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Genomic DNA from 192 Col x Ler F2 individuals was extracted using CTAB and used to generate sequencing libraries as described (Rowan et al; Yelina et al), with the following modifications. DNA was extracted from 3 rosette leaves of 5 week old plants and 150 ng of DNA used as input for each library. DNA shearing was carried out for 20 minutes at 37°C with 0.4U of DNA Shearase (Zymo research). The barcoded adapters used for library construction are listed in Rowan et al. Each set of 96 libraries was sequenced on one lane of an Illumina NextSeq500 instrument (300-cycle Mid Output run). Sequencing data was analysed to identify crossovers as previously reported, using the TIGER pipeline (Rowan et al; Yelina et al). Rowan BA, Patel V, Weigel D, Schneeberger K. Rapid and Inexpensive Whole-Genome Genotyping-by-Sequencing for Crossover Localization and Fine-Scale Genetic Mapping. G3 (Bethesda). 2015;5: 385–98. Yelina NE, Lambing C, Hardcastle TJ, Zhao X, Santos B, Henderson IR. DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis. Genes Dev. 2015;29: 2183–202. |