Abstract
Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in Vigna unguiculata is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in Vigna unguiculata. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in Vigna unguiculata, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume.
Highlights
The evolutionary relatedness of symbiosis receptor-like kinase (SYMRK) proteins is consistent with the phylogeny and estimated divergence times of the phaseoloid legumes, including soybean, common bean, and cowpea [5], whereas Arachis hypogaea and Aeschynomene evenia both belong to Dalbergieae of the Faboideae subfamily
To enhance the gene disruption efficiency, these three guide RNAs (gRNAs) were constructed into one binary vector, under the control of L. japonicus U6 small nuclear RNA promoter (LjU6 pro), with a co-expression of the CRISPR-associated protein 9 (Cas9) endonuclease and a GFP fluorescent marker [18] (Figure 2C)
These results demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was effective in causing gene mutations in cowpea
Summary
Walp) belongs to the genus Vigna and originated in Africa. In this species, there are ten wild subspecies In the last decade (2008–2017), among the 11 primary legumes harvested as dry seeds, cowpea was ranked the 4th highest production of dry grain across the world [4] (Figure 1A). In many African countries, cowpea is a significant source of cheap protein for human health due to its excellent capability of drought tolerance and growth in low-nutrient soils. Nigeria and Niger are the two largest cowpea producing countries, accounting for ~47.6% and ~24.6% of the global production, respectively (Figure 1B). Cowpea leaves can be used as livestock feed in many developing countries
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