Abstract
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 (ISBPs) are PCR markers designed based on the knowledge of the sequence flanking transposable element (TE) sequences. The two PCR primers are designed with 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 analyze the available HRM-ISBP assays for wheat 3B and 4A chromosomes, and update their applications in wheat diversity at drought and heat MQTL loci.
Highlights
Wheat is considered one of the most important crops worldwide [1]
Gene expression analysis was performed using the information previously published by Liu et al [29] [experimental seedling samples grown under controlled conditions (NCBI SRA ID SRP045409): control (IS), heat and PEG induced drought stress for 1 and 6 h (PEG1 and PEG6, respectively)], Ma et al [30] [experimental samples grown in a shelter (NCBI SRA ID SRP102636): anther stage irrigated leaf phenotype (AD_C), anther stage drought-stressed leaf phenotype (AD_S), tetrad stage irrigated developing spike phenotype (T_C), and tetrad stage drought-stressed developing spike phenotype (T_S)], and Gálvez et al [31] [flag leaf samples from field grown plants (NCBI SRA ID SRP119300): irrigated (IF), mild stress (MS), and severe stress (SS)]
Akhunov et al [32] and Munkvold et al [33] highlighted a positive gradient of gene density from the centromere to the telomeres in wheat, which is consistent with the low presence of insertion site-based polymorphism markers (ISBP) markers found in the MQTL3 [27] proximities
Summary
Wheat is considered one of the most important crops worldwide [1]. Its development and final yield are affected by abiotic stresses as drought [2,3] and heat [4,5], whose effects are being increased as consequence of predicted climate change [6,7]. Tolerance to these abiotic stresses are important aims in plant breeding to increase crops production [8] In this way, molecular markers as ISBP, can be developed and applied to identify genomic regions and genes of interest closely related to interesting traits as drought and heat tolerance [9]. ISBP are PCR markers designed based on the information of sequence flanking TE sequences [10] They appeared as powerful and interesting tools to apply in genomic and genetic studies in wheat [10,11,12], and have been used in marker-assisted selection (MAS) and as selecting tools in plant breeding programs [10]. We have assessed and updated the polymorphic HRM-ISBP assays developed for wheat chromosomes 4A and 3B, regarding their applications in wheat at drought and heat stresses MQTL loci
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