Climate change is significantly impacting agricultural production worldwide. Peanuts provide food and nutritional security to millions of people across the globe because of its high nutritive values. Drought and heat stress alone or in combination cause substantial yield losses to peanut production. The stress, in addition, adversely impact nutritional quality. Peanuts exposed to drought stress at reproductive stage are prone to aflatoxin contamination, which imposes a restriction on use of peanuts as health food and also adversely impact peanut trade. A comprehensive understanding of the impact of drought and heat stress at physiological and molecular levels may accelerate the development of stress tolerant productive peanut cultivars adapted to a given production system. Significant progress has been achieved towards the characterization of germplasm for drought and heat stress tolerance, unlocking the physiological and molecular basis of stress tolerance, identifying significant marker-trait associations as well major QTLs and candidate genes associated with drought tolerance, which after validation may be deployed to initiate marker-assisted breeding for abiotic stress adaptation in peanut. The proof of concept about the use of transgenic technology to add value to peanuts has been demonstrated. Advances in phenomics and artificial intelligence to accelerate the timely and cost-effective collection of phenotyping data in large germplasm/breeding populations have also been discussed. Greater focus is needed to accelerate research on heat stress tolerance in peanut. A suits of technological innovations are now available in the breeders toolbox to enhance productivity and nutritional quality of peanuts in harsh environments. A holistic breeding approach that considers drought and heat-tolerant traits to simultaneously address both stresses could be a successful strategy to produce climate-resilient peanut genotypes with improved nutritional quality.

Peanut ( Arachis hypogaea L.) seed is a rich source of edible oil, comprised primarily of monounsaturated oleic acid and polyunsaturated linoleic acid, accounting for 80% of its fatty acid repertoire. The conversion of oleic acid to linoleic acid, catalyzed by Fatty Acid Desaturase 2 (FAD2) enzymes, is an important regulatory point linked to improved abiotic stress responses while the ratio of these components is a significant determinant of commercial oil quality. Specifically, oleic acid has better oxidative stability leading to longer shelf life and better taste qualities while also providing nutritional based health benefits. Naturally occurring FAD2 gene knockouts that lead to high oleic acid levels improve oil quality at the potential expense of plant health though. We undertook a CRISPR/Cas9 based site-specific genome modification approach designed to downregulate the expression of two homeologous FAD2 genes in seed while maintaining regulation in other plant tissues. Two cis -regulatory elements the RY repeat motif and 2S seed protein motif in the 5′UTR and associated intron of FAD2 genes are potentially important for regulating seed-specific gene expression. Using hairy root and stable germ line transformation, differential editing efficiencies were observed at both CREs when targeted by single gRNAs using two different gRNA scaffolds. The editing efficiencies also differed when two gRNAs were expressed simultaneously. Additionally, stably transformed seed exhibited an increase in oleic acid levels relative to wild type. Taken together, the results demonstrate the immense potential of CRISPR/Cas9 based approaches to achieve high frequency targeted edits in regulatory sequences for the generation of novel transcriptional alleles, which may lead to fine tuning of gene expression and functional genomic studies in peanut.

Smash-ridging cultivation is an efficient farming method that was recently developed in China. The technique involves vertically cutting by using a spiral drill, causing the soil to ‘suspend’ as ridges, thereby breaking through the traditional plough pan, and thickening the plough layer up to 30–50 cm. Smash-ridging cultivation has effectively improved soil quality and has increased the water and nutrient retention capacity. Loose soil enables the plant’s root system to optimally develop and more efficiently absorb nutrients. This facilitates the growth of the above ground parts of plant, leading to a significant increase in crop yield. This method has been successfully applied on 40 crops and tested in 26 provinces. The yield increase within a single season ranges from 10.0% to 54.8%. This technique may have a positive and extensive impact on food safety and agricultural production in China and the rest of the world.

