Resequencing of 3,497 FlcDNA in wild emmer and cultivated durum wheat
Two accessions of tetraploid wheat where included in the sequence capture experiment: 1) wild emmer accession (Triticum dicoccoides, PI 428082-2 from Turkey) selected from the natural population grown at the putative site of wheat domestication in Turkey; 2) durum wheat cultivar Langdon (Triticum turgidum var durum) adapted to grow in the northern parts of US. Genomic DNA isolated from the 3-week seedlings was used for library construction. DNA concentration was determined spectrophotometrically using Nanodrop-1000 (Thermo Scientific). For each genotype, 3 µg of genomic DNA dissolved in 60 µl of deionized water was fragmented to an average size of 200 bp by 15 min of sonication on ice at maximum intensity (Virsonic 50, Virtis). The following steps were performed according to the standard protocol of Agilent with slight modifications. Fragment-end repairing, A-tailed ligation, adapter’s ligation and final PCR were performed using the NEBNext® DNA Sample Prep Reagent kit. Average fragments size and molar concentration of the genomic libraries following sonication were estimated using Bioanalyser (Agilent). Fragment end-repairing was carried out by incubation of reaction mix for 30 min at 20°C (100 ul reaction volume,10 µl T4 DNA ligase buffer supplemented with 10 mM ATP, 4 µl dNTP, 5 µl T4 DNA polymerase, 1 µl Klenow enzyme and 5 µl T4 polynucleotide kinase). A-overhangs were added by incubating the library for 30 min at 37°C in a 50 µl final volume with 5 µl Klenow enzyme, 10 µl dATP and 3 µl Klenow exo (3’5’ exo-). Samples were purified on QIAquick columns (Qiagen) after each of these three steps. Adapter pools with different sequence tags (barcodes) were ligated to the wild emmer and durum wheat libraries. Ligation reactions were performed for 15 min at room temperature using 5 µl of DNA ligase in a 50 µl final volume. Samples were purified using MinElute columns (Qiagen). Size-selection of 200-300 bp fragments was performed on a 2% agarose gel followed by elution of DNA using Qiaquick columns (Qiagen). Eluted DNA was amplified by 14 cycles of PCR in a 50 µl reaction mix containing 0.4 µM primer-A (CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT), 0.4 µM primer-B (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and 25 µl Phusion High-Fidelity PCR Master Mix. Finally, PCR products were purified on QIAquick columns (Qiagen) and the quality of the libraries was assessed using Bioanalyser (Agilent). DNA concentration was determined using Nanodrop (Thermo Scientific). The concentration of library was adjusted to 147 ng/µl.    Hybridization and sequencing  Solution phase hybridization was performed according to Agilent’s standard protocol. In a 200 µl dome cap PCR tube, 250 ng of each DNA library were pooled with blockers number 1, 2 and 3 (Agilent SureSelect Kit), denatured for 5 min at 95°C and incubated 5 min at 65°C. In parallel, the hybridization solution was prepared by mixing buffers 1, 2, 3 and 4 from SureSelect kit while keeping the solution at 65°C. We then mixed 13 µl of hybridization solution, 7 µl of the library, 5 µl of pre-warmed (65°C) mix of SureSelect Oligo Capture Library, 1 ul of water and 1 µl of RNase block. A drop of mineral oil (Sigma) was further added on the top of the reaction mix to prevent evaporation and sample was incubated at 65°C for 24 hours in GeneAmp PCR System 9700 thermocycler (Applied Biosystems). The capture targets were then selected by pulling down the biotinylated bait/target with streptavidin-coated magnetic beads (Dyna M270 Streptavidin, Invitrogen). The obtained capture solution was desalted using MinElute columns (Qiagen). Two separate 18 cycle PCR amplification steps were performed with 1 µl capture target, 2.5 µl Herculase II fusion DNA polymerase (Stratagene), 0.625 mM dNTP, and 2.5 µl SureSelect GA PCR primers in a 50 µl final volume. PCR products were pooled and purified on QIAquick columns (Qiagen). The quality and concentration of the capture sample was assessed on Bioanalyser prior to sequencing on the Illumina GAII instrument as single-end 40 bp reads.
Kansas State University
Resequencing of 3,497 FlcDNA in wild emmer and cultivated durum wheat