Genetic Diversity Analysis of the Innovative Black Rice RILs Developed by Interspecific Hybridization (O. sativa x O. rufipogon) Based on Agromorphology and Grain Quality

Rice (Oryza sativa L.) is an important staple grain because more than half of the world's population depends on it for their livelihood. White rice contributes to the daily caloric intake (20–40%) of Asian countries, but its nutritional quality is poor compared to that of pigmented rice. Pigmented rice varieties (black and red) are gaining popularity among consumers due to nutritional health benefits. Despite having nutritional importance, pigmented rice is usually low-yielding due to the narrow genetic base that occurred during domestication and artificial selection. Considering the potentials of wild rice, two distinct RIL populations (each 100 lines) were developed through interspecific hybridization BWF (Badshabhog x O. rufipogon) and CWF (Chenga x O. rufipogon) to break the yield plateaus. In the present study, we have created novel and inventive aromatic black lines from non-black parental lines. Significant genetic diversity prevailed in the RIL lines, which were assessed through genetic variability parameters, path coefficient, Mahalanobis D2 test, and PCA using 15 agromorphological traits. High heritability (>90%), high GA and GAM were found in RIL lines, suggesting additive gene action for the characteristics. The first four principal components together accounted for 73.74% of the variability in BWF, and the first six PCs showed 71.90% cumulative variability in CWF (eigenvalue >1). Total anthocyanin content was varying at 261 mg/100 g, 253 mg/100 g, and 259 mg/100 g in BW23, CW16, and control black, respectively. The DPPH activity was highest in BWF (78.37%) compared to control black (76.29%). Amylose content varies from 7.57 to 24.59%, and protein content was recorded at 8.76 to 8.81 g/100 g in the RIL lines. RIL lines (BW23, CW16) contain high-quality essential amino acids, including anthocyanin petunidin 3-O glucoside, quantified through HR-LCMS-QTOF. The Kala4 gene-specific PCR amplification product supports the acquired mutation with neo-functionalization activity through LINE1 insertional rearrangement near the Kala4 promoter, suggesting that the Kala4 gene is activated in black lines. Moreover, the RIL lines may provide some insight into the evolutionary and domestication history of black rice origin and can be used as important genetic resources for improving black rice for food and nutritional security.