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RFLP analysis of mitochondrial DNA was carried out with eight restriction enzymes BamHI, EcoRI, HaeIII, HindIII, MspI, PstI, SalI and XhoI, from which nine mitochondrial gene probes (atp6, atp9, atp1, cox1, nad3, nad6, nad9, pol-r, orf25) were hybridized, by means of digestion products, for seven species of the genus Secale. RFLP EcoRI/pol-r specific markers were determined for all the species of rye. To estimate the relationships among species, genetic pairwise similarities between them were estimated and a UPGMA dendrogram was constructed. The analysis separated the species into two groups. The first comprises the pair Secale sylvestre Host and S. cereale subsp. segetale Zhuk., exhibiting the greatest genetic similarity, that is, closest relationships. The second group is composed of S. strictum/Presl/Presl, S. strictum/Presl/Presl subsp. kuprijanovii/Grossh./ Hammer, S. strictum/Presl/Presl subsp. africanum/Stapf/Hammer, Secale cereale L. and S. vavilovii Grossh., with one clear subgroup comprising Secale strictum/Presl/Presl and S. strictum/Presl/Presl subsp. kuprijanovii/ Grossh./Hammer. The latter two species showed the highest genetic similarity to each other and relatively high genetic similarity to the remaining species in the group.
Employing FISH analysis as well as BLAST and CUSTAL W (1.82) programs, we investigated types of DNA nucleotide sequences building an additional heterochromatic band in 2R chromosomes of 3 lines of Secale vavilovii Grossh. The probes used in FISH analysis were designed based on the reverse transcriptase sequence of Ty 1-copia and Ту 3-gypsy retrotransposons and the 5S rRNA gene sequence. No hybridization signals from the reverse transcriptase probes were observed in the chromosome region where the additional band occurs. On the other hand, signals were observed after hybridization with the 5S rDNA probe, clearly suggesting the presence of that type of sequences in the analyzed heterochromatin band. Using BLAST and CUSTAL W programs, we revealed high similarity of the JNK1 sequence to the 5S rRNA gene from Hordeum chilense (HCH1016, HCH1018, 88%) and to a fragment of the 5S rRNA sequence of H. marinum (HMAR003, 97%). In addition, the same fragment of JNK1 was shown to be very similar to the part of the Angela retrotransposon (92%) as well as to the SNAC 426K20-1 transposon (89%) belonging to CACTA family, both from Triticum monococcum, and to Zingeria biebersteiniana pericentromeric sequences (78%). The similarity of JNK1 to those sequences may be accidental or the JNK1 may represent an ancient mobile genetic element that caught the 5S rRNA sequence. During the evolution those sequences might have been accumulated in the particular region on the 2R chromosome. Our results suggest that the additional heterochromatin band in chromosomes 2R of S. vavilovii is a collection of defective genes and/or mobile genetic elements.
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