Efficient SSR Development in Hazelnut from in silico Comparisons

The genome sequence of 'Jefferson' hazelnut (Corylus avellana L.) (115× coverage), assembled from Illumina reads, serves as the reference genome sequence and allows efficient development of DNA markers.  Genomic DNA of seven other cultivars was also sequenced using Illumina.  Alignment of reads from these seven cultivars with the 'Jefferson' sequence allowed in silico comparisons and identification of polymorphic SSRs.  A search of the 'Jefferson' sequence for four types of simple sequence repeats (SSRs) (tri-, tetra-, penta- and hexa-repeats) for repeat lengths >15 bp using the MISA tool identified 17,588 SSR-containing fragments.  Short fragments (<400 bp), repeat motifs containing only A’s and T’s, and repeats at the ends of the fragments were removed.  The remaining 2,075 unique fragments were trimmed, retaining 250 bp on either side of the repeat motif.  Paired-end Illumina sequences from the seven accessions were aligned with each trimmed 'Jefferson' sequence using MAQ software.  Aligned sequences were inspected using Tablet, and 489 microsatellites that showed variation in number of repeats but conserved flanking regions were identified.  Primer pairs were designed for these SSRs and used for PCR amplification of 24 diverse accessions.  Electrophoresis on agarose gels confirmed polymorphism of >300 of these.  Polymorphic SSRs will be characterized by amplification of 50 accessions with fluorescent primers and sizing with capillary electrophoresis.  New SSR loci will be assigned to linkage groups based on segregation in one of our mapping populations.  This approach used Illumina genome sequences to efficiently develop hundreds of new polymorphic SSR markers for hazelnut.