Computational protocol: Characterization of the FAD2 Gene Family in Soybean Reveals the Limitations of Gel-Based TILLING in Genes with High Copy Number

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Protocol publication

[…] Multiple sequence alignments were performed using the MEGA4 software package and the Clustal W algorithm. An unrooted phylogenetic tree was calculated with the neighbor-joining method (Saitou and Nei, ), and tree topology robustness was tested through bootstrap analysis of 5,000 replicates. [...] All M3 and M4 lines were analyzed for seed fatty acid composition using the two-step procedure as outlined by Kramer et al. (). At least four seeds per mutant from the M3 and M4 generations and 30 wild type Forrest seeds were randomly selected for fatty acid analysis. Briefly, each seed was broken/scratched and then loaded into one 16 mm × 200 mm tube with a Teflon-lined screw caps. Two milliliters of sodium methoxide was added into each tube and then the tube was incubated in a water bath at 50°C for 10 min. Afterward 3 ml of 5% (v/v) methanolic HCl was added into each tube after the tube was cooled for 5 min. Subsequently, the tube was incubated in a water bath at 80°C for 10 min, cooled for 7 min, and then 7.5 ml of 6% (w/v) potassium carbonate and 1 ml of hexane were added. Finally, the tubes were centrifuged at 1,200 g for 5 min to separate the layers. The upper layer was then transferred and used for the fatty acid analyses using a Shimadzu GC-2010 (Columbia, MD) gas chromatograph equipped with a flame ionization detector and a Supelco 60-m SP-2560 (Bellefonte, PA) fused silica capillary famewax column (0.25 mm i.d. × 0.25 μm film thickness). The helium carrier gas was maintained at a linear velocity of 23 cm/s. The oven temperature was programmed for 175°C for 22 min, then increased at 15°C/min to 225°C and held for 3 min. The injector and detector temperatures were set at 255°C. Peaks were identified by comparing the retention times with those of the corresponding standards (Nu-Chek-Prep., Elysian, MN; Supelco). The levels of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid were calculated using the statistical computing package (JMP Pro V12 software). [...] Young leaf tissue from mutants with high oleic acid content, as well as wild type Forrest, was used to extract DNA using the CTAB method (Rogers and Bendich, ) with small modifications. Specific primers were designed to amplify the fragments covering both the promoters and exons of two genes, FAD2-1A and FAD2-1B, using the extracted DNA as the template with 38 cycles of PCR amplification at 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min. The PCR products were purified by enzymatic clean-up, or the specific bands were cut from the agarose gels after electrophoresis using a Qiagen gel extraction kit (QIAquick). Then, the purified PCR fragments were sequenced at GENEWIZ (www.genewiz.com). The genotypes and specific mutations were determined by comparing the genomic cDNA and predicted protein sequences of the genes with wild type Forrest using DNASTAR Lasergene software. […]

Pipeline specifications

Software tools MEGA, Clustal W, JMP Pro, DNASTAR Lasergene
Applications Miscellaneous, Phylogenetics, Sanger sequencing
Chemicals Oleic Acid