*3.5. Candidate Genes Related with Mesocotyl Elongation*

According to the results of GWAS, 107 candidate genes were found to be related with mesocotyl elongation (Table S3). Also, considering that mesocotyl elongation is highly regulated by various phytohormones, such as SLs, CTK, BR, ABA, JAs, GA and IAA, five genes involved in the biological metabolism of the phytohormones, cell elongation and division were selected as the high-confidence candidate genes for mesocotyl elongation (Table 3). That is the zinc finger CCCH type family protein (*LOC\_Os05g10670*) and transcription factor jumonji (*LOC\_Os05g10770*) on chromosome 5 (5.6–6.1 Mb), a gene encoding cytokinin-O-glucosyl transferase 2 on chromosome 9 (1.2–1.5 Mb) and two genes (4.3–4.5 Mb and 14.1–14.5 Mb) encoding flavin monooxygenase and 9-cis-epoxycarotenoid dioxygenase 1, respectively.

The expression of five candidate genes were detected using qRT-PCR (Figure 5). The results showed that there was significant difference between accessions with extreme mesocotyl length, with the longer one exhibited higher expression abundance. In detail, five genes showed more than 4.4-fold (*LOC\_Os05g10670*), 2.8-fold (*LOC\_Os05g10770*), 3.0-fold (*LOC\_Os09g03140*), 8.0-fold (*LOC\_Os12g08780*) and 32.6-fold (*LOC\_Os12g24800*) higher expression in long-mesocotyl accessions compared to short-mesocotyl accessions, respectively.

**Figure 5.** Expression analysis of five candidate genes. Relative expressions of the five genes in long- and short -mesocotyl length rice accessions were tested by qRT-PCR. Transcription levels were normalized to that of *Osactin1* and compared to the short control. 1 to 5 represent five candidate genes, that is 1, *LOC\_Os05g10670*; 2, *LOC\_Os05g10770*; 3, *LOC\_Os09g03140*; 4, *LOC\_Os12g08780*; 5, *LOC\_Os12g24800*. Values of fold change are showed in mean ± SEM. Relative transcription levels were calculated by the log2-CT method. \* and \*\* indicate the significance at *p* ≤ 0.05 and *p* ≤ 0.01, respectively.


