Genetically directed differential subtraction chain products related to in vitro response of immature embryos of rye (Secale cereale L.): isolation, characterization, and expression analysis

摘要

Winter rye (Secale cereale L.) is known to be recalcitrant to tissue culture response (TCR). Moreover, the mechanisms controlling TCR are poorly recognized. In the present study, a Genetically Directed Differential Subtraction Chain (GDDSC) strategy was used to isolate genomic regions associated with TCR. Two pairs of bulks, R-NR and E > 90-E < 25, were prepared, and for each pair, the bulk was used both as a tester and as a driver. After eight rounds of subtraction, 45 unique GDDSC products were obtained. To verify the connection between GDDSCs and TCR two approaches were applied: Real-Time RT-PCR analysis and genetic mapping. The expression profiles of four out of six investigated products agrees with the phenotype and subtraction direction. Two from the developed GDDSC-SCAR markers showed polymorphism in lines L9 and L318, the parental components of a mapping population. The polymorphic SCAR GDDSC markers were mapped on the rye chromosome 4R (SCAR-GDDSC NR 440BamHI) and 6R (SCAR-GDDSC E < 25 340BamHI). The marker E < 25_340B9 was localized in the border region of QTL for embryogenic callus production.