Supplementary Figure 1. CRISPR/Cas9-induced targeted mutations in TaGASR7, TaDEP1, TaNAC2, TaPIN1, TaLOX2 and TaGW2 genes in wheat protoplasts. Lanes 1 and 2: digested CRISPR/Cas9-transformed protoplasts; lanes 3 and 4: digested and undigested wild type controls; M: marker. Sequences of CRISPR/Cas9-induced mutations are shown on the right. The wild type sequences are shown at the top of each sequence group. The numbers at the sides indicate the type of mutations and how many nucleotides are involved. 

Gene name

Target site Oligo-F Oligo-R Detection 2. PCR primers and their applications. 
LOX2-F LOX2-R CGTCTACCGCTACGACGTCTACAACG GGTCGCCGTACTTGCTCGGATCAAGT Amplifying the TaLOX2 target site GW2-F GW2-R ATGCCAACCCTTGCGTGTGCGT TCCTGCTTGTGGGAGCTTTATG Amplifying the TaGW2 target site GW2-A1-F CTGCCATTACTTTGTATTTTGGTAATA Amplifying the TaGW2-A1 target site GW2-B1-F GTTCAGATGGCAATCTAAAAGTT Amplifying the TaGW2-B1 target site GW2-D1-F GCATGTACTTTGATTGTTTGCGTGA Amplifying the TaGW2-D1 target site GW2-A1/B1/D1-R TCCTTCCTCTCTTACCACTTCCC Amplifying the TaGW2 target site T7-GW2-F sgRNA-PCR-R TAATACGACTCACTATAGGCAGGATGGGGTATT TCTAG GCACCGACTCGGTGCCACTT Amplifying the T7-GW2-sgRNA ZmUbi-5'UTR-F ZmUbi-KH-5'UTR-R TTCCCCAACCTCGTGTTGTTCGGAG ACGACCCATGACAAGCTTTAGCGGTACCCTTGA AGCGGAGGTGCCGACG Amplifying the 5'UTR of ZmUbi1 for pLZT7 cloning ZmUbi-KH-3'UTR-F ZmUbi-polyA-3'UTR -R CTCCGCTTCAAGGGTACCGCTAAAGCTTGTCAT GGGTCGTTTAAGCTGC TCTAGATTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTCGCACACACAACACAACCGGT Amplifying the 3'UTR of ZmUbi1 for pLZT7 cloning KpnI-zCas9-F HindIII-zCas9-R

GGGGGTACCATGGATTACAAGGACCACGACG CCCAAGCTTTCACTTCTTCTTCTTCGCCTGC

Amplifying the KpnI-zCas9-HindIII for pLZT7-zCas9 cloning Supplementary Table 3. Genotypes of the 80 T0 tagasr7 mutants with respect to mutations in the TaGASR7-A1, -B1 and -D1 homoeologs. 

