A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grapevine and olive cultivars

Abstract

Background: Microsatellites are popular molecular markers in many plant species due to their stable and highly polymorphic nature. A number of analysis methods have been described but analyses of these markers are typically performed on cumbersome polyacrylamide gels or more conveniently by capillary electrophoresis on automated sequencers. However post-PCR handling steps are still required. High resolution melting can now combine detailed sequence analysis with the closed-tube benefits of real-time PCR and is described here as a novel way to verify the identity of plant varieties such as grapevine and olive.

Results: DNA melting profiles for various plant variety and rootstock samples were compared to profiles for certified reference samples. Two closely related grapevine rootstocks differing by as little as a single di-nucleotide repeat could be rapidly differentiated while there was high reproducibility of melting profiles for identical cultivars.

Conclusion: This novel microsatellite analysis method allows high sample throughput with greatly reduced time to results for varietal certification and is amenable to other microsatellite analyses.

Figure 1

Rootstock differentiation. Difference plot of the ZAG62 microsatellite for 6 closely related rootstocks derived from Vitis berlandieri crosses and Vitis riparia crosses: 5C, SO4, 125AA, 161-49C, 3309C and 420A. 4 DNA extractions for each rootstock were amplified in order to show the reproducibility of melting curves.

Figure 2

Detecting a single di-nucleotide repeat difference. Two closely-related rootstocks (5C and SO4) share one allele size (259 bp) and differ by a single CT repeat at the other allele (236 bp for 5C and 234 bp for SO4) for microsatellite locus VVMD32. Five individual extractions for 5C and SO4 show the differentiation of these two rootstocks

Figure 3

Rapid detection of incorrectly identified rootstock. A rootstock sample (*) stated as Schwarzmann matched 101–14 Mgt at microsatellite VVMD5 when melting curves were adjusted along the temperature axis (A). The differences in the melting curves are highlighted in the difference plot (B) using the plot for Schwarzmann as the baseline reference (horizontal line).

Figure 4

Scion variety differentiation. Temperature-shifted melting curves (A) and difference plots (B) for the differentiation of scion varieties for Pinot (allele sizes 238:244 bp), Syrah (244:250 bp), Sauvignon blanc (244:246 bp) and Chardonnay (242:244 bp) at microsatellite locus ZAG79. Duplicate samples for two clones of each variety were extracted and amplified. Allele sizes from [28].

Figure 5

Addition of reference cultivar to query samples. Reference DNA was added to query samples and amplified together with the reference and query samples amplified individually. (1) reference Riesling (2) query Riesling 1 (3) query Riesling 1/reference Riesling mixed sample (4) query Riesling 2 (5) query Riesling 2/reference Riesling mixed sample.

Figure 6

Analysis of Olive cultivars. Five DNA extractions for each olive cultivar 'Frantoio' and 'Koroneiki' showing distinctive melting profiles with the microsatellites DCA-3 (A) and UDO-12 (B).