Retrotransposon-based markers from potato monoploids used in somatic hybridization

Abstract

In an attempt to remove lethal and deleterious genes and enhance the heterozygosity of the potato genome, we developed several diverse somatic hybrids through the electrofusion of selected monoploids. Somatic hybrids and somaclones resulting from fused and unfused protoplasts, respectively, were verified with microsatellites. Molecular markers anchored in the Tst1 retrotransposon were used to examine polymorphisms in the regenerated plants and to reveal any somaclonal variation. Inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon display (sequence-specific amplified polymorphism (S-SAP), anchored in a retransposon) were examined on an ALFexpress DNA sequencer. Because of inconsistencies in the number and quality of bands revealed by the combination of either class of marker in combination with the ALFexpress, we cloned and sequenced 11 S-SAP bands to use as restriction fragment length polymorphism (RFLP) probes in Southern blot analyses of genetic relationships in our potato populations and among related Solanaceae. Readily scorable bands (n = 27) that separated somatic hybrids from parental monoploids and somaclones and grouped monoploids according to known genetic relationships were produced. Some of the probes could be used to differentiate tomato and Datura from potato. Sequence analysis of 5 cloned IRAP and 11 cloned S-SAP markers confirmed that they were anchored in the Tst1 retrotransposon. BLAST searches within GenBank produced 10 highly significant hits (5 nucleotide, 4 expressed sequence tag (EST), and 1 protein) within closely related Solanaceae, suggesting that Tst1 represents an old retroelement that was inserted before the diversion of genera within Solanaceae; however, most sequences were undescribed.