1
Supplementary information
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Fig. S1 Subcellular localization of the BnAHAS1 protein. The SE5-GFP fusion protein or GFP alone
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expressed under the control of the CaMV 35S promoter in Arabidopsis protoplasts was observed using
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confocal microscopy. The photographs were taken in the blue channel (left), the red channel (middle), and
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merged (right). Scale bars represent 10 µm.
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7
9
35S::GFP
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GFP
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13
14
15
16
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18
19
20
21
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35S::BnAHAS1-GFP
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Chlorophyll
Merged
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Fig. S2 Diagram of the procedure of F1 seeds using the restore line 10M169 with IMI resistance to enhance
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hybrid seed purity in hybrid rapeseed production. The allelic composition of IMI resistant for the restore line
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10M169 and the CMS line Ning A7 were represented by RR and rr in round brackets, respectively, and the
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resulting F1 hybrid was represented by Rr; the off-type contaminants such as sibs/selfs produced by trace
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pollen and biological mixtures were also represented by rr.
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29
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The CMS line Ning A7 (rr) × The restore line 10M169 (RR)
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32
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Artificial hybridization
Hybridization in tents
Natural pollination
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35
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F1 (Rr)
biological mixtures (rr)
F1 (Rr)
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40
41
42
43
44
45
F1 (Rr), sibs/selfs (rr), and
After IMI treatment
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38
F1 (Rr) and sibs/selfs (rr)
0
F1 (Rr)
F1 (Rr)
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Table S1 List of primers used for amplification and genotyping of the AHAS genes
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Primer or probe
Sequence (5'-3')
Use
AHAS1-F1
TCAAGAACAGTTAGATCCAC
Gene cloning
AHAS1-R1
GATCACCAGCTTCATCTCT
Gene cloning
AHAS2-F1
AAGCAATTTCTCGCAACACTC
Gene cloning
AHAS2-R1
CAGAAGAGAGCATAGAATAATCAA
Gene cloning
AHAS3-F1
CTCTCTCTCTCTCATCTAACCAT
Gene cloning
AHAS3-R1
ACTGAAACTAAGTCTTTT ACCAT
Gene cloning
KpnI-AHAS1R-F*
CGCGGTACCTCATCTCTCTCTCCTCTAACC
Plasmid construction
EcoRI-AHAS1R-R*
GTCCGGAATTCTCAGTACTTAGTGCGACCATCCCCTTC
Plasmid construction
AHAS1-F2
TTCTCCTTAACCCCACAGAAAGA
RT-PCR
AHAS1-R2
GGGAGCGTAGCGGGAGAC
RT-PCR
AHAS1-P
Fam+CCGTCAATGTCGCACCTC CTTCC+Tamra
RT-PCR
AHAS2-F2
CTTCGTTTTCGTTCTTCGGC
RT-PCR
AHAS2-R2
AGACACGAGT AGCACGGCG
RT-PCR
AHAS2-P
Fam+CAAAAGCTTCCGTCTTCTCCCTGCC
RT-PCR
AHAS3-F2
CTCCTTAACCCCACAGAAACC
RT-PCR
AHAS3-R2
GGGAGCGTAGCGGGAGAT
RT-PCR
AHAS3-P
CAACTCACCCGTCAATGTCGCACC
RT-PCR
18s rRNA-F
AACGG CTACCACATCCA
RT-PCR
18s rRNA-R
CACCAGACTTGCCCTCCA
RT-PCR
18s rRNA-P
Fam+AGCAGG CGCGCAAATTACC
RT-PCR
NcoI-AHAS1-F*
AGCTCCATGGCGGCGGCAACATCGTC
Plasmid construction
SpeI-AHAS1-R*
CAAGACTAGTGGAGATGGCGAGTGGACGGTG
Plasmid construction
AHAS1-S**
CATCTTTGAAAGTGCCACAAC
AP-PCR
AHAS1-R**
CATCTTTGAAAGTGCCACAAT
AP-PCR
AHAS1-C
CTTTCGCTAGCAGGGCTAAA
AP-PCR
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* The underlining represents the enzyme sites.
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** The underlining shows mismatched bases. The bold bases indicate SNP loci.
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Table S2 Genotype frequencies at SNP (G/A) sites in the F2, BC1, and BC2 populations
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Genotype
Cross
χ2-value
P-value
39
154
0.20
0.50–0.75
117
0
227
0.45
0.25–0.50
98
113
201
0.37
0.50–0.75
G/A
A/A
F2 (3075R×M9)
35
80
BC1 [(3075R×M9) ×3075R]
110
0
BC2 [(3075R×M9) ×M9]
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No. of plants
G/G