Automated Design of PCR Primers and Polymorphism Discovery for Marker-Assisted Selection In Tomato

The objective of this project is to generate a set of well distributed molecular markers for whole genome selection in tomato breeding lines.  This was accomplished using the current assembly of the tomato genome sequence to design markers with known genomic positions.  Several checks for uniqueness were done to obtain PCR primers that produce a single band with a high success rate.  The assembled sequence was masked to exclude known repeats and blasted against itself to mask non-unique sequences.  The filtered sequences were divided into segments of approximately 100 kilobases and, where unmasked sequence was available, PCR primer pairs were designed for each segment.  Python scripts were used as a final check to ensure that the primers match unique sequence and iteratively search for additional primer pairs when the uniqueness check fails.  Primers were excluded that had blast hits to the assembled genome sequence greater than 75 percent of their length or hits matching the last five bases on the 3’ end.  A total of 480 primer pairs were synthesized and tested for amplification of a single band on the reference genotype.  The primer pairs with single band amplification were used on a panel of fresh market tomato lines.  The PCR products were pooled and SNPs were detected by ecotilling with Cel I enzyme.  Amplicons from primer pairs where SNPs were detected were sequenced and cleaved amplified polymorphic sequence (CAPS) markers were designed and validated.