Base Editing in Peanut Using CRISPR/nCas9.

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.

Genome-Wide Identification of Powdery Mildew Resistance in Common Bean (Phaseolus vulgaris L.).

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_010G1320001g and Phavu_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) in Arabidopsis. 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.

Genomics, genetics and breeding of common bean in Africa: A review of tropical legume project.

Common bean (Phaseolus vulgaris L.) is an important legume crop worldwide. The International Centre for Tropical Agriculture (CIAT) and its national partners in Africa aim to overcome production constraints of common bean and address the food, nutrition needs and market demands through development of multitrait bean varieties. Breeding is guided by principles of market-driven approaches to develop client-demanded varieties. Germplasm accessions from especially two sister species, P. coccineus and P. acutifolius, have been utilized as sources of resistance to major production constraints and interspecific lines deployed. Elucidation of plant mechanisms governing pest and disease resistance, abiotic stress tolerance and grain nutritional quality guides the selection methods used by the breeders. Molecular markers are used to select for resistance to key diseases and insect pests. Efforts have been made to utilize modern genomic tools to increase scale, efficiency, accuracy and speed of breeding. Through gender-responsive participatory variety selection, market-demanded varieties have been released in several African countries. These new bean varieties are a key component of sustainable food systems in the tropics.

Genomics, genetics and breeding of tropical legumes for better livelihoods of smallholder farmers.

Legumes are important components of sustainable agricultural production, food, nutrition and income systems of developing countries. In spite of their importance, legume crop production is challenged by a number of biotic (diseases and pests) and abiotic stresses (heat, frost, drought and salinity), edaphic factors (associated with soil nutrient deficits) and policy issues (where less emphasis is put on legumes compared to priority starchy staples). Significant research and development work have been done in the past decade on important grain legumes through collaborative bilateral and multilateral projects as well as the CGIAR Research Program on Grain Legumes (CRP-GL). Through these initiatives, genomic resources and genomic tools such as draft genome sequence, resequencing data, large-scale genomewide markers, dense genetic maps, quantitative trait loci (QTLs) and diagnostic markers have been developed for further use in multiple genetic and breeding applications. Also, these mega-initiatives facilitated release of a number of new varieties and also dissemination of on-the-shelf varieties to the farmers. More efforts are needed to enhance genetic gains by reducing the time required in cultivar development through integration of genomics-assisted breeding approaches and rapid generation advancement.

Viruses infecting common bean (Phaseolus vulgaris L.) in Tanzania: A review on molecular characterization, detection and disease management options.

Common bean (Phaseolus vulgaris L.) is a major legume crop, serving as a main source of dietary protein and calories and generating income for many Tanzanians. It is produced in nearly all agro-ecological zones of Tanzania. However, the average yields are low (<1000 kg/ha), which is attributed to many factors including virus diseases. The most important viruses of common bean in Tanzania are Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) but other viruses have also been reported. There has never been a review of common bean virus diseases in the country, and the lack of collated information makes their management difficult. Therefore, this review focuses on (1) occurrence of different viruses of common bean in Tanzania, (2) molecular characterization of these viruses, (3) detection tools for common bean viruses in Tanzania and (4) available options for managing virus diseases in the country. Literature and nucleotide sequence database searches revealed that common bean diseases are inadequately studied and that their causal viruses have not been adequately characterized at the molecular level in Tanzania. Increased awareness on common bean virus diseases in Tanzania is expected to result into informed development of strategies for management of the same and thus increased production, which in turn has implication on nutrition and income.