach in dissecting important traits in non-model plants.

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.

Translational genomics for achieving higher genetic gains in groundnut.

KEY MESSAGE: Groundnut has entered now in post-genome era enriched with optimum genomic and genetic resources to facilitate faster trait dissection, gene discovery and accelerated genetic improvement for developing climate-smart varieties. Cultivated groundnut or peanut (Arachis hypogaea), an allopolyploid oilseed crop with a large and complex genome, is one of the most nutritious food. This crop is grown in more than 100 countries, and the low productivity has remained the biggest challenge in the semiarid tropics. Recently, the groundnut research community has witnessed fast progress and achieved several key milestones in genomics research including genome sequence assemblies of wild diploid progenitors, wild tetraploid and both the subspecies of cultivated tetraploids, resequencing of diverse germplasm lines, genome-wide transcriptome atlas and cost-effective high and low-density genotyping assays. These genomic resources have enabled high-resolution trait mapping by using germplasm diversity panels and multi-parent genetic populations leading to precise gene discovery and diagnostic marker development. Furthermore, development and deployment of diagnostic markers have facilitated screening early generation populations as well as marker-assisted backcrossing breeding leading to development and commercialization of some molecular breeding products in groundnut. Several new genomics applications/technologies such as genomic selection, speed breeding, mid-density genotyping assay and genome editing are in pipeline. The integration of these new technologies hold great promise for developing climate-smart, high yielding and more nutritious groundnut varieties in the post-genome era.

Discovery of two novel and adjacent QTLs on chromosome B02 controlling resistance against bacterial wilt in peanut variety Zhonghua 6.

KEY MESSAGE: Two novel and adjacent genomics and candidate genes for bacterial wilt resistance were identified on chromosome B02 in peanut variety Zhonghua 6 using both traditional QTL mapping and QTL-seq methods. Peanut (Arachis hypogaea) is an important oilseed crop worldwide. Utilization of genetic resistance is the most economic and effective approach to control bacterial wilt, one of the most devastating plant diseases, in peanut production. To accelerate the genetic improvement of bacterial wilt resistance (BWR) in peanut breeding programs, quantitative trait locus (QTL) mapping has been conducted for two resistant varieties. In this context, we deployed linkage mapping as well as sequencing-based mapping approach, QTL-seq, to identify genomic regions and candidate genes for BWR in another highly resistant variety Zhonghua 6. The recombination inbred line population (268 progenies) from the cross Xuhua 13 × Zhonghua 6 was used in BWR evaluation across five environments. QTL mapping using both SSR- and SNP-based genetic maps identified a stable QTL (qBWRB02-1) on chromosome B02 with 37.79-78.86% phenotypic variation explained (PVE) across five environments. The QTL-seq facilitated further dissection of qBWRB02-1 into two adjacent genomic regions, qBWRB02-1-1 (2.81-4.24 Mb) and qBWRB02-1-2 (6.54-8.75 Mb). Mapping of newly developed Kompetitive allele-specific PCR (KASP) markers on the genetic map confirmed their stable expressions across five environments. The effects of qBWRB02-1-1 (49.43-68.86% PVE) were much higher than qBWRB02-1-2 (3.96-6.48% PVE) and other previously reported QTLs. Nineteen putative candidate genes affected by 49 non-synonymous SNPs were identified for qBWRB02-1-1, and ten of them were predicted to code for disease resistance proteins. The major and stable QTL qBWRB02-1-1 and validated KASP markers could be deployed in genomics-assisted breeding (GAB) to develop improved peanut varieties with enhanced BWR.

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.

Optimization of extraction of total trans-resveratrol from peanut seeds and its determination by HPLC.

