Direct multiplex PCR for grapevine genotyping and varietal identification

Abstract

Grapevine cultivar identification is based mainly on two complementary methodologies: microsatellite (simple sequence repeat (SSR)) DNA analysis and traditional ampelography. Here, we report a direct multiplex PCR approach that allows the simultaneous amplification of 11 SSR loci from crude samples, i.e. bypassing DNA extraction, by using an engineered DNA polymerase improved to tolerate plant PCR inhibitors. Many different plant tissues were successfully amplified: leaf, root, wood, berry flesh and skin, stalk and must, from wine and table grape varieties, and rootstocks. The direct multiplex PCR that we propose is quicker and cheaper than the methodologies used until now, and provides accurate results. Thus, SSR DNA analysis becomes economically more accessible to a larger number of potential users in addition to research institutes.

Whole-genome genotyping of grape using a panel of microsatellite multiplex PCRs

“…Microsatellite and SNP markers have become in the last years the markers of choice for genetic analyses in grapevine, including genetic mapping and QTL identification (Huang et al 2012;Doligez et al 2013;Battilana et al 2013;Barba et al 2014), linkage disequilibrium and association analyses (Emanuelli et al 2010;Barnaud et al 2010;Cardoso et al 2012;Vargas et al 2013), and varietal identification (Myles et al 2010;Cabezas et al 2011;Laucou et al 2011;Migliaro et al 2012). As in many other woody plant species, genetic mapping in grapevine has been carried out using mainly the double pseudo test-cross strategy (Grattapaglia et al 1995), which is based on the study of allelic segregation of markers found in heterozygosis in one or both progenitors of an F 1 progeny.…”

“…For fragment analysis, another round of multiplexing was needed to develop 22 multi-loading sets combining up to four multiplex PCRs per load. Additionally, there are a few published studies in which multiplex PCR protocols have been developed and used for cultivar identification and/or germplasm characterization (Ibáñez et al 2009;Laucou et al 2011;Moreno-Sanz et al 2011;Migliaro et al 2012). Any case, none of these tools allows to carry out genome-wide studies in grapevine, such as genetic mapping or SSR-assisted backcrossing as proposed by Herzog et al (2013).…”

Development of SNAP markers based on nucleotide variability of WRKY genes in coconut and their validation using multiplex PCR

“…Multiplex PCR techniques have previously been developed to amplify either SSR or SNP markers (Hayden et al 2008;Kim et al 2012;Liu and Wu 2012;Wen and Zang 2012;Mousset et al 2015). Researchers also used multiplex PCR for plant genotyping, linkage and parental analysis of many plants (Masi et al 2003;Chaerani et al 2009;Migliaro et al 2012;Larekeng et al 2015a,b).…”

Direct DNA amplification from virgin olive oil for traceability and authenticity

“…Various enzymes have been tested; however, the results were not always efficient or successful. Unlike all procedures present in the literature [9,12,13,17,21], in our study we performed an easy protocol that just by filtration, without adding chemical solutions, allowed us to make direct amplification of DNA.…”

“…It was shown that the direct DNA amplification from substrates such as grape must and juice is possible [12], using a particular TaqDNA polymerase capable of tolerating PCR inhibitors. In our study, we employed a new experimental procedure for physical separation of DNA from VOO followed by PCR amplification with the DNA polymerase.…”