Table 4.

Mutation detection methods applied in rice.

Method	Type of mutagenesis applied	Type of mutation detected	Pros	Cons	Reference
TILLING	EMS, MNU, SA, γ-ray	SNPs	High sensitivity; Provides the approximate location of the induced mutation; Detect induced and naturally occurring homozygous and heterozygous SNPs; Suitable for polyploids.	Require celery CEL I endonuclease for the mismatch detection; False negatives and positives (are low but exist).	McCallum et al., 2000; Till et al., 2003; reviewed in Barkley and Wang, 2008 and Taheri et al., 2017
TILLING-NGS	MNU, SA	SNPs	No require enzymatic digestion; High throughput; Time saving; Efficient in polyploids; Mutation detection in pools deeper than eight individuals.	Expensive; Needs multi-dimensional pooling; Can incorrectly identify DNA bases with high frequency which is not easy to identify due the amount of data produced; It is laborious to process, storage and analyze the data.	Kumar et al., 2017; reviewed in Taheri et al., 2017
TILLING-HRM	γ-ray	SNPs, indels	No require enzymatic digestion; High sensitivity; Time and cost saving.	Depends on good PCR instruments and dyes; Needs multi-dimensional pooling; More difficult to detect indels than substitutions; Sensitivity limited to amplicons <450 bp.	Li et al., 2018b; reviewed in Taheri et al., 2017
Exome capture	EMS	SNPs, indels	Large-scale mutation discovery; High-throughput; Cost-effective; Applicable in polyploids.	It is laborious to process, storage and analyze the data; Need transcriptome assembly in cases a reference genome is not available.	Henry et al., 2014; reviewed in Taheri et al., 2017
Eco-TILLING	Natural mutations	SNPs	Provides the approximate location within a few base pairs of the induced mutation; Detect induced and naturally occurring homozygous and heterozygous SNPs;	Require CEL I endonuclease mismatch detection celery; False negatives and positives (are low but do exist).	reviewed in Barkley and Wang, 2008
MutMap	EMS	SNPs	Minimizes the number of crosses in crop species and required mutant F2 progeny.	Not suitable for plants without reference genome sequence (now improved by MutMap-GAP).	Abe et al., 2012; Takagi et al., 2013; Taheri et al., 2017
CRISPR-S	CRISPR/Cas9	−	Enable a PCR-free, phenotype-based identification of genome-edited T0 plants, and a subsequent selection of transgene-free T1 plants.	Require a RNAi expression element incorporated into the CRISPR/Cas9.	Lu et al., 2017
PCR-based	CRISPR/Cas9	short indels (± 1pb)	Accurately identify indel sizes down to ± 1 bp	Efficiency is affected by target sequence; Applicable only for mutation upstream to PAM	Biswas et al., 2019
Amplicon labelingbased	CRISPR/Cas9	short indels (± 1pb)	Accurately identify indel sizes down to ± 1 bp	Sometimes could not detected the exact nucleotide change needing sequencing to confirm.	Biswas et al., 2019