Plants have numerous fatty acid desaturase (FAD) enzymes regulating the unsaturation of fatty acids, which are encoded by a FAD gene family. The FAD2 genes belong to such family and play a vital role in converting monounsaturated oleic acid to polyunsaturated linoleic acid. Oleic acid has the health benefits for humans, such as reduction in cholesterol level, antioxidation property, and industrial benefits like longer shelf life. The development of genotypes with high oleic acid content in seeds has become one of the primary goals in breeding oilseed plants. The identification and characterization of the FAD2 genes in plants have been an important step to better manipulate gene expression to improve the seed oil quality. The induction of mutations in FAD2 genes to reduce FAD2 enzyme activity has been an integral approach to generate genotypes with high oleic acid. This chapter will describe the FAD2 gene family in the model organism soybean and the correction of mutations in FAD2 genes with the increase of oleic acid content. Leveraging advanced research of FAD2 gene family in soybean promotes the study of FAD2 genes in other legume species, including peanut. The future perspectives and challenges associated with mutations in FAD2 genes will be discussed.

Peanut (Arachis hypogaea L.), an allotetraploid legume of the Fabaceae family, is able to thrive in tropical and subtropical regions and is considered as a promising oil seed crop worldwide. Increasing the content of oleic acid has become one of the major goals in peanut breeding because of health benefits such as reduced blood cholesterol level, antioxidant properties and industrial benefits such as longer shelf life. Genomic sequencing of peanut has provided evidence of homeologous AhFAD2A and AhFAD2B genes encoding Fatty Acid Desaturase2 (FAD2), which are responsible for catalyzing the conversion of monounsaturated oleic acid into polyunsaturated linoleic acid. Research studies demonstrate that mutations resulting in a frameshift or stop codon in an FAD2 gene leads to higher oleic acid content in oil. In this study, two expression vectors, pDW3873 and pDW3876, were constructed using Cas9 fused to different deaminases, which were tested as tools to induce point mutations in the promoter and the coding sequences of peanut AhFAD2 genes. Both constructs harbor the single nuclease null variant, nCas9 D10A, to which the PmCDA1 cytosine deaminase was fused to the C-terminal (pDW3873) while rAPOBEC1 deaminase and an uracil glycosylase inhibitor (UGI) were fused to the N-terminal and the C-terminal respectively (pDW3876). Three gRNAs were cloned independently into both constructs and the functionality and efficiency were tested at three target sites in the AhFAD2 genes. Both constructs displayed base editing activity in which cytosine was replaced by thymine or other bases in the targeted editing window. pDW3873 showed higher efficiency compared to pDW3876 suggesting that the former is a better base editor in peanut. This is an important step forward considering introgression of existing mutations into elite varieties can take up to 15 years making this tool a benefit for peanut breeders, farmers, industry and ultimately for consumers.

Peanuts are an economically important crop cultivated worldwide. However, several limitations restrained its productivity, including biotic/abiotic stresses. CRISPR/Cas9-based gene-editing technology holds a promising approach to developing new crops with improved agronomic and nutritional traits. Its application has been successful in many important crops. However, the application of this technology in peanut research is limited, probably due to the lack of suitable constructs and protocols. In this study, two different constructs were generated to induce insertion/deletion mutations in the targeted gene for a loss of function study. The first construct harbors the regular gRNA scaffold, while the second construct has the extended scaffold plus terminator. The designed gRNA targeting the coding sequence of the FAD2 genes was cloned into both constructs, and their functionality and efficiency were validated using the hairy root transformation system. Both constructs displayed insertions and deletions as the types of edits. The construct harboring the extended plus gRNA terminator showed a higher editing efficiency than the regular scaffold for monoallelic and biallelic mutations. These two constructs can be used for gene editing in peanuts and could provide tools for improving peanut lines for the benefit of peanut breeders, farmers, and industry.

Most agronomic traits in plants are quantitatively inherited. Quantitative trait lociQuantitative trait loci (QTL) (QTL) are chromosome regions associated with a particular phenotypic trait. QTL mappingQTL mapping is used to gain insight into the genetic architecture of complex quantitative traits in plants. Genome-wide association study (GWASGenome-wide association studies (GWAS)) has become a routine strategy to understand the genetic basis of quantitative traits. QTLQuantitative trait loci (QTL) mappingQTL mapping and GWAS study requires genotypic and phenotypic data that have been scored across a large number of individuals within a population. Utilization of statistical methods is a crucial step for identifying QTLsQuantitative trait loci (QTL) or causal genes associated with traits to explain the genetic basis of complex traits. In this chapter, we review current advances and strategies in the QTLQuantitative trait loci (QTL) mappingQTL mapping such as the creation of various populations, application of different types of DNAMarkers markersDNA marker, collection of phenotypic data, and selection of appropriate statistical software to ensure the execution of association mapping. Association studies between DNA regions and traits can provide an optimistic assessment of the prospects of marker-assisted selectionMarker-assisted selection (MAS) (MAS) employed in plant breedingBreeding programs. Candidate genes associated with complex traits can be discovered and cloned after validation of QTLsQuantitative trait loci (QTL) through the functional study by gene editing or the combination of QTL mappingQTL mapping with RNA-seqRNA-seq.

