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. 2022 Dec 28;12(1):22467.
doi: 10.1038/s41598-022-26989-z.

Development of a simple multiple mutation detection system using seed-coat flavonoid pigments in irradiated Arabidopsis M1 plants

Satoshi Kitamura  1 , Shoya Hirata  2   3 , Katsuya Satoh  2 , Rie Inamura  2   3 , Issay Narumi  3 , Yutaka Oono  2
Affiliations

Development of a simple multiple mutation detection system using seed-coat flavonoid pigments in irradiated Arabidopsis M1 plants

Satoshi Kitamura et al. Sci Rep. .

Abstract

Ionizing radiation induces genetic variations in plants, which makes it useful for plant breeding. A theory that the induced mutations occur randomly in the genome has long been accepted, but is now controversial. Nevertheless, a comparative analysis of the mutations at multiple loci has not been conducted using irradiated M1 genomes that contain all types of mutations. In this study, we identified Arabidopsis mutants (pab2 and pab3) in a mutagenized population of an anthocyanin-positive seed mutant (ban). Both pab2 and pab3 were revealed to be double mutants (tt4 ban and tt8 ban, respectively) that produced similar anthocyanin-less immature seeds, but differentially colored mature seeds. These features enabled the seed color-based detection of de novo M1 mutations in TT4 or TT8 following the irradiation of double heterozygous plants (TT4/tt4 TT8/tt8 ban/ban). Most of the irradiated double heterozygous plants produced anthocyanin-positive immature seeds, but 19 plants produced anthocyanin-less immature seeds. Of these 19 mutants, 2 and 17 exhibited tt4- and tt8-type mature seed coloration, respectively. The molecular analysis of the seed coat DNA from randomly selected anthocyanin-less seeds detected mutations at the locus predicted on the basis of the phenotype. Thus, the simple system developed in this study can reliably detect radiation-induced mutations at multiple loci in irradiated Arabidopsis M1 plants.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Characterization of pab2-1 ban and pab3-1 ban. Seed color at the immature (a) and mature (b) stages in ban, pab2-1 ban, and pab3-1 ban. (c) Schematic representation of mutations in the TT4 and TT8 regions for pab2-1 ban (top) and pab3-1 ban (bottom), respectively. Pale blue and black boxes represent exons and untranslated regions, respectively. Mutations in pab2-1 ban and pab3-1 ban are indicated in red. Green bars indicate the region used for the qPCR analysis. Gray and white boxes below the gene (labeled with a–i) indicate the amplified and non-amplified fragments, respectively, for the PCR performed using site-specific primers and template DNA from the pab2-1 ban and pab3-1 ban mutants.
Figure 2
Figure 2
Strategy for detecting mutations at multiple loci in the M1 generation. The cross-pollination of pab2-1 ban and pab3-1 ban generated double heterozygous plants for TT4 and TT8. The double heterozygotes were irradiated with ion beams. When the wild-type allele at the heterozygous TT4 or TT8 locus was mutated by irradiation, anthocyanin-less immature seeds were produced by the tissues with the mutated tt4 or tt8 allele in the M1 plants. These seeds were easily discriminated from the red immature seeds produced by the tissue with non-mutated TT4 and TT8 alleles. The M1 plants producing anthocyanin-less immature seeds were grown until the seeds matured to determine whether their mature seeds were pale yellow (tt4-type) or pale brown (tt8-type).
Figure 3
Figure 3
Quantification of the TT4 and TT8 fragments in DNA from anthocyanin-less seed coat-enriched tissues on the basis of a qPCR analysis. The TT4:TT8 fragment ratios normalized against the corresponding ratio in the non-irradiated double heterozygous plants (red dotted line) are provided for the four mutants. Data are presented as the mean value and standard error from three biological replicates.
Figure 4
Figure 4
Dosage variations on chromosomes 4 and 5 for the four anthocyanin-less seed coat-enriched DNA samples (123-1 (blue circles), 49-4 (orange circles), 39-2 (gray triangles), and 43-7 (gold crosses)). The approximate positions of the heterozygous TT8 and TT4 loci are indicated by pale orange bars on chromosomes 4 and 5, respectively.
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