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  Eur.J.Hortic.Sci. 81 (6) 327-338 | DOI: 10.17660/eJHS.2016/81.6.6
ISSN 1611-4426 print and 1611-4434 online | © ISHS 2016 | European Journal of Horticultural Science | Original article

A multiplexed microsatellite fingerprinting set for hazelnut cultivar identification

M. Akin1, A. Nyberg2, J. Postman2, S. Mehlenbacher1 and N.V. Bassil2
1Oregon State University, Department of Horticulture, Corvallis, OR 97331, USA
2USDA-ARS, National Clonal Germplasm Repository, Corvallis, OR 97333, USA

The objective of this study was to develop a robust and cost-effective fingerprinting set for hazelnuts using microsatellite markers, also known as simple sequence repeat (SSR) markers. Twenty SSRs containing repeat motifs of three or more nucleotides distributed throughout the hazelnut genome were screened on eight cultivars to assess polymorphism, allele size range, and ease of scoring. Six SSRs were discarded after genotyping 96 hazelnut samples either due to large allele bin widths and/or alleles that do not match the motifs, which complicates allele scoring. Fourteen polymorphic, easy-to-score SSRs were selected and amplified in a single multiplex. The 14-SSR multiplexed set generated the same alleles that were obtained when amplifying each SSR individually in the eight test accessions. SSR primer concentrations were then optimized to generate a clear signal for each locus. This 14-SSR fingerprinting set was used to genotype 102 hazelnut accessions from different origins. The fingerprinting set distinguished unique accessions mainly according to parentage and in some cases based on geographic origin. They identified each of the cultivars released from the Oregon State University breeding program and confirmed the parentage of six cultivars. Tools for DNA fingerprinting of clonally propagated horticultural crops like hazelnut are in demand and this multiplexed set constitutes a reliable, less-time consuming and cost-effective procedure for identity and parentage confirmation in hazelnut.

Keywords Corylus avellana L., genetic resources, identity, markers, simple sequence repeats, trueness-to-type

Significance of this study

What is already known on this subject?

  • Microsatellite markers are available in hazelnut and can distinguish hazelnut cultivars.
What are the new findings?
  • We found that microsatellite markers with a minimum of three core repeats have fewer artifacts than the more abundant dinucleotide-containing markers. After evaluating 20 SSRs in hazelnut, we developed a DNA test that contains 14 such SSRs and that can be amplified in a single PCR reaction. This DNA test distinguished all unique hazelnut cultivars tested with the exception of clonal duplicates in the field or in tissue culture.
What is the expected impact on horticulture?
  • This DNA test provides hazelnut growers, nurserymen, propagators, breeders, curators of collections and other scientists with an economical and reliable tool to confirm identity and parentage, detect propagation errors and enforce intellectual property.

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  • Baldoni, L., Cultrera, N., Mariotti, R., Ricciolini, C., Arcioni, S., Vendramin, G., Buonamici, A., Porceddu, A., Sarri, V., Ojeda, M., et al. (2009). A consensus list of microsatellite markers for olive genotyping. Mol. Breed. 24, 213–231.

  • Bassil, N.V., Boccacci, P., Botta, R., Postman, J., and Mehlenbacher, S.A. (2013). Nuclear and chloroplast microsatellite markers to assess genetic diversity and evolution in hazelnut species, hybrids and cultivars. Genet. Resources Crop Evol. 60, 543–568.

  • Bassil, N.V., Botta, R., and Mehlenbacher, S.A. (2005a). Microsatellite markers in hazelnut: isolation, characterization and cross-species amplification. J. Am. Soc. Hortic. Sci. 130, 543–549.

  • Bassil, N.V., Botta, R., and Mehlenbacher, S.A. (2005b). Additional microsatellite markers of the European hazelnut. Acta Hortic. 686, 105–110.

  • Bassil, N.V., Hummer, K., Botu, M., and Sezer, A. (2009). SSR fingerprinting panel verifies identities of clones in backup hazelnut collection of USDA genebank. Acta Hortic. 845, 95–102.