Genome-wide association studies (GWAS) have been utilized to detect genetic variations related to several agronomic traits and disease resistance in common bean. However, its application in the powdery mildew (PM) disease to identify candidate genes and their location in the common bean genome has not been fully addressed. Single-nucleotide polymorphism (SNP) genotyping with a BeadChip containing 5398 SNPs was used to detect genetic variations related to PM disease resistance in a panel of 211 genotypes grown under two field conditions for two consecutive years. Significant SNPs identified on chromosomes Pv04 and Pv10 were repeatable, ensuring the phenotypic data’s reliability and the causal relationship. A cluster of resistance genes was revealed on the Pv04 of the common bean genome, coiled-coil-nucleotide-binding site–leucine-rich repeat (CC-NBS-LRR, CNL), and Toll/interleukin-1 receptor-nucleotide-binding site–leucine-rich repeat type (TIR-NBS-LRR, TNL)-like resistance genes were identified. Furthermore, two resistance genes,Phavu_010G1320001gandPhavu_010G136800g, were also identified on Pv10. Further sequence analysis showed that these genes were homologs to the disease-resistance protein (RLM1A-like) and the putative disease-resistance protein (At4g11170.1) inArabidopsis. Significant SNPs related to two LRR receptor-like kinases (RLK) were only identified on Pv11 in 2018. Many genes encoding the auxin-responsive protein, TIFY10A protein, growth-regulating factor five-like, ubiquitin-like protein, and cell wall RBR3-like protein related to PM disease resistance were identified nearby significant SNPs. These results suggested that the resistance to PM pathogen involves a network of many genes constitutively co-expressed.

Powdery mildew disease caused by Oidium arachidis poses a threat to peanut production in Africa. Loss of function mutants of specific Mlo (Mildew Locus O) genes have provided broad-spectrum and durable resistance against pathogen in many crop species. Since there is huge potential to utilize susceptibility gene-mediated resistance in crop improvement, genome-wide mining of susceptibility genes is required for further researches. However, the susceptibility genes have not been characterized in peanut genome. In this research study, the genome of the cultivated peanut was used as reference to identify the AhMlo loci. Our results revealed that 25 AhMlo loci were identified and distributed on the chromosomes of the cultivated peanut. Eleven AhMlo loci were located on the A-genome while the remaining 14 on the B-genome. Variable number of inserted intron sequences (4–14) and transmembrane helix (4–8) were observed in the coding sequence of the AhMlo loci. Furthermore, phylogenetic analysis of the AhMlo loci along with homologs from other species has clustered the AhMlo loci into six clades. Three AhMlo loci were clustered in the clade V known to regroup the powdery susceptibility loci in dicots. Additionally, four core promoters were predicted on the promoter region of the specific AhMlo along with cis-regulatory elements related to PM susceptibility. These results provided strong evidence of the identification and distribution of the Mlo loci in the cultivated peanut genome and the identified specific AhMlo loci can be used for loss of susceptibility study.

Objective Early leaf spot (ELS) caused by Cercospora arachidicola (Hori) is a serious foliar disease in peanut worldwide, which causes considerable reduction of yield. Identification of resistance genes is important for both conventional and molecular breeding. Few resistance genes have been identified and the mechanism of defense responses to this pathogen remains unknown. Results We detected several genes involved in disease resistance to ELS through transcriptome analysis. Using RNA-seq technology, one hundred thirty-three differentially expressed genes (DEGs) were identified between resistant and susceptible lines. Among these DEGs, coiled coil-nucleotide binding-leucine rich repeat (NLR) type resistance genes were identified as duplicated R genes on the chromosome B2. Peanut phytoalexin deficient 4 (PAD4) regulator of effector-triggered immunity mediated by NLR resistance proteins and polyphenol oxidase (PPO) genes play important roles in early leaf spot resistance. Our study provides the useful information on plant response to C. arachidicola infection in peanut. The results suggest that a few major genes and several factors mediate the resistance to ELS disease, showing the characteristics of quantitative trait in defense responses.

