Submitted to: Journal of the American Society for Horticultural Science
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/5/2003
Publication Date: 9/20/2003
Citation: Rowland, L.J., Smriti, M., Dhanaraj, A., Ehlenfeldt, M.K., Ogden, E.L., Slovin, J.P. 2003. Development of est-pcr markers for dna fingerprinting and mapping in blueberry (vaccinium, section cyanococcus). Journal of American Society for Horticultural Science. 128 (5):682-690. Interpretive Summary: The problem this research addresses is the need to develop a new, more reliable DNA marker sytem for blueberry useful for distinguishing (fingerprinting) cultivars, determining genetic relatedness, and for mapping purposes. To solve this problem, we have developed a new DNA marker system for blueberry based on DNA sequences of expressed genes, called EST-PCR (expressed sequence tag-polymerase chain reaction) markers. By testing the markers in a variety of different blueberry plants, we found that they were very effective at DNA fingerprinting and very useful for genetic relatedness and mapping studies. These markers can be used by scientists for breeding new blueberry cultivars and by scientists, breeders, and the nursery industry for DNA fingerprinting of cultivars.
Technical Abstract: Expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants, were used to develop EST-PCR markers for blueberry (Vaccinium spp). Thirty clones, picked at random from the cDNA library, were single-pass sequenced from the 5 and 3 ends. Thirty PCR primer pairs were designed from the ends of the best quality sequences that were generated and tested in amplification reactions with DNA from 19 blueberry genotypes, including two wild selections (the original parents of a mapping population), and 17 cultivars. Fifteen of the 30 primer pairs resulted in amplification of polymorphic fragments that were detectable directly after ethidium bromide staining of agarose gels. Several of the monomorphic amplification products were digested with the restriction enzyme AluI and approximately half resulted in polymorphic-sized fragments (cleaved amplified polymorphic sequences or CAPS markers). The polymorphic EST-PCR and CAPS markers developed in this study distinguished all the genotypes and many were polymorphic between the parent plants of the mapping population, indicating that these markers should have general utility for DNA fingerprinting and mapping in blueberry. Similarity values were calculated based on the molecular marker data, and a dendrogram was constructed based on the similarity matrix. Coefficients of coancestry were calculated for each pair of genotypes from complete pedigree information. A fair correlation between similarity coefficients calculated from marker data and coefficients of coancestry was found.