Lowbush blueberry (Vaccinium angustifolium, section Cyanococcus A. Gray, Ericaceae) is a unique agricultural crop of northeastern North America in that it is wild in origin. Plants are not sown but have been left to reclaim burned and cleared stretches of land over several centuries. In a sense, they have been domesticated not by breeding, but by cultural management practices. It is Maine's most important fruit crop and grows on over 24,000 ha (Yarborough, 1998). Management has focused on optimizing growth conditions, deterring competition from weeds, and minimizing the losses incurred by fungal diseases and insect damage (Yarborough, 1998). These cultural practices have resulted in a 4-fold increase in yield during the last several decades (Yarborough, 2004), but these kinds of increases are unlikely to continue in the absence of detailed knowledge of the reproductive genetics of this highly polymorphic species.
Lowbush blueberry is a complex tetraploid (2n = 4x = 48) proposed to be an autotetraploid of V. boreale (Camp, 1945) or an allotetraploid of V. boreale × V. pallidum or V. boreale × V. myrtilloides (Vander Kloet, 1977, 1978) but with tetrasomic inheritance (Hokanson and Hancock, 1998). It is morphologically and genetically polymorphic (Burgher et al., 2002; Hokanson and Hancock, 1998) and shows greatly varying yields among individual plants. The causes of these differences are unknown. Individual plants reproduce sexually on a 2-year cropping cycle in managed fields and asexually by underground rhizomes. Individual plants can span several meters in diameter, are almost certainly of different ages within fields, and appear as a mosaic patchwork of clones. The species is bee pollinated [rented honeybees (Apis mellifera L.)], which in managed fields with a very high density and acreage of lowbush blueberry clones likely results in a localized pollen distribution based on foraging behaviors and patterns of bees. Thus, a given clone is expected to receive significant amounts of its own pollen and that of its near neighbors (F.A. Drummond, personal communications). Native pollinators include more than 100 species of bees, including solitary bees (Andrenidae, Halictidae, Megacilidae) and bumble bees (Bombus Latreille) (Stubbs et al., 1996). In Maine, however, due to the recent decline of native pollinators, rented honeybees are generally stocked at approximately two to four hives per acre to achieve adequate pollination (Drummond, 2002). V. angustifolium is predominantly viewed as self-infertile, requiring outcrossing to achieve good yields (Aalders and Hall, 1961; Hall et al., 1979). A physical separation of male and female function within the same flower (herkogamy) may further enhance outcrossing and reduce pollen discounting (Harder and Wilson, 1998; Nagylaki, 1976).
Although managed intensively, the clones are of wild origin and their spatial genetic structure and relationship patterns are completely unknown. This knowledge is critical to test hypotheses that proximal clones who likely exchange pollen may experience inbreeding depression due to nearness of relationship and thus lower yields. Inbreeding depression has been clearly demonstrated in V. angustifolium through pollen chase experiments in which self pollen was “chased” with outcross pollen after 1 d and then 3 d (Hokanson and Hancock, 2000). The increased numbers of aborted seeds in the 3-d treatment versus the 1-d treatment indicates that self pollen does fertilize the ovules but often seeds do not develop properly. This is interpreted as early-acting inbreeding depression (Hokanson and Hancock, 2000) and has therefore been hypothesized as a possible cause of the depression of yields in patches within fields comprised of closely related individuals (Myra et al., 2004). The identification of molecular markers that are informative across multiple and varied blueberry genotypes would allow future testing of the inbreeding hypothesis by characterizing relatedness among participants in controlled field crosses.
Rowland et al. (2003b) developed express sequence tag-polymerase chain reaction (EST-PCR) markers from EST libraries derived from floral buds of cold acclimated and nonacclimated highbush blueberry plants (V. corymbosum). Surprisingly, ≈50% of the primers designed from the ends of the ESTs amplified polymorphic fragments among the tested genotypes that were detectable directly on agarose gels and thus represented scoreable length polymorphisms. This high level of polymorphism was explained as likely due to blueberry species being primarily outcrossing and, thus, highly heterozygous. The Cyanococcus taxon has been considered a very difficult one from a systematics perspective throughout the 20th century due to “intermittent but strong gene flow” (Vander Kloet, 1973) among populations in the section and the prevalence of polyploidy (Lyrene et al., 2003; Vander Kloet, 1978). Until now, the only molecular markers used in lowbush blueberry have been RAPD markers, which are anonymous markers amplified from short random primers, 10 bases in length (Burgher et al., 2002). Although EST technology is generally viewed first as a gene discovery tool for expression profiling, the development and use of EST-PCR products for phylogenetics and studies of intrapopulation genetic variation have been recently shown to be effective in other taxa such as Rhododendron L. (Wei et al., 2006) and Picea abies L. (Schubert et al., 2001).
For a variety of reasons, including better reproducibility than RAPDs and derivation from inferred coding regions, we asked whether we could reliably import these EST-PCR markers originally developed for highbush blueberry to fingerprint and estimate genetic similarity among clones within fields of lowbush blueberry. The study described here was designed to test whether these markers would reliably discriminate among five related species of blueberry that are represented in Maine and, in particular, whether they would be useful for estimating intraspecific variation within the wild, commercial V. angustifolium.
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