Application of Restriction Fragment Length Polymorphism (RFLP).

Restriction fragment length polymorphism (RFLP) was the first reliable genetic marker used in plant molecular studies. It is a DNA-based marker that detects variation in DNA sequence at restriction enzyme sites and produces codominant, locus-specific markers that differentiate homologous and heterozygous genotypes. It provides greater accuracy than trait-based methods. The application of RFLP markers includes developing genetic linkage maps, conducting QTL analysis, characterizing germplasm, and detecting somaclonal variations. Despite being the availability of new molecular markers, RFLP still remained highly valued for its reproducibility and clear Mendelian inheritance patterns. This chapter presents optimized, nonradioactive protocols for DNA isolation, Southern blotting, DIG-labeled probe hybridization, and chemiluminescent detection, offering a practical guideline for integrating RFLP markers into modern plant breeding and genomic research.