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

Xuping Feng,Yue Chen,Xiaodan Liu,Yong He,Cheng Peng,Xujun Feng

doi:10.1038/s41598-017-16254-z

Abstract

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.

Highlights

IntroductionCrop breeding has come to the stages of targeted genome editing that has benefited from the development of the CRISPR/Cas[9] (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9, Cas9) gene editing system
Crop breeding has come to the stages of targeted genome editing that has benefited from the development of the CRISPR/Cas[9] gene editing system
According to Ishimaru et al.[19], a deletion THOUSAND-GRAIN WEIGHT 6 (TGW6) mutant had greater grain length and higher thousand-grain weight (TGW) but did not influence grain width compared with its transformation receptors

Summary

Introduction

Crop breeding has come to the stages of targeted genome editing that has benefited from the development of the CRISPR/Cas[9] (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9, Cas9) gene editing system. Several approaches have been developed for identifying the mutants produced by the CRISPR/ Cas[9] system, such as T7 endonuclease I (T7EI) assay, high-resolution melting curve analysis (HRAM), restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)/restriction enzyme (RE) assay[4,5,6,7,8]. These DNA- and protein-based techniques need the complex process of extractions are timeand labour-intensive, costly, and require expensive capital equipment. The main objectives of this study were (1) to study the feasibility of screening CRISPR/Cas9-induced mutant rice seeds using NIR hyperspectral imaging and chemometrics analysis; (2) to identify important wavelengths that can be attributed to the differences between wild-type (WT) and mutant rice strains; (3) to build an optimal discrimination model based on important wavelengths to simplify the prediction model and to speed up the operation; and (4) to visualize the number and locations of mutants by developing imaging processing algorithms

Objectives

Methods

Results

Conclusion