Mutations that provide environment-dependent selective advantages drive adaptive divergence among species. Many phenotypic differences among related species are more likely to result from gene expression divergence rather than from non-synonymous mutations. In this regard, cis-regulatory mutations play an important part in generating functionally significant variation. Some proposed mechanisms that explore the role of cis-regulatory mutations in gene expression divergence involve microsatellites. Microsatellites exhibit high mutation rates achieved through symmetric or asymmetric mutation processes and are abundant in both coding and non-coding regions in positions that could influence gene function and products. Here we tested the hypothesis that microsatellites contribute to gene expression divergence among species with 50 individuals from five closely related Helianthus species using an RNA-seq approach. Differential expression analyses of the transcriptomes revealed that genes containing microsatellites in non-coding regions (UTRs and introns) are more likely to be differentially expressed among species when compared to genes with microsatellites in the coding regions and transcripts lacking microsatellites. We detected a greater proportion of shared microsatellites in 5′UTRs and coding regions compared to 3′UTRs and non-coding transcripts among Helianthus spp. Furthermore, allele frequency differences measured by pairwise F_ST at single nucleotide polymorphisms (SNPs), indicate greater genetic divergence in transcripts containing microsatellites compared to those lacking microsatellites. A gene ontology (GO) analysis revealed that microsatellite-containing differentially expressed genes are significantly enriched for GO terms associated with regulation of transcription and transcription factor activity. Collectively, our study provides compelling evidence to support the role of microsatellites in gene expression divergence.

• Premise of the study: Research on the evolutionary role of exonic microsatellites currently lacks an understanding of the evolutionary pressures that promote or limit their expansion. Contrasting levels of variability and genetic structures at anonymous and transcribed microsatellite loci of varying lengths are likely to provide useful insights regarding the relative strength of selection acting on different classes of microsatellites. We have developed primers for long transcribed microsatellites in Helianthus annuus to make these comparisons.• Methods and Results: Eight relatively long microsatellites from sequences in the expressed sequence tag database of H. annuus were characterized. A total of 63 individuals from three populations in Kansas were genotyped. The number of alleles per locus ranged from four to 11 with an average observed heterozygosity of 0.723.• Conclusions: Our study has generated suitable tools for studying the population genetics of long transcribed microsatellites that are potentially influenced by selection.

Microsatellites are common in most species. While an adaptive role for these highly mutable regions has been considered, little is known concerning their contribution towards phenotypic variation. We used populations of the common sunflower ( Helianthus annuus ) at two latitudes to quantify the effect of microsatellite allele length on phenotype at the level of gene expression. We conducted a common garden experiment with seed collected from sunflower populations in Kansas and Oklahoma followed by an RNA-Seq experiment on 95 individuals. The effect of microsatellite allele length on gene expression was assessed across 3325 microsatellites that could be consistently scored. Our study revealed 479 microsatellites at which allele length significantly correlates with gene expression (eSTRs). When irregular allele sizes not conforming to the motif length were removed, the number of eSTRs rose to 2379. The percentage of variation in gene expression explained by eSTRs ranged from 1–86% when controlling for population and allele-by-population interaction effects at the 479 eSTRs. Of these, 70.4% are in untranslated regions (UTRs). A Gene Ontology (GO) analysis revealed that eSTRs are significantly enriched for GO terms associated with cis- and trans-regulatory processes. These findings suggest that a substantial number of transcribed microsatellites can influence gene expression.