Methodology to identify dwarfing gene d60 that complements gamete lethal gene gal by Next-generation DNA sequencing analysis

Abstract

The author previously discovered dwarfing gene, d60, and itcomplements the gametic lethal gene, g al, to cause gameticlethality in rice. Namely, in the F1 hybrid (genotypeD60d60Galgal) of Koshihikari (D60D60galgal) × Hokuriku100 (d60d60GalGal), male and female gametes having bothgal and d60 become gametic lethal and the pollen and seedfertility de crease to 75%. Therefore , F2 progeny shows aunique mode of inheritance that is segregated into a ratio of6 fertile long culm (4D60D60: 2D60d60GalGal) : 2partially fertile long culm (D60d60Galgal = F1 type) : 1dwarf (d60d60GalGal).Prior to Next Generatio nSequencing analysis targeting d60, it is required to developisogenic genome of reference e xcept for target d60 gene.When the F1 (D60d60Galgal) progenies of ‘Koshihikari’ בHokuriku 100’ were backcrossed to ‘Koshihikari’(D60D60galgal), BC1 F1 individ uals segregated in a ratioof 1 tall and 25% sterile (D60d60galGal) 2 tall (1D60D60Galgal 1 D60D60galgal). Here,D60d60Galgal heterozygous plants can be recognized bypollen sterility prior to anthesis. Then Tall and 25% sterileBC1F1 plants (D60d60galGa l) were selected by 25% pollensterility and backcrossed with ‘Koshihikari’ as femaleparent to produce BC2F1 seeds. Hereafter,D60d60Galgal heterozygous plants were selected in thefirst generation of each backcross (BCnF1) and immediatelybackcrossed rep eatedly with Koshihikari to obtain isogenicgenome useful for next generation sequencing. That is tosay, the target d60 DNA region narrowed down with eachback cross generation, enabling identification of the targetDNA mutation point by whole genomic seq uencing.