Christine E. Edwards

Genetic architecture of the circadian clock and flowering time in Brassica rapa

The circadian clock serves to coordinate physiology and behavior with the diurnal cycles derived from the daily rotation of the earth. In plants, circadian rhythms contribute to growth and yield and, hence, to both agricultural productivity and evolutionary fitness. Arabidopsis thaliana has served as a tractable model species in which to dissect clock mechanism and function, but it now becomes important to define the extent to which the Arabidopsis model can be extrapolated to other species, including crops. Accordingly, we have extended our studies to the close Arabidopsis relative and crop species, Brassica rapa. We have investigated natural variation in circadian function and flowering time among multiple B. rapa collections. There is wide variation in clock function, based on a robust rhythm in cotyledon movement, within a collection of B. rapa accessions, wild populations and recombinant inbred lines (RILs) derived from a cross between parents from two distinct subspecies, a rapid cycling Chinese cabbage (ssp. pekinensis) and a Yellow Sarson oilseed (ssp. trilocularis). We further analyzed the RILs to identify the quantitative trait loci (QTL) responsible for this natural variation in clock period and temperature compensation, as well as for flowering time under different temperature and day length settings. Most clock and flowering-time QTL mapped to overlapping chromosomal loci. We have exploited micro-synteny between the Arabidopsis and B. rapa genomes to identify candidate genes for these QTL.

Quantitative Variation in Water-Use Efficiency across Water Regimes and Its Relationship with Circadian, Vegetative, Reproductive, and Leaf Gas-Exchange Traits

Drought limits light harvesting, resulting in lower plant growth and reproduction. One trait important for plant drought response is water-use efficiency (WUE). We investigated (1) how the joint genetic architecture of WUE, reproductive characters, and vegetative traits changed across drought and well-watered conditions, (2) whether traits with distinct developmental bases (e.g. leaf gas exchange versus reproduction) differed in the environmental sensitivity of their genetic architecture, and (3) whether quantitative variation in circadian period was related to drought response in Brassica rapa. Overall, WUE increased in drought, primarily because stomatal conductance, and thus water loss, declined more than carbon fixation. Genotypes with the highest WUE in drought expressed the lowest WUE in well-watered conditions, and had the largest vegetative and floral organs in both treatments. Thus, large changes in WUE enabled some genotypes to approach vegetative and reproductive trait optima across environments. The genetic architecture differed for gas-exchange and vegetative traits across drought and well-watered conditions, but not for floral traits. Correlations between circadian and leaf gas-exchange traits were significant but did not vary across treatments, indicating that circadian period affects physiological function regardless of water availability. These results suggest that WUE is important for drought tolerance in Brassica rapa and that artificial selection for increased WUE in drought will not result in maladaptive expression of other traits that are correlated with WUE.

Assembly, Gene Annotation and Marker Development Using 454 Floral Transcriptome Sequences in Ziziphus Celata (Rhamnaceae), a Highly Endangered, Florida Endemic Plant

Large-scale DNA sequence data may enable development of genetic resources in endangered species, thereby facilitating conservation efforts. Ziziphus celata, a federally endangered, self-incompatible plant species occurring in Florida, USA, is one species for which genetic resources are necessary to facilitate new introductions and augmentations essential for recovery of the species. We used 454 pyrosequencing of a Z. celata normalized floral cDNA library to create a genomic resource for gene and marker discovery. A half-plate GS-FLX Titanium run yielded 655 337 reads averaging 250 bp. A total of 474 025 reads were assembled de novo into 84 645 contigs averaging 408 bp, while 181 312 reads remained unassembled. Forty-seven and 43% of contig consensus sequences had BLAST matches to known proteins in the Uniref50 and TAIR9 annotated protein databases, respectively; many contigs fully represented orthologous proteins in TAIR9. A total of 22 707 unique genes were sequenced, indicating substantial coverage of the Z. celata transcriptome. We detected single-nucleotide polymorphisms and simple sequence repeats (SSRs) and developed thousands of SSR primers for use in future genetic studies. As a first step towards understanding self-incompatibility in Z. celata, we identified sequences belonging to the gene family encoding self-incompatibility. This study demonstrates the efficacy of 454 transcriptome sequencing for rapid gene and marker discovery in an endangered plant.

