Development of EST-PCR Markers for DNA Fingerprinting and Genetic Relationship Studies in Blueberry (Vaccinium, section Cyanococcus)

Abstract

<jats:p>Because randomly amplified polymorphic DNA (RAPD) is the only type of molecular marker that has been used extensively in blueberry (<jats:italic>Vaccinium</jats:italic> spp.) for mapping and DNA fingerprinting of cultivars, there is a need to develop a new, robust marker system. 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. 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 were tested in amplification reactions with genomic 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 <jats:italic>Alu</jats:italic>I 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 indicating that these markers should have general utility for DNA fingerprinting and examination of genetic relationships 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.</jats:p>

Spatial genetic structure of lowbush blueberry,Vaccinium angustifolium, in four fields in Maine

“…The protocols for genomic DNA extraction from leaf tissue, primer design from ESTs for generation of EST-PCR markers, PCR, and gel electrophoresis have been described in detail previously for highbush blueberry (Rowland et al 2003a(Rowland et al , 2003b. Briefly, frozen leaves were ground with dry ice in a coffee grinder and stored at -80 8C.…”

“…EST-PCR markers were initially developed for use in highbush blueberry (Vaccinium corymbosum L.) (Rowland et al 2003a(Rowland et al , 2003b. Primers were designed from EST libraries derived from cold-acclimated and non-acclimated floral buds of the highbush blueberry cultivar 'Bluecrop'.…”

“…Using random amplified polymorphic DNA (RAPDs) for fingerprinting and estimating genetic relationships among 26 select clones of lowbush blueberry from northern and western Nova Scotia, New Brunswick, and Maine, Burgher et al (2002) found weak association by geographical origin for the accessions from Nova Scotia and New Brunswick, but the Maine accessions shared a greater degree of similarity. More recently, we have demonstrated that expressed sequence tag -polymerase chain reaction (EST-PCR) markers developed from highbush blueberry (Rowland et al 2003a(Rowland et al , 2003b are useful for discerning genetic relationships among species of Vaccinium and also for estimating intraspecific genetic variability within and among clones in fields of lowbush blueberry (Bell et al 2008).…”

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“…3). The database was extremely useful in the identification and characterization of transcripts that are highly expressed during cold acclimation [8,9] and in the development of EST-based markers for mapping cold tolerance in blueberry [10]. A Blast application (fig.…”

Microsatellite markers for Vaccinium from EST and genomic libraries

“…Bluecrop DNA. EST libraries were constructed from cold acclimated (CA) and nonacclimated (NA) floral buds (Rowland et al . 2003; Dhanaraj et al .…”