A frameshift mutation of the chloroplast matK coding region is associated with chlorophyll deficiency in the Cryptomeria japonica virescent mutant Wogon-Sugi

Abstract

Wogon-Sugi has been reported as a cytoplasmically inherited virescent mutant selected from a horticultural variety of Cryptomeria japonica. Although previous studies of plastid structure and inheritance indicated that at least some mutations are encoded by the chloroplast genome, the causative gene responsible for the primary chlorophyll deficiency in Wogon-Sugi, has not been identified. In this study, we identified this gene by genomic sequencing of chloroplast DNA and genetic analysis. Chloroplast DNA sequencing of 16 wild-type and 16 Wogon-Sugi plants showed a 19-bp insertional sequence in the matK coding region in the Wogon-Sugi. This insertion disrupted the matK reading frame. Although an indel mutation in the ycf1 and ycf2 coding region was detected in Wogon-Sugi, sequence variations similar to that of Wogon-Sugi were also detected in several wild-type lines, and they maintained the reading frame. Genetic analysis of the 19 bp insertional mutation in the matK coding region showed that it was found only in the chlorophyll-deficient sector of 125 full-sibling seedlings. Therefore, the 19-bp insertion in the matK coding region is the most likely candidate at present for a mutation underlying the Wogon-Sugi phenotype.

Fig. 1

Reading frameshift mutation in the matK coding region of the Wogon-Sugi chloroplast genome. The nucleotide and deduced amino acid sequences of part of the matK gene of Wogon-Sugi and wild-type chloroplast genomes are shown. Blue nucleotides show the insertion sequence in Wogon-Sugi, and red nucleotides show the introduced stop codon

Fig. 2

Repetitive indel mutations in the ycf2 coding region of Wogon-Sugi and four wild-type chloroplast genomes. The nucleotide and deduced amino acid sequences of part of the ycf2 gene of Types I–IV, and of Wogon-Sugi, showing variation in the number of 66 bp insertions. Colored nucleotides show the 33-bp repetitive units

Fig. 3

Repetitive indel mutations in the ycf1 coding region of Wogon-Sugi and four wild-type chloroplast genomes. The nucleotide and deduced amino acid sequences of part of the ycf1 gene of Types I–IV, and of Wogon-Sugi, showing variation in the number of 198 bp insertions. Colored nucleotides show the 66-bp repetitive units

Fig. 4

The phenotypes obtained from an artificial cross between Wogon-Sugi (paternal parent) and wild-type (maternal parent). aWogon-Sugi type with yellowish-white leaves. b Wild-type with normal green leaves. c Chimeric type with yellowish-white sectors and normal green sectors. In the chimeric type, the yellowish-white sector, the green sector, and the variegated sectors are shown as c-i, c-ii, and c-iii, respectively. Red arrows show the sample used for genetic analysis

Fig. 5

Fluorescently labeled PCR products from the three seedling types of Fig. 4. The expected PCR product size of Wogon-Sugi type and wild-type (Yoshiki 1) is 196 bp and 177 bp, respectively. a Yellowish-white leaves with the Wogon-Sugi type matK indel. b Normal green leaves with the wild-type matK indel. c-i Yellowish-white sector of chimeric seedling with the Wogon-Sugi type matK indel; c-ii normal green sector of chimeric seedling with the wild-type matK indel; c-iii variegated sector of chimeric seedling with the Wogon-Sugi type matK indel and the wild-type matK indel. The rightmost peak of each group corresponds to the expected PCR product (196 bp and 177 bp). On the left, peaks corresponding to slippage of the Taq polymerase are visible (195 bp and 176 bp)