The Genetic Architecture of Ecophysiological and Circadian Traits in Brassica rapa

Developmental mechanisms that enable perception of and response to the environment may enhance fitness. Ecophysiological traits typically vary depending on local conditions and contribute to resource acquisition and allocation, yet correlations may limit adaptive trait expression. Notably, photosynthesis and stomatal conductance vary diurnally, and the circadian clock, which is an internal estimate of time that anticipates diurnal light/dark cycles, may synchronize physiological behaviors with environmental conditions. Using recombinant inbred lines of Brassica rapa, we examined the quantitative-genetic architecture of ecophysiological and phenological traits and tested their association with the circadian clock. We also investigated how trait expression differed across treatments that simulated seasonal settings encountered by crops and naturalized populations. Many ecophysiological traits were correlated, and some correlations were consistent with expected biophysical constraints; for example, stomata jointly regulate photosynthesis and transpiration by affecting carbon dioxide and water vapor diffusion across leaf surfaces, and these traits were correlated. Interestingly, some genotypes had unusual combinations of ecophysiological traits, such as high photosynthesis in combination with low stomatal conductance or leaf nitrogen, and selection on these genotypes could provide a mechanism for crop improvement. At the genotypic and QTL level, circadian period was correlated with leaf nitrogen, instantaneous measures of photosynthesis, and stomatal conductance as well as with a long-term proxy (carbon isotope discrimination) for gas exchange, suggesting that gas exchange is partly regulated by the clock and thus synchronized with daily light cycles. The association between circadian rhythms and ecophysiological traits is relevant to crop improvement and adaptive evolution.

Molecular Phylogeny of Conradina and Other Scrub Mints (Lamiaceae) from the Southeastern USA: Evidence for Hybridization in Pleistocene Refugia?

Abstract Conradina (Lamiaceae) consists of six allopatric species endemic to the southeastern United States, four of which are federally endangered or threatened. The limits and status of several taxa have been contested based on morphological grounds, and clarification of these limits is necessary for the design and implementation of effective and fiscally responsible protection and management plans. The objectives of this study were to investigate the monophyly of Conradina and its relationship to other endemic mints of the southeastern United States, to understand the patterns of diversification in Conradina, and to clarify species relationships. A molecular phylogeny was inferred by sequencing ITS and plastid regions from multiple accessions of each species of Conradina (except for a single accession of C. verticillata) and multiple individuals from species of Clinopodium, Dicerandra, Piloblephis, Stachydeoma, Monarda, Pycnanthemum, and Mentha. ITS sequence data strongly support the monophyly of Conradina, in agreement with evidence from morphology. In contrast, plastid sequence data do not support a monophyletic Conradina and place the genus as paraphyletic to Clinopodium, Stachydeoma, and Piloblephis. Similar plastid haplotypes are shared by different genera, perhaps due to shared ancestral polymorphisms, or more likely, introgression that occurred recently or during the Pleistocene. Within Conradina, ITS sequence data do not resolve species relationships, while plastid sequence data do not support the monophyly of most traditionally defined species of Conradina that are distinguishable morphologically. Species relationships in the plastid data set may also be obscured by introgression or ancestral polymorphism. More rapidly evolving sequence data from nuclear markers will be necessary to clarify relationships in Conradina and related mints from the southeastern United States.

Genetic architecture of life history traits and environment‐specific trade‐offs

Life history theory predicts the evolution of trait combinations that enhance fitness, and the occurrence of trade‐offs depends in part on the magnitude of variation in growth rate or acquisition. Using recombinant inbred lines, we examined the genetic architecture of age and size at reproduction across abiotic conditions encountered by cultivars and naturalized populations of Brassica rapa. We found that genotypes are plastic to seasonal setting, such that reproduction was accelerated under conditions encountered by summer annual populations and genetic variances for age at reproduction varied across simulated seasonal settings. Using an acquisition–allocation model, we predicted the likelihood of trade‐offs. Consistent with predicted relationships, we observed a trade‐off where early maturity is associated with small size at maturity under simulated summer and fall annual conditions but not under winter annual conditions. The trade‐off in the summer annual setting was observed despite significant genotypic variation in growth rate, which is often expected to decouple age and size at reproduction because rapidly growing genotypes could mature early and attain a larger size relative to slowly growing genotypes that mature later. The absence of a trade‐off in the winter setting is presumably attributable to the absence of genotypic differences in age at reproduction. We observed QTL for age at reproduction that jointly regulated size at reproduction in both the summer and fall annual settings, but these QTL were environment‐specific (i.e. different QTL contributed to the trade‐off in the fall vs. summer annual settings). Thus, at least some of the genetic mechanisms underlying observed trade‐offs differed across environments.

