Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage map

Inter-simple sequence repeat (ISSR) analysis was evaluated for its usefulne ss in generating markers to extend the genetic linkage map of Citrus using a backcross population previously mapped with restriction fragment length p olymorphism (RFLP), random amplified polymorphic DNA (RAPD) and isozyme mar kers. ISSR markers were obtained through the simple technique of PCR follow ed by analysis on agarose gels, using simple sequence repeat (SSR) primers. Optimization of reaction conditions was achieved for 50% of the SSR primer s screened, and the primers amplified reproducible polymorphic bands in the parents and progeny of the backcross population. Mendelian segregation of the polymorphic bands was demonstrated, with an insignificant number of ske wed loci. Most of the SSR primers produced dominant loci; however co-domina nce was observed with loci derived from three primers. A new genetic map wa s produced by combining the segregation data for the ISSR markers and data for the RFLP, RAPD and isozyme markers from the previous map and creating g enetic linkages among all the markers using JoinMap 2.0 mapping software. T he new map has an improved distribution of markers along the linkage groups with fewer gaps, and marker order showed partial or complete conservation in the linkage groups. The incorporation of ISSR markers into the genetic l inkage map demonstrates that ISSR markers are suitable for genetic mapping in Citrus.