**4. Discussion**

Baegilmi is an extremely early heading Korean rice cultivar that can be incorporated in diverse double cropping systems to improve land use efficiency [14]. To genetically dissect the early heading characteristics of Baegilmi, we used a RIL population derived from the cross between Koshihikari and Baegilmi and identified the chromosomal locations harboring the genes controlling DH. We identified three major QTLs for DH, of which the allele for early heading was contributed by Baegilmi at *qDH6* and *qDH7*, and by Koshihikari at *qDH3*. As pyramiding of the alleles for early heading at each QTL was effective in accelerating heading, the Koshihikari × Baegilmi RIL population provides useful breeding lines with different allelic combinations of the three QTLs conferring varying DH, ranging from 71 days to 98 days under the natural long day condition in Wanju (36◦ N), Korea (Figure 3b). Therefore, the RIL population would be useful for selecting promising breeding lines potentially inheriting the good eating quality of Koshihikari with various heading dates that can be adapted in different environments and cropping patterns. To support this idea, we are currently evaluating the RILs with different allelic combinations of the three DH QTLs in terms of important agronomic traits such as yield performance and grain quality.

All three DH QTLs identified in this study harbor previously cloned heading date genes, i.e., *Hd1* [7], *Ghd7* [10], and *Hd16* [23] at *qDH6*, *qDH7*, and *qDH3*, respectively. *Hd1*, the ortholog of Arabidopsis *CO*, is the first heading date gene cloned in rice [7]. While *Hd1* accelerates heading under short day by upregulating *Hd3a*, it downregulates *Hd3a* under long day and represses heading [6–8,25]. Due to this dual function of *Hd1* depending on daylength, nonfunctional *hd1* alleles confer early heading phenotype under long day as the loss-of-function of *Hd1* de-represses *Hd3a* and promotes heading [26,27]. The nonfunctional *hd1* alleles have played an important role in expanding rice cultivation in high latitude areas with natural long day conditions where early heading is critical for rice plants to mature before the cold winter [27]. *Hd1* sequencing revealed that Baegimli carries a nonfunctional *hd1* allele due to the 43 bp deletion in exon 1 causing a frameshift (Figure 5b). The 43 bp deletion in *Hd1* was first reported in the γ ray mutant cultivar HS66 [7], and has been observed in many rice cultivars grown in high latitude areas such as Jilin (44◦ N) and Liaoning (41◦ N) of China [28] and Italy (35–47◦ N) [27]. Among the 295 Korean rice cultivars screened in this study, 21 including Baegilmi carried the nonfunctional *hd1* allele due to the 43 bp deletion and showed earlier heading phenotype compared to those without the 43 bp deletion (Figure 5g). As there are many other nonfunctional *hd1* alleles arising from frameshift or premature stop codon at di fferent genic positions [22,26–28], further work is underway to define additional allelic variation in *Hd1* among the Korean rice cultivars.

Similar to *Hd1*, *Ghd7* encoding a CCT (CO, CO-LIKE, and TIMING OF CAB1) domain protein delays heading under long day by downregulating *Hd3a* [10]. In addition, similar to nonfunctional *hd1*, nonfunctional *ghd7* alleles such as *ghd7-0* (full gene deletion) and *ghd7-0a* (premature stop codon) accelerate heading under long day and contribute to the adaption of rice in high latitude areas [10,28]. Relative to Koshihikari, Baegilmi has a 1901 bp insertion in the promoter region (−228 bp from the start codon) of *Ghd7* (Figure 5c). The 1901 bp insertion is identical to that reported in *Ghd7* from a Japanese cultivar Sorachi (GenBank ID LC472533) with 449 bp long terminal repeats at each end and 5 bp (AGGTA) target site duplication (Fujino and Yamanouch 2020). This allele has been mainly found in rice cultivars bred in Hokkaido (42–45◦ N) of Japan, providing an important genetic source for rice adaption in high latitude [24,29]. Among the 295 Korean rice cultivars screened in this study, Baegilmi was the only cultivar carrying the loss-of-function *ghd7* allele due to the 1901 bp retrotransposon insertion (Figure 5g), suggesting that this allele would provide a valuable genetic source to accelerate heading in breeding programs. Interestingly, the same retrotransposon insertion has been also found in exon 2 of *Hd1* from a Taiwanese landrace Muteka (GenBank ID KR230393), creating a loss-of-function *hd1* allele [30]. This illustrates that transposable elements can play important roles in functionally diversifying the heading date genes.

*Hd16* encoding casein kinase I delays heading under long day by activating *Ghd7* through phosphorylation, and the missense mutation in exon 10 of *Hd16* from Koshihikari decreases the kinase activity of Hd16, thus accelerating heading under long day [23]. The screening of over 300 worldwide rice cultivars and 30 wild rice accessions revealed that the Koshihikari-type *Hd16* allele is found only in Japanese *japonica* cultivars [23]. Among the 295 Korean rice cultivars, only eight (i.e., Manan, Boseok, Jinkwang, Pungmi, Pungmi 1, Cheonga, Jungsaenggold, and Jopum) carried the Koshihikari-type *Hd16* and showed earlier heading phenotype compared to those carrying the Nipponbare-type *Hd16* (Figure 5g). These cultivars would provide useful genetic sources for developing early heading rice cultivars.

We previously reported that Baegilmi was developed by chemically mutagenizing Koshihikari [14]. However, it is extremely unlikely that the EMS mutagenesis would have created the above-mentioned sequence polymorphisms in *Hd1*, *Ghd7*, and *Hd16* that had been reported previously. This indicates that in the initial stages of breeding Baegilmi, the Koshihikari seed stock used for mutagenesis might have been contaminated during seed handling (e.g., threshing) with a di fferent germplasm which may carry all or some of the previously known alleles, i.e., the HS66-type *Hd1*, the Sorachi-type *Ghd7*, and the Nipponbare-type *Hd16*. We currently do not know the exact source of the potential contamination. Although lacking a clear parentage, the practical value of Baegilmi as a cultivar is una ffected because Baegilmi carries a unique allelic composition of the three heading date genes and exhibits the second earliest heading date among the 295 Korean rice cultivars released in 1979–2017 [31]. Our study also suggests that developing cultivars with earlier heading would be possible by pyramiding the alleles for early heading at *Hd1*, *Ghd7*, and *Hd16*, as none of the 295 cultivars screened in this study carried the alleles for early heading at all three genes.
