Whole-genome resequencing of BC1 populations from Arabidopsis (Col x Ler) x Col crosses (wild type and ctf18)
This experiment aims at analyzing crossover distribution in the Arabidopsis spf2, ctf18 and sgo2 compared to wild type and each other. T1 plants were selfed in the T2 generation, and we selected for mutants that are Cas9-free based on selecting non-fluorescent seeds and validating the T2 plants by PCR, genotyping for the mutation in the gene, and validating via sequencing. If the T2 plant is homozygous for the mutation, then the plant was backcrossed to Ler before crossing it to sgo2-1, ctf18-mp193, and spf2-2 alleles in Col-0. If the T2 Ler plant had the heterozygous mutation, then the plant was crossed to its allele in Col-0. This way we produced, ctf18-mp193/ctf18-del at G181, spf2-3/spf2-del at L90 and sgo2-1/sgo2-del at K74 Col/Ler F1 hybrids. These F1 were validated by PCR for the mutations in both Col and Ler backgrounds and were selfed to produce F2 progeny, which were sequenced. Leaf samples from the self-crossed population were used for DNA purification and library preparation for Illumina sequencing (HiSeq 3000 2 × 150 bp), performed at the Max Planck-Genome-center (https://mpgc.mpipz.mpg.de/home/).
This experiment aims at analyzing crossover distribution in the Arabidopsis spf2, ctf18 and sgo2 compared to wild type and each other. T1 plants were selfed in the T2 generation, and we selected for mutants that are Cas9-free based on selecting non-fluorescent seeds and validating the T2 plants by PCR, genotyping for the mutation in the gene, and validating via sequencing. If the T2 plant is homozygous for the mutation, then the plant was backcrossed to Ler before crossing it to sgo2-1, ctf18-mp193, and spf2-2 alleles in Col-0. If the T2 Ler plant had the heterozygous mutation, then the plant was crossed to its allele in Col-0. This way we produced, ctf18-mp193/ctf18-del at G181, spf2-3/spf2-del at L90 and sgo2-1/sgo2-del at K74 Col/Ler F1 hybrids. These F1 were validated by PCR for the mutations in both Col and Ler backgrounds and were selfed to produce F2 progeny, which were sequenced. Leaf samples from the self-crossed population were used for DNA purification and library preparation for Illumina sequencing (HiSeq 3000 2 × 150 bp), performed at the Max Planck-Genome-center (https://mpgc.mpipz.mpg.de/home/).
European Bioinformatics Institute