Development of an efficient DNA test for genetic identity confirmation in blueberry

Cultivated highbush blueberries were first domesticated in the early 20^th century. Since then they have become not only a major US fruit crop but one that is grown in North America, South America, Europe, China, Japan, Australia and New Zealand. The United States Department of Agriculture (USDA) National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon, preserves the genetic diversity of this important crop and maintains a national collection of over 1,750 accessions representing 81 taxa from around the world. Clonal identity of the blueberries in this collection needs to be verified. DNA analysis can be useful to determine incorrect identities or duplicate germplasm. The objective of this study was to develop a minimal and effective blueberry fingerprinting set of reliable primer pairs. These primer pairs must be able to be multiplexed into a single reaction and yet differentiate genetic variants. Sixteen microsatellite or simple sequence repeat (SSR) primer pairs flanking core repeats of three nucleotides were screened for polymorphism and ease of scoring in simplex PCR using eight blueberry cultivars. Eight of these 16 SSRs were easy to score and generated well-spaced alleles that differed by 3 base pairs, the size of the repeat. When tested in multiplex PCR, each of five SSRs selected to amplify non-overlapping alleles generated the same alleles as those obtained by simplex PCR indicating lack of primer interaction. Furthermore, this 5 SSR-multiplexed set distinguished each of the eight cultivars and clustered them as expected based on their pedigrees. The ability to use these primers in a single PCR reaction instead of five separate reactions decreased the PCR cost and time needed for identity verification. This 5-SSR multiplexed set will be tested further for ability to distinguish each of the genetic variants in the diverse NCGR blueberry collection.