SNP discovery and genetic mapping of T-DNA insertional mutants in Fragaria vesca L

As part of a program to develop forward and reverse genetics platforms in the diploid strawberry [Fragaria vesca L.; (2n = 2x = 14)] we have generated insertional mutant lines by T-DNA mutagenesis using pCAMBIA vectors. To characterize the T-DNA insertion sites of a population of 108 unique single copy mutants, we utilized thermal asymmetric interlaced PCR (hiTAIL-PCR) to amplify the flanking region surrounding either the left or right border of the T-DNA. Bioinformatics analysis of flanking sequences revealed little preference for insertion site with regard to G/C content; left borders tended to retain more of the plasmid backbone than right borders. Primers were developed from F. vesca flanking sequences to attempt to amplify products from both parents of the reference F. vesca 815 x F. bucharica 601 mapping population. Polymorphism occurred as: presence/absence of an amplification product for 16 primer pairs and different size products for 12 primer pairs, For 46 mutants, where polymorphism was not found by PCR, the amplification products were sequenced to reveal SNP polymorphism. A cleaved amplified polymorphic sequence/derived cleaved amplified polymorphism sequence (CAPS/dCAPS) strategy was then applied to find restriction endonuclease recognition sites in one of the parental lines to map the SNP position of 74 of the T-DNA insertion lines. BLAST search of flanking regions against GenBank revealed that 46 of 108 flanking sequences were close to presumed strawberry genes related to annotated genes from other plants.