Dna analyzer

Mutations in SlFOLK were identified by screening an EMS-mutagenized tomato population (Just et al., 2013 ) essentially as described in Okabe et al. (2011) (link). TILLING (Targeting Induced Local Lesions In Genomes) unlabeled external primers and internal primers 5′ labeled with IRDye 700 and IRDye 800 dye are listed in Supplementary Table S1 at JXB online. Induced point mutations were identified using the mismatch-specific endonuclease ENDO 1. Digested DNA fragments were separated on a Li-Cor DNA analyzer (LI-Cor, Lincoln, NE, USA). The mutation analysis was performed using PARSESNP (Taylor and Greene, 2003 (link)) and SIFT (Ng and Henikoff, 2003 (link)) software. Homozygous mutant plants were identified by sequencing of the tilled M₃ family. Phenotypic characterization was performed in M₄ plants homozygous for the folk-1 allele using the corresponding segregating individuals homozygous for the FOLK wild-type allele as control genotype.

AFLP analyses were carried out with the IRDye fluorescent AFLP kit for large plant genome analysis (LI-COR, Lincoln, NE, USA). We analyzed 64 AFLP selective primer combinations: EcoRI + AX₁X₂/MseI + CX₃X₂ (X₁ = A or C; X₂ = A, C, G or T; X₃ = A or T). The PCR products were separated by electrophoresis through 6% (w v⁻¹) denaturing acrylamide gels in a LI-COR DNA analyzer (LI-COR), according to the manufacturer’s instructions.

The AFLP analysis was performed as described previously (Vos et al., 1995 (link)), with minor modifications according to Klie et al. (2013) (link). For each sample, 100 ng of DNA was digested with 9 U HindIII (Fisher Scientific – Germany GmbH, Schwerte, D) and 3.5 U MseI (Fisher Scientific - Germany GmbH, Schwerte, D). The preamplification reactions were performed with specific primers that had an A as a selective base at the 3′ end [HindIII (5′-AGACTGCGTACCAGCTT-A-3′) and MseI (5′-GACGATGAGTCCTGAGTAA-A-3′)]. HindIII (5′-AGACTGCGTACCAGCTT-ANN-3′) primers with two extra selective bases and MseI (5′-GACGATGAGTCCTGAGTAA-ANNN-3′) primers with three extra selective bases were used for the final amplification. The HindIII primers were end-labeled with an infrared dye (either IRD 700 or IRD 800; Eurofins MWG, Ebersberg, D). In a single PCR reaction, labeled primers were used either as single primers or in combinations of two differently labeled primers (IRD 700 and IRD 800). In total, 21 selective primer combinations were analyzed (Table 1). The fragments were separated on 6 % polyacrylamide gels (Sequagel XR, Hessle, UK) using a DNA analyzer (LI-COR, Lincoln, Nebraska, USA) and automatically processed using the e-Seq-Software (V3.0, LI-COR, Lincoln, Nebraska, USA).