A Simple and Efficient Method for CRISPR/Cas9-Induced Rice Mutant Screening
FENG Xu-ping1, 2, PENG Cheng3, ZHANG Chu1, 2, LIU Xiao-dan1, 2, SHEN Ting-ting1, 2, HE Yong1, 2*, XU Jun-feng3
1. College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China
2. Key Laboratory of Spectroscopy, Ministry of Agriculture, Hangzhou 310058, China
3. Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences，Hangzhou 310021, China
Abstract：Mutant screening is an important step for CRISPR/Cas9 gene editing technology employed in crop breeding program. The present study proposes a visual identification method of CRISPR/Cas9-induced rice mutants based on near-infrared hyperspectral image technology. A total of 1 200 samples of rice seeds were collected, comprising 600 wide types and 600 CRISPR/Cas9-induced mutant samples. The whole data set was divided into two groups according to the Kennard-Stone algorithm, a calibration set (400 samples) and a prediction set (200 samples) for each class. 24 optimal wavelengths were selected by 2nd spectra algorithm after preprocessing the selection spectral region with absolute noises by wavelet transform. Radial basis function neural network (RBFNN), extreme learning machine (ELM) and K-nearest neighbor (KNN) were used to build discrimination models based on the preprocessed full spectra and feature wavelengths. The results demonstrated that neural networks models achieved good recognition ability. The RBFNN model calculated on the optimal wavelength showed classification rates of 92.25% and 89.50% for calibration set and prediction set, respectively. Finally, the classification of mutant seeds could be visualized on prediction maps by predicting the features of each pixel on individual hyperspectral image based on 2nd derivative-RBFNN model. It was concluded that hyperspectral imaging together with chemometric data analysis was a promising technique to identify CRISPR/Cas9-induced rice mutants, which offered a powerful tool for evaluating large number of samples from CRISPR/Cas9 gene editing performance trials and breeding programs.
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