A comparison of freezer-stored DNA and herbarium tissue samples for chloroplast assembly and genome skimming.

Premise: The use of DNA from herbarium specimens is an increasingly important source for evolutionary studies in plant biology, particularly in cases where species are rare or difficult to obtain. Here we compare the utility of DNA from herbarium tissues to their freezer-stored DNA counterparts via the Hawaiian Plant DNA Library. Methods: Plants collected for the Hawaiian Plant DNA Library were simultaneously accessioned as herbarium specimens at the time of collection, from 1994-2019. Paired samples were sequenced using short-read sequencing and assessed for chloroplast assembly and nuclear gene recovery. Results: Herbarium specimen-derived DNA was statistically more fragmented than freezer-stored DNA derived from fresh tissue, leading to poorer chloroplast assembly and overall lower coverage. The number of nuclear targets recovered varied mostly by total sequencing reads per library and age of specimen, but not by storage method (herbarium or long-term freezer). Although there was evidence of DNA damage in the samples, there was no evidence that it was related to the length of time in storage, whether frozen or as herbarium specimens. Discussion: DNA extracted from herbarium tissues will continue to be invaluable, despite being highly fragmented and degraded. Rare floras would benefit from both traditional herbarium storage methods and extracted DNA freezer banks.

Transposase expression, element abundance, element size, and DNA repair determine the mobility and heritability of PIF/Pong/Harbinger transposable elements.

Introduction: Class II DNA transposable elements account for significant portions of eukaryotic genomes and contribute to genome evolution through their mobilization. To escape inactivating mutations and persist in the host genome over evolutionary time, these elements must be mobilized enough to result in additional copies. These elements utilize a "cut and paste" transposition mechanism that does not intrinsically include replication. However, elements such as the rice derived mPing element have been observed to increase in copy number over time. Methods: We used yeast transposition assays to test several parameters that could affect the excision and insertion of mPing and its related elements. This included development of novel strategies for measuring element insertion and sequencing insertion sites. Results: Increased transposase protein expression increased the mobilization frequency of a small (430 bp) element, while overexpression inhibition was observed for a larger (7,126 bp) element. Smaller element size increased both the frequency of excision and insertion of these elements. The effect of yeast ploidy on element excision, insertion, and copy number provided evidence that homology dependent repair allows for replicative transposition. These elements were found to preferentially insert into yeast rDNA repeat sequences. Discussion: Identifying the parameters that influence transposition of these elements will facilitate their use for gene discovery and genome editing. These insights in to the behavior of these elements also provide important clues into how class II transposable elements have shaped eukaryotic genomes.

The Agavoideae: an emergent model clade for CAM evolutionary biology.

Crassulacean acid metabolism - or CAM photosynthesis - was described in the early to mid-20th century, and our understanding of this metabolic pathway was later expanded upon through detailed biochemical analyses of carbon balance. Soon after, scientists began to study the ecophysiological implications of CAM, and a large part of this early work was conducted in the genus Agave, in the subfamily Agavoideae of the family Asparagaceae. Today, the Agavoideae continues to be important for the study of CAM photosynthesis, from the ecophysiology of CAM species, to the evolution of the CAM phenotype and to the genomics underlying CAM traits. Here we review past and current work on CAM in the Agavoideae, in particular highlighting the work of Park Nobel in Agave, and focusing on the powerful comparative system the Agavoideae has become for studying the origins of CAM. We also highlight new genomics research and the potential for studying intraspecific variation within species of the Agavoideae, particularly species in the genus Yucca. The Agavoideae has served as an important model clade for CAM research for decades, and undoubtedly will continue to help push our understanding of CAM biology and evolution in the future.

Transcriptomics of developing wild sunflower seeds from the extreme ends of a latitudinal gradient differing in seed oil composition.

Seed oil composition, an important agronomic trait in cultivated sunflower, varies latitudinally across the native range of its wild progenitor. This pattern is thought to be driven by selection for a higher proportion of saturated fatty acids in southern populations compared with northern populations, likely due to the different temperatures experienced during seed germination. To investigate whether these differences in fatty acid composition between northern and southern populations correspond to transcriptional variation in the expression of genes involved in fatty acid metabolism, we sequenced RNA from developing seeds of sunflowers from Texas, USA, and Saskatchewan, Canada (the extreme ends of sunflower's latitudinal range) grown in a common garden. We found 4,741 genes to be differentially expressed between Texas and Canada, including several genes involved in lipid metabolism. Several differentially expressed lipid metabolism genes also colocalized with known oil quantitative trait loci (QTL). The genes producing stearoyl-ACP-desaturases (SAD) were of particular interest because of their known role in the conversion of fully saturated into unsaturated fatty acids. Two SAD genes were more highly expressed in seeds from Canadian populations, consistent with the observation of increased levels of unsaturated fatty acids in seeds from that region. We also constructed a gene co-expression network to investigate regional variation in network modules. The results of this analysis revealed regional differentiation for eight of 12 modules but no clear relationship with oil biosynthesis. Overall, the differential expression of SAD genes offers a partial explanation for the observed differences in seed oil composition between Texas and Canada, while the expression patterns of other metabolic genes suggest complex regulation of fatty acid production and usage across latitudes.

