**2.6. Targeting Induced Local Lesions in Genomes (TILLING) and NGS-based mutation detection**

One of the factors contributing to slow genetic gain in breeding of vegetatively propagated crops is the narrow genetic base of the source population. This is a result of clonal propagation as opposed to sexual reproduction, which limits recombination. TILLING (Targeting Induced Local Lesions in Genomes) [85,86] provides an alternative approach for creating novel variation in these crops [87,88]. Rare alleles harbored in germplasm collections and wild species can be accessed by TILLING and EcoTILLING by sequencing. TILLING may lead to the development of functional markers for screening-associated traits through marker-assisted selection (MAS). The technique of TILLING using high-throughput mutation discovery has already been applied successfully to more than 20 plant species [89].

A wide spectrum of mutation detection assays, ranging from heteroduplex analysis with highpressure liquid chromatography (HPLC), screening with labeled primers, electrophoresis, microarray, the use of fluorescent dye-labeled primers assayed on ABI genetic analyzer have been used. However, these methods are generally slow, costly, and labor intensive. Applica‐ tion of NGS has been shown to be a cost-effective mutation detection system by re-sequencing the gene of interest in mutagenized plants [90,91]. The availability of genome sequence enables the use of reverse genetic approaches to identify mutations in specific target genes, thereby accelerating the generation of novel phenotypes. Comparative genome analysis methods offer the opportunity to select target genes involved in biosynthetic pathways and networks of traits/phenotypes of economic importance. The use of multidimensional pooling of DNA samples enables screening of DNA pools for multiple independent mutations in any target gene using NGS, which provides a cost-effective assay. This has led to the discovery of rare mutations in rice and wheat, termed TILLING by sequencing [92], tef [93], and in animals [94]. Different sample pooling schemes for NGS, which further enhance the power of NGS in processing multiple samples in parallel have been developed [95]. In light of the rapidly evolving sequencing technology together with a plethora of sample pooling schemes, com‐ bined with bar coding, it is feasible and imperative to apply TILLING by sequencing to understudied crops of Africa. A direct application of NGS to detect mutant regions in a segregating population of rice has been demonstrated in a method called MutMap [96].
