Characterization of T-DNA integration sites within a population of insertional mutants of the diploid strawberry Fragaria vesca L.

Cultivated strawberry (Fragaria × ananassa) is an octoploid (2n=8x=56) species that belongs to the Rosaceae family and the high ploidy level makes genetic and molecular studies difficult. However, its commercial success because of its unique flavor and nutritious qualities has increased interest in the development of genomic resources. Fragaria vesca L. is a diploid (2n=2x=14) species with a small genome size (206 Mbp), short reproductive cycle, and facile vegetative and seed propagation that make it an attractive model for genomic studies. The availability of an efficient transformation methodology for Fragaria vesca has facilitated the use of a T-DNA mutagenesis system to develop a collection of several hundred insertional T-DNA mutants at Virginia Tech, using either of two commercially available vectors, pCAMBIA 1302 and 1304. In this study, we have used expression of the green fluorescent protein (GFP) as a tool to identify homozygous mutant lines. Three different approaches were conducted, first we identified 11 homozygous lines by PCR, then another 55 homozygous lines by absence of segregation of GFP expression in T2 seedlings, and finally we attempted to distinguish homozygous from hemizygous lines by relative GFP expression measured using a commercially available GFP meter. The latter methodology was unsuccessful due to uncontrolled variability in the readings. Continuing the characterization of our mutant population, we used thermal asymmetric interlaced PCR (TAIL-PCR) to obtain the nucleotide sequence of the genomic DNA regions that flank the T-DNA insertion sites in independent transgenic strawberry lines. Primers were designed that would amplify the derived strawberry flanking sequences in the two parents of an interspecific mapping population between the two diploid species, F. vesca x F. bucharica. The amplified products were sequenced and examined for the occurrence of SNPs (single nucleotide polymorphisms). The same primers were then used on the F2 mapping population. Segregation of SNP markers with previously mapped genetic markers allowed us to position 74 SNP markers, and hence their corresponding insertional mutants, on a well-populated genetic linkage map for the diploid strawberry. Finally, we analyzed the insertion site from more than 190 mutants looking at both the right and left borders of the T-DNA where microsimilarities of a few base pairs between ends of T-DNA and genomic DNA were observed, indicating that T-DNA integration had not occurred randomly in strawberry. We have also characterized the insertion sites through gene annotation found in the strawberry genome database.