Genotyping Tomato Chromosome 9 Markers

For saturation of the shr locus, 14 SSR markers from Kazusa DNA Research Institute (http://www.kazusa.or.jp/tomato/, Shirasawa et al., 2010a (link),b (link)),10 SSR markers from Veg Marks a DNA marker database for vegetables (http://vegmarks.nivot.affrc.go.jp, Last accessed in 2013) and 7 CAPS markers viz., At3g63190, C2_At4g02580, C2_At2g29210, C2_At4g02680, C2_At1g02910, C2_At4g03200, and U228448 (http://solgenomics.net) that were specific to chromosome 9 were selected and screened for polymorphism between the parental lines of mapping population (Supplementary Tables 2–4 for marker details). For amplification of CAPS region, 30 ng genomic DNA, 1 μL of 5 pM/ μL primer, 1X PCR buffer (10 mM Tris, 5 mM KCl, 1.5 mM MgCl₂, 0.1% (w/v) gelatin, 0.005% (v/v) Tween-20, 0.005% (v/v) Np-40, pH 8.8, 0.2 mM dNTPs and 1 μL Taq polymerase were used. After confirming PCR amplification for CAPS locus by agarose gel electrophoresis, the PCR amplicons of the CAPS markers were digested using ApoI, Hinf I, DraI and MspI (Fermentas) enzymes. Digestion reactions performed according to the supplier's manual and the products were separated on 3.5% (w/v) agarose gels.