Developing molecular markers for marker assisted selection for resistance to raspberry bushy dwarf virus (RBDV) in red raspberry

A modified Bulked Segregant Analysis (BSA) was applied using eight RBDV resistant and eight susceptible raspberry plants. The best markers from this effort were applied to sixty seedlings of a 'Nootka' x WSU1499 population segregating for resistance to RBDV conferred by the Bu resistance gene. ELISA was used to identify resistant and susceptible genotypes in this population following graft inoculations. Three markers showed promise for use in Marker Assisted Selection (MAS); BC615_550, BC002_590 and BC296_425. BC615_550 proved to be the most predictive and reproducible marker and was cloned and sequenced. Specific primers were developed for this marker, resulting in the BC615_553_AluI Cleaved Amplified Polymorphic Sequence (CAPS) marker. This marker was applied to the segregating population and correctly identified the resistant plants in 58 out of 60 cases (96.7% accuracy). This marker was not present in other notable resistant sources, such as that derived from ‘Haida’ so a comparative approach based on assumed microsynteny between raspberry and strawberry was implemented. A BLAST search identified the putative location of the BC615_550 polymorphism in the diploid strawberry genome and primers were designed in candidate genes physically linked to this locus. Of note, six primer sets were designed to amplify different parts of a gene containing motifs homologous to the tobacco N-gene, a known virus resistance gene. One of the primer sets resulted in a Sequence Characterized Amplified Region (SCAR) marker, called rasp_N_gene_1202, that amplified in the resistant plants 'Nootka', 'Haida', and 'Willamette' in initial tests, while failing to amplify in the susceptible WSU1499. In the test population, this marker segregates perfectly with the original BC615_553_AluI marker. In addition to 'Nootka', 'Haida', and 'Willamette', it is present in a diverse set of known resistant genotypes including WSU 78117-1, 'Newburgh', 'Cowichan', 'Chilcotin', 'Malling Promise', 'Heritage' and 'Latham'. The marker is absent in known susceptible genotypes including 'Chilliwack', 'Tulameen', WSU 1276, 'Meeker', 'Autumn Bliss', 'Malahat' and WSU 1098. Screening of additional test populations is in progress for the evaluation of the marker’s utility in MAS for RBDV resistance in red raspberry.