Computational protocol: Genome wide SNP identification in multiple morphotypes of allohexaploid tall fescue (Festuca arundinacea Schreb)

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Protocol publication

[…] The amplicons sequenced were selected based upon their likelihood to be evenly distributed throughout the tall fescue genome, as determined by a comparative genome analysis involving wheat (Triticum aestivum L.), perennial ryegrass (Lolium perenne L.) and Brachypodium distachyon. The comparative sequence analysis between both wheat deletion bin-mapped ESTs [] (Additional File ) and perennial ryegrass ESTs [] and the B. distachyon 8X genome release [] was performed using the BLASTN search tool (best hits retained with e-value < 10-20) []. All primers were subsequently designed based on either wheat or perennial ryegrass EST template sequences using Primer3 software [], and were intended to amplify regions approximately 500 bp in length.PCRs were performed in a total volume of 10 μl, and contained 1 x Immolase PCR buffer, 1.5 mM MgCl2, 200 μM dNTPs, 0.25 μM each primer, 0.2 units Immolase DNA polymerase (Bioline) and approximately 5 ng of genomic DNA template. Cycling conditions for the perennial ryegrass EST-derived amplicons were as follows: an enzyme activation step of 95°C for 15 min, 30 cycles of 95°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute and a final extension of 72°C for 7 minutes. Amplicons generated using primers designed from wheat EST sequence template used an alternative annealing temperature of 60°C.Individual PCR products were purified with the addition of Agencourt AMPure® (Beckman Coulter) solution equivalent to 1.7 x the prior reaction volume. The magnetic beads were subsequently washed, and DNA was eluted according to the manufacturer’s protocol. A 30-fold dilution of the purified product was quantified using PicoGreen® (Life Technologies) following the manufacturer’s instructions. Amplicons were subsequently pooled to obtain an approximate equimolar concentration of each amplicon, based on the relative DNA concentration as estimated by PicoGreen® (Life Technologies) and the amplicon length. A separate pool of amplicons was generated for each genotype processed, resulting in a total of 13 pools. […]

Pipeline specifications

Software tools BLASTN, Primer3
Organisms Festuca arundinacea