Discrimination of CRISPR/Cas9-induced mutants of rice seeds using near-infrared hyperspectral imaging

Identifying individuals with target mutant phenotypes is a significant procedure in mutant exploitation for implementing genome editing technology in a crop breeding programme. In the present study, a rapid and non-invasive method was proposed to identify CRISPR/Cas9-induced rice mutants from their acceptor lines (huaidao-1 and nanjing46) using hyperspectral imaging in the near-infrared (NIR) range (874.41-1733.91 nm) combined with chemometric analysis. The hyperspectral imaging data were analysed using principal component analysis (PCA) for exploratory purposes, and a support vector machine (SVM) and an extreme learning machine (ELM) were applied to build discrimination models for classification. Meanwhile, PCA loadings and a successive projections algorithm (SPA) were used for extracting optimal spectral wavelengths. The SVM-SPA model achieved best performance, with classification accuracies of 93% and 92.75% being observed for calibration and prediction sets for huaidao-1 and 91.25% and 89.50% for nanjing46, respectively. Furthermore, the classification of mutant seeds was visualized on prediction maps by predicting the features of each pixel on individual hyperspectral images based on the SPA-SVM model. The above results indicated that NIR hyperspectral imaging together with chemometric data analysis could be a reliable tool for identifying CRISPR/Cas9-induced rice mutants, which would help to accelerate selection and crop breeding processes.