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TILLING (Targeting Induced Local Lesions IN Genomes) results and mutation frequencies.

 
Sample No. plants or spikes Fragment (bp screened) Total mutants Mutation type Frequency
Missense Silent Truncation Intron Unknown
QA 288 Wx7A3 (743) 18 7 7 2 2
576 Wx7D3 (710) 18 5 5 2 1 5
288 Wx7A4 (708) 5 2 1 1 1
864 Pina (397) 11 7 2 2
576 Pinb (397) 8 4 3 1
1,398,528 60 1/23.3 kb
QB 768 Wx7D3 (710) 18 6 5 2 3 2
545,280 18 1/30.3 kb
VA 332 Wx7A3 (743) 7 3 2 2
332 Wx7A2 (498) 5 2 2 1
332 Wx7D3 (710) 6 3 3
647,732 18 1/36.0 kb
VB 288 Wx7A3 (743) 8 3 3 1 1
288 Wx7A4 (708) 7 3 2 2
288 Wx7A2 (498) 3 2 1
288 Wx7D3 (710) 6 2 3 1
756,792 24 1/31.9 kb
VC 384 Wx7A3 (743) 7 4 1 1 1
384 Wx7A2 (498) 4 1 2 1
384 Wx7D3 (710) 9 2 2 1 1 3
749,184 20 1/37.5 kb
Q, QAL2000; V, Ventura; A, B, and C, 0.7, 0.6, and 0.5% ethylmethane sulfonate treatments, respectively.
Amplicon size multiplied by the total number of samples screened.
§Sequence data were not good enough to determine the nucleotide change.
Total number of mutations divided by the total base pairs screened.



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Molecular changes in the mutations identified in the TILLING (Targeting Induced Local Lesions IN Genomes) population.

 
Sequenced mutants Silent mutations Nonsilent mutations Intron mutations Homozygous mutants Heterozygous mutants G to A change C to T change A to C change
Pin: 19 5 (26.3%) 14 (73.7%) 0 5 (26.3%) 14 (73.7%) 13 (68.4%) 5 (26.3%) 1 (5.3%)
Waxy: 100 31 (31%) 51 (51%) 18 (18%) 37 (37%) 63 (63%) 55 (55%) 45 (45%) 0
Total 119 36 (30.3%) 65 (54.6%) 18 (15.1%) 42 (35.3%) 77 (64.7%) 68 (57.2%) 50 (42.0%) 1 (0.8%)



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Mutants identified in QAL2000 treated with 0.7% ethylmethane sulfonate (EMS) and QAL2000 treated with 0.6% EMS sister spikes with repeated or multiple mutations.

 
Gene/fragment Line Zygosity Nucleotide change Codon change Amino acid change Type
Wx7A3 QA49.5 Het C1372T ATC-ATT I287I Repeat
QA49.4 Het C1372T ATC-ATT I287I
QA49.3 Het G1860A GTG-ATG V420M
QA49.2 Hom G1860A GTG-ATG V420M
QA49.1 Het G1860A GTG-ATG V420M
QA53.2 Hom G1762A GGG-GAG G387E Multiple
QA53.3 Hom C1485T TCT-TTT S325F
QA61.4 Het C1874T GAC-GAT D424D Repeat
QA61.2 Hom C1874T GAC-GAT D424D
QA152.4 Het G1339A CTG-CTA L276L Multiple
QA152.5 Het C1372T ATC-ATT I287I
QA161.3 Het C1342T CCC-CCT P277P Multiple
QA161.2 Het G1413A TGG-TAG W301Stop
QA191.3 Het G1872A GAC-AAC D424N Repeat
QA191.4 Het G1872A GAC-AAC D424N
Wx7D3 QA154.2 Hom C1561T CTC-TTC L323F Multiple
QA154.4 Het G1748A Intron
QA201.1 Hom G1885A AGG-AGA R402R Multiple/
QA201.4 Hom C1703T ACC-ATC T370I Repeat
QA201.5 Het C1703T ACC-ATC T370I
QB3.3 Het G1474A GTG-ATG V294M Repeat
QB3.4 Het G1474A GTG-ATG V294M
QB1.1 Het G1912A GTG-GTA V411V Multiple
QB1.3 Hom G1656A TGG-TGA W354Stop
QB163.1 Het G1800A GGG-GAG G374E Repeat
QB163.5 Het G1800A GGG-GAG G374E
Pina QA209.3 Het G206A TGG-TAG W41Stop Multiple
QA209.4 Het G65A AGC-AAC S(-7)N
QA158.1 Het C118T CCT-TCT P12-S12 Multiple
Pinb QA158.4 Hom G127A GAG-AAG E14-K14
QA164.1 Hom G375A CAG-CAA Q96Q Multiple
QA164.2 Het A318C CAA-CAC Q77H
Het, heterozygote; Hom, homozygote.
C1372T and C1342T were detected >10 times in 288 samples, considered as natural variations.



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Single-Kernel Characterization System hardness and grain size measurements of control wheat cultivars and mutant lines.

 
Sample Hardness index (HI) Diameter (mm) Genotype
Avg. SD Avg. SD
Australian Hard 67.99 19.25 2.46 0.51 Hard
Sunco 86.96 14.72 2.51 0.42 Hard, Pina-D1a, Pinb-D1b
Janz 84.52 16.64 2.48 0.41 Hard, Pina-D1a, Pinb-D1b
Rosella 40.05 13.31 2.60 0.35 Soft, Pina-D1a, Pinb-D1a
Tincurrin 47.37 17.22 2.32 0.35 Soft, Pina-D1a, Pinb-D1a
QA51.2 WT 43.1 15.4 3.05 0.59 Pina-D1a, Pinb-D1a
QA51.2 M 44.73 13.92 3.05 0.56 Pina-D1a, Pinb-T(-13)I
QA150.4 WT 43.06 18.33 2.63 0.53 Pina-D1a, Pinb-D1a
QA150.4 M 42.54 12.58 3.31 0.54 Pina-C11Y, Pinb-D1a
QA158.1 WT 34.29 13.87 2.44 0.52 Pina-D1a, Pinb-D1a
QA158.1 M 45.1 15.71 2.24 0.55 Pina-P12S, Pinb-D1a
QA232.2 WT 69.69 16.39 2.60 0.45 Pina-D1a, Pinb-D1a
QA232.2 M 87.46 18.23 2.31 0.47 Pina-D1a, Pinb-R74Stop
***Significant difference of HI between M and WT at the 0.001 probability level.
Australian Hard is an Australian marketing standard sample. It could be a mixture or a single cultivar but is certified to only contain hard wheat. Sunco and Janz are both hard cultivars that carry the Pinb-D1b allele. Rosella and Tincurrin are soft cultivars. Mutant seeds with segregated genotypes, homozygous mutant (M) and wild type (WT) at the mutation site, were tested.
Hardness index is recorded as both an average and standard deviation.



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Single-Kernel Characterization System hardness and grain size measurement of wheat seeds from genotyped homozygous mutant (M) and nonmutant (WT) F2 plants of the cross QA232.2 × QAL2000.

 
Sample Hardness index Diameter (mm) Genotype
Avg. SD Avg. SD
QAL2000 41 15 2.63 0.51 Pina-D1a, Pinb-D1a
F2 WT 45 14 2.58 0.50 Pina-D1a, Pinb-D1a
F2 M 75 16 2.60 0.34 Pina-D1a, Pinb-R74Stop
***Significant difference between mutant and wild-type lines at the 0.001 probability level.
QAL2000 wild type grown in a greenhouse at the same condition and the same time as for the mutant lines.