Microsatellite Development and Characterization in Hazelnut

The genome sequences of ‘Jefferson’ and seven other hazelnut (Corylus avellana L.) cultivars allowed efficient in silico comparisons and identification of polymorphic SSRs. The ‘Jefferson’ genome was searched using MISA and 17,588 SSRs >15bp were identified. Removal of duplicates, short fragments, repeats at ends, and repeats containing only A’s and T’s reduced the number of unique fragments to 2069. The ‘Jefferson’ sequences were aligned with reads  from the seven other cultivars using MAQ and the results visualized in Tablet.  The 489 SSRs showed variation in repeat number and primers were designed for them. The PCR amplification and separation on agarose gels confirmed that 360 were polymorphic. Fluorescent forward primers were ordered and were used to amplify 48 diverse accessions plus the parents of the mapping population. After post-PCR multiplexing, samples were submitted for sizing using capillary electrophoresis. Allele sizes were determined using GeneMapper software, entered in a spreadsheet and analyzed using PowerMarker and Cervus software. A total of 1906 alleles were present at the 360 loci. The number of alleles per locus ranged from 2 to 15 with an average of 5.29. The mean observed heterozygosity, expected heterozygosity, polymorphism information content, and the frequency of null alleles were 0.52, 0.54, 0.49, and 0.03, respectively. These newly developed polymorphic microsatellite markers will be mapped and used for diversity studies, cultivar fingerprinting, and MAS.