Platinum superfi dna polymerase invitrogen kit

RNA was extracted from the shoot of young seedlings at 10 DAS (3 seedlings for both WT and mutant) using the “GeneJET Plant RNA Purification Mini Kit” (ThermoFisher Scientific, Waltham, MA, USA) after powdering the plant tissue with liquid nitrogen. All the extractions were performed following the protocol provided by the manufacturer. RNA samples were long-stored at −80 °C. After extraction, the integrity of RNA was checked on 1% agarose gel. RNA was then treated with DNAse (TURBO DNA-free™ Kit, Invitrogen, Waltham, MA, USA).
RNA was retrotranscripted using the Thermo Fisher Scientific “Maxima First Strand cDNA Synthesis” kit, following the protocol provided by the manufacturer. Specific primers for the housekeeping gene orange pericarp-1 (orp-1) were used to standardize the cDNA concentration. Specific primers were designed to evaluate the gene expression level of the candidate gene GRMZM2G569948. The gene-specific primers are listed in Table S1. PCR was performed using the Platinum™ SuperFi™ DNA Polymerase Invitrogen Kit with 34 cycles of amplification. Amplification products were visualized on 1.2% (w/v) agarose gels with ethidium bromide staining.

The physical position of the molecular markers umc1060 and umc1221, and of GRMZM2G569948, was determined using the Maize GDB, which refers to B73 RefGen_v3 (https://www.maizegdb.org/ accessed on 01 October 2021) [46 (link)]. For the sequence analysis of the candidate gene, the genomic DNA was extracted from normal and homozygous stocky1 seedlings (3 seedlings for both WT and mutant). Two pairs of specific primers indicated in Table S1 were used to perform PCR using the Platinum™ SuperFi™ DNA Polymerase Invitrogen Kit. The reaction mix underwent an initial denaturation step at 94 °C for 2 min, 30 cycles of denaturation at 94 °C for 15 s, annealing at the specific primer temperature for 30 s, and extension at 72 °C for 30 s. Extension at 72 °C for 5 min was performed to complete the reaction. Amplification products were visualized on 1% (w/v) agarose gels with ethidium bromide staining. The PCR products were sequenced with Sanger method and the sequences were compared with the coding sequence of KCS of the B73 line (ZEAMMB73_Zm00001d016438) obtained from www.maizegdb.org/ on 20 October 2021 using this tool: https://www.ebi.ac.uk/Tools/msa/clustalo/ accessed on 20 October 2021.