*3.2. Candidate Gene Analysis*

In the present study, three new QTLs were detected in all six germination parameters, namely *qDOM3.1*, *qDOM6.2*, and *qDOM10.2*. Through a CSSL-derived F2 population, the major effect of *qDOM3.1* on seed dormancy was validated for the first time using the six seed dormancy-related parameters and delimited to a 90 kb region (Figures 4 and 5 and Figure S3), which included 19 candidate genes. As the heterozygous line had the same phenotype as ZS97 (Figure 3), the candidate gene in ZS97 should be dominant. Based on the chromosomal synteny analysis and the gene expression profile in different tissues, we deducted that eight genes left as candidate genes. For the eight candidate genes, LOC\_Os03g01540 was annotated as DNA-binding protein, LOC\_Os03g01530 was annotated as tubulin/FtsZ domain-containing protein, and the other six candidate genes were annotated as expressed protein (based on the RGAP database: http://rice.plantbiology.msu.edu/, Release 7).

Hormonal regulation may be a highly conserved mechanism of seed dormancy among many species. ABA plays an essential role in the regulation of seed dormancy [4,24,37]. In the present study, we investigated the ABA content in freshly harvested NIL-NIP and NIL-ZS97, and found out the ABA content was almost five times higher in NIL-NIP than in NIL-ZS97 (Figure 7). Subsequently, the ABA sensitivity assay demonstrated that NIL-NIP was indeed more sensitive to high ABA solutions than NIL-ZS97 (Figure 8C). Based on those results, we assumed that our candidate gene could be responsive to ABA treatment, and the two alleles in NIP and ZS97 should respond differently. Thus, four candidate genes were selected (Figure 9), which were two expressed proteins (LOC\_Os03g01442 and LOC\_Os03g01470), one tubulin/FtsZ domain-containing protein (LOC\_Os03g01530), and one DNA-binding protein (LOC\_Os03g01540).

The DNA-binding protein (LOC\_Os03g01540) had 60% of protein sequence similarity with AT-hook motif DNA-binding family protein in *Arabidopsis* and 56.35% of similarity with AT-hook protein 1 in *Oryza sativa*. A recent study showed that the DNA-binding protein AT-Hook-Like 10 (AHL10) could be dephosphorylated by a protein phosphatase Highly ABA-Induced1 (HAI1), which was involved in abiotic stress and abscisic acid signaling. AHL10 phosphorylation was crucial for hormone-related genes during drought stress [44]. Therefore, we thought our candidate gene LOC\_Os03g01540 was somehow involved in the ABA signaling pathway to regulate seed dormancy. However, further research will be needed to prove our hypothesis.

LOC\_Os03g01530 is one of the isotype genes controlling β-tubulin, which is a basic component of microtubules. Proteomic analysis of rice embryo showed that LOC\_Os03g01530 was upregulated during seed germination, suggesting its possible role in seed germination [45]. LOC\_Os03g01490 was annotated as expressed protein. A research study showed that it is a functional new chimerical gene for *Oryza sativa* ssp. *japonica* by comparing *Oryza sativa* ssp. *japonica* and its five wild progenitors [46], although its function was not revealed. Another research paper identified LOC\_Os03g01490 and LOC\_Os03g01470 as phosphopeptides [47]. The other four candidate genes were annotated as expressed protein, and very limited information was available based on a literature search.