Background: Micronutrient deficiencies caused by lack of Iron (Fe), Zinc (Zn) and Vitamin A have negative effects on human health worldwide. Even though common bean meets Fe and Zn deficiencies based on recommended dietary allowances, the cooking time remains an important challenge. Understanding the genome organization, the loci and genes localization controlling multiple traits is crucial for developing enhanced micronutrient content and improved common bean with short cooking time. Results: In this study, GWAS method was used to determine SNPs associated with four traits studied, including water uptake, cooking time, Fe and Zn concentration level, using ~5,000 SNPs for a panel of 206 genotypes. By the analysis of population structure, 206 common bean genotypes were formed in three groups, the Mesoamerican gene pool, the Andean gene pool, and the admixture gene pool using the Bayesian method. With the significance levels between p = 2.94 x 10 -08 to p = 8.33 x 10 -03 and phenotypic variation of 8.26% to 18.19%, total of 10 SNPs was found to be significantly associated with traits on chromosomes Pv03, Pv04 and Pv10. Among of these SNPs, four SNPs were associated with water uptake, another four SNPs with the cooking time, one SNP with Zn concentration and one of less significant SNP associated with Fe concentration. Within the range or nearby the peak of significant SNP, putative loci encoding pectin degradation, cell structure and cellular exchange/transport were identified, suggesting these loci may play important roles in modulating expression of traits. Conclusion: The resultant data in this study contributed on deciphering the molecular mechanisms associated with water uptake, cooking time and micronutrient contents in common bean. The investigation of association between phenotypic and genotypic data has showed that GWAS approach is a powerful tool to dissect genetic architecture of complex traits and cooperate molecular breeding for advanced genotypes with enhanced micronutrients and short cooking time.

Fungal, bacterial and viral diseases are economic foliar diseases that cause yield losses, between 40 and 100%, in commonly grown dry bean cultivars in the world. Development of disease resistance genotypes is a complex interaction between genetic and environmental factors. This study focused on determining the natural infection of disease-causing pathogens of angular leaf spot, powdery mildew, bacterial blight and bean common mosaic virus in different agro-ecologies in relation to grain yield. Diversity of 211 bean genotypes were tested at two different disease hot spots areas under incomplete block design, with two replications for two cropping seasons in Tanzania. Diseases severity was significantly different (p<0.001) for genotypes and their interactions with the environment and season. Higher disease severity was observed at Lyamungo site than Selian site. Effects of genotypes by environment were observed with maximum yield of 2170 kg/ha to low yield of 398 kg/ha with the grand mean of 1151.54 kg/ha. High annual rainfall and relative humidity contributed to disease development among the tested environment. Five genotypes (FEB 189, A774, NUA 16, KG 71-4 and DOR 766) expressed trait of resistance to above diseases and are advised to be incorporated in breeding programs for enhancing dry bean productivity. Key words: Diseases, losses, productivity, G*E interaction.

Background Long noncoding RNAs (lncRNAs) have several known functions involving various biological regulatory processes in plant. However, the possible roles of lncRNAs during peanut seed development have not been fully explored. Results In this study, two peanut recombinant inbred lines (RIL8) that differ in seed size were used to investigate comprehensive lncRNA profiles derived from the seed development at 15 and 35 days after flowering (DAF). We identified a total of 9388 known and 4037 novel lncRNAs, from which 1437 were differentially expressed lncRNAs. Interestingly, the expression patterns of a number of lncRNAs can be very different between two closely related inbred lines and these lncRNAs were expressed predominantly in only one RIL at 35 DAF. Some differentially expressed lncRNAs were found related to putative cis-acting target genes and predicted to be involved in transcription, transport, cell division, and plant hormone biosynthesis. The expression patterns of several representative lncRNAs and 12 protein-coding genes were validated by qPCR. Same expression pattern was observed between most lncRNAs and their target genes. 11 lncRNAs, XR_001593099.1, MSTRG.18462.1, MSTRG.34915.1, MSTRG.41848.1, MSTRG.22884.1, MSTRG.12404.1, MSTRG.26719.1, MSTRG.35761.1, MSTRG.20033.1, MSTRG.13500.1, and MSTRG.9304.1 and their cis-acting target genes may play key roles in peanut seed development. Conclusions These results provided new information on lncRNA-mediated regulatory roles in peanut seed development, contributing to the comprehensive understanding of the molecular mechanisms involved in peanut seed development.

