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Supporting Experimental Procedures
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Methods S1: hiTAIL PCR to Determine Location of Transgene Insertion Site
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High-efficiency thermal asymmetric interlaced (hiTAIL) PCR (Liu and Chen, 2007) was
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utilized to determine the T-DNA flanking sequences for each Sbmyb60 overexpression line to
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identify the point of insertion into the genome. DNA was extracted from one plant from each
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transgenic line using a cetyl-trimethyl-ammonium bromide-based DNA extraction buffer
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(Rogers and Bendich, 1985). A series of nested left (LB) and right (RB) border primers were
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designed using Primer3 Plus (version 2.3.6) (Untergasser et al., 2012), which included RB0: 5′-
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GGCACTGGCCGTCGTTTTACA-3′, RB1: 5′-GGAAAACCCTGGCGTTACCCAAC-3′, and
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RB2: 5′-GCAGCCTGAATGGCGAATGC-3′ or LB0: 5′-AGTCCCTATAGATCCCCCGA-3′,
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LB1: 5′-AAAAACGTCCGCAATGTGTTA-3, and LB2 5′AGCGTCAATTTGTTTACACCAC-
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3′. The pre-amplification, primary TAIL, and secondary TAIL reactions and thermal cycling
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parameters were carried out as described previously (Liu and Chen, 2007) in a Biometra thermal
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cycler (Göttingen, Germany) using ExTaq PCR master mix (Takara, Mountain View, CA).
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Secondary TAIL products were sequenced using the M13F and M13R primers (Operon,
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Huntsville, AL). The consensus sequence generated from each line was aligned to the Sorghum
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bicolor v2.1 genome using BLAT (version 35) (Kent, 2002).
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Methods S2: Construction of SbMyb60 T-DNA
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SbMyb60 was amplified with the gene specific primers SbMyb60F 5'-
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GAGTAATCAGAGCCACGAAC-3' and SbMyb60R 5'- CCTAGACATACGAACTTGCC-3'
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from genomic DNA collected from Tx623 sorghum plants. The PCR product was re-amplified
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with the following primers: SbMyb60NcoI-F 5'-
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GAGCCATGGGGCGAGGGCGAGCGCCGTG-'3 and SbMyb60XbaI-R 5'-
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TTAGAGCTCAGCCTTCGATCTCAACGGGACG-3', to introduce restriction sites and cloned
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into the NcoI and SacI restriction sites of the pCR™-Blunt vector using the Zero Blunt Cloning
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Kit (Invitrogen, Carlsbad, CA). The fidelity of the insert was confirmed through DNA
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sequencing using the M13F and M13R primers. The insert containing SbMyb60 was extracted
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from pCR™-Blunt as a NcoI/SacI fragment, and then ligated into NcoI/SacI sites of the pRTL2
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vector using T4 ligase. The recombinant pRTL2 vector was digested with HindIII to extract a
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fragment containing the SbMyb60 sequence under the control of cauliflower mosaic virus (CMV)
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E35S promoter. The HindIII fragment was cloned directionally into pZP211 (Hajdukiewicz et
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al., 1994).
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