Microsatellite Markers: Valuable in Vaccinium L.

Abstract

The United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, Corvallis, Oregon, maintains a collection of Vaccinium L. that contains >1,700 accessions representing 66 species from 33 countries. The mission of the National Clonal Germplasm Repository is to acquire, preserve, evaluate, document, and freely distribute crop germplasm. Microsatellite (syn. simple sequence repeat) markers provide a unique fingerprint for identifying germplasm accessions and are useful for genetic diversity analysis and in saturating genetic maps. The National Clonal Germplasm Repository Genetics Lab was the first to develop 39 simple sequence repeat primer pairs from expressed sequence tags and 10 simple sequence repeat primer pairs from a genomic microsatellite-enriched library of ‘Bluecrop’. Simple sequence repeat markers from these 49 primer pairs were identified for providing unique fingerprints for blueberry, cranberry, and ohelo berry; evaluating genetic diversity of wild and cultivated blueberry and southern highbush blueberry cultivars; assessing the effect of wide hybridization on genetic diversity and heterozygosity of the cultivated blueberry; populating the diploid Fl #10 (Fla4B x W85-20) x W85-23 and the tetraploid ‘Draper’ x ‘Jewel’ linkage maps; supplying a source of useful markers in 22 species representing nine Vaccinium sections; and use in population genetic analyses in diploid Vaccinium section Myrtillus taxa, such as V. calycinum, V. reticulatum, V. scoparium, and V. praestans. Over 450 simple sequence repeat primer pairs were recently designed from newly generated blueberry expressed sequence tags as part of a Specialty Crop Research Initiative project to develop genomic tools for blueberry, and will provide a valuable resource for the Vaccinium research and breeding community.

Keywords:

• blueberry
• cranberry
• ohelo berry
• simple sequence repeat markers

INTRODUCTION

The genus Vaccinium L. of the Ericaceae family contains more than 400 species of terrestrial or epiphytic long-lived woody shrubs or small trees (Vander Kloet, 1988). It is traditionally divided into 33 sections (Sleumer, 1941) Sections Cyanococcus (blueberries), Oxycoccus (cranberries), Vitis-idaea (lingonberries), Myrtillus (ohelo berry), and Vaccinium contain species that are either cultivated or managed from native stands (Luby et al., 1991). The United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository (USDA, ARS, NCGR), Corvallis, Oregon maintains a collection of these Vaccinium species. This collection contains >1,700 accessions representing 66 species from 33 countries. The mission of the NCGR is to acquire, preserve, evaluate, document, and freely distribute crop germplasm. DNA-based markers are valuable tools that can be used for germplasm characterization and efficient management of such a large Vaccinium collection.

Microsatellite [syn. simple sequence repeat (SSR)] markers are DNA-based markers that are sequence-specific and, as such, must be developed in the organism under study, in this case, Vaccinium. SSRs are valuable markers for genebanks, breeding programs, growers, and the nursery industry because they can be used for establishing a unique genetic fingerprint for each accession, validating cultivar identity, evaluating genetic diversity, and saturating genetic maps. Once a genotype is associated with the presence of a trait characteristic that is economically important, use of this marker enables marker-assisted breeding. The NCGR Genetics Lab was the first to develop 39 SSR primer pairs from an expressed sequence tag (EST) library of the V. corymbosum blueberry cultivar ‘Bluecrop’ [described in Dhanaraj et al. (2004)] and 10 SSR primer pairs from a genomic microsatellite-enriched library of ‘Bluecrop’ (Boches et al., 2005). This short review summarizes the use of these markers for germplasm characterization in sections Cyanococcus, Oxycoccus, and Myrtillus at the NCGR. Current progress on developing additional SSR markers and future directions of this work are also described.