OT1-2

GTTGCCGccGGTGCCCGGTGG GTTCGTTCTCACGACGGTGGAGAC TAGGTGCCCGTCTCTTTCGTACAG 

OT1-3

GgaGCCGTcGGTGCCCGGAGG TTCCGATGGCCGAAACTCGACTTC TTGTCGCTGCCTTCGCATCTCAAG 

OT1-4

GTcGCCGTccGTGCCCGGCGG GAGGAGCTGATCCTCTGTTCAATC GAAGGAGAGGATCCCCGAGCTGCT 

OT1-5

GTTcCCGTgGGcGCCCGGGGG TCGCGTGTTGGTGCTTGGGAAG TCCATGCTAACTTACACCCAATTG 

OT1-6

cTTGgCGTAcGTGCCCGGCGG AAGTCCGCCGCATGCATGAGTTCC GCACCTTGCAGTGACATATACTCC 

OT1-7

GTTcCCaTgGGTGCCCGGGGG TGAGGTCTGGTCGCTACACGAA GTGTGCCACTGAGATGTAGCAT 

OT1-8

GTTGaCcTtGGTGCCCGGCGG ACTTGTCCTCGCCCTTCTGCTT ATGGTCACTATCACCGACAAGA 

OT1-9

GTTGCaGcAcGTGCCCGGCGG GGTTATCAAAGGTCTCCAACAACA ATCGCTTACCGCTGTCAGAACG 

OT1-10

cTcGCCGTAtGTGCCCGGCGG CGCTTCATCTTCAAGTCAATGTTC TGGACTGGTGTTTACCCTTTGTT 

OT1-11

GTgGtCGgAGGTGCCCGGAGG GGAACGATGGCAGTGACGATGA TCCCTTTGTTCCCGCACTTAGT 

OT1-12

tTTGCCcTAGGaGCCCGGAGG CATAAATGGAGTGAGGATGAACTG CGTTTATTCTTACCAGGCGGAC 

OT1-13

GTTGaCGgAGaTGCCCGGAGG ACGCTTCCTCTGGGCTCTTAGG CAAACTTGCTGACCCACCTGTC 

OT1-14

cTTGtCGgAGaTGCCCGGCGG CCTTCACTATCCGCCGTCCAGA CGCATCAAACGCAGCAACTTGT 

OT1-15

GgcGCgcTAGGTGCCCGGCGG GATGCCCAGAACAAGGAGGTCG GCCGCACAGGTCGTAGTTGTCA 

OT1-16

GTTcaCcTtGGTGCCCGGCGG GGTGTGGCATCATCATCATCATCT ATCGTCACCATCACCGACAAGA 

OT1-17

GTcGCgcTAcGTGCCCGGCGG AATGGACGCCCAGAACAAGGAG AGGACGTAGGAGCTGAACGGCA 

OT1-18

GacGCgGTAGGaGCCCGaGGG GAGGAGGAGGAACACGTTGTCC AAAGCAAATGATGCCTCCCAGC 

OT1-19

GTcGaaGTgGGTGCgCGGCGG CCATTGCCCTTCACCCACATCT CCTTGTTATGCTGATGTATCGTGC 

OT1-20

cgTcgCGTAcGTGCCCGGCGG CACCGTGTTCAAGGTCAAGGAC GAAGTAAGTGGCGAGGCACCCT 

OT1-21

GgcGCaGgAGGaGCCCGGCGG GGAAGGACGAGGAGAAGAAGAG GAACTCGGACAGCATCTACGAC 

OT1-22

agaGaCGgAGGTGCCCGGCGG AGGTCCTCACCGCAACTCGTAT CGTTTGTGGACTGGCAGGAAAT 

OT1-23

GcTcCtGTAGcTGCgCGGCGG CAGCGGCAGCCTCCATAAAGAG CGCCGTTCAGTTGGCATACAAG 

OT1-24

GaTGtaGTAcGTcCCCGGCGG GGTCTTTCACCCACCCGAACTC CAACTACTGCCGATTTCGTCGT 



  ., not available, these mutant types were not obtained from the experiments. N.D., not detected. a "-" indicates deletion of the indicated number of nucleotides; "+" indicates insertion of the indicated number of nucleotides; "-/+" indicates simultaneous deletion and insertion of the indicated number of nucleotides at the same site. b Based on the absence of mutant plants harboring CRISPR DNA construct or not. 




  




  




  




  




  




  


Table 4 .

 4 Potential off-target sites for TaGASR7-sgRNA and TaGW2-sgRNA in wheat. 


	Supplementary Target Site name	Sequence	Forward primer sequence	Reverse primer sequence
	Target 1	GASR7	CCGCCGGGCACCTACGGCAAC		
	(GASR7)	OT1-1	GTcGtCGTAGGTGCCCGGCGG	CTCGCTCAGGTTCCTGGGCTTCTT	CGGGTCCCATTATCCTCTCTCCTA

Table 5 .

 5 Molecular and genetic analysis of CRISPR/Cas9-induced mutations in TaGASR7, TaDEP1 and TaLOX2 homoeologs and their transmission to the T1 generation. 


	Target 2	GW2	CCTCTAGAAATACCCCATCCTG		
	(GW2)	OT2-1	tAaGATGGGtTATTTCTAGAGG	AAACTCAGCGTAAATCTGGCG	CCGAGACAAACAATGGACCAA
		OT2-2	gAGGATGGGaTtTTTCTAGAGG	GATGACAACGGGTGGCAAGAG	TGCCATCACCTCCCTACATCG
		OT2-3	aAGaATGGGccATTTCTAGAGG	CCCTCTGGTAACTCTTCGTTC	TCTCTGATGTGCTATGTTCCA
		OT2-4	aAGaATGGGtcATTTCTAGAGG	CTGCCTCTTGTTACCATCCAC	TCATCATTTGCTTCCTCTCTG
		OT2-5	CgGcATGGaGTATaTCTAGAGG	AATACCAACTAAGCCTGAGCAAA	ACTCCCTAGACCACGAACGAC
		OT2-6	CcGGAgGGaGTATgTCTAGAGG	GCGTATTACATTCGTGTCAAGT	TCAAACAACAGTTACCGCAAC
		OT2-7	ttGGAaGGGGgATTTCTAGAGG	AAAGCAATGGGCACAAAGTCT	GTATTCGGTGATTTCTGCGTT
		OT2-8	gAGGAcaGaGgATTTCTAGAGG	GCAGAAGCAGCAACTAATAAGAAG	TCATTCAGAGCAGTCAGAAGC

(DD) 16 (Dd) 5 (dd) 75.0 d 

Supplementary Table 1. SgRNA target loci and sequences. 5. Molecular and genetic analysis of CRISPR/Cas9-induced mutations in TaGASR7, TaDEP1 and TaLOX2 homoeologs and their transmission to the T1 generation (continued). 

Supplementary Table

Hetero, heterozygous; Homo, homozygous. 
a "-" indicates deletion of the indicated number of nucleotides; "+" indicates insertion of the indicated number of nucleotides; "-/+" indicates simultaneous deletion and insertion of the indicated number of nucleotides at the same site. b Based on the number of plants carrying the observed mutations over the total number of plants tested. c 
Based on the number of mutant plants not harboring CRISPR DNA over the total number of mutant plants tested. d Segregation of the heterozygous lines conforms to a Mendelian 1:2:1 ratio according to the χ2 test (P > 0.05). 

Gene name


Wheat variety

Plant ID