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ARS Home » Southeast Area » Mayaguez, Puerto Rico » Tropical Crops and Germplasm Research » Research » Research Project #438360

Research Project: Sensitive qPCR Screening of U.S. Cacao Germplasm Collection in Puerto Rico for Presence of Emergent Badnaviruses

Location: Tropical Crops and Germplasm Research

Project Number: 6090-21000-057-06-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: May 1, 2020
End Date: Sep 30, 2021

Objective:
Research Objectives: 1. Screen the USDA cacao germplasm collections in Miami and Puerto Rico, and any symptomatic trees observed in the Trinidad and CATIE-Costa Rica collections for CaMMV/CYVBV using recently developed qPCR amplification assays to determine badnavirus distribution and simultaneously guide replacement of infected clones with uninfected trees, and removal of infected commercial trees; 2. Confirm identity of badnaviral species in representative samples found positive for CaMMV or CYVBV presence by PCR amplification via cloning and DNA sequencing; 3. Genotype cacao samples using cacao SNPs analysis (other funds, available) found to be CaMMV or CYVBV-infected.

Approach:
Primer and probe sets were previously designed, and validated to specifically detect CaMMV and CYVBV, and a cacao internal control gene, through quantitative PCR (qPCR) using the TaqMan system. The qPCR assay will be performed on collected samples. Virus-free cacao ‘amelonado’ plant DNA will be the negative control. Cacao samples will be tested with CaMMV specific primers, and symptomatic trees negative for CaMMV will be tested for CYVBV presence. Virus identification will be confirmed for selected samples using CaMMV or CYVBV-specific primers that amplify the RT-RNaseH gene. Virus identification will be accomplished by BLASTn and phylogenetic analysis of the RT-RNase H gene. Symptomatic cacao samples will be genotyped with 10,000 SNPs. The SNP data will be used to genotype and classify accessions based on 10 genetic cacao established genetic groups, with levels of genetic admixture using Structure software.