MATERIALS AND METHODS

DNA was extracted from actively-growing leaves using a modified Puregene (Gentra Systems Inc., Minneapolis, MN, USA) protocol, used routinely in the NCGR lab. SSR primer development and screening was as previously described by Boches et al. (2005). For identifying SSR primer pairs that can be used outside the source species, V. corymbosum, Polymerase Chain Reaction (PCR) products were separated on a 2% agarose gel and visualized after ethidium bromide staining. The presence of a PCR product near the expected size indicated cross transference. Higher resolution capillary electrophoresis separation of the PCR products on a Beckman CEQ 8000 genetic analyzer (Beckman Coulter Inc., Fullerton, CA, USA) was used for most fingerprinting. The ABI 3100 (Applied Biosystems, Foster City, CA, USA) capillary electrophoresis system (OSU's Central Services Laboratory) was used for PCR product separation for a few primers (Boches et al. 2006a). Allele sizing and visualization were performed using the fragment analysis module of the CEQ 8000 software, or GeneScan and Genotyper for the ABI 3100 instrument.

RESULTS AND DISCUSSION

The goal of cross-amplification studies is to identify those SSR primer pairs developed in one species that can be applied to related species. An initial cross-amplification study of 36 V. corymbosum ‘Bluecrop’ EST-SSRs in a limited number of accessions (23) from 10 sections of the genus Vaccinium resulted in amplification in 17 to 100% of the genotypes (Boches et al., 2005). Cross transference was high at an average of 83% (Boches et al., 2005, 2006b). In fact, 16 loci amplified in all genotypes indicating possible usefulness across the whole genus Vaccinium.

In section Cyanococcus, SSR markers were used to identify genotypes (Boches et al., 2006a; Hinrichsen et al., 2008) and to evaluate genetic diversity of wild as opposed to cultivated blueberry (Boches et al., 2006a; Brevis et al., 2008). Blueberry SSR markers were able to distinguish 69 unique blueberry accessions irrespective of ploidy or species designation and group them according to pedigree (Boches et al., 2006a). Microsatellite analysis reflected a high level of heterozygosity in the 69 blueberry accessions as indicated by an average of 17.7 alleles per single locus and a Shannon's index (H) of 9.77. To develop a minimal fingerprinting set that is economical for cultivar identification, Hinrichsen et al. (2008) evaluated 12 of these SSRs in 75 blueberry cultivars. The use of two informative primer pairs (NA1040 + CA421) allowed the differentiation of each of the 75 cultivars (Hinrichsen et al., 2008). This information was used to establish a fingerprinting service in Chile that can allow nurserymen and growers to verify the identity of the genotypes they are handling. SSR-based analysis indicated a statistically significant decrease in genetic diversity among cultivated blueberries compared to wild blueberries (Boches et al., 2006a). However, substantial genetic diversity was still found in the cultivated blueberry gene pool. A similar conclusion was also reported by Brevis et al. (2008), where 21 single-locus SSRs were used to evaluate genetic relationships of southern highbush blueberry cultivars and to assess the effects of wide hybridization on the genetic diversity of these cultivars. Pedigree-based genetic distances and the SSR-based distance estimator, the proportion of shared alleles, were significantly correlated (r = 0.57, P < 0.0001), indicating that microsatellite markers are a reliable tool to assess the genetic relationships among southern highbush cultivars. The relative genetic contributions of V. angustifolium, V. corymbosum, V. darrowii, V. elliottii, V. tenellum, and V. virgatum clones to 38 southern highbush cultivars were also determined in this study.

