Application of real-time PCR-based SNP detection for mapping of <i>Net2</i>, a causal D-genome gene for hybrid necrosis in interspecific crosses between tetraploid wheat and <i>Aegilops tauschii</i>

Abstract

Available information on genetically assigned molecular markers is not sufficient for efficient construction of a high-density linkage map in wheat. Here, we report on application of high resolution melting (HRM) analysis using a real-time PCR apparatus to develop single nucleotide polymorphism (SNP) markers linked to a hybrid necrosis gene, Net2, located on wheat chromosome 2D. Based on genomic information on barley chromosome 2H and wheat expressed sequence tag libraries, we selected wheat cDNA sequences presumed to be located near the Net2 chromosomal region, and then found SNPs between the parental Ae. tauschii accessions of the synthetic wheat mapping population. HRM analysis of the PCR products from F 2 individuals' DNA enabled us to assign 44.4% of the SNP-representing cDNAs to chromosome 2D despite the presence of the A and B genomes. In addition, the designed SNP markers were assigned to chromosome 2D of Ae. tauschii. The order of the assigned SNP markers in synthetic hexaploid wheat was confirmed by comparison with the markers in barley and Ae. tauschii. Thus, the SNP-genotyping method based on HRM analysis is a useful tool for development of molecular markers at target loci in wheat.

The cuticular wax inhibitor locus Iw2 in wild diploid wheat Aegilops tauschii: phenotypic survey, genetic analysis, and implications for the evolution of common wheat

“…The primer sequences for each SNP marker and any relevant restriction enzymes are summarized in Additional file 1. PCR and subsequent analyses were performed as described previously [31,32,35]. …”

Quantitative trait locus analysis for flowering-related traits using two F₂ populations derived from crosses between Japanese common wheat cultivars and synthetic hexaploids

“…tauschii . PCR and analysis of the CAPS and HRM markers were performed according to our previous studies Matsuda et al, 2012).…”

Discovery of High-Confidence Single Nucleotide Polymorphisms from Large-Scale De Novo Analysis of Leaf Transcripts of Aegilops tauschii, A Wild Wheat Progenitor

“…tauschii . 32 Primers for HRM analysis were designed for the remaining 17 contigs (including the three contigs that failed in the dCAPS analysis) to obtain PCR products of ∼100 bp. Only two of the 17 HRM primer sets failed in polymorphism detection, although both amplified genomic regions contained HC SNPs between PI476874 and IG47182.…”

“…All these markers were successfully mapped onto a linkage map constructed using 87 publicly available SSR markers. 26,32 The total map length was 1282.1 cM, with an average interval of 9.5 cM between markers. In total, 12 markers were distorted on chromosome 1D, 24 markers on chromosome 2D, 6 markers on chromosome 4D, and 10 markers on chromosome 5D.…”

“…31 The polymerase chain reaction (PCR) conditions for CAPS and dCAPS markers were 1 cycle of 94°C for 2 min and 40 cycles of 94°C for 30 s, 60°C (for CAPS) or 56°C (for dCAPS) for 30 s, and 68°C for 30 s. HRM analysis was performed using a LightCycler 480 Real-Time PCR System and LightCycler 480 HRM Master 2× reagents (Roche Diagnostics) according to our previous study. 32 For HRM analysis, all samples were spiked with 10–50% of PI476874 DNA to facilitate discrimination of the homozygous genotype. All PCR products were checked on a 1.5% agarose gel to ensure the presence of a single band.…”