Kompetitive allele specific PCR marker development for Budbreak date and validation

Kompetitive allele specific PCR (KASP) markers are based on the dual Fluorescence Resonance Energy Transfer (FRET) method, in which the sample DNA is amplified with allele specific primers conjugated to fluorometric dyes HEX and FAM at their 5′ end. Based on GWAS results regarding the budbreak date, the following KASP primers were developed (LGC Genomics, Hoddesdon, UK) to target the most significantly associated SNP with this trait:

Allele A: 5′-AGGACAGCAATAAACTCAATCACACA-3′.

Allele C: 5′-GGACAGCAATAAACTCAATCACACC-3′.

Allele A tail (FAM tail): 5′-GAAGGTGACCAAGTTCATGCT-3′.

Allele C tail (HEX tail): 5′-GAAGGTCGGAGTCAACGGATT-3′.

Common reverse: 5′-AGGTTCTGCCAAGCTACAGGGTATA-3′.

These primers were developed by the BioGEVES laboratory (Beaucouzé, France) on the complementary strand. The KASP reaction and its components are explained at https://www.biosearchtech.com/support/education/kasp-genotyping-reagents/how-does-kasp-work. The KASP reaction was prepared as follows: 1.95 μL of PCR mix PACE (3CR Biosciences Limited, Harlow, UK), 2 μL of genomic DNA (2 ng/μL), and 0.053 μL of the three primers (Integrated DNA Technologies, Leuven, Belgium), for a total volume reaction of 4.003 μL. Amplification was performed in a hydrocycler, starting with 15 min at 94 °C, a touchdown phase of 10 cycles at 94 °C for 20 s and at 61 °C for 60 s with a 0.6 °C decrease in temperature per cycle, followed by 35 cycles of 94 °C for 20 s and 55 °C for 60 s. Once the cycle was complete, Fluostar Omega reader (BMG Labtech, Paris, France) was used to read the fluorescence signal. The KASP method was tested using DNA from a set of 96 unreleased breeding line accessions from the Walnut Improvement Program of the University of California, Davis.