Selection during crop diversification involves correlated evolution of the circadian clock and ecophysiological traits in Brassica rapa

Crop selection often leads to dramatic morphological diversification, in which allocation to the harvestable component increases. Shifts in allocation are predicted to impact (as well as rely on) physiological traits; yet, little is known about the evolution of gas exchange and related anatomical features during crop diversification. In Brassica rapa, we tested for physiological differentiation among three crop morphotypes (leaf, turnip, and oilseed) and for correlated evolution of circadian, gas exchange, and phenological traits. We also examined internal and surficial leaf anatomical features and biochemical limits to photosynthesis. Crop types differed in gas exchange; oilseed varieties had higher net carbon assimilation and stomatal conductance relative to vegetable types. Phylogenetically independent contrasts indicated correlated evolution between circadian traits and both gas exchange and biomass accumulation; shifts to shorter circadian period (closer to 24 h) between phylogenetic nodes are associated with higher stomatal conductance, lower photosynthetic rate (when CO2 supply is factored out), and lower biomass accumulation. Crop type differences in gas exchange are also associated with stomatal density, epidermal thickness, numbers of palisade layers, and biochemical limits to photosynthesis. Brassica crop diversification involves correlated evolution of circadian and physiological traits, which is potentially relevant to understanding mechanistic targets for crop improvement.

Modeling development and quantitative trait mapping reveal independent genetic modules for leaf size and shape.

Improved predictions of fitness and yield may be obtained by characterizing the genetic controls and environmental dependencies of organismal ontogeny. Elucidating the shape of growth curves may reveal novel genetic controls that single-time-point (STP) analyses do not because, in theory, infinite numbers of growth curves can result in the same final measurement. We measured leaf lengths and widths in Brassica rapa recombinant inbred lines (RILs) throughout ontogeny. We modeled leaf growth and allometry as function valued traits (FVT), and examined genetic correlations between these traits and aspects of phenology, physiology, circadian rhythms and fitness. We used RNA-seq to construct a SNP linkage map and mapped trait quantitative trait loci (QTL). We found genetic trade-offs between leaf size and growth rate FVT and uncovered differences in genotypic and QTL correlations involving FVT vs STPs. We identified leaf shape (allometry) as a genetic module independent of length and width and identified selection on FVT parameters of development. Leaf shape is associated with venation features that affect desiccation resistance. The genetic independence of leaf shape from other leaf traits may therefore enable crop optimization in leaf shape without negative effects on traits such as size, growth rate, duration or gas exchange.

Using Population Genetic Data as a Tool to Identify New Species: Conradina cygniflora (Lamiaceae), a New, Endangered Species from Florida

Abstract Understanding species limits in recent radiations is often difficult because sufficient time may not have elapsed since speciation to allow for the accumulation of unique species-specific traits. However, because population genetic markers evolve rapidly, patterns of genetic structure based on these markers can often discern genetically isolated population groups, even when other markers, such as DNA sequences, can not. In this study, we analyzed patterns of genetic structure based on microsatellites in Conradina and identified a group of plants in Dunns Creek State Park (Putnam County, Florida) that are genetically divergent from all other described Conradina species. We then carried out a morphological study that revealed several unique characters in these populations, most notably thin-walled unicellular hairs, epidermis features, and larger calyces. Because these populations are genetically and morphologically diagnosable from all other Conradina species, we thus consider them to be specifically distinct, and name this taxon Conradina cygniflora. Conradina cygniflora is endemic to Dunns Creek State Park in south-central Putnam County, Florida, where it occupies nine tightly-clustered sites that probably form around two to four self-sustaining populations. Due to its extremely limited geographic distribution and few individuals, we recommend that Conradina cygniflora be listed as federally endangered.

Novel microsatellite loci for Agave parryi and cross-amplification in Agave palmeri (Agavaceae)

PREMISE OF THE STUDY To examine the foraging behavior of nectarivorous bats in southeastern Arizona, we developed microsatellite primers in Agave parryi. These markers were also tested for cross-amplification and applicability to assess patterns of genetic diversity and structure in A. palmeri. METHODS AND RESULTS Utilizing DNA sequence data from 454 shotgun sequencing, we identified seven novel polymorphic microsatellite loci in A. parryi and screened them for cross-amplification in A. palmeri. These markers were characterized in two populations of 30 individuals each for each species. In A. parryi, all primers were polymorphic and amplified between three and 12 alleles per population. In A. palmeri, all primers amplified, six were polymorphic, and allelic diversity ranged from one to 16 alleles per population. CONCLUSIONS Our results demonstrate the applicability of these microsatellite primers for population genetics studies in both A. parryi and A. palmeri.