Analyses of diverse low alkaloid tobacco germplasm identify naturally occurring nucleotide variability contributing to reduced leaf nicotine accumulation.

Recent suggestions for mandated lowering of nicotine content in cigarettes have prompted tobacco breeders to search for N. tabacum germplasm with allelic variability contributing to low alkaloid accumulation. In this research, we phenotyped a series of 81 selected diverse tobacco introductions (TIs) to identify a sub-group with authentic low alkaloid phenotypes. We also genotyped these materials for sequences associated with the Nic1 and Nic2 loci previously reported to influence tobacco alkaloid biosynthesis. Only five low alkaloid TIs possessed previously described deletions of Ethylene Response Factor (ERF) genes at the Nic2 locus that contribute to lower alkaloid accumulation. Eleven TIs possessed an apparent deletion of ERF199, a gene recently reported to underlie the effect at the Nic1 locus. Quantitative trait locus (QTL) mapping was performed using populations derived from three selected low alkaloid TIs to possibly identify new genomic regions affecting alkaloid accumulation. A major QTL was identified on linkage group 7 in all three populations that aligned with the Nic1 locus. A newly discovered 5 bp deletion in the gene MYC2a on linkage group 5 was found to likely partially underlie the ultra-low alkaloid phenotype of TI 313. This new information is useful for tobacco breeders attempting to assemble novel genetic combinations with the potential for meeting future levels of tolerance for nicotine concentration in cigarette tobacco. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01274-5.

Transcribed microsatellite allele lengths are often correlated with gene expression in natural sunflower populations.

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 trans-regulatory processes. Our findings suggest that a substantial number of transcribed microsatellites can influence gene expression.

Patterns of microsatellite evolution inferred from the Helianthus annuus (Asteraceae) transcriptome.

The distribution of microsatellites in exons, and their association with gene ontology (GO) terms is explored to elucidate patterns of microsatellite evolution in the common sunflower, Helianthus annuus. The relative position, motif, size and level of impurity were estimated for each microsatellite in the unigene database available from the Compositae Genome Project (CGP), and statistical analyses were performed to determine if differences in microsatellite distributions and enrichment within certain GO terms were significant. There are more translated than untranslated microsatellites, implying that many bring about structural changes in proteins. However, the greatest density is observed within the UTRs, particularly 5'UTRs. Further, UTR microsatellites are purer and longer than coding region microsatellites. This suggests that UTR microsatellites are either younger and under more relaxed constraints, or that purifying selection limits impurities, and directional selection favours their expansion. GOs associated with response to various environmental stimuli including water deprivation and salt stress were significantly enriched with microsatellites. This may suggest that these GOs are more labile in plant genomes, or that selection has favoured the maintenance of microsatellites in these genes over others. This study shows that the distribution of transcribed microsatellites in H. annuus is nonrandom, the coding region microsatellites are under greater constraint compared to the UTR microsatellites, and that these sequences are enriched within genes that regulate plant responses to environmental stress and stimuli.

Gene expression assays for actin, ubiquitin, and three microsatellite-encoding genes in Helianthus annuus (Asteraceae).

PREMISE OF THE STUDY: The "tuning knob" model of King et al. (Endeavor 21: 36-40, 1997) postulates that microsatellite mutations can alter phenotypes in a stepwise fashion. Some proposed mechanisms involve regulation of gene expression. To study the effect of microsatellites harbored in untranslated regions on gene expression in Helianthus annuus, we have developed TaqMan assays for three microsatellite-encoding genes, and two constitutively expressed genes, actin and ubiquitin, to serve as standards. METHODS AND RESULTS: All five TaqMan assays yielded strong log-linear relationships between cycle threshold (C(T)) values and cDNA concentrations (R(2) = 0.98-0.99). Standard curves were based on five concentrations for each of five individuals. Efficiencies ranged from 0.83 to 1.03. CONCLUSIONS: The developed tools will allow for relative quantification of gene expression across individuals. Genotyping these loci will allow for testing the "tuning knob" hypothesis. Further, the actin and ubiquitin assays should be generally applicable to gene expression studies in H. annuus.

Characterization of long transcribed microsatellites in Helianthus annuus (Asteraceae).

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.