| Sumario: | To Editor Yam (Dioscorea spp.) is a multi-species tuber crop providing food and income to millions of people worldwide, particularly in Africa (Price et al., 2016). The 'yam belt' in West Africa, including Nigeria, Benin, Togo, Ghana, and Côte d'Ivoire, accounts for 92% of 72.6 million tons of global yam production (FAOSTAT, 2018). Despite the economic importance, yam cultivation is plagued by several biotic and abiotic factors. Yam genetic improvement via conventional breeding has not achieved substantial progress mainly due to the dioecy nature, long breeding-cycle, polyploidy, heterozygosity, poor seed set, and non-synchronous flowering (Mignouna et al., 2008). A precise genome-engineering holds the potential to overcome some of these limitations. CRISPR/Cas9 is the most popular genome-editing system applied extensively for crop improvement, wherein yam is lagging far behind other crop species. The genetic transformation technologies and genome sequences, only recently available, made it possible to realize the potential of CRISPR-based genome editing for basic and applied research in yam (Manoharan et al., 2016; Nyaboga et al., 2014; Tamiru et al., 2017). Here, we report, for the first time, the successful establishment of a CRISPR/Cas9-based genome-editing system and validation of its efficacy by targeting the phytoene desaturase gene (DrPDS) in a West African farmer-preferred D. rotundata accession Amola. The PDS gene is involved in converting phytoene into carotenoid precursors phytofluene and ζ-carotene (Mann et al., 1994). It is commonly used as a visual marker to validate genome editing in plants, as disruption of its function causes albinism. We first sought to identify the promoters for expressing guide RNAs (gRNAs) in yam. Five U6 genes from D. alata were identified, and the respective promoters (~300 bp) were synthesized. To identify the best DaU6 promoters, a gRNA targeting a mutated green fluorescence protein gene (GFP + 1) was constructed under each DaU6 promoter. Individual gRNAs and Cas9 (pCas9-DaU6
|
|---|