ISSR-PCR for Genetic Diversity Analysis in Faba Bean

Molecular marker-based diversity assessment is essential for crop improvement strategies in faba bean (Vicia faba L.), a diploid grain legume with limited genomic resources relative to model species such as Medicago truncatula or major crops like soybean (Glycine max). ISSR markers provide a PCR-based, multilocus fingerprinting approach requiring no prior sequence information, making them particularly suitable for orphan crops. This protocol provides comprehensive, step-by-step procedures covering the complete analytical pipeline from genomic DNA isolation to statistical interpretation. This chapter includes: (1) CTAB DNA extraction optimized for polyphenol removal, (2) ISSR-PCR with optimization guidelines, (3) gel electrophoresis protocols, (4) manual and software-based fragment sizing with Rf-based interpolation methods, (5) binary matrix construction and band scoring, and (6) statistical analysis for diversity parameters calculation. Comprehensive notes provide troubleshooting guidance for common problems and data interpretation standards. While optimized for faba bean, the protocol approach allows ready adaptation to other plant species through tissue-specific modifications to extraction procedures and PCR conditions.