In order to expand ongoing Rosaceae genomics studies [29
], the original goal of this work was to use existing informatics resources to devise a PCR-based strategy to obtain plastid DNA sequence for cultivated strawberry and peach. This information would assist in identifying indel (insertion/deletion) polymorphisms or SNPs (single nucleotide polymorphisms) that could serve as an additional tool for phylogenetic analysis [30
] and also allow the design of vectors useful for strawberry plastid engineering. A schematic explanation of the technique is shown in Figure . If a given primer pair fails to generate an amplicon in initial PCR trials, the forward primer can then be paired with the reverse primer from the next primer pair to obtain coverage of that region. Figure demonstrates proof-of-concept, as universal primer set derived from the IR of five sequenced eudicots is sufficient to amplify the corresponding ~30 Kb region in commercial strawberry. The 27 primer pairs generate amplicons spanning this region. The corresponding PCR products were sequenced, and the sequence was immediately deposited to public databases. Here we proceeded from computational analyses to finished strawberry and peach IR sequence in one week for <$500.
Schematic representation of the ASAP approach. Three rounds of PCR allow for 100% coverage of a given region. F and R suffix to the numbers represent forward and reverse universal primers.
ASAP profile from Fragaria × ananassa. A. Round 1 touchdown PCR. B. Round 2 touchdown PCR and C. Round 3 touchdown extension PCR.
Since the method proved useful in strawberry and peach, its applicability across plant species was assessed. Total genomic DNA was derived from 13 diverse plant species and subjected to the ASAP protocol using the primers listed in Table and the conditions stated in Table . The ASAP primer set effectively generated expected amplicons from all eudicot species tested. Expected results were obtained in Nicotiana and Arabidopsis. Complete coverage was obtained with the first round of PCR and the amplicon sizes were consistent with predictions (Table ). Comparison of agarose gel electrophoresis profiles from the IR region revealed clearly discernible amplified fragment length polymorphisms (AFLPs) in the regions amplified with primer pairs 11, 17 and 27 (Figure ). The profiles for these two species are in complete agreement with the calculated sizes.
ASAP PCR primers. Primer sequences, annealing site and the relative position in tobacco (Nt), Arabidopsis (At) and maize (Zm) are presented, along with the anticipated amplicon size. * represent the primers with low sequence similarity in maize.
ASAP PCR conditions. The thermalcycler parameters used to generate ASAP amplicons in successive rounds of PCR are presented.
Figure 3 Composite ASAP PCR profiles from 8 plant species. At – Arabidopsis thaliana, Nt – Nicotiana tabacum, Cs – Citrus sinensis, Pp – Prunus persica, Le – Lycopersicon esculentum, Ah – Amaranthus hypochondriacus (more ...)
The maize plastid genome lacks the ycf2 open reading frame in the IR region, therefore primer pairs 3 to 9 failed to produce any amplicons, as anticipated (Figure ). Similarly, primer pairs 11, 26 and 27 did not produce any amplicons. Using bl2Seq program the maize IR region was compared with the associated primers and no significant sequence similarity was found between them. Interestingly, one primer each in primer pairs 1 and 10 had very low sequence similarity and yet the amplicons were obtained. Using three rounds of PCR (Table ) 100% coverage was obtained even in the monocot plastome (Figure ), indicating the applicability of eudicot-based primer designs to this taxonomic group. Absence of amplicons from primer pair 26 and 27 could be due to the fact that the primers annealed in the spacer region which could be unique to the eudicots. The results of the reactions are presented in both Table and Figure . Table presents the conditions required to produce the amplified regions from individual species, whether the products were obtained from PCR round 1, round 2 or round 3. Figure shows the complete array of amplified products corresponding to the amplification conditions presented in Table .
Zea mays ASAP PCR profiles. A. Round 1 and 2 touchdown PCR. B. Round 3 touchdown extension PCR and C. Composite profile from A and B.
ASAP coverage. Percentage coverage of the IR B region using ASAP method in 10 different genera and unique features of 4 diverse genera used in the study.
