|Botta, Roberto - UNIVERSITY OF TORINO, ITA|
|Mehlenbacher, Shawn - OREGON STATE UNIVERSITY|
Submitted to: Journal of the American Society for Horticultural Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 20, 2004
Publication Date: July 20, 2005
Citation: Bassil, N.V., Botta, R., Mehlenbacher, S. 2005. Microsatellite markers in hazelnut: isolation, characterization and cross-species amplification. Journal of the American Society for Horticultural Science. 130(4):543-549. Interpretive Summary: The objective of this study was to develop molecular techniques known as microsatellite markers, for identifying different types of hazelnut. Groups of reference DNA pieces, which contained mostly these repeated sequences, were made from hazelnut. These reference pieces are known as libraries. Each library contained different types of these simple repeated pieces of DNA that scientists call 'simple sequence repeats,' or SSRs. Specific DNA sequences (or primers) were made from regions on either side of the repeats. These primers were then used in a polymerase chain reaction (PCR) to amplify or make many copies of the specific repeated DNA sequence. Thirty-three primers were marked with fluorescence and used to investigate differences among 20 types of European hazelnut as well as seven other species. Twenty-five of 32 primer pairs were useful for detecting genetic differences among twenty cultivars of hazelnut. Using these primer pairs, we could distinguish between all the hazelnut species irrespective of geographical origin. These DNA markers will continue to be helpful in identifying different species and cultivated types of hazelnut plants.
Technical Abstract: Three microsatellite-enriched libraries of the European hazelnut, Corylus avellana L., were constructed: Library A for CA repeats, library B for GA repeats, and library C for GAA repeats. Twenty-five primer pairs amplified easy-to-score single loci and were used to investigate polymorphism among twenty C. avellana genotypes and to evaluate cross-species amplification in seven Corylus L. species. Microsatellite alleles were estimated by fluorescent capillary electrophoresis fragment sizing. The number of alleles per locus ranged from 2 to 12 (average = 7.16) in C. avellana and from 5 to 22 overall (average = 13.32). With the exception of CAC-B110, di-nucleotide SSRs were characterized by a relatively large number of alleles per locus (' 5), high average observed and expected heterozygosity (Ho and He > 0.6), and a high mean polymorphic information content (PIC ' 0.6) in C. avellana. In contrast, tri-nucleotide microsatellites were more homozygous (Ho = 0.4 on average) and less informative than di-nucleotide SSRs as indicated by a lower mean number of alleles per locus (4.5), He (0.59) and PIC (0.54). Cross-species amplification in Corylus was demonstrated. Microsatellites were highly heterozygous and polymorphic and differentiated among genotypes of Corylus avellana irrespective of geographical origin. These microsatellite markers will aid in fingerprinting genotypes of the European hazelnut and other Corylus species, genome mapping, and genetic diversity assessments.