A breeding line “Chogokuwase” (extreme earliness in Japanese) was developed from an early transgressive plant in a segregating population from a cross of middle-early varieties, a Japanese variety “Minaminokomugi” and a Korean variety “Geurumil”. Flowering time of “Chogokuwase” is much earlier, by two to four weeks, compared to an intermediate flowering variety "Norin 61" which has been a leading variety for a long time in southwestern Japan. To identify causal genes for extreme earliness in “Chogokuwase”, we conducted map-based gene identification.

Genetic analysis using a RILs population of “Chogokuwase” x “Kinuiroha” (a middle-early variety) showed a segregation of early and middle-early plants at a ratio of 1:3, indicating the involvement of two major genes. By bulked segregant analysis, they were localized on the distal part of 3BL and 3DL chromosomes. According to the study of early mutant of einkorn wheat, earliness is conferred by the homoeologous region in 3AmL chromosome which contains a circadian clock gene PCL1-3Am (Mizuno et al. 2012, Gawronski et al. 2014). Then, the PCL1 was considered a good candidate in “Chogokuwase”. As expected, “Chogokuwase” was found to carry non-functional alleles for all three PCL1 homoeologs and they were closely associated with extreme earliness in the segregating populations (Mizuno et al. 2016). The fact that “Kinuiroha” carries the nonfunctional allele for PCL1-3A only was consistent with the genetic analysis in RILs population. To validate this result by mapbased study, we focused on PCL1-3B due to rich sequence information and polymorphism in chromosome 3B. Mapping populations were derived from a cross of “Chogokuwase” x “CKRIL54” (RIL carrying the functional allele for PCL1-3B and non-functional alleles for PCL1-3A and PCL1-3D). In the mapping populations, segregation of early and middle-early plants fitted to a ratio of 1:3, clearly confirming the involvement of a single gene. The earliness effect of the gene was estimated to be around ten days. In the initial mapping, the gene was mapped to a 10.8cM region in the distal part of 3BL, which was consistent with previous studies. Subsequently, the candidate region was narrowed down to a 445kb region through the screening of recombinants and their progeny tests. According to the gene annotation in Chinese Spring genome sequence, this region contained seven predicted genes including PCL1-3B. Among them, PCL1-3B was the only gene which transcribed intact mRNA in RNAseq analysis. Thus, we concluded that the earliness gene on 3BL chromosome is PCL1-3B.