Fragaria and Prunus (Rosaceae)
Complete coverage of the plastid IR region from Fragaria was obtained after proceeding through three rounds of ASAP PCR (Table ) with the 27 pairs of primers (Figure ). These amplicons were generated using Pfu Turbo DNA polymerase (Stratagene Inc., Carlsbad, CA) in order to minimize potential errors generated during PCR reactions. These amplicons were directly sequenced in a 96-well format. The sequence was assembled and annotated as described in Methods.
Interestingly, in Prunus complete coverage was obtained with Round 2 PCR. Sequence comparison with Fragaria revealed that Prunus and Fragaria share considerable sequence similarity in the IR region as expected being from the same phylogenetic group. This is another demonstration of the utility of this technique where two members of the same taxonomic group were sequenced and compared in a very short time frame and in a cost-effective manner.
Other eudicots and identification of a variable region
The ASAP protocol was attempted in other eudicot species for which plastid genome sequence has not been reported. In Citrus and Lycopersicon complete coverage was obtained after Round 1 PCR and for the remaining species almost 99 – 100% coverage was obtained using Round 2 PCR conditions. Electrophoresis profiles revealed highly discernible AFLPs amongst different plant species. The most consistently variable region was represented by primer pair 11. In tobacco this amplicon represents sequences for orf87/ycf15, orf92, orf115, trnL and orf79. Gel electrophoresis profiles of amplicons generated from this primer pair revealed a great range of variability across all species tested (Figure ). AFLPs were discernable by gel electrophoresis between two solanaceous species, Nicotiana and Lycopersicon. Sequencing and alignment of this region from two members of Solanaceae, tobacco and tomato revealed a 95% – 98% sequence similarity in the aligning sequences. Tomato had two deletions in the region coding for orf92 and ycf15 in tobacco, which reconciles the smaller amplicon size. On the other extreme is the representative member of Caryophyllaceae, Amaranthus, where sequencing and subsequent alignments with tobacco revealed absence of ORFs between ycf2 and orf92 – trnL-CAA region (Figure ). Thus the ASAP method provides the advantage of analyzing a large region from a number of species and identifying a highly variable region at the same time.
Figure 5 A. PCR amplicons generated from primer pair 11 in 9 plant species. Fa – Fragaria × ananassa/Rosaceae, Pp – Prunus persica/Rosaceae, At – Arabidopsis thaliana/Crucifereae, Cs – Citrus sinensis/Rutaceae, Ch – (more ...)
To test the limits of this methodology, the same 27 pairs of primers were used against total DNA from Pisum sativum, Ginkgo biloba, Pinus taeda and Equisetum hyemale. These species represent a unique member of Fabaceae (Pisum – largest deletion resulting in removal of the rRNA cluster; has only one IR), an ancient and contemporary gymnosperm, and a pteridophyte. The primer pairs designed for eudicot plastid genomes were able to amplify the regions corresponding to primer pairs 14 to 25 in Pisum. In tobacco these primer pairs amplify the 98494 – 110052 nt region of the IR that includes the rrn operon. The bl2Seq program was used to determine the sequence similarity between the 27 primer pairs and the Pisum chloroplast genome sequence (Kindly provided by John Gray, John Innes Institute, UK). The observed amplicon patterns are consistent with what is anticipated from the sequence data. Primer pairs that failed to generate an amplicon do not share a significant sequence similarity with the Pisum plastid genome sequence (Figure ). In the two gymnosperms, similar amplicon patterns were generated from the rrn operon region. Again in the pteridophyte only the primer pairs corresponding to the rrn operon produced an amplicon. The Equisetum chloroplast genome does possess ycf2 gene but comparative sequence analysis with higher plant ycf2 revealed no significant sequence similarity. Interestingly the amino acid sequence similarity was almost 94% (data not shown).
Composite ASAP PCR profiles from 4 unique plant species. Ps – Pisum sativum, Gb – Gingko biloba, Pt – Pinus taeda, Eh – Equisetum hyemale.