Differential-Display Reverse Transcription-PCR for Unraveling the Molecular Mechanisms in Foot Rot (Phytophthora capsici sp. nov.) Tolerance in Black PepperPiper nigrum L.

Foot rot, caused by the oomycete Phytophthora capsici is the most devastating disease in black pepper. Understanding the genes and pathways involved in the field tolerance to foot rot is important in deciding the breeding, gene editing, and transgenesis strategies. Differential-display reverse transcription-PCR (DDRT-PCR) is a reverse genetic approach for gene finding. The protocol involves isolating the foot rot pathogen from infected plant material, preparing the inoculum, inoculatingboth tolerant and susceptible plants, collecting leaf samples at regular intervals, isolating RNA, generating the first-strand complementary DNA by reverse transcription, amplifying double-stranded DNA fragments using independent PCR reactions with common anchored and different arbitrary primers, resolving them through electrophoresis in denaturing urea polyacrylamide gel, developing the gel by silver staining, identifying the differentially expressed bands, eluting DNA from those bands, and sequencing to identify the genes related to foot rot tolerance in black pepper. This chapter details the materials and procedures involved from the selection of tolerant and susceptible cultivar to the identification of differentially expressed genes. Compared to the widely used RNA-Seq, DDRT-PCR is a less technology-intensive but cost-effective strategy, which can be practiced in a medium-class laboratory for identifying the major genes and pathways contributing to a specific trait.