  • Beltramo, C., Valentini, N., Portis, E., Torello Marinoni, D., Boccacci, P., Sandoval Prando, M.A., and Botta, R. (2016). Genetic mapping and QTL analysis in European hazelnut (Corylus avellana L.). Mol. Breed. 36, 1–17.

  • Bhattarai, G. (2015). Microsatellite marker development, characterization and mapping in European hazelnut (Corylus avellana L.), and investigation of novel sources of eastern filbert blight resistance in Corylus. Dissertation, Oregon State University, Corvallis, USA.

  • Boccacci, P., Akkak, A., Bassil, N.V., Mehlenbacher, S.A., and Botta, R. (2005). Characterization and evaluation of microsatellite loci in European hazelnut (Corylus avellana L.) and their transferability to other Corylus species. Molec. Ecol. Notes 5, 934–937.

  • Boccacci, P., Aramini, M., Valentini, N., Bacchetta, L., Rovira, M., Drogoudi, P., Silva, A.P., Solar, A., Calizzano, F., Erdoğan, V. et al. (2013). Molecular and morphological diversity of on-farm hazelnut (Corylus avellana L.) landraces from southern Europe and their role in the origin and diffusion of cultivated germplasm. Tree Genetics & Genomes 9, 1465–1480.

  • Boccacci, P., Beltramo, C., Sandoval Prando, M.A., Lembo, A., Sartor, C., Mehlenbacher, S.A., Botta, R., and Torello Marinoni, D. (2015). In silico mining, characterization and cross-species transferability of EST-SSR markers for European hazelnut (Corylus avellana L.). Mol. Breed. 35, 1–14.

  • Boccacci, P., and Botta, R. (2010). Microsatellite variability and genetic structure in hazelnut (Corylus avellana L.) cultivars from different growing regions. Scientia Hortic. 124, 128–133.

  • Boccacci, P., Botta, R., and Akkak, A. (2006). DNA typing and genetic relations among European hazelnut (Corylus avellana L.) cultivars using microsatellite markers. Genome 49, 598–611.

  • Boccacci, P., Rovira, M., and Botta, R. (2008). Genetic diversity of hazelnut (Corylus avellana L.) germplasm in northeastern Spain. HortScience 43, 667–672.

  • Botstein, D., White, R.L., Skolnick, M., and Davis, R.W. (1980). Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics 32, 314–331.

  • Botta, R., Akkak, A., and Boccacci, P. (2005). DNA-typing of hazelnut: a universal methodology for describing cultivars and evaluating genetic relatedness. Acta Hortic. 686, 117–124.

  • Brookfield, J.F.Y. (1996). A simple new method for estimating null allele frequency from heterozygote deficiency. Mol. Ecol. 5, 453–455.

  • Campa, A., Trabanco, N., Pérez-Vega, E., Rovira, M., and Ferreira, J.J. (2011). Genetic relationship between cultivated and wild hazelnuts (Corylus avellana L.) collected in northern Spain. Plant Breeding 130, 360–366.

  • Dakin, E.E., and Avise, J.C. (2004). Microsatellite null alleles in parentage analysis. Heredity 93, 504–509.

  • FAOstat (2016). Agriculture data. (accessed May 25, 2016).

  • Gilmore, B.S., Bassil, N.V., and Hummer, K.E. (2011). DNA extraction protocols from dormant buds of twelve woody plant genera. J. Amer. Pom. Soc. 65, 201–207.

  • Gökirmak, T., Mehlenbacher, S.A., and Bassil, N.V. (2009). Characterization of European hazelnut (Corylus avellana) cultivars using SSR markers Genet. Resour. Crop Ev. 56, 147–172.

  • Gürcan, K., and Mehlenbacher, S.A. (2010a). Development of microsatellite marker loci for European hazelnut (Corylus avellana L.) from ISSR fragments Mol. Breed. 26, 551–559.

  • Gürcan, K., and Mehlenbacher, S.A. (2010b). Transferability of microsatellite markers in the Betulaceae. J. Amer. Soc. Hortic. Sci. 135, 159–173.

