The mechanisms by which natural populations generate adaptive genetic variation are not well understood. Some studies propose that microsatellites can function as drivers of adaptive variation. Here, we tested a potentially adaptive role for transcribed microsatellites with natural populations of the common sunflower (Helianthus annuus L.) by assessing the enrichment of microsatellites in genes that show expression divergence across latitudes. Seeds collected from six populations at two distinct latitudes in Kansas and Oklahoma were planted and grown in a common garden. Morphological measurements from the common garden demonstrated that phenotypic variation among populations is largely explained by underlying genetic variation. An RNA-Seq experiment was conducted with 96 of the individuals grown in the common garden and differentially expressed (DE) transcripts between the two latitudes were identified. A total number of 825 DE transcripts were identified. DE transcripts and nondifferentially expressed (NDE) transcripts were then scanned for microsatellites. The abundance of different motif lengths and types in both groups were estimated. Our results indicate that DE transcripts are significantly enriched with mononucleotide repeats and significantly depauperate in trinucleotide repeats. Further, the standardized mononucleotide repeat motif A and dinucleotide repeat motif AG were significantly enriched within DE transcripts while motif types, C, AT, ACC and AAC in DE transcripts, are significantly differentiated in microsatellite tract length between the two latitudes. The tract length differentiation at specific microsatellite motif types across latitudes and their enrichment within DE transcripts indicate a potential functional role for transcribed microsatellites in gene expression divergence in sunflower.

Microsatellites are common in genomes of most eukaryotic species. Due to their high mutability, an adaptive role for microsatellites has been considered. However, little is known concerning the contribution of microsatellites 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 3,325 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 2,379. 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 eSTRs, 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 transregulatory processes. Our findings suggest that a substantial number of transcribed microsatellites can influence gene expression. K E Y W O R D S gene expression, Helianthus annuus, microsatellite, sunflower | 1705 RANATHUNGE ET Al. S U PP O RTI N G I N FO R M ATI O NAdditional supporting information may be found online in the Supporting Information section.

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 FST 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.

ABSTRACTMicrosatellites 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.

Microsatellites have long been considered non-functional, neutrally evolving regions of the genome. Recent findings suggest that they can function as drivers of rapid adaptive evolution.Previous work on common sunflower identified 479 transcribed microsatellites where allele length significantly correlates with gene expression (eSTRs) in a stepwise manner. Here, a population genetic approach is used to test whether eSTR allele length variation is under selection. Genotypic variation among and within populations at 13 eSTRs was compared with that at 19 anonymous microsatellites in 672 individuals from 17 natural populations of sunflower from across a cline running from Saskatchewan to Oklahoma. Expected heterozygosity, allelic richness, and allelic diversity were significantly lower at eSTRs, a pattern consistent with higher relative rates of purifying selection. Further, an analysis of variation in microsatellite allele lengths (lnRV), and heterozygosities (lnRH), indicate recent selective sweeps at the eSTRs. Mean microsatellite allele lengths at four eSTRs within populations are significantly correlated with latitude consistent with the predictions of the tuning knob model which predicts stepwise relationships between microsatellite allele length and phenotypes. This finding suggests that shorter or longer alleles at eSTRs may be favored in climatic extremes. Collectively, our results imply that eSTRs are likely under selection and that they may be playing a role in facilitating local adaptation across a well-defined cline in the common sunflower.

Microsatellites have long been considered non-functional, neutrally evolving regions of the genome. Recent findings suggest that they can function as drivers of rapid adaptive evolution. Previous work on common sunflower identified 479 transcribed microsatellites where allele length significantly correlates with gene expression (eSTRs) in a stepwise manner. Here, a population genetic approach is used to test whether eSTR allele length variation is under selection. Genotypic variation among and within populations at 13 eSTRs was compared with that at 19 anonymous microsatellites in 672 individuals from 17 natural populations of sunflower from across a cline running from Saskatchewan to Oklahoma. Expected heterozygosity, allelic richness, and allelic diversity were significantly lower at eSTRs, a pattern consistent with higher relative rates of purifying selection. Further, an analysis of variation in microsatellite allele lengths (lnRV), and heterozygosities (lnRH), indicate recent selective sweeps at the eSTRs. Mean microsatellite allele lengths at four eSTRs within populations are significantly correlated with latitude consistent with the predictions of the tuning knob model which predicts stepwise relationships between microsatellite allele length and phenotypes. This finding suggests that shorter or longer alleles at eSTRs may be favored in climatic extremes. Collectively, our results imply that eSTRs are likely under selection and that they may be playing a role in facilitating local adaptation across a well-defined cline in the common sunflower.

Summary We present promor, a comprehensive, user-friendly R package that streamlines label-free (LFQ) proteomics data analysis and building machine learning-based predictive models with top protein candidates. Availability and implementation promor is freely available as an open source R package on the Comprehensive R Archive Network (CRAN)(https://CRAN.R-project.org/package=promor) and distributed under the Lesser General Public License (version 2.1 or later). Development version of promor is maintained on GitHub (https://github.com/caranathunge/promor) and additional documentation and tutorials are provided on the package website (https://caranathunge.github.io/promor/).

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 and are abundant in both coding and non-coding regions and could influence gene function and products. Here we tested the hypothesis that microsatellites contribute to gene expression divergence among species with 50 individuals from nine 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. Further, allele frequency differences measured by pairwise FST 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.