Submitted to: HortScience
Publication Type: Abstract only
Publication Acceptance Date: 1/31/2003
Publication Date: 8/20/2003
Citation: BOCHES, P., ROWLAND, L.J., BASSIL, N.V. CHARACTERIZATION OF EST-DERIVED MICROSATELLITE MARKERS IN BLUEBERRY. HORTSCIENCE. 2003. v.38(5): Abstract p. 737-738. Interpretive Summary: Blueberry DNA markers can now be used to verify the identity of blueberries and to confirm parentage. Our program examines tiny pieces of DNA made of sequence repeats that are 1 to 6 units long. The number of repeated units changes greatly between different blueberries, forming a marker. We initially tested 25 markers in ten samples of wild blueberry relatives. Five of them varied in size. These markers could distinguish between these types of wild blueberry. We applied these markers to some historical blueberry varieties, (more than 80 years old) including 'Pioneer', 'Rubel', 'Stanley', 'June', and 'Weymouth.' These varieties are ancestors of modern blueberries. Five markers were able to distinguish between these varieties, and the fingerprint of each variety reflected its origin. 'Weymouth' produced different sized markers than did 'Pioneer' or 'Rubel'. 'Rubel' was a wild selection from New Jersey obtained in 1911 and 'Pioneer' was a 1920 cross from wild selections in New Hampshire and New Jersey. Although 'Weymouth' shares one ancestor with 'Pioneer,' one of 'Weymouth's' other parents is an entirely different species, the lowbush blueberry. We were also able to distinguish between the closely related varieties 'Earliblue' and 'Bluecrop'. 'Earliblue' is a cross between 'Stanley' and 'Weymouth'; 'Bluecrop' has 'Stanley' as a grandparent, and shares many of the same wild ancestors as 'Weymouth.' The success of these markers in distinguishing the ancestry of blueberry varieties indicates that they will be useful for determining diversity and verifying identity of many modern cultivars.
Technical Abstract: Twenty-eight microsatellite-containing EST sequences were obtained from a highbush blueberry, Vaccinium corymbosum L. 'Bluecrop' cDNA library made from flower buds. The most common motif was dinucleotide repeat CT/GA. Trinucleotide repeat motifs were highly variable. Except for two CTT repeats, the remaining trinucleotide repeats were unique. Most of the SSR sequences were simple and the ratio of simple to compound to interrupted repeats was approximately 3:1:1. A BlastN search identified significant homology to known genes for 14 EST sequences while 14 sequences had no known homologues. Based on ClustalW alignment of homologous sequences, six of the simple sequence repeats (SSRs) were predicted to reside in an exon while eight were located in the 5' untranslated region (5'UTR). To date, 25 primer pairs were designed from the EST's and initially amplified in 10 Vaccinium species: V. corymbosum, V. corymbosum L. f. ashei, V. ovatum Pursh., V. arboreum Marshall, V. oldhami Miq., V. elliotii Chapm., V. simulatum Small, V. virgatum Aiton, V. bracteatum Thunb., as well as in V. hybrid 'Flordablue'. Of the 19 primer pairs that amplified fragments of predicted size, seven amplified in all species, and 12 amplified in at least 80% of species. Five primer pairs detected highly polymorphic loci (VcEST-23F, VcEST-112F, VcEST-169F, VcEST-236F and VcEST-247NAF). They were subsequently tested in a group of historically important cultivars of V. corymbosum, including 'Pioneer', 'Rubel', 'Stanley', 'June', 'Earliblue', 'Weymouth' and 'Bluecrop'. 'Pioneer', 'Rubel', and 'Stanley' had different alleles than 'Weymouth' for primers 23F, 112F, and 169F. This result agrees with the pedigree of 'Weymouth' which has two wild selections in its background ('Russell' and 'Chatsworth') that are not in the background of 'Rubel', 'Pioneer' and 'Stanley'. Primer pairs 112F and 236F successfully distinguished between the closely related 'Earliblue' and 'Bluecrop'. Polymorphism was also detected in two wild-collected V. corymbosum accessions. Blueberry ESTs provided SSR loci that will be useful for genetic diversity assessments as well as for cultivar and species identification.