20-Plex Gene Expression Analysis Using Hyperplex PCR (hpPCR) Toward the Development of Salt-Tolerant Wheat.

Climate change exacerbates the challenge of soil salinity, directly threatening the agricultural yields of staple crops such as wheat. Salinity currently affects at least 20% of total arable land and this figure is expected to worsen depending on future climate change trends. Aiming at improving the salt tolerance of wheat and ensuring future food security, researchers use, for example, multi-omics tools and genome-wide association studies (GWAS) to understand plant stress tolerance, with transcription factors playing a crucial role in regulating stress responses. Here, we describe the use of a next-generation PCR method, Hyperplex PCR (hpPCR), to analyze a 20-plex transcription factor panel focusing on three underexplored wheat transcription factor families-bHLH, C3H, and RAV-to identify their involvement in salt stress. All measured transcription factors, except one of the RAV (related to ABI3 and VP1) targets in shoot tissue, were observed to be downregulated in the presence of 100 mM NaCl. Compared to 2-step RT-qPCR, hpPCR measured the same upregulation and downregulation trends for all targets with up to 18 fewer PCR cycles.