314 inbred lines 541 and Ot1-3 is different and the genetic distance between them is significant (Myśków et al. 2001). The line 2020LM was kindly provided by L. Madej from the Institute of Plant Breeding and Acclimatization, Radzików. Its pedigree is unknown, however it proved to be genetically distant from line 541 (Myśków et al. 2010). Maternal line of both mapping populations (line 541) possessed normal cytoplasm, so this type of cytoplasm was present in all RILs analyzed. All recombinant inbred lines were genotyped with the use of Diversity Array Technology essentially as described by Bolibok-Brągoszewska et al. (2009). Genomic repre- sentations of individual RILs were prepared using the PstI/ TaqI complexity reduction method, Cy3 or Cy5 labeled and hybridized to the rye genotyping array 2.0. Two differently labeled representations were hybridized to each slide. Tiff images of slides acquired with fluorescent microarray scanner (Tecan LS300,Grödig, Salzburg, Austria) were automatically analyzed with dedicated software (DArTsoft version 7.3, DArT P/L, Yarralumla, Australia, unpublished) to identify and classify polymorphic markers. Markers with Q> 80% and a call rate of at least 90% were selected for subsequent analyses. Additionally, a set of polymorphic PCR-based sequence specific markers (SCAR, STS) with known chromosomal localization were analyzed in both populations. Between them, there were markers described by Stracke et al. (2003), Milczarski et al. (2007) and two newly developed SCAR markers obtained by sequencing RAPD fragments located on the 4RL chromosome (Stojałowski et al. 2004). Sequences of primers for these markers are as follows: Xscsz23L500 – CGAAGCAGACCCGTACACAT / CGAAGCAGACGAGGATATTT Xscsz670L900 – GGACTGGAGTGATAAGGACG / GGACTGGAGTCTAATATATG. Segregations of all the markers significantly deviating from the 1:1 ratio (by Chi2 test, at P<0.01) were not taken under consideration. In order to determine whether a given recombinant line carries the non-restorer or restorer allele in the Rfc1 locus, each genotype of both mapping populations was testcrossed with the male sterile genotype. As a source of sterility- inducing cytoplasm, the male sterile single cross between two related inbred lines (CMS-C line 585/92-1-2 and non- restorer line 585/92-6-1) was used. Male fertility/sterility of plants obtained as a result of the testcrosses between the male sterile source of CMS-C and a given recombinant inbred line was assessed in the field conditions in Szczecin. The necessary amount of hybrid seeds was gained for crosses between source of CMS and 81 RILs of population 541×2020LM and 107 RILs 541×Ot1-3. From 5 to 12 plants for each of the 188 cross combinations were grown J Appl Genetics (2011) 52:313–318 during the vegetation season 2009/2010 with 25×25cm spacing and at the flowering time all plants were assessed visually with the use of the Geiger and Morgenstern (1975) scale. Additionally, results of visual scoring were verified by evaluation of seed setting on spikes bagged on each plant before flowering. Male fertile progeny of a testcross indicated that the analyzed RIL had a restorer allele in the Rfc1 locus, while male sterile one – a non-restorer allele. Occasionally, observed segregation for male fertile and male sterile plants indicated the heterozygous character of the RIL with respect to the Rfc1 locus. Identification and localization of markers tightly linked with the Rfc1 gene were performed with the use of the JoinMap 3.0 software (Van Ooijen and Voorrips 2001). First, for both mapping populations linkage groups were constructed at the LOD = 15. Then, within the group containing the Rfc1 locus the “show strong linkages” command was applied for the selection of markers with recombination frequency to the Rfc1 not exceeding the 0.05 level. Segregations of the selected markers as well as that of the Rfc1 gene, were used for map construction. In order to allow for comparisons of localization of the markers present in the constructed maps to the other, formerly published genetic maps of rye, a STS marker Xpsr119 was included into the analysis. This PCR-based marker was developed from a RFLP probe (Milczarski et al. 2007) and it was polymorphic in both studied RILs populations. Molecular markers common for both mapping popula- tions were applied for construction of an integrated genetic map and estimation of average frequencies of recombina- tion per meiosis between these markers and Rfc1 locus. The assessment of male fertility in the testcrosses has revealed a distribution of progenies into two main pheno- typic classes: male sterile and male fertile, indicating that analyzed RILs belonged to the genotypic classes of maintainers and restorers, respectively (Table 1). Partial restoration was observed occasionally in individual plants, but never in whole progenies. The proportion of both genotypic classes (maintainers: restorers) in studied hybrids remained in agreement with monogenic model of inheri- tance in a RIL population (1:1). There were, however, some exceptions found in both mapping populations. Five RILs in the population 541×Ot1-3 and four RILs in the 541×2020LM showed heterozygous character giving seg- regating progenies in the testcrosses. These nine lines were excluded from mapping analyses. Restoration of male fertility in plants with the C cytoplasm stays under control of at least 2–3 nuclear genes (Łapiński 1990; Stojałowski et al. 2004). On the other hand, monogenic inheritance of male fertility restoration in rye with CMS-C was reported in some cross combinations (Łapiński 1990). Likely, in both mapping populations analyzed in this study, only one restorer gene showed