DCARv2 2017 MITE insertion markers JBrowse GFF3

Coordinates of 4028 DcSto insertions belonging to 14 families were compared to coordinates of ca. 32 thousand genes annotated in the carrot reference DH1 genome assembly (Iorizzo et al., 2016; NCBI accession LNRQ01000000). 609 gene-associated DcSto insertion sites localized in introns were identified, of which 209 were manually selected for development of ILP markers. The criteria for initial selection were as followed: insertion sites were (1) free from any other annotated repetitive sequences, (2) present in introns not longer than 3.7 Kb, and (3) evenly distributed over each chromosome. Primer3 (Untergasser et al., 2012) and Primer-BLAST (Ye et al., 2012) were used to design PCR primer pairs anchored in exons flanking introns harboring the selected DcSto insertions. Primer pairs were designed to amplify fragments in a 400–3,700-bp range. The optimal annealing temperature was set to 58°C; and the size and GC content ranged from 18 to 23 bases and 40 to 60%, respectively.

Primer pairs from supplementary table 2 were located on the carrot genome by in silico PCR.

Data from this analysis can be viewed in JBrowse here.