FIGURE 3. Example of HRM mutations screening (amplicon SBE2#1920) in individual and 4× pooled
samples with an additional parental SNP. (A) Individual M1 plants segregate in three classes for the parental alleles; two homozygous classes (blue and light blue) and a heterozygous class (grey). Novel mutations have a distinct heterozygous melting pattern (red line). (B) Pools of four plants can have parental allelic copy-numbers varying from zero to eight and classification of the genotypes is not possible. Detection of a novel mutation (red line) in these pools is problematic.
The pollen-treated EMS populations were screened for six amplicons of four candidate genes, all located on different chromosomes (Table 2). Three amplicons were screened individually and three amplicons were screened using pooled DNA (four plants per pool) of all plants (Table S2). On average, 88% of the individually screened plants produced a good PCR product suitable for HRM screening. In total we screened 2,717 kb of the four candidate genes (Table 3). We detected and confirmed only one mutation, a C to T transition mutation found in an intron of the ZEP candidate gene (Table S4). The mutation was found in the 0.625% EMS pollen-treated population.
TABLE 3. Summary of mutation detection in candidate genes of the pollen-treated EMS populations.
EMS
concentration Population size (% of total) Screened targeta Verified mutations (intron mutations)
0.625% 414 plants (22%) 597 kb 1 (1)
1.000% 1,269 plants (67%) 1,830 kb 0 (0)
1.500% 201 plants(11%) 290 kb 0 (0)
Total 1,884 plants (100%) 2,717 kb 1 (1)
a Calculated as target length (amplicon length - length of primers) × number. of screened plants. In case of pooled plants the number of screened plants was multiplied by the percentage of successfully screened plants in the non-pooled plants. For the seed-treated EMS population leaf DNA of all 1,824 M1 seedlings was extracted and
screened for mutations. Six candidate genes, located on five different chromosomes (Table 2), were screened for eleven amplicons. Three amplicons were screened individually for all plants, six amplicons were completely screened using pooled DNA and two amplicons were individually screened for only part of the population (Table S3). On average 97% of the individual screened plants produced a good PCR product suitable for HRM screening. We screened over four million DNA base pairs of the seed-treated population and confirmed 65 mutations by sequencing the corresponding amplicons (Table 4). Of the 45 non-synonymous amino acid changes, 21 were predicted to have a conceivable impact on protein function. These included five stop codons in four different candidate genes. All but one mutation, a G to T transversion, were C-to-T or G-to-A transitions (Table S4). The overall mutation detection frequency was 1 mutation per 65.3 kb of screened bases. Mutation detection frequency of individually screened plants, pooled plants and individually screened plants with natural polymorphisms between the G254 alleles were comparable and in the range of 1 mutation/22- 220 kb (Table S3).
TABLE 4. Summary of mutation detection in candidate genes of the seed-treated EMS population.
a Calculated as target length (amplicon length - length of primers) × number of screened plants. In case of pooled plants the number of screened plants was multiplied by the percentage of successfully screened plants in the non-pooled plants.
Development of genetically stable M
2and M
3mutants
Using in vitro culture we tried to propagate twenty-six M1 mutants, containing 27 of the most
interesting mutations identified in the M1 seedlings (one seedling contained mutations in two
of the screened genes). Ten plants were dead by the time we started the in vitro propagation. Seventeen potentially chimeric mutant plants of the M1 seed-treated EMS population,
containing 18 putatively interesting mutations, were successfully propagated (Table 5). For
Gene ampliconsScreened Total target size (% CDS) Screened target a Verified mutations (intron mutations) Mutation detection density Non- synonymous mutations Putative damaging
mutations Stop codon mutations
GWD 3 798 bp (78%) 1,035 kb 20 (1) 1/52 kb 14 6 2 PAIN 2 474 bp (100%) 842 kb 16 (0) 1/53 kb 12 4 1 SBE2 3 645 bp (78%) 759 kb 4 (0) 1/190 kb 4 1 0 PWD 1 331 bp (63%) 588 kb 9 (3) 1/65 kb 6 4 1 SBE1 1 288 bp (100%) 512 kb 12 (0) 1/43 kb 6 3 0 SSS3 1 289 bp (100%) 510 kb 4 (0) 1/128 kb 3 3 1 Total 11 2,825 bp (84%) 4,244 kb 65 (4) 1/65 kb 45 21 5
Chapter 5 – Results
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each successfully propagated mutant plant an average of eight independent in vitro clones were made. Of the 139 in vitro clones, 97 contained the identified mutation. For eleven mutants all clones contained the mutation, two mutations were lost in all clones, and for four mutants only a number of the in vitro clones contained the mutation. The vegetatively propagated clones originating from the mutant harbouring two of the identified mutations co-segregated for these mutations, with two of the clones having both mutations and the other nine clones having neither of the mutations. To obtain a genetically stable M2 generation of the mutants,
39 in vitro clones containing 16 different mutations (1-4 plants per mutation) were used for selfing or for crossing with non-mutagenized RH. Selfing the mutant plants failed and crosses with the mutants as pollen donor were not attempted. Crosses with RH failed repetitively for six of the mutations, and included the clones harbouring the double mutation. For 10 of the 27 initially selected mutations, M2 (M1×RH) seeds were obtained (Table 5).
