Identification of AFLP fragments linked to seed coat colour in Brassica juncea and conversion to a SCAR marker for rapid selection

A Brassica juncea mapping population was generated and scored for seed coat colour. A combination of bulked segregant analysis and AFLP methodology wa s employed to identify markers linked to seed coat colour in B. juncea. AFL P analysis using 16 primer combinations revealed seven AFLP markers polymor phic between the parents and the bulks. Individual plants from the segregat ing population were analysed, and three AFLP markers were identified as bei ng tightly linked to the seed coat colour trait and specific for brown-seed ed individuals. Since AFLP markers are not adapted for large-scale applicat ion in plant breeding, our objective was to develop a fast, cheap and relia ble PCR-based assay. Towards this goal, we employed PCR-walking technology to isolate sequences adjacent to the linked AFLP marker. Based on the seque nce information of the cloned flanking sequence of marker AFLP8, primers we re designed. Amplification using the locus-specific primers generated bands at 0.5 kb and 1.2 kb with the yellow-seeded parent and a 1.1-kb band with the brown-seeded parent. Thus, the dominant AFLP marker (AFLP8) was convert ed into a simple codominant SCAR (Sequence Characterized Amplified Region) marker and designated as SCM08. Scoring of this marker in a segregating pop ulation easily distinguished yellow- and brown-seeded B. juncea and also di fferentiated between homozygous (BB) and heterozygous (Bb) brown-seeded ind ividuals. Thus, this marker will be useful for the development of yellow se ed B. juncea cultivars and facilitate the mapbased cloning of genes respons ible for seed coat colour trait.