Advances in mutant characterization for detecting causal mutations in crop plants

Induced mutagenesis creates novel allelic variants to improve crop yield, climate resilience, and nutritional profile. However, utilizing these mutants effectively in breeding programs requires identification of the exact genetic lesions responsible for target traits. This review covers structural DNA mapping techniques, which are divided into two primary categories, whole-genome resequencing (WGS) frameworks (like MutMap, MutMap + , and QTL-seq) and cost-effective reduced-representation sequencing approaches (such as GBS, RAD-seq, ddRAD-seq, and SLAF-seq). Whole-genome methods use bulked segregant analysis of extreme plant phenotypes to isolate single-nucleotide polymorphisms, while reducing representation libraries (RRL) make high-density genotyping affordable for complex, polyploid crops. Moving past structural DNA changes, the manuscript explores how RNA transcriptomic profiling reveals modified gene networks and alternative splicing in mutants. It explores multi-omics tools, like expression quantitative trait loci (eQTL) mapping, which help filter out non-expressing gene fragments. Once candidate genes are identified, subsequent validation is imperative to confirm their functional roles in the target phenotype. Accordingly, this review encompasses several methods of pre-validation like target exome capture, kompetitive allele-specific PCR (KASP) markers, transient gene silencing to screen targets for marker-assisted breeding or amplicon-based TILLING. Finally, it discusses using targeted gene editing tools, specifically TALENs, CRISPR/Cas9, and base editing systems to validate candidate gene action and sufficiency in elite crop backgrounds. Overall, this manuscript reviews recent phenotypic, genomic, and transcriptomic advances, emphasizing their role in efficient mutant characterization for utilization in crop improvement programs.