KASP assay for Soil-borne wheat mosaic virus (SBWMV) resistance gene Sbwm1 in wheat
Contributed by Shubing Liu and Guihua Bai
Soil-borne wheat mosaic virus (SBWMV) is an important pathogen in winter wheat worldwide. In the U.S. soil-borne wheat mosaic is a serious wheat disease in the Great Plains and eastern U. S. winter wheat growing regions (1). SBWMV is a member in Furovirus genus, and can be transmitted to wheat roots by the plasmodiophorid vector Polymyxa graminis to cause the disease. SBWMV-infected plants show yellow to light green leaves with dark green mottling, stunting, reduced tillers, and low kernel and test weights (2). Reported yield losses due to SBWMV infection range from 10% to 30% and was reported to be up to 80% in seriously infected fields in the U. S. and 50% in Brazil (3-5).
Sbwm1, a gene for SBWMV resistance, has been mapped on the long arm of chromosome 5D of different populations (6-8). Using association mapping with 9K Infinium iSelect Beadchips, Liu et al (9) identified two SNP markers, wsnp_CAP11_c209_198467 and wsnp_JD_c4438_5568170 that were highly associated with SBWMV resistance and mapped within 3 cM from Sbwm1 in linkage mapping.
Two KASP assays, KASPwsnp_CAP11_c209_198467 and KASPwsnp_JD_c4438_5568170, were developed for the two SNPs. PCR can be run in a regular PCR thermal cycler and PCR product can be read in a Real-Time PCR Cycler or a fluorescence scanner following manufacturer instruction (http://www.kbioscience.co.uk/reagents/KASP_manual.pdf). A pre-plate scan before PCR is required to remove background fluorescence.
KASP assay for KASPwsnp_CAP11_c209_198467:
Forward primers: CACGCCATTAGCAGACGTACGTA -FAM ACGCCATTAGCAGACGTACGTG -HEX Reverse primer: GGGGAGTTCCCGTGTATATGTAAATAAAT
KASP assay for KASPwsnp_JD_c4438_5568170:
Forward primers: GCCATCAGAAGTATGGGCGACT -FAM CCATCAGAAGTATGGGCGACC -HEX Reverse primer: AAATGACTGGTCATCACCTTGTATCCTT
The diagnostic value of the two SNP was evaluated in a set of 205 SBWMV resistant and susceptible cultivars. In KASPwsnp_CAP11_c209_198467, ‘A’ allele co-segregated with SBWMV resistance and G allele co-segregated with SBWMV susceptibility (Figure 1). In KASPwsnp_JD_c4438_5568170, ‘A’ allele present in 96.5% of the resistant and ‘G’ allele present in 95% of the susceptible accessions.
Conditions presented here should be considered only as a starting point of the PCR optimization for individual laboratories.
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