Genotyping and transgenic lines

Genotyping of brx alleles was performed as described (Mouchel et al., 2004; Rodriguez-Villalon et al., 2014). Genotyping of other loci by analysis of PCR-amplified genomic DNA fragments was performed as follows. To detect the bri1-116 allele, a 552 bp genomic DNA fragment was amplified using oligonucleotides 5′-AAGGAGAGATCCCTCAGGAG-3′ and 5′-TGTCCAGAAACATCATCGAAC-3′. Restriction digest with PmeI indicated presence of the bri1-116 allele if amplicons could not be cut into 314 bp and 238 bp fragments. To detect the BRL1 alleles, oligonucleotide 5′-CTGTAAAGCGCCATGACTAGC-3′ was combined with either 5′-ATATGGATGTTGCCGAATCTG-3′ (to detect the BRL1 wild-type allele) or 5′-ATTTTGCCGATTTCGGAAC-3′ (to detect the brl1 T-DNA insertion). To detect the BRL3 alleles, oligonucleotide 5′-TTTATCGAACACTTTGTGGGC-3′ was combined with either 5′-CCAGTGAACTCGTTTGAGCTC-3′ (to detect the BRL3 wild-type allele) or 5′-ATTTTGCCGATTTCGGAAC-3′ (to detect the brl3 T-DNA insertion). To detect the bri1-1 allele, a 224 bp genomic DNA fragment was amplified using oligonucleotides 5′-GCTAACAACACCAATTGGAAG-3′ and 5′-CTAACATGAATCAGTTCTTGATAT-3′. Restriction digest with EcoRV indicated presence of the bri1-1 allele if amplicons were cut into 201 bp and 23 bp fragments. PCR annealing temperature was 56°C, with an extension time of 1 min. The MAKR5::BRI1-CITRINE, BAM3::BRI1-CITRINE and CVP2::BRI-CITRINE constructs were created by replacing the respective coding sequences in described vectors (Kang and Hardtke, 2016; Rodriguez-Villalon et al., 2014, 2015) with the BRI1 coding sequence. The constructs were then transformed into homozygous brl1 brl3 double mutants that were heterozygous for the bri1-116 allele, and similar brl1^−/− brl3^−/− bri1^+/− plants that carried the transgenes were selected by genotyping in the T2 generation. Phenotypic analyses were performed in the subsequent T3 generation.