Ir2 4300 4200 dna analyzer

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).

Phenotypic screening of the DM resistance was performed in the parents, HA 89 and HA 458, and in the selected H.pra 1 to H.pra 8 of H. praecox ILs using the North America (NA) Plasmopara halstedii race 734. This is a highly virulent race identified in USA in 2010 [44 ]. HA 458 is a known carrier of DM R-gene, Pl₁₇. Resistance for DM in these lines was tested using the whole seedling immersion method in the greenhouse under control conditions [35 (link),45 ]. The susceptible plants produced numerous white fungal spores on the abaxial surface of the cotyledons and true leaves, while the resistant plants lacked spores.
Genotyping of the parental lines, HA 89 and HA 458, and the eight selected ILs, H.pra 1 to H.pra 8 was performed using a simple sequence repeat (SSR) marker ORS963, and two single nucleotide polymorphism (SNP) markers, SFW04052 and SFW08268. These markers are tightly linked to the DM resistance gene Pl₁₇ [35 (link)]. A polymerase chain reaction (PCR) for the SSR and SNP markers was performed as described by Qi et al. [46 (link)] and Qi et al. [35 (link)], respectively. The PCR reactions were run on a Peltier thermocycler (Bio-Rad Lab, Hercules, CA, USA) and the products were size segregated in an IR² 4300/4200 DNA Analyzer with denaturing polyacrylamide gel electrophoresis (LI-COR, Lincoln, NE, USA).

The introgression lines of the BC₂F₅, along with the parental lines HA 89 and HA 458 (carrying Pl₁₇), were screened for resistance to downy mildew using the North America (NA) downy mildew race 734, a virulent race identified in USA in 2010 (Gulya et al., 2011 ). The whole seedling immersion method was used for the seedling tests as described by Gulya et al. (1991 ) and Qi et al. (2015 (link)). The susceptible plants showed an abundant white sporulation on the underside of the cotyledons and true leaves, while the resistant plants had no sporulation.
Simple sequence repeat (SSR) marker ORS963 and two single nucleotide polymorphism (SNP) markers, SFW04052 and SFW08268 that are linked to the Pl₁₇ downy mildew resistance gene were used to screen the introgression lines (Qi et al., 2015 (link)). Polymerase chain reaction (PCR) for SSR primers was performed on a Peltier thermocycler (Bio-Rad Lab, Hercules, CA, USA) with a touchdown program as described by Qi et al. (2011 (link)). Genotyping of the SNPs was performed using a newly developed technique of converting the SNPs into length polymorphism markers described by Qi et al. (2015 (link)). The PCR products were diluted 40–60 times and size segregated using an IR² 4300/4200 DNA Analyzer with denaturing polyacrylamide gel electrophoresis (LI-COR, Lincoln, NE, USA).