Differential timing of gene expression and recruitment in independent origins of CAM in the Agavoideae (Asparagaceae).

Crassulacean acid metabolism (CAM) photosynthesis has evolved repeatedly across the plant tree of life, however our understanding of the genetic convergence across independent origins remains hampered by the lack of comparative studies. Here, we explore gene expression profiles in eight species from the Agavoideae (Asparagaceae) encompassing three independent origins of CAM. Using comparative physiology and transcriptomics, we examined the variable modes of CAM in this subfamily and the changes in gene expression across time of day and between well watered and drought-stressed treatments. We further assessed gene expression and the molecular evolution of genes encoding phosphoenolpyruvate carboxylase (PPC), an enzyme required for primary carbon fixation in CAM. Most time-of-day expression profiles are largely conserved across all eight species and suggest that large perturbations to the central clock are not required for CAM evolution. By contrast, transcriptional response to drought is highly lineage specific. Yucca and Beschorneria have CAM-like expression of PPC2, a copy of PPC that has never been shown to be recruited for CAM in angiosperms. Together the physiological and transcriptomic comparison of closely related C3 and CAM species reveals similar gene expression profiles, with the notable exception of differential recruitment of carboxylase enzymes for CAM function.

Development of mPing-based activation tags for crop insertional mutagenesis.

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing-based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

Hybridization History and Repetitive Element Content in the Genome of a Homoploid Hybrid, Yucca gloriosa (Asparagaceae).

Hybridization in plants results in phenotypic and genotypic perturbations that can have dramatic effects on hybrid physiology, ecology, and overall fitness. Hybridization can also perturb epigenetic control of transposable elements, resulting in their proliferation. Understanding the mechanisms that maintain genomic integrity after hybridization is often confounded by changes in ploidy that occur in hybrid plant species. Homoploid hybrid species, which have no change in chromosome number relative to their parents, offer an opportunity to study the genomic consequences of hybridization in the absence of change in ploidy. Yucca gloriosa (Asparagaceae) is a young homoploid hybrid species, resulting from a cross between Yucca aloifolia and Yucca filamentosa. Previous analyses of ∼11 kb of the chloroplast genome and nuclear-encoded microsatellites implicated a single Y. aloifolia genotype as the maternal parent of Y. gloriosa. Using whole genome resequencing, we assembled chloroplast genomes from 41 accessions of all three species to re-assess the hybrid origins of Y. gloriosa. We further used re-sequencing data to annotate transposon abundance in the three species and mRNA-seq to analyze transcription of transposons. The chloroplast phylogeny and haplotype analysis suggest multiple hybridization events contributing to the origin of Y. gloriosa, with both parental species acting as the maternal donor. Transposon abundance at the superfamily level was significantly different between the three species; the hybrid was frequently intermediate to the parental species in TE superfamily abundance or appeared more similar to one or the other parent. In only one case-Copia LTR transposons-did Y. gloriosa have a significantly higher abundance relative to either parent. Expression patterns across the three species showed little increased transcriptional activity of transposons, suggesting that either no transposon release occurred in Y. gloriosa upon hybridization, or that any transposons that were activated via hybridization were rapidly silenced. The identification and quantification of transposon families paired with expression evidence paves the way for additional work seeking to link epigenetics with the important trait variation seen in this homoploid hybrid system.

Range-wide phenotypic and genetic differentiation in wild sunflower.

