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. 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. 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.

Cultivated peanut (Arachis hypogaea L.) is an important oilseed crop that is grown extensively in Africa, Asia and America. The diseases early and late leaf spot severely constrains peanut production worldwide. Because multiple genes control resistance to leaf spot diseases, conventional breeding is a time-consuming approach for pyramiding resistance genes into a single genotype. Marker-assisted selection (MAS) would complement and accelerate conventional breeding once molecular markers tightly associated with the resistance genes are identified. In this study, we have generated a large number of SNPs through genotyping by sequencing (GBS) and constructed a high-resolution map with an average distance of 1.34 cM among 2,753 SNP markers distributed on 20 linkage groups. QTL mapping has revealed that major QTL within a confidence interval could provide an efficient way to detect putative resistance genes. Analysis of the interval sequences has indicated that a major QTL for resistance to late leaf spot anchored by two NBS-LRR resistance genes on chromosome B05. Two major QTLs located on chromosomes A03 and B04 were associated with resistance genes for early leaf spot. Sequences within the confidence interval would facilitate identifying resistance genes and applying marker-assisted selection for resistance.

Peanut (Arachis hypogaeaL.) 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 occurringFAD2gene 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 homeologousFAD2genes in seed while maintaining regulation in other plant tissues. Twocis-regulatory elements the RY repeat motif and 2S seed protein motif in the 5′UTR and associated intron ofFAD2genes 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.

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.

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.