PCR-based length polymorphic SNP primers were designed by using the Primer 3-based Primer-BLAST suite embedded within the NCBI website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed on 16 August 2019)). The artificial mismatches and length polymorphisms for the SNP primers were created (Supplementary Table S6 ) as described by Qi et al. (2016) [33 (link)] and Long et al. (2017) [85 (link)] based on SNP flanking sequences. Polymerase chain reaction (PCR) for SNPs was conducted as described by Ma et al. (2020) [86 (link)], and the amplicons were separately visualized and scored on 6.5% polyacrylamide gel using an IR2 4300/4200 DNA analyzer (LI-COR, Lincoln, NE, USA).
After scoring each marker, the genotype data were chi-square (χ2) tested for goodness-of-fit to evaluate whether the segregation ratio for each marker fit the Mendelian ratios, e.g., 1:3 for dominant and 1:2:1 for codominant. Markers fitting the Mendelian ratios were used for linkage analysis with either the respective rust or DM phenotype data by using JoinMap 4.1 software, in which a regression mapping algorithm and Kosambi’s mapping function were selected [87 ]. The cutoffs for the linkage analysis among markers were set at a likelihood of odds (LOD) ≥ 3.0 and maximum genetic distance ≤ 50 centimorgans (cM).
After scoring each marker, the genotype data were chi-square (χ2) tested for goodness-of-fit to evaluate whether the segregation ratio for each marker fit the Mendelian ratios, e.g., 1:3 for dominant and 1:2:1 for codominant. Markers fitting the Mendelian ratios were used for linkage analysis with either the respective rust or DM phenotype data by using JoinMap 4.1 software, in which a regression mapping algorithm and Kosambi’s mapping function were selected [87 ]. The cutoffs for the linkage analysis among markers were set at a likelihood of odds (LOD) ≥ 3.0 and maximum genetic distance ≤ 50 centimorgans (cM).
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