Template Preparation for TVL-PCR
DNA was isolated from unexpanded leaf tissue of Fragaria virginiana accession L2 (CFRA 1995) as described [6], except that no chloroform:octanol solution was included in the microfuge tube to which CTAB slurry was transferred. Per reaction, 400 ng of genomic DNA was digested with 20 U of Eco RI, Bam HI, or Hin dIII (New England Biolabs, Ipswich, Massachusetts) in a 40 μl reaction that was incubated overnight at 37°C. Employed restriction enzymes must produce recessed 3' (5' overhangs) or blunt ends. Digestion was verified by electrophoresis of 100 ng digested genomic DNA and undigested comparator on a 1% agarose TBE gel.
Repair of Fragment Ends and Addition of 3'A Overhang
End-repair employed 20 μl (200 ng) of each digested DNA sample with 1 μl of 10 mM dNTP mix, 1.3 μl sterile water, 2.5 μl of EconoTaq® buffer (Lucigen, Middleton, Wisconsin) and 0.2 μl (1 U) EconoTaq DNA polymerase. Reactions were incubated at 72°C for 30 minutes to fill in recessed 3' ends of cut sites, add a 3' adenosine overhang, and heat-inactivate the restriction enzyme.
Ligation
End-repaired DNA was ligated to the pCR4-TOPO vector (Invitrogen) using 4.5 μl (36 ng) end-repaired DNA solution, with 0.5 μl (5 ng) of TOPO vector and 1 μl of supplied salt solution. The reaction was gently mixed and incubated at room temperature for one hour.
Primer Design
The design of the gene-specific primers relied on alignments comprised of Superman-like sequences from four heterologous sources (Petunia, Nicotiana, Arabidopsis, and Malus), and strawberry Superman-like sequences obtained via conventional PCR amplification using degenerate primers targeted to sites conserved among the four heterologous sequences (results not shown). Two strategies were employed. For paralog mining of new Superman-like genes, degenerate primers were targeted to genomic sites identified as conserved among the heterologous and strawberry sequences. For sequence-specific walking to extend initially obtained gene segments, sequence-specific primers were targeted to genomic sites that were not conserved among the aligned heterologous and strawberry sequences. Genomic primers were always sited at least 50 bases upstream of the transition point from known to unknown sequence, to provide a sufficient read of known sequence in the resulting TVL-PCR product to confirm sequence identity and continuity.
TVL-PCR
The thermocycler profile used for both the first and second rounds of TVL-PCR was: initial denaturation at 94°C for 1 min; then 35 cycles of 94°C for 30 sec, 52°C for 30 sec, 68°C for 4 min; followed by a final extension at 68°C for 10 minutes. For each ligated template, two first-round amplification reactions were employed for the initial step of TVL-PCR, because the genomic fragment can ligate to the vector in alternate orientations (Figure 2). In one first-round TVL-PCR reaction (Figure 1C), the distal genomic primer (G1) was paired with the M13F or T3 vector primer, while in the other the G1 primer was paired with the M13R or T7 vector primer. In the first round, use of a distal M13_ (M13F or M13R) vector primer allows for subsequent vector primer nesting, while use of a proximal T_ (T3 or T7) vector primer precludes subsequent vector primer nesting (and is therefore recommended only if incompatibility exists between M13 primers and the distal genomic primer). For the second-round of TVL-PCR (Figure 1D), each first-round product was used as a template, and the nested genomic primer (G2) was paired with the appropriate T_ vector primer (T3 or T7, respectively - see below).
The 25 μl first-round TVL-PCR reactions contained 3 μl ligation reaction template, 0.5 μl of 20 μM G1 genomic primer, 0.5 μl vector primer, 0.1 μl (0.5 U) AccuPrime™ Taq DNA Polymerase High Fidelity (Invitrogen), and 2.5 μl 10× AccuPrime PCR buffer II. The first-round product (10 μl) was visualized on a 2% agarose 1% TBE gel run at 4.1 V/cm for 80 minutes. The second-round of TVL-PCR was performed with 1 μl of first-round product as the template, and 0.5 μl of 20 μM stock of both the nested G2 genomic primer and the appropriate vector primer. If the first-round vector primer was M13F or T3, then T3 was used as the second-round vector primer. If the first-round vector primer was M13R or T7, then T7 was used as the second-round vector primer.
Cloning and Sequencing of TVL-PCR Products
The amplification products from each second round TVL-PCR reaction were cloned using the Invitrogen TOPO TA Cloning® kit for sequencing, per the manufacturer's instructions. Transformed cells were plated on LB agar plates containing 50 μg/ml ampicillin and were grown at 37°C overnight. Colony PCR was performed using M13 primers provided with the TOPO cloning kit to confirm insert size. Plasmids were isolated from clones of interest using the Promega Wizard®Plus SV Minipreps DNA Purification System (Promega, Madison, WI). The plasmid inserts were then sequenced bidirectionally on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA), using M13 forward and M13 reverse sequencing primers. The T3 and T7 sequencing primers cannot be used for this purpose, because their priming sites may be represented twice in the final TOPO clone: once in the cloning vector itself and once in the cloned product of second round TVL-PCR (Figure 2).