BACKGROUND:Increasing the content of oleic acid in peanut seeds is one of the major goals in peanut breeding due to consumer and industry benefits, such as anti-oxidation and long shelf-life. Homeologous ahFAD2A and ahFAD2B genes encode fatty acid desaturases, which are the key enzymes for converting oleic acid to linoleic acid that oxidizes readily. To date, all high oleic acid peanut varieties result from natural mutations occurred in both genes. A method to induce mutations in the genes of other elite cultivars could speed introgression of this valuable trait. The gene-editing approach utilizing CRISPR/Cas9 technology was employed to induce de novo mutations in the ahFAD2 genes using peanut protoplasts and hairy root cultures as models.RESULTS:The hot spot of natural mutation in these genes was selected as the target region. Appropriate sgRNAs were designed and cloned into a CRISPR/Cas9 expression plasmid. As a result of CRISPR/Cas9 activity, three mutations were identified - G448A in ahFAD2A, and 441_442insA and G451T in ahFAD2B. The G448A and 441_442insA mutations are the same as those seen in existing high oleate varieties and the G451T is new mutation. Because natural mutations appear more often in the ahFAD2A gene than in the ahFAD2B gene in subspecies A. hypogaea var. hypogaea, the mutations induced in ahFAD2B by gene editing may be useful in developing high oleate lines with many genetic backgrounds after validation of oleic acid content in the transformed lines. The appearance of the G448A mutation in ahFAD2A is a further benefit for high oleic acid oil content.CONCLUSIONS:Overall, these results showed that mutations were, for the first time, induced by CRISPR-based gene editing approach in peanut. This research demonstrated the potential application of gene editing for mutagenesis in peanut and suggested that CRISPR/Cas9 technology may be useful in the peanut breeding programs.

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.

Like many important crops, peanut is a polyploid that underwent polyploidization, evolution, and domestication. The wild allotetraploid peanut species Arachis monticola (A. monticola) is an important and unique link from the wild diploid species to cultivated tetraploid species in the Arachis lineage. However, little is known about A. monticola and its role in the evolution and domestication of this important crop. A fully annotated sequence of ≈2.6 Gb A. monticola genome and comparative genomics of the Arachis species is reported. Genomic reconstruction of 17 wild diploids from AA, BB, EE, KK, and CC groups and 30 tetraploids demonstrates a monophyletic origin of A and B subgenomes in allotetraploid peanuts. The wild and cultivated tetraploids undergo asymmetric subgenome evolution, including homoeologous exchanges, homoeolog expression bias, and structural variation (SV), leading to subgenome functional divergence during peanut domestication. Significantly, SV-associated homoeologs tend to show expression bias and correlation with pod size increase from diploids to wild and cultivated tetraploids. Moreover, genomic analysis of disease resistance genes shows the unique alleles present in the wild peanut can be introduced into breeding programs to improve some resistance traits in the cultivated peanuts. These genomic resources are valuable for studying polyploid genome evolution, domestication, and improvement of peanut production and resistance.

The aim of the this study was to optimize peanut transformation system to improve the transformation efficiency.Several steps and components were studied for their effect on transformation efficiency mediated by Agrobacterium tumefaciens through detecting the transgenic gene bar in the genomic DNA of transformed plants.The results showed that the transformation efficiency could be significantly increased by collecting the Agrobacterium at a concentration of OD600 =0.7,then using an Agrobacterium infection medium AB supplemented with 1 mmol/L dithiothreitol (DTT) to infect the half-seed explants.The Agrobacterium transformation efficiency for peanut variety EXP27-1516 (69.03％) was higher than that of variety 14AU01 (56.67％).The transformation system de scribed here is an efficient approach that yield vigorous plants within 8 weeks,compared to 13 weeks required in the transformation using cotyledonary nodes,and is the fastest method available for peanut transformation.This study provided an optimized protocol for A.tumefaciens-mediated transformation in peanut as a useful reference to perform rapid transgenic research.