plant_genotyping.md

**lwd1 T0 and T1 plants **

• DNA was extracted from leaves of all mutants using QIAGEN_DNeasy-Plant_mini protocol
• plants were tested for possible mutations in the CDS of LWD1 by amplifying the full genomic sequence using the primers LWD1_71us_F (5'-ATCAGACCCCTCCAACGACT-3') and LWD1_21ds_R (5'-TTCCTCTTTCTACCCCACCA-3')
• the PCR fragments were Sanger sequenced using different primers:
  • plants transformed with pMGE599/7-97 (approach 1): LWD1_71us_F
  • plants transformed with pMGE599/182-1222 (approach 2): LWD1_qPCR114F (5'-CAGCTCATAGCCCATGACAA-3')
• mutations and the zygosity of the mutations were determined relative to the start codon of LWD1

only in T1 plants:

• PCR was performed on transformation vector to check presence/absence of Cas9 using the primers pMGE599_M13F (5'-GTTTTCCCAGTCACGAC-3') and pMGE599_R (5'-TGTCGACATCGATAAGCTTGAA-3')