Fine-tuning of heading time by earliness per se effect due to multi-allelic variants in VRN-B3 locus of hexaploid wheat.

Main conclusion: Wheat VRN-B3 contains multi-allelic variants conferring earliness per se effects and can generate a new allele by recombining multiple alleles, highlighting its importance for the fine-tuning of heading time. Fine-tuning of heading time is required for breeding well-adapted varieties of wheat in regional environments. The VRN-B3 locus, which encodes the FT-B1 gene, is known as a vernalization gene. In this study, we analyzed two alleles in the VRN-B3 region: QHt-7B_Zen of a Japanese variety (Zenkouji-komugi) and QHt-7B_spelt of a spelt wheat strain (st. Rumania). Phenotypic evaluation of near-isogeneic lines (NILs) of 'Chinese Spring' (CS) under long-day (16 h) conditions showed that QHt-7B_Zen and QHt-7B_spelt conferred approximately 3.9 d earlier and 3.0 d later heading time compared with CS, respectively. Differences in heading times among NILs were also observed for fully vernalized plants under long-day conditions, indicating their earliness per se effect. Both QTLs behaved as single genes with incomplete dominant effects, and fine-mapping showed that FT-B1 was responsible for heading time. Droplet digital PCR analysis revealed that QHt-7B_Zen contained three copies of FT-B1, similar to the CS. QHt-7B_spelt had one FT-B1 copy with 14 substitutions, a 15 bp insertion in the 4.8 kb promoter region, and one amino acid substitution in the third exon, which could be designated as a novel allele, Vrn-B3f. Furthermore, a new allele with two FT-B1 copies conferring an intermediate heading time between the parents was created by the recombination of FT-B1 copies of CS and NIL for QHt-7B_spelt. Our findings indicate that fine-tuning heading time is possible through the versatility of the VRN-B3 locus, which can generate multi-allelic variants that have both vernalization and earliness per se effects in wheat.