*3.1. Phenotypic Variation of Mesocotyl Elongation*

Mesocotyl length of 208 rice accessions were evaluated in water, sand and soil (2, 4 and 6 cm). Continuous variation was observed across all environments, presenting a wide range of mesocotyl length and indicating that this diversity panel was ideal for conducting GWAS (Figure 1). In sand culture, the mesocotyl lengths ranged from 0 to 5.03 cm, with an average of 1.67 cm; while in water, the mesocotyl lengths ranged from 0 to 3.57 cm, with an average of 1.21 cm. The seeds under soil were sown at a depth of 2, 4 and 6 cm, with the average mesocotyl lengths of 0.33 cm (0–0.71 cm), 0.51 cm (0–1.66 cm) and 2.47 cm (0–4.51 cm), respectively. Among different sowing depth, most mesocotyl lengths of 6 cm sowing depth were significantly longer than those of 2 cm or 4 cm sowing depth (Figure 1, Figure 2a). The maximum of mesocotyl lengths were 0.70 cm, 1.66 cm and 4.51 cm at 2 cm, 4 cm and 6 cm sowing depths in soil, respectively, indicating that mesocotyl elongation of these accessions had larger variation at 6 cm sowing depth than those at 2 cm or 4 cm. Furthermore, there was only a small proportion, about 32.4%, 7.2% and 70.0% accessions with longer mesocotyls (≥ 2.0 cm) in sand, water and soil (6 cm), respectively.

The correlation analysis showed that moderate correlations were observed among different culture media and high correlations were found among different sowing depth under soil. Mesocotyl length evaluated in sand showed active correlation with that under water (*r* = 0.70), soil (2 cm) (*r* = 0.74), soil (4 cm) (*r* = 0.75) and soil (6 cm) (*r* = 0.78). Also, mesocotyl length evaluated in water showed active correlation with that under soil (2 cm) (*r* = 0.76), soil (4 cm) (*r* = 0.78) and soil (6 cm) (*r* = 0.75). High correlations were observed among different sowing depth under soil. Mesocotyl length evaluated in soil (2 cm) showed significant active correlation with mesocotyl length at soil (4 cm) (*r* = 0.93) and soil (6 cm) (*r* = 0.85), whereas the correlation coefficient (*r*) between soil (4 cm) and soil (6 cm) was 0.86.

*Genes* **2020**, *11*, 49

**Figure 1.** Summary of the phenotypic variations of mesocotyl length among different culture conditions, including correlations between mesocotyl lengths under different culture conditions and mesocotyl length distribution under varied culture conditions. Soil\_2: soil culture with 2 cm sowing depth; Soil\_4: soil culture with 4 cm sowing depth; Soil\_6: soil culture with 6 cm sowing depth, numbers in blue boxes are correlation coefficients (*r*), \* indicates the significance at *p* ≤ 0.05.

Analysis of variance (ANOVA) for mesocotyl length in 208 rice accessions revealed significant differences (*P*≤0.001) among genotypes (G), environments (E), and genotype×environment interactions (GEI) (Table 1). The VE is much larger than VG and VGEI, indicating that mesocotyl length were greatly affected by environments. Besides, the broad sense heritability (*h*b2) estimate for mesocotyl length across all environments was 0.49. Thus, identifying the loci significantly associated with mesocotyl length under multiple environments is crucial for MAS breeding.

**Figure 2.** Phenotypic variations of mesocotyl elongation among three culture media. (**a**) Mesocotyl length of 208 rice accessions measured in soil culture with three sowing depths. (**b**) Mesocotyl length variation in sand, soil culture with 6 cm sowing depth and water.



\*\*\* indicates the significance at *p* ≤ 0.001.
