Fig. 1
From: The utility of flow sorting to identify chromosomes carrying a single copy transgene in wheat
Experimental workflow. a Monovariate flow karyotype is dissected into small regions. From each region, 200 chromosomes are sorted. b Flow-sorted chromosomes are used as template for PCR with transgene-specific marker. c The region representing chromosome with the transgene identified on monovariate flow karyotype is dissected by sorting chromosomes from sort regions on bivariate flow karyotype. d From the sort gates on the bivariate flow karyotype, 100 chromosomes are sorted for PCR with transgene-specific marker. Moreover, 1000 chromosomes are sorted immediately afterwards onto the microscopic slides to identify flow-sorted chromosomes by FISH. e From the sort gate most enriched for the transgene-bearing chromosome (region G in this example), single chromosomes are sorted individually into PCR tubes. DNA of single-flow sorted chromosomes is amplified and resulting DNA is used as template for PCR to identify the presence of multiple transgene- and chromosome-specific sequences. This step unambiguously confirms the chromosome with integrated transgene