Genome-wide distribution of H3K4me3 in Medicago truncatula wild type R108 and Mting2-1 mutant.

Medicago truncatula (Medicago) flowering is promoted by winter cold (vernalization) followed by long day photoperiods (VLD). By analysing a Tnt1 insertion mutant and 17 CRISPR-Cas9 gene-edited lines with delayed flowering, we identified INHIBITOR OF GROWTH (ING) 2 (MtING2) encoding a plant homeodomain (PHD) zinc finger and predicted H3K4me2/3 epigenome reader in Medicago. There are two ING genes in most plants, but their physiological role has not been described. Mting2 mutants had many developmental abnormalities including delayed flowering particularly in VLD, compact architecture, abnormal leaves with extra leaflets but no trichomes and smaller seeds and seed barrels. RNA-Seq experiments indicated that >7000 genes are mis-expressed in the Mting2 mutant consistent with its strong mutant phenotypes. Mting2 did not show reduced accumulation of the strong floral promoter FLOWERING LOCUS T-LIKE gene, MtFTa1, but did have increased expression of MtTFL1c, consistent with the delayed flowering of the mutant. ChIP-Seq analysis identified >5000 novel H3K4me3 locations in the genome of Mting2-1 mutant compared to wild type, which overlapped with some differentially expressed genes. Overall, our study has uncovered an important physiological role of a plant ING gene in development, flowering and gene expression, which likely involves an epigenetic mechanism. Overall design: Germinated seeds from wild type R108 and Mting2-1 mutant plants were grown under vernalised long day conditions in sterile culture. Whole aerial tissues were harvested from 14–17-day-old plants at 4 h after dawn (ZT4). Three biological replicates for each genotype were harvested. Chromatin Immunoprecipitated DNA were extracted and a total of 6 ChIP-seq libraries were constructed using NEBNext ChIP-Seq Library Prep Kit for Illumina (NEB, USA) and sequenced.