Mapping of Genomic Regions Underlying the Early Flowering Trait in ‘RE2’, a Mutant Derived from Flax (Linum usitatissimum L.) Cultivar ‘Royal’

Canada is a world leader in flax production, and the expansion of the crop into the northern region of the prairies requires early flowering, consequently early maturing cultivars to overcome the frost damage. New sources of variation for flowering time thus hold great interest. Flax genomics resources including chromosome level assembly are now sufficiently developed to examine traits with complex inheritance. An early flowering mutant ‘RE2’ was selected from cultivar ‘Royal’ after treatment with 5-Azacytidine (5-AzaC). The mutant line flowered nearly seven to 13 days earlier than the progenitor ‘Royal’. A large recombinant inbred line (RIL) population encompassing 656 lines, derived from ‘Royal’ x ‘RE2’ was used to identify the potential genomic region underlying the trait. Firstly, the RIL population was phenotyped for early vigour, days to- start of flowering, full flowering, maturity and height in three field seasons (2015, 2016 and 2017) using a modified augmented design type 2, and once in the growth-cabinet. Secondly, the distributional extremes for flowering time identified from the RIL population were subjected to sequencing based bulked segregant analysis. Thirdly, the QTL-seq bioinformatics pipeline (Takagi et al. 2013) was used for the identification of SNP, which were annotated using SnpEff. QTL-seq pipeline identified a SNP upstream of the flax gene homologous to Arabidopsis LUMINIDEPENDENS. Later, the sequencing data were reanalysed with customized variant calling steps succeeded by statistical analysis using QTLseqr (Mansfeld and Grumet 2018), a recent improved pipeline. QTLseqr detected two genomic regions having significant association with early flowering trait on chromosomes 9 and 12. The variants in these regions were found to be associated with genes encoding LATE EMBRYOGENESIS ABUNDANT (LEA) HYDROXYPROLINE-RICH GLYCOPROTEIN FAMILY, MAINTENANCE OF MERISTEMS-LIKE, CYTOCHROME P450 87A3 and PHLOEM PROTEIN 2-A12, based on homology analysis. As ‘RE2’ was derived from the population resulting from the treatment of ‘Royal’ with the demethylating agent 5-AzaC, whole genome bisulfite sequencing data were generated to identify variation in methylation patterns and its association with early flowering. A total of 260,193 cytosines were transformed from methylated state in the late flowering bulk to the unmethylated state in the early flowering bulk, potentially owing to the hypomethylating action of 5-AzaC. Out of the 127 significant differentially methylated regions (DMRs) detected, 59 were overlapping with genes, and 35 DMRs and 33 DMRs were within the upstream- (5kb interval) and intergenic regions, respectively. Interestingly, a cluster of significant DMRs were also present on chromosome 12. Three DMRs (on chromosomes 1, 6 and 7) were overlapping the genes whose homologues encode FASCICLIN-LIKE ARABINOGALACTAN group of proteins, and two DMRs (on chromosome 12) were present upstream to SUPPRESSOR OF FRI 4 and FRIGIDA-ESSENTIAL 1. This study is first of its kind in flax, providing the basis for identifying novel epialleles underlying the early flowering phenotype