In this study, a MiSeq next-generation sequencer was used to achieve 5× rice genome coverage; namely, 4-5 ng of five DNA libraries was applied in one MiSeq run. Using this approach, the targeted gene mutation in Jukkoku_sd1 was successfully detected as the SNP (G to T) in the defective GA20-oxidase gene, which had reduced the culm length by a loss-of-function mutation of GA synthesis, localized at position 38,382,746 from the end of the short arm of chromosome 1 of Koshihikari. This would be the minimum scale to detect Jukkoku_sd1 in practical breeding. In Japan, genetically modified organisms are not acceptable to consumers; thus the target SNP in Jukkoku_sd1 would be effectively tracked by using NGS of 5× coverage scale in each backcrossed generation with Koshihikari. On the other hand, as the DNA sequence and function of sd1 have been deciphered [15–18, 37], new breeding methods, such as RNAi gene silencing  and genome editing , are available in other countries to retard culm length by knockout of Sd1.