RAD Sequence Data from OWB Genetic Map
OWB genomic DNA from the selected mapping population was digested with the restriction endonuclease SbfI and processed into RAD libraries similarly to the method of Baird et al 2008. Briefly, P0 (parental genotypes) and DH (progeny) genomic DNA (~300 ng; from pooled samples) was digested for 60 min at 37°C in a 50 µL reaction with 20 units (U) of SbfI (New England Biolabs [NEB]). Samples were heat-inactivated for 20 min at 65°C. 2.0 µL of 100 nM P1 Adapter(s), a modified Solexa© adapter (2006 Illumina, Inc., all rights reserved). SbfI P1 adapters each contained a unique multiplex sequence index (barcode) which is read during the first four nucleotides of the Illumina sequence read. 100 P1 nM adaptor were added to each sample along with 1 µL of 10 mM rATP (Promega), 1 µL 10× NEB Buffer 4, 1.0 µL (1000 U) T4 DNA Ligase (high concentration, Enzymatics, Inc), 5 µL H2O and incubated at room temperature (RT) for 20 min. Samples were again heat-inactivated for 20 min at 65°C, pooled and randomly sheared with a Bioruptor (Diagenode) to an average size of 500 bp. Samples were then run out on a 1.5% agarose (Sigma), 0.5× TBE gel and DNA 300 bp to 700 bp was isolated using a MinElute Gel Extraction Kit (Qiagen). End blunting enzymes (Enzymatics, Inc) were then used to polish the ends of the DNA. Samples were then purified using a Minelute column (Qiagen) and 15 U of Klenow exo- (Enzymatics) was used to add adenine (Fermentas) overhangs on the 3' end of the DNA at 37°C. After subsequent purification, 1 µL of 10 µM P2 adapter, a divergent modified Solexa© adapter (2006 Illumina, Inc., all rights reserved), was ligated to the obtained DNA fragments at 18°C. Samples were again purified and eluted in 50 µL. The eluate was quantified using a Qubit fluorimeter and 20ng of this product was used in a PCR amplification with 20 µL Phusion Master Mix (NEB), 5 µL of 10 µM modified Solexa© Amplification primer mix (2006 Illumina, Inc., all rights reserved) and up to 100 µL H2O. Phusion PCR settings followed product guidelines (NEB) for a total of 18 cycles. Samples were gel purified, excising DNA 300–650 bp, and diluted to 1 nM.