Toward the identification of dwarfing gene d60 that complements gametic lethal gene gal by Next-Generation Sequencer oriented genome analysis

Abstract

The author previously discovered dwarfing gene, d60, and it complements the gametic lethal gene, gal, to cause gametic lethality in rice. Namely, in the F1 hybrid (genotype D60d60Galgal) of Koshihikari (D60D60galgal) × Hokuriku 100 (d60d60GalGal), male and female gametes having both gal and d60 become gametic lethal and the pollen and seed fertility decrease to 75%. Therfore, F2 progeny shows a unique mode of inheritance that is segregated into a ratio of 6 fertile long-culm (4D60D60: 2D60d60GalGal) : 2 partially fertile long-culm (D60d60Galgal = F1 type) : 1 dwarf (d60d60GalGal).Prior to Next-Generation Sequencing analysis targeting d60, it is required to develop isogenic genome of reference except for target d60 gene. When, the F1 (D60d60Galgal) progenies of ‘Koshihikari’ × ‘Hokuriku 100’ were backcrossed to ‘Koshihikari’ (D60D60galgal), BC1 F1 individuals segregated in a ratio of 1 tall and 25% sterile (D60d60galGal):2 tall (1 D60D60Galgal:1 D60D60galgal). Here, D60d60Galgal-heterozygous plants, can be recognized by pollen sterility prior to anthesis. Then Tall and 25% sterile BC1F1 plants (D60d60galGal) were selected by 25% pollen sterility and backcrossed with ‘Koshihikari’ as female parent to produce BC2F1 seeds. Hereafter, D60d60Galgal-heterozygous plants were selected in the first generation of each backcross (BCnF1) and immediately backcrossed repeatedly with Koshihikari to obtain isogenic genome useful for next-generation sequencing. That is to say, the target d60 DNA region narrowed down with each back cross generation, enabling identification of the target DNA mutation point by whole genomic sequencing.