BACKGROUND: Divergent phenotypes and genotypes are key signals for identifying the targets of natural selection in locally adapted populations. Here, we used a combination of common garden phenotyping for a variety of growth, plant architecture, and seed traits, along with single-nucleotide polymorphism (SNP) genotyping to characterize range-wide patterns of diversity in 15 populations of wild sunflower (Helianthus annuus L.) sampled along a latitudinal gradient in central North America. We analyzed geographic patterns of phenotypic diversity, quantified levels of within-population SNP diversity, and also determined the extent of population structure across the range of this species. We then used these data to identify significantly over-differentiated loci as indicators of genomic regions that likely contribute to local adaptation. RESULTS: Traits including flowering time, plant height, and seed oil composition (i.e., percentage of saturated fatty acids) were significantly correlated with latitude, and thus differentiated northern vs. southern populations. Average pairwise FST was found to be 0.21, and a STRUCTURE analysis identified two significant clusters that largely separated northern and southern individuals. The significant FST outliers included a SNP in HaFT2, a flowering time gene that has been previously shown to co-localize with flowering time QTL, and which exhibits a known cline in gene expression. CONCLUSIONS: Latitudinal differentiation in both phenotypic traits and SNP allele frequencies is observed across wild sunflower populations in central North America. Such differentiation may play an important adaptive role across the range of this species, and could facilitate adaptation to a changing climate.

Population Genetics of the Rubber-Producing Russian Dandelion (Taraxacum kok-saghyz).

The Russian dandelion, Taraxacum kok-saghyz (TKS), is a perennial species native to Central Asia that produces high quality, natural rubber. Despite its potential to help maintain a stable worldwide rubber supply, little is known about genetic variation in this species. To facilitate future germplasm improvement efforts, we developed simple-sequence repeat (SSR) markers from available expressed-sequence tag (EST) data and used them to investigate patterns of population genetic diversity in this nascent crop species. We identified numerous SSRs (1,510 total) in 1,248 unigenes from a larger set of 6,960 unigenes (derived from 16,441 ESTs) and designed PCR primers targeting 767 of these loci. Screening of a subset of 192 of these primer pairs resulted in the identification of 48 pairs that appeared to produce single-locus polymorphisms. We then used the most reliable 17 of these primer pairs to genotype 176 individuals from 17 natural TKS populations. We observed an average of 4.8 alleles per locus with population-level expected heterozygosities ranging from 0.28 to 0.50. An average pairwise FST of 0.11 indicated moderate but statistically significant levels of genetic differentiation, though there was no clear geographic patterning to this differentiation. We also tested these 17 primer pairs in the widespread common dandelion, T. officinale, and a majority successfully produced apparently single-locus amplicons. This result demonstrates the potential utility of these markers for genetic analyses in other species in the genus.

Molecular evolution of candidate genes for crop-related traits in sunflower (Helianthus annuus L.).

Evolutionary analyses aimed at detecting the molecular signature of selection during crop domestication and/or improvement can be used to identify genes or genomic regions of likely agronomic importance. Here, we describe the DNA sequence-based characterization of a pool of candidate genes for crop-related traits in sunflower. These genes, which were identified based on homology to genes of known effect in other study systems, were initially sequenced from a panel of improved lines. All genes that exhibited a paucity of sequence diversity, consistent with the possible effects of selection during the evolution of cultivated sunflower, were then sequenced from a panel of wild sunflower accessions an outgroup. These data enabled formal tests for the effects of selection in shaping sequence diversity at these loci. When selection was detected, we further sequenced these genes from a panel of primitive landraces, thereby allowing us to investigate the likely timing of selection (i.e., domestication vs. improvement). We ultimately identified seven genes that exhibited the signature of positive selection during either domestication or improvement. Genetic mapping of a subset of these genes revealed co-localization between candidates for genes involved in the determination of flowering time, seed germination, plant growth/development, and branching and QTL that were previously identified for these traits in cultivated × wild sunflower mapping populations.

Mitochondrial gene diversity associated with the atp9 stop codon in natural populations of wild carrot (Daucus carota ssp. carota).

Mitochondrial genomes extracted from the wild populations of Daucus carota have been used as a genetic resource by breeders of cultivated carrot, yet little is known concerning the extent of their diversity in nature. Of special interest is an SNP in the putative stop codon of the mitochondrial gene atp9 that has been associated previously with male-sterile and male-fertile phenotypic variants. In this study, either the sequence or PCR/RFLP genotypes were obtained from the mitochondrial genes atp1, atp9, and cox1 found in D. carota individuals collected from 24 populations in the eastern United States. More than half of the 128 individuals surveyed had a CAA or AAA, rather than TAA, genotype at the position usually thought to function as an atp9 stop codon in this species. We also found no evidence for mitochondrial RNA editing (Cytosine to Uridine) of the CAA stop codon in either floral or leaf tissue. Evidence for intragenic recombination, as opposed to the more common intergenic recombination in plant mitochondrial genomes, in our data set is presented. Indel and SNP variants elsewhere in atp9, and in the other 2 genes surveyed, were nonrandomly associated with the 3 atp9 stop codon variants, though further analysis suggested that multilocus genotypic diversity had been enhanced by recombination. Overall the mitochondrial genetic diversity was only modestly structured among populations with an F(ST) of 0.34.