Magextractor

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.

Total DNAs were extracted from blood samples by using a standard phenol-chloroform extraction. A fragment of 546-bp from the mtDNA D-loop region was amplified using PCR. The primers used were L16750 (5′-AGGACTACGGCTTGAAAAGC-3′; Akishinonomiya et al., 1994 (link)) and H522 (5′-ATGTGCCTGACCGAGGAACCAG-3′; Liu et al., 2006 (link)). The numbers in the primer names indicate the homologous positions of the 3′ end of the primers on the mtDNA sequence described by Desjardins and Morais (1990) (link). The PCR reaction was performed using the GeneAmp PCR System 9700 (Applied Biosystems, CA, USA) and the following mixture consisted of 100 ng of template DNA, 1×PCR reaction buffer, 4 pmol of each primer, 400 μmol of each dNTPs, and 1 U of exTaq polymerease (TaKaRa, Otsu, Japan). The thermal profile included an initial denaturation at 94°C for 1 min followed by 30 cycles, each of which included denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min, with a final extension step at 72°C for 7 min. The PCR products were isolated from 1% agarose gels and purified with Mag Extractor (TOYOBO, Osaka, Japan). The BigDye terminator cycle sequencing kit v. 3.1 (Applied Biosystems) and ABI Prism 3130xl genetic analyzer sequencer (Applied Biosystems) were used for sequence analysis.