PolyMarker

PolyMarker generates genome-specific Kompetitive Allele-Specific PCR (KASP) SNP assays for polyploid wheat by identifying genome-informative positions through alignments to International Wheat Genome Sequencing Consortium (IWGSC) chromosome survey sequences.

Key Features:

• Multiple Alignment Generation: Constructs multiple alignments between target SNP sequences and IWGSC chromosome survey sequences for each of the three bread wheat genomes to identify genome-specific differences.
• Genome-Informative Mask Creation: Produces a mask that highlights informative positions relative to the target genome to guide assay specificity.
• Automated KASP Assay Conversion: Converts identified SNPs into genome-specific Kompetitive Allele-Specific PCR (KASP) assays.
• Primer Selection via Local Alignments and Standard Tools: Leverages local alignments and standard primer design tools to select candidate primers specific to the target genome.

Scientific Applications:

• Marker development for disease resistance (Yr15): Enabled development of genome-specific SNP assays that mapped closely to the Yr15 yellow rust resistance locus, producing 28 polymorphic assays associated with the gene.
• RNA-Seq-based SNP discovery in segregating populations: Supported analysis of RNA-Seq data from bulked F2 pools to identify and classify over 27,000 genes with SNPs between parental lines.
• Candidate filtering and mapping: Applied stringent filtering including a bulk frequency ratio (BFR) threshold to reduce candidates to 175 genes and achieve high-resolution mapping within a 0.77-cM interval for marker-assisted selection.

Methodology:

Creates multiple alignments between target SNP sequences and IWGSC chromosome survey sequences for each of the three wheat genomes, generates a mask of genome-informative positions, converts identified SNPs into genome-specific KASP assays, and selects candidate primers using local alignments and standard primer design tools; in the Yr15 application, RNA-Seq on bulked F2 pools was used to identify >27,000 genes with SNPs, followed by BFR filtering to 175 candidates and generation of 28 polymorphic assays mapping within 0.77 cM.