title: R-ISSR - for fingerprinting, mapping and identification of new genomic loci in rye (secale cereale l.).
abstract: The results of the research confirming the possibility of applying various combinations of RAPD and ISSR primers in one multiplex PCR and the generation of a new type of R-ISSR products for the rye genome were presented in this work. The following was applied in the research: five rye genotypes including two inbred lines (153/79-1 and Otl-3), hybrid F1 and two bulks (tolerant and susceptible) formed from recombinant inbred lines - RILs (F9) varying in the response to abiotic stress caused by nutrient deficiencies at the seedling stage. While evaluating the possibility of applying R-ISSR to the assessment of the rye variability, five of its genotypes were amplified separately with the RAPD and ISSR primers in each PCR reaction. These primers were combined in R-ISSR amplifications. The products of RAPD, ISSR and R-ISSR amplification were separated in 1.5% agarose gel. 32 R-ISSR combinations were examined, combining 20 and 8 selected RAPD and ISSR primers, respectively. 658 loci were amplified, including 230 RAPD, 180 ISSR and 271 R-ISSR, including 157 new loci. Over 91 loci were found, with an identical electrophoretic mobility for three methods. It was shown that R-ISSR products with electrophoretic mobility on agarose gels, identical to the co-migrating RAPD or ISSR, are not products of RAPD or ISSR, but they possess sequences of heteroamplicons - R-ISSR. The occurrence of sequences of primers used to R-ISSR was demonstrated while sequencing seven selected products of the above type. The ISSR primers with a low Tm were proven to generate repeatable fingerprints in the thermal profile of the reaction specific for RAPD and combined with the RAPD primer - repeatable R-ISSR profiles. A similar range of variability as described in RAPD or ISSR was observed in the R-ISSR profiles. The correlation coefficient between genetic similarity matrices for five rye genotypes, calculated with the Mantel test, amounted to rABC = 0.870.
authors.affiliation: Department of Plant Genetics, Breeding and Biotechnology West Pomeranian University of Technology in Szczecin, 71-434 Szczecin, Poland msmolik@zut.edu.pl
authors.forename: M
authors.initials: M
authors.lastname: Smolik
mesh_terms: D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods; D017931:DNA Primers; D056426:Genetic Loci; D005819:Genetic Markers / Q000235:genetics; D005838:Genotype; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods*; D012434:Secale / Q000235:genetics*
chemical_list: D017931:DNA Primers; D005819:Genetic Markers