In section Oxycoccus, amplification and polymorphism of ‘Bluecrop’-derived SSRs were higher in V. oxycoccos as compared to V. macrocarpon leading to 29 potential markers in the little-leaf cranberry and to 23 possible markers in the American cranberry (Bassil et al., 2008). Polymorphism in the EST-SSR loci that cross-amplified in cranberry ranged from 46.2% in V. macrocarpon to 66.7% in V. oxycoccos. Polymorphism in the genomic blueberry SSRs that cross-amplified in cranberry ranged from 83.3% in V. macrocarpon to 100% in V. oxycoccos. EST-SSRs resulted in a higher rate of amplification and a lower rate of polymorphism than those observed in genomic SSRs in both species, as previously observed in other plants (reviewed by Varshney et al. 2005). Nine of these SSRs identified multiple genotypes or variants in five cranberry cultivars (‘Stevens’, ‘Crowley’, ‘McFarlin’, ‘Olson's Honkers’, and ‘Pilgrim’) collected from 11 Oregon bogs and in two cultivars (‘Bergman’ and ‘Stevens’) collected from Vancouver. The most polymorphic and easy to score blueberry SSR primer pairs in cranberry were: CA794, NA1040, VCC_J3, and VCC_J9 (Bassil, unpublished). This small set can be used in two multiplexes to minimize the cost of cranberry identification.

Fruits of wild plants from section Myrtillus are highly aromatic and harvested mostly for home use as fresh or processed products. The fruit pulp, in addition to the skin, is intensely colored in Myrtillus, as opposed to green or white in blueberries (section Cyanococcus). In an attempt to develop sustainable ohelo (V. reticulatum, section Myrtillus) berry production in Hawaii, three clones of ohelo were selected for their highly attractive foliage and berries. On the islands of Hawaii and Maui, residents gather berries in the summer from the wild in disturbed, open sites at 640 to 3,700-meter elevation for use in jam, jelly, and pie fillings. Ten blueberry SSR primer pairs clearly identify the three ohelo berry clones (Bassil et al., 2010), now named ‘Nene’, ‘Kilauea’, and ‘Red Button’. The three selected ohelo clones will be released for nursery or foliage plant production. Conserved blueberry primers that amplify what appear to be single loci SSRs were recommended for population genetic analyses in the diploid Vaccinium section Myrtillus taxa including V. calycinum, V. reticulatum, V. scoparium, and V. praestans. SSR-based analysis indicated that six species within section Myrtillus included in this study are highly diverse and that the Hawaiian species, V. reticulatum and V. calycinum, seem to be more recently derived than other species of section Myrtillus. More representatives from each species and additional SSR- or sequence-based markers will be needed for clear taxonomic resolution.

CONCLUSIONS

SSR markers developed in blueberry have proved useful for identification of blueberry as well as cranberry and ohelo berry. These primer pairs have supplied a source of useful markers in 22 species representing nine Vaccinium sections and for future use in population genetic analyses in diploid Vaccinium section Myrtillus taxa, such as V. calycinum, V. reticulatum, V. scoparium, and V. praestans. They have detected high genetic diversity in cultivated blueberry and southern highbush blueberry cultivars. Their current use in populating the diploid Fl #10 (Fla4B x W85-20) x W85-23 and the tetraploid ‘Draper’ x ‘Jewel’ linkage maps is a prerequisite for enabling marker-assisted breeding in blueberry.

ACKNOWLEDGMENTS

This article is a concise review of the work of many individuals who were authors or co-authors on the original article that described the work and are cited herein. The author would like to acknowledge: Lisa J Rowland for sharing her EST sequences and expertise to enable SSR development; Peter Boches for developing and evaluating SSRs in blueberry; Adrienne Oda for genotyping cranberry; Patricio Hinrichsen for developing a minimal SSR fingerprinting set; Patricio Brevis for testing the impact of wide species hybridization in blueberry; Dorrie Main for developing an SSR development pipeline; Barbara Gilmore, April Nyberg, and Ted Bunch for technical assistance. Thanks also go to the author's many colleagues (Jim Ballington, Jim Hancock, and Kim Hummer) for collaborating on many of these studies. This research has been funded by the British Columbia Cranberry Growers Association, the Oregon Growers Association, the Washington Cranberry Alliance, the Northwest Center for Small Fruit Research, the USDA-ARS CRIS 5358-21000-033D, and the USDA-National Institute of Food and Agriculture-Specialty Crop Research Initiative Projects # 1275-21000-180-08R and # 2008-51180-04873.

Notes

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