The LD222 NILs were grown in 1 L pots in the glasshouse under long day conditions (16 h light: 8 h dark). We harvested flag leaf, as well as glume, lemma, palea, and anthers at Waddington stage 7.5–8 (Waddington et al. 1983 (link)) from central spikelets of the main spike of four plants for each NIL (four biological replicates). We also collected grains from florets one and two at 3, 10, and 20 dpa from the four central spikelets (eight grains per sample) of the main spike of four plants for each NIL (four biological replicates). All tissues were immediately frozen in liquid nitrogen and stored at − 80 °C.
Grains were homogenized using mortar and pestle with liquid nitrogen, while other tissues were homogenized in SPEX CertiPrep 2010–230 Geno/Grinder (Cat No.: 12605297, Fischer Scientific) using 5-mm steel beads (Cat No.: 69989, Qiagen). For grain samples, RNA was extracted following the protocol described in Adamski et al. (2021 (link)). For non-grain tissues, we used the Spectrum Plant Total RNA kit (Cat No.: STRN250-1KT, Sigma) following the manufacturer’s protocol.
Grains were homogenized using mortar and pestle with liquid nitrogen, while other tissues were homogenized in SPEX CertiPrep 2010–230 Geno/Grinder (Cat No.: 12605297, Fischer Scientific) using 5-mm steel beads (Cat No.: 69989, Qiagen). For grain samples, RNA was extracted following the protocol described in Adamski et al. (2021 (link)). For non-grain tissues, we used the Spectrum Plant Total RNA kit (Cat No.: STRN250-1KT, Sigma) following the manufacturer’s protocol.
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