The aim of this work was to evaluate the efficiency of real-time RT-PCR for detection of different isolates of ten important virus species that infect grapevines in Brazil: Grapevine leafroll-associated virus (GLRaV-1, -2, -3 and -5), Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fleck virus (GFkV) and Grapevine fanleaf virus (GFLV). The reactions consisted of individual (simplex) and simultaneous (duplex) virus detections. Thirty six grapevine accessions, regenerated after thermotherapy and tissue culture treatments, have been analysed. All the above-mentioned viruses were sensitively detected in simplex reactions in samples infected with different virus isolates. Specifically to GLRaV-1 it was necessary to mix reagents refered by different sources to achieve the amplification. GVA, GRSPaV, GLRaV-2 and GLRaV-3 combined with GVB, GFLV, GFkV, GVD and GLRaV-5 were accurately detected in duplex trials. It was shown, that real-time RT-PCR (TaqMan) is able to efficiently detect different local virus species and isolates.
This work aimed to develop and validate individual SNP molecular markers previously identified in a genetic association study of resistance to Meloidogyne incognita in soybean using a microarray panel. The markers identified in the array were converted in single TaqMan® markers. The single markers were used to create an SNP genotyping protocol and establish a marker-assisted selection (MAS) routine associated with resistance to M. incognita in soybean. Out of the eight TaqMan® assays tested, three were validated for use in MAS. The MAS protocol developed in this study uses sequential selection. Initially, molecular markers are used to identify susceptible plants; subsequently, the phenotypic evaluation of plants expressing resistance genotype for the markers is carried out, resulting in the accurate identification of resistant plants. The accuracy of this approach for MAS sequential for M. incognita varied from 94 to 96%.
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