2014 ASHS Annual Conference

Researchers with limited budgets and expertise may find it challenging to isolate and genotype microsatellite loci from plants, especially plants for which no prior genomic information is available.  Next-generation sequencing promises to be a rapid, economical approach to microsatellite isolation from plants, although enrichment of genomic DNA for simple sequence repeat (SSR) motifs often is required.  Such enrichment adds time and expense, and it may yield a biased sample of genomic SSRs.  Therefore, we evaluated the suitability of the Illumina Hiseq 2500 platform for isolation of microsatellites from raw genomic DNA of an obscure shrub, Dirca palustris L. (Thymelaeaceae), by next-generation sequencing without prior enrichment.  We used a Perl script developed by others to mine raw sequence data for SSRs and to conduct quality control of SSR loci.  We also evaluated the use of fluorescently tagged universal primers and multiplexed genotyping to decrease costs further.  Sequence data from 1/5 of a single lane comprised 26.89 million fragments, 19,200 of which contained di-, tri-, or tetra- nucleotide SSRs.  Among all SSR fragments, 5,280 contained flanking sequences suitable for stringent primer design.  Screening for copy number yielded 496 loci, most of which (89%) contained di-nucleotide SSRs.  We evaluated 38 tri-nucleotide loci for amplification and polymorphism, which yielded 12 loci that were polymorphic among plants from one population of D. palustris.  Several universal primers were used as tags for fluorescence-based genotyping of amplification products, which eliminated costs associated with fluorescently labeling a primer at each locus.  Multiplexing amplification products into groups of four loci for genotyping generated a 75% savings in genotyping costs compared with genotyping amplification products individually.  These approaches facilitate rapid and economical microsatellite identification and genotyping from plants with no prior genomic information available and with no SSR enrichment required.  Compared with traditional approaches, the time and cost of a microsatellite project can be reduced substantially by using these techniques.