High-resolution melting analysis (HRM) is a resolutive technique, using PCR amplification and in-tube detection, which is based on the PCR product's melting analysis. It is a promising technique for breeding analysis, as it does not require dedicated sequencing equipment. It can be performed using QRT-PCR equipment that can be available in small-medium molecular biology laboratories or locally by the breeders, and it does not require an electrophoretic step to analyze the amplified DNA fragments. To develop effective HRM assays, the search for highly polymorphic sites amenable to PCR amplification is a prerequisite, which is not an easy task in wheat due to its genome complexity. The insertion site-based polymorphism markers (ISBP) are PCR markers designed based on the knowledge of the sequence flanking transposable element (TE) sequences. The two PCR primers are designed one in the transposable element and the other in the flanking DNA sequence. TEs are very abundant and nested in the wheat genome, with unique (genome-specific) insertion sites that are highly polymorphic. In this work, we have developed HRM-ISBP assays for wheat 3B and 4A chromosomes, and used them to analyze wheat diversity at previously defined drought and heat MQTL Loci. A candidate gene analysis of these loci derived key drought and heat responsive genes, validated by their differential expression patterns under stress conditions.

Funding support from projects P18-RT-992 from Junta de Andalucía (Andalusian Regional Government), Spain (Co-funded by FEDER), and by the Spanish Ministry of Science and Innovation project PID2019-109089RB-C32 are gratefully acknowledged.