TABLE 5. Mutants selected for crossing with RH to obtain genetically stable M2 and M3 families.
a Double mutant.
b Dead by the time in vitro propagation started.
* = stop codon
Mutant Amino acid change In vitro clones (M(Mutant:Wildtype)1) M2(M1×RH) seeds
Segregation in M2
(Mutant:Wildtype) M3(M2×M2) seeds
PAIN-13H02 T248I 8:0 191 38:11 Yes
PAIN-16H01 W299* 17:0 475 17:26 Yes
PWD-02H10 W579* 7:0 635 24:24 Yes
SBE1-06G12 G447R 5:0 621 1:64 In progress
GWD-10B07 W306* 10:0 18 In progress In progress
GWD-11E07 R344W 2:0 548 In progress In progress
SBE1-03C04 G440E 6:0 13 In progress In progress
SBE2-11A10 D161N 10:0 582 In progress In progress
SSS3-17F04 W392* 4:1 24 In progress In progress
SSS3-05F10 A370V 11:0 225 In progress In progress
GWD-16G10 E335* 3:0 No M2seed No M2seed No M2seed
PWD-07F12 S612L 8:4 No M2seed No M2seed No M2seed
SBE2-01C12 G413E 1:0 No M2seed No M2seed No M2seed
SBE2-04H02 G127R 1:4 No M2seed No M2seed No M2seed
PAIN/GWD -15A01a D245N/H287Y 2:9 No M2seed No M2seed No M2seed PWD-03H10 E602K 0:15 No in vitromutants No in vitromutants No in vitromutants SBE1-16B10 T432I 0:11 No in vitromutants No in vitromutants No in vitromutants GWD-05E11b E322K No in vitroplants No in vitroplants No in vitroplants No in vitroplants GWD-12E03b E397K No in vitroplants No in vitroplants No in vitroplants No in vitroplants GWD-15H11b G350E No in vitroplants No in vitroplants No in vitroplants No in vitroplants GWD-01B11b G262S No in vitroplants No in vitroplants No in vitroplants No in vitroplants PWD-16D03b L610F No in vitroplants No in vitroplants No in vitroplants No in vitroplants SBE1-02A10b S433F No in vitroplants No in vitroplants No in vitroplants No in vitroplants SBE1-09C08b S454N No in vitroplants No in vitroplants No in vitroplants No in vitroplants PAIN-17B03b G270D No in vitroplants No in vitroplants No in vitroplants No in vitroplants SSS3-09G04b L388F No in vitroplants No in vitroplants No in vitroplants No in vitroplants
For four of the mutations M2 seedlings were grown in 2011 and tested for presence of the
mutations. Only one M2 family showed the expected 1:1 segregation. Offspring of the other
three crosses showed a (severely) distorted segregation (Table 5). Even so, M2 plants
containing the mutation were obtained for all four mutations. The single M2 plant containing
mutant SBE1-6G12 was too weak for immediate use in crossing towards an M3 population. For
the other three mutants, eight M2 plants containing the mutation were crossed among each
other and a large number of M3 (M2×M2) seeds for genotypic and phenotypic evaluation were
obtained.