Genotyping and Marker Development for Strawberry MAGIC Population

Genotyping was performed using a set of 105 EST-SSR markers (Supplemental Table 1) for the F₁ and IC₁ populations during the development of the MAGIC population; the developed MAGIC population, which was composed of 338 IC₂ plants, was also genotyped with a set of 336 EST-SSR markers (Supplemental Table 2) that were polymorphic among the six founder parental cultivars. The 105 EST-SSR markers, which were used for genotyping the F₁ and IC₁ populations, were selected while focusing on the equal distribution of the strawberry linkage groups (Isobe et al. 2013 (link)); the average marker density was 15 markers per chromosome in F. vesca. Genomic DNA of F₁ and IC₁ was extracted from young leaves of each plant with a DNeasy plant mini kit (Qiagen, Valencia, CA, USA); genomic DNA of IC₂ was extracted using a Mag extractor (Toyobo, Osaka, Japan). PCR was performed in a 5-μL reaction volume using 0.6 ng of genomic DNA in 1× PCR buffer (Bioline, London, UK), 3 mM MgCl₂, 0.08 U of BIOTAQ DNA polymerase (Bioline), 0.8 mM dNTPs, and 0.4 mM of each primer. The PCR products were separated with an ABI 3730xl fluorescent fragment analyzer (Applied Biosystems, MA, USA), according to the polymorphic fragment sizes of the PCR amplicons. Polymorphisms were investigated using the Gene Marker software (Softgenetics, PA, USA), based on the presence and absence of the relevant peak.