  • Gürcan, K., Mehlenbacher, S.A., Botta, R., and Boccacci, P. (2010a). Development, characterization, segregation, and mapping of microsatellite markers for European hazelnut (Corylus avellana L.) from enriched genomic libraries and usefulness in genetic diversity studies. Tree Genetics and Genomes 6, 513–531.

  • Gürcan, K., Mehlenbacher, S.A., and Erdogan, V. (2010b). Genetic diversity in hazelnut cultivars from Black Sea countries assessed using SSR markers. Plant Breeding 129, 422–434.

  • Jombart, T. (2008). adegenet: a R package for the multivariate analysis of genetic markers. Bioinformatics 24, 1403–1405.

  • Kalinowski, S.T., Taper, M.L., and Marshall, T.C. (2007). Revising how the computer program Cervus accommodates genotyping error increases success in paternity assignment. Mol. Ecol. 16, 1099–1106.

  • Kalinowski, S.T., Taper, M.L., and Marshall, T.C. (2010). Corrigendum. Mol. Ecol. 19, 1512–1512.

  • Kamvar, Z.N., Tabima, J.F., and Grünwald, N.J. (2014). Poppr: an R package for genetic analysis of populations with clonal, partially clonal, and/or sexual reproduction. PeerJ 2, e281.

  • Kasapligil, B. (1972). A bibliography on Corylus (Betulaceae) with annotations. Ann. Rep. North Nut Grow. Assoc. 63, 107–162.

  • Liu, K., and Muse, S. (2004). PowerMarker: new genetic data analysis software. Version 3.0.

  • Marshall, T.C., Slate, J., Kruuk, L.E.B., and Pemberton, J.M. (1998). Statistical confidence for likelihood-based paternity inference in natural populations. Mol. Ecol. 7, 639–655.

  • Mehlenbacher, S.A. (2014). Geographic distribution of incompatibility alleles in cultivars and selections of European hazelnut. J. Amer. Soc. Hortic. Sci. 139, 191–212.

  • Mehlenbacher, S.A., Brown, R.N., Nouhra, E.R., Gökirmak, T., Bassil, N.V., and Kubisiak, T.L. (2006). A genetic linkage map for hazelnut (Corylus avellana L.) based on RAPD and SSR markers. Genome 49, 122–133.

  • Nei, M. (1972). Genetic distance between populations. The American Naturalist 106, 283–292.

  • Nei, M. (1978). Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89, 583–590.

  • Paradis, E. (2010). pegas: an R package for population genetics with an integrated–modular approach. Bioinformatics 26, 419–420.

  • Paradis, E., Claude, J., and Strimmer, K. (2004). APE: Analyses of phylogenetics and evolution in R language. Bioinformatics 20, 289–290.

  • Peterschmidt, B. (2013). DNA markers and characterization of novel sources of eastern filbert blight resistance in European hazelnut (Corylus avellana L.). Dissertation, Oregon State University, Corvallis, USA.

  • Powell, W., Machray, G.C., and Provan, J. (1996). Polymorphism revealed by simple sequence repeats. Trends in Plant Science 1, 215–222.

  • R Core Team (2014). R: A language and environment for statistical computing (Vienna, Austria: R Foundation for Statistical Computing),

  • Sathuvalli, V.R., and Mehlenbacher, S.A. (2012). Characterization of American hazelnut (Corylus americana) accessions and Corylus americana × Corylus avellana hybrids using microsatellite markers. Genetic Resources and Crop Evolution 59, 1055–1075.

  • Sathuvalli, V.R., and Mehlenbacher, S.A. (2013). De novo sequencing of hazelnut bacterial artificial chromosomes (BACs) using multiplex Illumina sequencing and targeted marker development for eastern filbert blight resistance. Tree Genetics & Genomes 9, 1109–1118.

  • Testolin, R., and Cipriani, G. (2010). Molecular markers for germplasm identification and characterization. Acta Hortic. 859, 59–72.

Received: 20 September 2016 | Accepted: 21 November 2016 | Published: 23 December 2016 | Available online: 23 December 2016

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