S1 analysis of long PCR heteroduplexes: detection of chloroplast indel polymorphisms in Fragaria

S1 analysis of long PCR heteroduplexes was found to be an effective method for detecting phylogenetically informative indel (insertion/deletion) polym orphisms in the highly conserved strawberry chloroplast genome. In this bro adly applicable method, long-range PCR products containing heteroduplex DNA molecules generated from mixed-template amplifications are subjected to SI nuclease digestion followed by separation and visualization on an agarose gel. The presence of fragments resulting from S1 digestion of mismatch loop s in heteroduplex molecules is indicative of indel polymorphism between the template sources. Upon analysis of 13-kb heteroduplex-containing PCR produ cts spanning the petA-psbB chloroplast genome region in Fragaria vesca and Fragaria moschata, two indels distinguishing these species were detected, a nd subsequently localized to the psbJ-psbL and rpl20-rps18 intergenic regio ns. Comparative sequencing of these regions revealed that F: moschata resem bled Fragaria viridis, but differed from F: vesca, Fragaria nubicola, and a closely related outgroup representative, Duchesnea indica, by a IO-bp dele tion in the psbJ-psbL region and a IO-bp insertion in the rpl20-rps18 regio n. Thus, of the three diploids (2n = 2x = 14) examined, F. viridis is favor ed over F: vesca and F: nubicola as the most likely chloroplast genome dono r to hexaploid (2n = 6x = 42) F: moschata.