Whole panicles from the field grown plants with two biological replicates from each of the three sub-plots for both WT and mutant, were sampled on dry ice at panicle initiation (28 DBH), heading, 5 DAH, 10 DAH, 20 DAH, 30 DAH and harvest (40 DAH).
Roots of Col-0 wild-type (WT), mto1, and tt4 mutants were sampled and analyzed.
The mutant transcripts were sampled from the inflorescence, and thus a priori may have had high ranks in tissues with transcriptomes more like the inflorescence than other tissues.
Young panicles (~ 3 4 cm in length) from WT, mutant and C4 were sampled in liquid nitrogen and pulverized cryogenically using a Multi-beads shocker (Yasui Kikai, Osaka, Japan) at 2000 rpm for 15 s.
Both morphant and mutant larval zebrafish were sampled at 120 hpf and fixed in 1.5% glutaraldehyde/1% PFA and 70 mM NaPO4, pH 7.2.
Spikelet hulls for WT, mutant and C4, 2 DBH were sampled and processed as previously described (Li et al. 2011) followed by dehydration in isoamyl alcohol.
Further study is required to determine if the KS1B genotype is common and stably fixed in the natural population or if it is a rare mutant that was sampled by chance or if it results from a recent mutation that occurred during maintenance of the virus in the laboratory.
For the fur mutant, cells were also sampled at 1, 5, 10, 20, 40, and 60 minutes after adding 2, 2'-dipyridyl to a final concentration of 160 uM.
If the wild-type progenitor of the mutant is available, this sample should be used to obtain the wild-type sequence.
In each case, S1 (5 µg) and P2 (20 µg) fractions for each mutant are boiled in Laemmli sample buffer that contains 5% β-mercaptoethanol (βME) and electrophoresed in 18% SDS PAGE gels.
Once mutants were identified, individual samples were prepared for sequencing.
