Development of High Resolution Melting Assay for quick and accurate molecular screening for the presence of functional Rpg5 alleles in barley.
A. Badea, B. Steffenson, W.G. Legge, and R. Brueggeman. 2016. Development of High Resolution Melting Assay for quick and accurate molecular screening for the presence of functional Rpg5 alleles in barley. 2016. 12th International Barley Genetics Symposium, Minneapolis, Minnesota, USA, June 26-30, 2016, Poster#14, Pp. 14. (poster presentation and abstract)
The highly virulent stem rust Puccinia graminis f. sp. tritici race TTKSK has emerged as a potentially serious threat to global wheat and barley production. Previous studies reported that susceptible barley genotypes such as Golden Promise (GP) contain a single cytosine insertion within the first exon resulting in a frame shift and a predicted truncated RPG5 protein. The molecular markers (Rpg5-LRK) that amplify across the leucine rich repeat (LRR) region to protein kinase domain in the resistant cultivars and from the LRR to protein phosphatase domain in the susceptible cultivars are used for routine screening for the presence of the functional Rpg5 gene. However, GP-like susceptible alleles will also amplify in this assay. Thus, the accessions producing this amplicon still need to be further analyzed for the presence of the GP allele that gives false positive results. This is performed by sequencing across the region, which is laborious and time consuming. Thus, a high-throughput method to identify the cytosine insertion is needed. High Resolution Melting (HRM) analysis is a relatively new, post-PCR analysis method that is gaining popularity for genotyping. To apply this methodology, primers for HRM assay were designed and validated by screening 26 barley accessions. These accessions were previously phenotyped for reaction to the TTKSK and QCCJ races. They were also screened with the Rpg5-LRK molecular markers and sequenced for the presence of the cytosine insertion. The HRM assay consistently and accurately distributed the 26 accessions into two independent clusters, with and without cytosine insertion, that perfectly match the sequencing results. Our findings suggest that the designed HRM assay is a robust genotyping method that can be used for high-throughput screening of germplasm for the presence of functional Rpg5 alleles and thus alleviating the need for direct sequencing of the amplicons.
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