Maping the thm1 mutation via bulked segregation analysis (BSA)

Phenotypes in the second filial generation (BC1F2) progeny were analyzed for their ability to trigger HSCE in response to heat shock. Three DNA pools, each consisting of 25 plants showing thm1‑like HSCE, 25 plants showing WT‑like HSCE, and 18 WT reference individuals, were sequenced.Approximately 50 BC1F2 lines exhibiting the lowest and highest HSCE phenotypes were selected and pooled to generate thm1-like and WT-like bulks, respectively.

Genomic DNA was extracted from rosette leaves of the bulks and WT using a 2% cetyltrimethylammonium bromide (CTAB) method. The WT DNA was used as an internal reference.