Resveratrol, a stilbene phytoalexin in plants, is believed to benefit human health. In this study, an optimized enzyme-assisted method was developed to extract the total content of trans-resveratrol (free or combined with glucose) in peanut seeds, followed by detection using high-performance liquid chromatography. The extraction process was optimized by Box-Behnken design and response surface methodology. The optimized enzyme concentration, digestion time, pH, and temperature were 3.02 g/L, 57.06 min, 5.88, and 51.05°C, respectively. Validation tests indicated that the experimental yield of trans-resveratrol was 0.183 ± 0.007 µg/g with a relative standard deviation of 3.87% (n = 5) under the optimal condition, which was closely agreed with the predicted value (0.182 µg/g). The recoveries obtained from the spiked samples were varied from 89.4 to 103.9%. Therefore, this study will provide a useful method for quantification of total trans-resveratrol in peanut seeds.

Genome-wide systematic characterization of bZIP transcription factors and their expression profiles during seed development and in response to salt stress in peanut.

BACKGROUND: Plant basic leucine zipper (bZIP) transcription factors play crucial roles in plant growth, development, and abiotic stress responses. However, systematic investigation and analyses of the bZIP gene family in peanut are lacking in spite of the availability of the peanut genome sequence. RESULTS: In this study, we identified 50 and 45 bZIP genes from Arachis duranensis and A. ipaensis genomes, respectively. Phylogenetic analysis showed that Arachis bZIP genes were classified into nine groups, and these clusters were supported by several group-specific features, including exon/intron structure, intron phases, MEME motifs, and predicted binding site structure. We also identified possible variations in DNA-binding-site specificity and dimerization properties among different Arachis bZIPs by inspecting the amino acid residues at some key sites. Our analysis of the evolutionary history analysis indicated that segmental duplication, rather than tandem duplication, contributed greatly to the expansion of this gene family, and that most Arachis bZIPs underwent strong purifying selection. Through RNA-seq and quantitative real-time PCR (qRT-PCR) analyses, the co-expressed, differentially expressed and several well-studied homologous bZIPs were identified during seed development stages in peanut. We also used qRT-PCR to explore changes in bZIP gene expression in response to salt-treatment, and many candidate bZIPs in groups A, B, and S were proven to be associated with the salt-stress response. CONCLUSIONS: This study have conducted a genome-wide identification, characterization and expression analysis of bZIP genes in Arachis genomes. Our results provide insights into the evolutionary history of the bZIP gene family in peanut and the funcntion of Arachis bZIP genes during seed development and in response to salt stress.

Discovery of genomic regions and candidate genes controlling shelling percentage using QTL-seq approach in cultivated peanut (Arachis hypogaea L.).

Cultivated peanut (Arachis hypogaea L.) is an important grain legume providing high-quality cooking oil, rich proteins and other nutrients. Shelling percentage (SP) is the 2nd most important agronomic trait after pod yield and this trait significantly affects the economic value of peanut in the market. Deployment of diagnostic markers through genomics-assisted breeding (GAB) can accelerate the process of developing improved varieties with enhanced SP. In this context, we deployed the QTL-seq approach to identify genomic regions and candidate genes controlling SP in a recombinant inbred line population (Yuanza 9102 × Xuzhou 68-4). Four libraries (two parents and two extreme bulks) were constructed and sequenced, generating 456.89-790.32 million reads and achieving 91.85%-93.18% genome coverage and 14.04-21.37 mean read depth. Comprehensive analysis of two sets of data (Yuanza 9102/two bulks and Xuzhou 68-4/two bulks) using the QTL-seq pipeline resulted in discovery of two overlapped genomic regions (2.75 Mb on A09 and 1.1 Mb on B02). Nine candidate genes affected by 10 SNPs with non-synonymous effects or in UTRs were identified in these regions for SP. Cost-effective KASP (Kompetitive Allele-Specific PCR) markers were developed for one SNP from A09 and three SNPs from B02 chromosome. Genotyping of the mapping population with these newly developed KASP markers confirmed the major control and stable expressions of these genomic regions across five environments. The identified candidate genomic regions and genes for SP further provide opportunity for gene cloning and deployment of diagnostic markers in molecular breeding for achieving high SP in improved varieties.