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 it
complements the gametic lethal gene, g al, 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 de crease to 75%. Therefore , 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 Generatio n
Sequencing analysis targeting d60, it is required to develop
isogenic genome of reference e xcept for target d60 gene.
When the F1 (D60d60Galgal) progenies of ‘Koshihikari’ ×
‘Hokuriku 100’ were backcrossed to ‘Koshihikari’
(D60D60galgal), BC1 F1 individ uals 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 (D60d60galGa l) 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 rep eatedly 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 seq uencing.