PCR-BASED GENOTYPING OF SNP MARKERS IN THEOBROMA CACAO
Authors: Roslina M.S., Nuraziawati M.Y, Aizat J., Ahmad K.M.J., Navies M., Alias A, Rosmin K., Neoh J.
Journal Issue: Malaysian Cocoa Journal, Volume 13(1)/2021
Keywords: Tetra ARM, PCR, SNPs, Cocoa, Traits
Published On: 1/11/2021
Abstract
The availability of reference sequence and sophisticated software does not always guarantee that the discovered SNP can be converted into a valid marker. Validation needs to be performed to ensure that the discovered SNP is in the Mendelian locus. The validation of a marker is the process of designing an assay based on the discovered polymorphism and then genotyping a panel of diverse germplasm and segregating population. Apart from that, the goal of the study was to develop a reliable, rapid, and inexpensive polymerase chain reaction (PCR)-based method to genotype for SNPs previously associated with desirable phenotypes in cocoa. Tetra-primer amplification refractory mutation system PCR (ARMS-PCR) is a simple and sufficient method for detecting different alleles in SNP locus. The allele-specific gene-tagged markers for the target genes are more effective than the genomic random markers as they will not show polymorphism in some recipient backgrounds and sometimes give up results as a false-positive marker. Allele-specific primer designs for the selected SNP from each trait. The primer is designed by using the software Primer1. DNA extracted from cocoa leaves was submitted to PCR amplification followed by agarose gel electrophoresis and determination of banding pattern. Tetra-primer ARMS-PCR was successfully optimized after changes in annealing temperature; annealing and extension times; concentration of MgCl2 and DNA; ratios of inner, outer, forward and reverse primer; and addition of adjuvants. There will be two types of product size expected if using an allele-specific primer set; one indicates that there is no SNP and another size will indicate there will be SNP/SNP is confirmed present in that specific sample. If there is any mismatch (SNP), a specific band size will be produced as compared to no mismatch (no nucleotide changes).