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Department of Plant Sciences University of Cambridge

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Namespace:Department of Plant Sciences University of Cambridge

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Protocols: Plants were grown at 20°C, 60% humidity with a long day photoperiod (16 hours light) and light intensity of 150 μmols. We applied a method to identify crossovers from low coverage (~0.5-2×) sequencing, using 96 barcoded genomic DNA libraries generated from recombinant individuals (Rowan et al. 2015), with the following modifications. DNA was digested with 0.3 units of dsDNA Shearase (Zymo Research) in a final volume of 15 l. The resulting DNA fragments were end-repaired with 3 units of T4 DNA polymerase (New England Biolabs), 10 units of T4 polynucleotide kinase (Thermo Fisher Scientific) and 1.25 units of Klenow Fragment (New England Biolabs), in the presence of 0.4 mM dNTPs in a reaction volume of 30 l for 30 min at 20 oC. DNA fragments were cleaned as described (Rowan et al. 2015), and the protocol followed until the DNA fragment size selection step. To size-select, following barcoded Illumina adaptor ligation, 30 l of a mixture of eight concentrated DNA libraries were combined in a tube containing 48 l of a 1:1 mix of AMPure XP magnetic SPRI beads (Beckman-Coulter. Hereafter, SPRI beads) in water. After 5 minutes incubation at room temperature, the samples were placed on a magnetic rack and allowed to clear before supernatant was transferred to a fresh tube and mixed with 0.12 volumes of undiluted SPRI beads. After 5 minutes incubation at room temperature, the tubes were placed on a magnetic rack and allowed to clear. The supernatants were discarded and the beads washed twice with 80% ethanol. DNA was eluted in 20 l of 10 mM Tris, pH 8.0. 12 l of the eluate was used for PCR amplification in a reaction volume of 50 l using KAPA HiFi HotStart ReadyMIx PCR Kit (Kapabiosystems) and the reported DNA oligonucleotides (Rowan et al. 2015). 12 cycles of amplification were performed and PCR products were then purified on SPRI beads (1× ratio) and quantified with a Bioanalyzer.